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