1) The study examined nasal lesion distribution patterns in rats from one inhalation study, two drinking water studies, and three gavage studies conducted by the NTP. 2) Nasal lesions from the inhalation study were predominantly bilateral, dorsal, and located in the anterior nasal sections. Lesions from the drinking water studies were uniformly located in the middle and posterior nasal sections and affected only the olfactory epithelium. 3) Nasal lesions from the gavage studies varied in character and distribution, tended to be more posterior and ventral, and were predominantly unilateral. Lesions in the nasopharyngeal duct only occurred in the gavage studies. 4) Differences in nasal lesion distribution provide clues

1. The Route of Exposure Influences Nasal Lesion Distribution in Rats in NTP Studies
Karen Y. Cimon1, Rebecca R. Moore2, Rodney A. Miller1, Gabrielle A. Willson1, Arun R. Pandiri3, and David E. Malarkey3
1Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, USA, 2 Integrated Laboratory Systems, Research Triangle Park, NC, USA,
3Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
Introduction: Nasal toxicity is not only observed in inhalation exposure
studies but also in drinking water and gavage studies. Nasal toxicants may
act systemically, directly, or both and the occurrence and distribution may
provide clues to the pathogenesis. This review focuses on nasal lesion
occurrence, features, and distribution as seen in a chronic whole body
inhalation study compared to two drinking water and three gavage studies
with nasal toxicity conducted by the NTP.
Methods: We examined all 3 routine sections of nose from all control and
high dose male and female F344N rats. The general distribution of nasal
and nasopharyngeal duct lesions was recorded and mapped as
predominately anterior or posterior; dorsal or ventral; and unilateral or
bilateral.
Results: Nasal lesions in the inhalation study were of varying severity,
occurred predominately as bilateral and dorsal; more frequently in the
anterior sections (98%) of the nose and less often (18%) in the most
posterior section of the nasal cavity. In the 2 drinking water studies, the
nasal lesions were uniformly observed in the middle and posterior nasal
sections; only olfactory epithelium was affected; and lesions had a dorsal
and bilateral distribution in 90% or 100% of the rats. In the 3 gavage
studies, the nasal lesions varied in character and distribution; tended to be
more posterior and ventral, and were predominately unilateral. Lesions in
the nasopharyngeal duct occurred irregularly and only in the gavage
studies. The changes in the gavage studies were similar in male and
female rats.
Impact Statement: Most inhalation or systemically induced nasal effects
are uniform in tissue specificity and/or distribution, thus affecting tissues in
anatomically consistent manner. The nasal lesions in the 3 gavage studies
resembled those described as related to gavage-related reflux. When
patterns of distribution of nasal lesions are not uniform or not consistent
with known patterns of induced lesions, then one must consider an
alternative pathogenesis, e.g., gastric reflux, gavage accidents or
aspiration.6,7,8
Abstract
Figure 3
Inhaled toxicants often induce nasal lesions in a site-specific manner.
Irritants like formaldehyde often cause lesions largely restricted to the
anterior nose where it symmetrically affects the respiratory and transitional
epithelia and the effect progressively lessens posteriorly. In contrast,
inhaled methyl bromide affects olfactory epithelium preferentially, leaving
the more anterior epithelia unaffected.1 Mapping lesions can effectively
demonstrate the unique distribution of nasal lesions.2,3 Lesion distribution
can be a function of airflow patterns delivering a local tissue dose or a
unique tissue susceptibility related to local metabolism. Compounds
administered parenterally can result in nasal lesions via systemic delivery
of toxic metabolites to susceptible nasal tissues or from systemic delivery
and subsequent local metabolism of the compound by the nasal, often,
olfactory epithelium.4 Most observed nasal effects are uniform, affecting
tissues in anatomically consistent and locally specific manners. When
patterns of distribution of nasal lesions are not uniform or not consistent
with known patterns of induced lesions, then one must consider an
alternative pathogenesis, e.g., gastric reflux, gavage accidents or
aspiration.6,7,8 The working hypothesis for this review was that there can
be a difference in nasal lesion distribution and quality, that may vary with
the route of exposure. The objective of this review was to revisit studies
having various routes of exposure and to record and evaluate nasal lesion
patterns to see if differences could be seen.
Introduction
Figure 2
1. The Nose Revisited: A Brief Review of the Comparative Structure, Function, and Toxicologic
Pathology of the Nasal Epithelium. Harkema et al. Toxicol Pathol. 2006:34(3):252-69.
2. Nasal Dosimetry, Lesion Distribution, and the Toxicologic Pathologist: A Brief Review. Morgan,
K. T. Inhalation Toxicol. 1994:6:41-57.
3. Histopathology of Nasal Olfactory Mucosa from Selected Inhalation Toxicity Studies Conducted
with Volatile Chemicals. Hardisty et al. Toxicol Pathol. 1999:27(6):618-627.
4. Respiratory Tract Lesions in Noninhalation Studies. Sells et al. Toxicol Pathol. 2007:35(1):170-
77.
5. Unexpected Nasal Changes in Rats Related to Reflux after Gavage Dosing. Damsch et al.
Toxicol Pathol. 2011:39(2):337-47.
6. Gavage-Related Reflux in Rats: Identification, Pathogenesis, and Toxicologic Implications
(Review). Damsch et al. Toxicol Pathol. 2011:39(2):348-60.
7. Impact of Gavage Dosing Procedure and Gastric Content on Adverse Respiratory
Effects and Mortality in Rat Toxicity Studies. Eichenbaum et al. J Appl Toxicol. 2011:31(4):342-
64.
8. National Toxicology Program Technical Report Series, 1990 March;377:1-211.
9. NTP TOX 40. Toxicity Studies of β-Bromo-β-Nitrostyrene (CAS No. 7166-19-0) Administered
by Gavage to F344/N Rats and B6C3F1 Mice.
10. NTP TR-399. Toxicology and Carcinogenesis Studies of Titanocene Dichloride (CAS No. 1271-
19-8) in F344/N Rats (Gavage Studies).
11. Drug Metabolism in the Nasal Cavity: Relevance to Toxicology. Reed, C. J. Drug Metab Rev.
1993:25(1-2):173-205.
References
To elucidate the importance of lesion distribution, we examined all
histologic sections of nose from all control and high dose rats from six
NTP studies; 1 inhalation, 2 drinking water, and 3 gavage studies. The
incidences of final diagnoses can be found in the NTP technical reports for
each study. Terms have been devised to reflect the expressions of
distribution and general pathological processes.
The studies reviewed included a chronic inhalation study, o-
chlorobenzalmalononitrile (CS2), methyl ethyl ketoxime and dipropylene
glycol (two subchronic drinking water studies), β-bromo-β-nitrostyrene (a
4-week gavage study), titanocene dichloride (a chronic gavage study), and
the green tea extract (a chronic gavage study).
The standard three NTP nose sections were evaluated in all control and
high dose rats except for titanocene, an older study where only anterior
and middle nose sections were present (the typical third nose section with
the ethmoid turbinates and nasopharyngeal duct was not present).
Treatment-related lesions noted in the noses were designated as
unilateral, bilateral, anterior, posterior, dorsal, or ventral. The site-specific
distribution of nose and nasopharyngeal duct lesions was depicted on
diagrams of transverse sections of the three nose levels, and on a
diagrammatic depiction of a longitudinal nose section.
Methods
Figure 1
This review highlights the importance of recognizing nasal lesion
distribution patterns. Nasal toxicants tend to express themselves in
somewhat unique and consistent patterns of distribution.1 Variance
from expected patterns, irregular lesion incidences across groups, or
lesions in unanticipated locations should alert us to consider
alternate pathogeneses or different interpretations.
Inhalation exposure of formaldehyde and other nasal irritants induce
lesions with an anterior to posterior nasal gradient affecting
transitional and respiratory epithelium preferentially, while inhaled
methyl bromide affects only olfactory epithelium, both toxicant
effects being expressed bilaterally.1
Occasionally, lesions observed in the nose can be the result of
gastric reflux or regurgitation. In this instance, the nasal lesions tend
to be less uniform in terms of distribution and symmetry. Lesions in
the nose related to reflux tended to be more unilateral, more ventral
and more posterior. Acute lesions of epithelial necrosis overlying a
chronically altered epithelium and suppurative lesions in the
nasopharyngeal duct may also signal reflux as a possible cause.5,6,7
Chemicals administered parenterally can affect the respiratory
epithelium but more often they affect the olfactory epithelium.4
Significant metabolic activity has been observed in the nasal
epithelium and may explain some regional susceptibility. The
distribution of the metabolic enzymatic activity tends to be bilateral
and in the case of the olfactory epithelium, in the posterior nose
sections.11
Lesion distribution can be attributed to delivered local dose and or
specific tissue susceptibility.2 Local dose can be affected by airflow,
mucous flow, systemic delivery via vascular perfusion,
physicochemical properties of the agent, and metabolism.1
Discussion
Grateful thanks for assistance with this poster to Emily Singletary and
David Sabio of EPL; and to Lois Wyrick of Image Associates.
Acknowledgements
Male and female rats had similar lesions and lesion distribution related to
the various routes of exposure; therefore, for space conservation, only
data from the high dose female rats was used for illustrative purposes.
*There was no third nasal section in this study
*There was no third nasal section in this study
Inhalation of CS2, a tear gas, elicited an irritant response in the nose in a
dose-related manner. The rats had bilateral hyperplasia and squamous
metaplasia of the respiratory epithelium and chronic focal inflammation in
primarily the first nasal section; and a lesser incidence of bilateral olfactory
epithelial degeneration and metaplasia in the second and third nasal
sections, effectively establishing a lessening of lesions with an anterior to
posterior gradient.8
Methyl ethyl ketoxime (drinking water) caused degeneration only of the
olfactory epithelium lining the bilateral dorsal meatuses of the second and
third nose sections in the two highest exposure groups and no lesions in
control or lower exposure groups.
Dipropylene glycol (drinking water) also caused degeneration of the
olfactory epithelium bilaterally in the dorsal meatus of the second and third
nasal sections. The highest exposure group only was affected, with no
lesions observed in the control or lower exposure groups.
β-bromo-β-nitrostyrene (gavage) was associated with a low incidence of
nasal lesions, including inflammation and olfactory epithelial degeneration,
plus suppurative lesions in the nasopharyngeal duct in two females. The
lesions were mainly posterior and ventral. Review of the technical report
showed that the incidence of nasal lesions varied widely among the
control and five treated groups with some of the lower dose groups having
more lesions than the high dose group. The technical report attributed the
nasal lesions to gastric reflux and aspiration.9
Titanocene dichloride (gavage) was associated with a prominent increase
in necrosis, degeneration and inflammation of the nasal mucosa.
Occasional mycotic infections occurred. These nose lesions were
somewhat evenly distributed anterior to posterior, but clearly had a ventral
and unilateral predisposition. The technical report attributed the nasal
lesions to gastric reflux and aspiration.10
Green tea extract (gavage) was associated with a prominent nasal lesion
complex including, but not limited to, degeneration of olfactory epithelium,
necrosis, suppuration, chronic inflammation, and epithelial hyperplasia
and squamous metaplasia of respiratory and olfactory epithelium. Foreign
bodies, (hair, plant), were observed. These lesions were present in a
predominately, but not exclusively, ventral, posterior and unilateral
manner. Acute lesions of epithelial necrosis overlaid chronic areas of
inflammation at times. The nasopharyngeal duct frequently experienced
necrosis, suppurative inflammation, chronic inflammation and epithelial
hyperplasia.
Figures 1-4 are typical lesions that were associated with exposure to
these chemicals. Figures 5-10 depict the relative lesion distribution
associated with these 6 exposures.
Results
Chemical Study type
Rats with
lesions - Nose
level 1
Rats with
lesions - Nose
level 2
Rats with lesions -
Nose level 3
Rats with lesions -
Nasopharyngeal duct
CS2 Chronic inhalation 49/50 25/50 9/50 0/50
Methyl ethyl
ketoxime
90-Day
Subchronic In
water
0/10 10/10 10/10 0/10
Dipropylene
glycol
90-Day
Subchronic In
water
0/10 10/10 9/10 0/10
β-bromo-β-
nitrostyrene
28-Day
Gavage
0/10 1/10 1/10 2/10
Titanocene
dichloride*
Chronic
Gavage
7/50 9/50 NA* NA*
Green tea extract
Chronic
Gavage
19/60 42/50 40/60 34/50
Chemical Study type
Lesions
mainly
anterior
Lesions
mainly
posterior
Lesions
mainly
dorsal
Lesions
mainly
ventral
Lesions
mainly
unilateral
Lesions mainly
bilateral
CS2
Chronic
inhalation
+ - + - - +
Methyl ethyl
ketoxime
90-Day
Subchronic
in water
- + + - -
+
Dipropylene glycol
90-Day
Subchronic
In water
- + + - - +
β-bromo-β-
nitrostyrene
28-Day
Gavage
- + - + - +
Titanocene
dichloride*
Chronic
Gavage
-
- - + + -
Green tea extract
Chronic
Gavage
- + - + + -
Figure 4
Lesions suggestive of gastric reflux are present in of the
third nasal section from a male Wistar Han rat. There is
suppurative inflammation in the ventral nasal cavity and
nasopharyngeal duct. Chronic exposure to green tea
extract by gavage. (H&E)
Irritant response in the nasal turbinates in the first nasal
section from a male F344/N rat exposed via inhalation to
CS2 (chronic study). Shown are hyperplasia and
squamous metaplasia of the respiratory epithelium.
(H&E)
Bilateral degeneration of the olfactory epithelium in the
dorsal meatuses in the second nose section from a
female F344/N rat exposed to dipropylene glycol via
drinking water (subchronic study). (H&E)
Lesions similar to those seen with gastric reflux are
present in the dorsal lateral portion of the third nasal
section in a male Wistar Han rat. There is unilateral
suppurative inflammation associated with chronic
exposure to green tea extract by gavage. (H&E)
Figure 5 Figure 6
Figure 7 Figure 8
Figure 9 Figure 10
Relative Lesion Distribution (Red)
CS2, Chronic Inhalation, Female Rats
Relative Lesion Distribution (Red)
Methyl Ethyl Ketoxime, Subchronic in Water, Female Rats
Relative Lesion Distribution (Red)
Dipropylene Glycol, Subchronic in Water, Female Rats
Relative Lesion Distribution (Red)
β-Bromo-β-Nitrostyrene, 4 Week Gavage, Female Rats
Relative Lesion Distribution (Red)
Titanocene dichloride, Chronic Gavage, Female Rats
Relative Lesion Distribution (Red)
Green Tea Extract, Chronic Gavage, Female Rats