mean severity grade 1-5

5.1: 0.1

52: 0.1

156: 0.5

“Results

Test substance

The purity of the naphthalene test substance was checked pre- and post-exposure. Results of both analyses indicated that the purity was in excess of 99.9%, demonstrating that the material was stable over the course of the study.

Inhalation exposure concentration measurements

The coefficient of variation of naphthalene concentration measurements made at nine different locations in the exposure chamber was less than 8% for all exposure chambers and the naphthalene vapor was considered to be uniformly distributed within the chamber. The means for each 6-h exposure day’s naphthalene concentration were compiled and the grand mean and standard deviation of those means (n=65–66) were calculated. Grand means ± standard deviations were 0.00±0.00, 0.10±0.00, 0.97±0.06, 9.97±1.2 and 29.8±1.7ppm, for target concentrations of 0, 0.1, 1.0, 10 and 30ppm naphthalene, respectively. The higher than expected standard deviation for the 10ppm exposure group was due to one high concentration event on one day of exposure. On that day, there was a momentary spike in concentration, reaching as high as 105ppm. The generation system was shut down and disconnected. The time-weighted-average concentration for this 6-h exposure was 20.7ppm. Grand means and standard deviations were also calculated for environmental parameters during the inhalation exposures. In general, inhalation chamber temperature and relative humidity ranges were 73–75° F and 45–56%, respectively, throughout the 90-day study. Inhalation chamber airflow was 300L/min (approximately 18 air changes per hour). Results of aerosol measurements using the SMPS with animals in the exposure chambers without naphthalene, showed that the concentration of particles among the five exposure chambers was approximately the same, ranging from 153 to 194 p/cm3 , and was lower than the concentration of particles in the room housing the inhalation chambers (376 p/cm3 ). Aerosol measurements were also taken during naphthalene exposure from the control (0ppm) and high (30ppm) naphthalene concentration chambers with animals present. Particle concentrations in the 30ppm chamber were slightly above or below particle concentrations measured in the control chamber, and for both exposure chambers, were lower than the room housing the chambers (data not shown). Thus, measures of aerosol formation, aerosol count in chambers during an exposure, and aerosol mass collection on a filter all indicated that there was no condensation of naphthalene vapor to an aerosol during the inhalation exposures.

Clinical observations, body weights, food and water consumption, and organ weights There were no clinical observations associated with NA exposure throughout the study. Rat body weights at the end of the 90-day study demonstrated a naphthalene concentration-related decrease compared to respective control groups. For example, at week 14 male body weights were 4–8% lower than the control mean in the 10 and 30ppm groups (p<0.05) (Figure 1). Decreases in female body weight means at the conclusion of the study were of similar magnitude (2–7%) compared to control value for NA exposure groups of 0.1ppm to 30ppm (p><0.05) (data not shown). Body weights of all recovery animals (including controls) showed a slight decrease in weight immediately following the end of exposure likely due to a shift in housing environment upon cessation of exposure and transfer to the recovery environment. However, body weights of all recovery animals increased in subsequent weeks (Figure 1 shows male rat data). In general, decreases (compared to controls) in body weights in NA-exposed animals during the 90-day exposure regimen were no longer observed, indicating mildly greater gains in body weights for NA-exposed animals during the recovery period compared to air only-exposed controls. Food consumption was lower for male rats exposed to 30ppm, and often times statistically significant (p><0.05) (data not shown). Males exposed to 10ppm occasionally had statistically significantly lower food consumption when compared with controls. After week 14, when the animals were no longer being exposed to NA, food consumption means between groups were not statistically significant. Female rats showed a similar pattern, though differences were not quite as pronounced (data not shown). Water consumption was lower for both male and female rats exposed to 30ppm (data not shown). In summary, food and water consumption was decreased in animals exposed at 30ppm, and the decreased consumption appears to be correlated with the observed decreased body weights in animals at the higher exposure concentrations of NA. No gross observations were noted at necropsy that could be related to NA exposure. The absolute mean weights of the spleen, left testis, and thymic region of male rats exposed to 30ppm were statistically significantly lower (p><0.05 or p><0.01) than the control mean values (Table 3). Mean right testis weight was slightly lower than control, but not statistically significant. The magnitude of the organ weight decreases compared to control means was approximately 5% (spleen and left testis) and 17% (thymus). When expressed relative to body weight, these organ weights were similar to control values (Table 3). Similar observations were observed in the organ weights of the 10 ppm-exposed males. In female rats, the absolute mean weights of the heart, liver, and thymic region were statistically significantly lower (p><0.05 or p><0.01) than the control mean weights in all NA-exposed groups (except the heart weight of the 10ppm group, the liver weight of the 1ppm group, and the thymus weight of the 0.1ppm group) (Table 4). The>< 0.05) (Figure 1). Decreases in female body weight means at the conclusion of the study were of similar magnitude (2–7%) compared to control value for NA exposure groups of 0.1ppm to 30ppm (p<0.05) (data not shown). Body weights of all recovery animals (including controls) showed a slight decrease in weight immediately following the end of exposure likely due to a shift in housing environment upon cessation of exposure and transfer to the recovery environment. However, body weights of all recovery animals increased ><0.05) (data not shown). Body weights of all recovery animals (including controls) showed a slight decrease in weight immediately following the end of exposure likely due to a shift in housing environment upon cessation of exposure and transfer to the recovery environment. However, body weights of all recovery animals increased in subsequent weeks (Figure 1 shows male rat data). In general, decreases (compared to controls) in body weights in NA-exposed animals during the 90-day exposure regimen were no longer observed, indicating mildly greater gains in body weights for NA-exposed animals during the recovery period compared to air only-exposed controls. Food consumption was lower for male rats exposed to 30ppm, and often times statistically significant (p<0.05) (data not shown). Males exposed to 10ppm occasionally had statistically significantly lower food consumption when compared with controls. After week 14, when the animals were no longer being exposed to NA, food consumption means between groups were not statistically significant. Female rats showed a similar pattern, though differences were not quite as pronounced (data not shown). Water consumption was lower for both male and female rats exposed to 30ppm (data not shown). In summary, food and water consumption was decreased in animals exposed at 30ppm, and the decreased consumption appears to be correlated with the observed decreased body weights in animals at the higher exposure concentrations of NA. No gross observations were noted at necropsy that could be related to NA exposure. The absolute mean weights of the spleen, left testis, and thymic region of male rats exposed to 30ppm were statistically significantly lower (p><0.05 or p><0.01) than the control mean values (Table 3). Mean right testis weight was slightly lower than control, but not statistically significant. The magnitude of the organ weight decreases compared to control means was approximately 5% (spleen and left testis) and 17% (thymus). When expressed relative to body weight, these organ weights were similar to control values (Table 3). Similar observations were observed in the organ weights of the 10 ppm-exposed males. In female rats, the absolute mean weights of the heart, liver, and thymic region were statistically significantly lower (p><0.05 or p><0.01) than the control mean weights in all NA-exposed groups (except the heart weight of the 10ppm group, the liver weight of the 1ppm group, and the thymus weight of the 0.1ppm group) (Table 4). The><0.05) (data not shown). Males exposed to 10ppm occasionally had statistically significantly lower food consumption when compared with controls. After week 14, when the animals were no longer being exposed to NA, food consumption means between groups were not statistically significant. Female rats showed a similar pattern, though differences were not quite as pronounced (data not shown). Water consumption was lower for both male and female rats exposed to 30ppm (data not shown). In summary, food and water consumption was decreased in animals exposed at 30ppm, and the decreased consumption appears to be correlated with the observed decreased body weights in animals at the higher exposure concentrations of NA. No gross observations were noted at necropsy that could be related to NA exposure. The absolute mean weights of the spleen, left testis, and thymic region of male rats exposed to 30ppm were statistically significantly lower (p< 0.05 or p<0.01) than the control mean values (Table 3). Mean right testis weight was slightly lower than control, but not statistically significant. The magnitude of the organ weight decreases compared to control means was approximately 5% (spleen and left testis) and 17% (thymus). When expressed relative to body weight, these organ weights were similar to control values (Table 3). Similar observations were observed in the organ weights of the 10 ppm-exposed males. In female rats, the absolute mean weights of the heart, liver, and thymic region were statistically significantly lower (p<0.05 or p<0.01) than the control mean weights in all NA-exposed groups (except the heart weight of the 10ppm group, the liver weight of the 1ppm group, and the thymus weight of the 0.1ppm group) (Table 4). The magnitude of these decreases (5–7% for heart and liver; 16% for thymus) was not exposure concentration-dependent and, similar to the males, when these organ weights were expressed relative to body weight, they were similar to control values (Table 4). A decrease in mean weight of adrenal glands in the 10ppm exposed females only was considered spurious. All statistically significant differences in organ weight values (absolute, relative to body weight or to brain) observed immediately following the 90-day exposure regimen were diminished in magnitude and no longer statistically significantly different from control values following 4 weeks recovery (data not shown).

Histopathology

The nose was removed during necropsy, nasal cavity processed, and stained (H&E) at six levels (Dodd et al., 2010). Microscopic evaluation showed NA-induced lesions in the respiratory and olfactory epithelium. Minimal to slightly mild inflammation of the squamous epithelium or transitional/respiratory epithelium was observed in the nasal tip and level 1 areas of all exposure groups, including controls (Table 1 for male data). Low grade inflammation was also observed in all exposure groups, including controls, after a 4-week recovery period (Table 2 for male data). This observation was not considered to be related to NA exposure. Lesions related to NA exposure were observed in the respiratory epithelium beginning at level 2 in the nose. At a concentration of 1ppm, hyperplasia of the transitional/respiratory epithelium was observed in males and females with an average severity of 1.0 (minimal). The severity of the lesion progressed to mild hyperplasia at 10ppm (average severity 1.4 in males, average severity of 1.6 in females) and 30ppm (average severity of 1.4 in males and average severity of 1.5 in females) (Figures 2 and 3; Table 1 for male data). No respiratory epithelial lesions were observed in animals exposed to 0.1ppm NA. After the 4-week recovery period, hyperplasia of the transitional/respiratory epithelium was observed in only 1 male exposed at 10ppm (Table 4) suggesting resolution of the lesion during the 4-week recovery period (Figure 4). Minimal squamous metaplasia of the transitional/respiratory epithelium was also observed in level 2 sections of male and female nasal cavities with average severity of 0.9 and 0.8 for 10ppm and 30ppm, respectively (Table 1 for male data). This lesion was not observed at level 2 in the recovery animals (Table 2 for male data). Nasal cavity lesions related to NA exposure were also observed in the olfactory epithelium. Olfactory epithelial degeneration and necrosis was observed at the 10- and 30-ppm exposure concentrations in males and females. The average lesion severity in 10 ppm-exposed males was 1.5, 1.4, 2.0 and 1.1 for levels 2 through 5, respectively (Table 1). For females at 10ppm, the average severity score was 1.9, 1.7, 2.3 and 1.5 for levels 2 through 5, respectively (data not shown). At 30ppm, average severity scores were mildly higher in both males (Figures 5 and 6; Table 1) and females. Additionally, and associated with the degenerative olfactory epithelium, there was a prominent basal cell hyperplasia characterized by an increase in the number of basal cells resulting in thickening of this layer of cells immediately under the degenerative epithelium (Figure 6). Table 1 shows the average severity scores of this lesion for all male exposure groups. The incidence and severity of both the olfactory epithelium degeneration and basal cell hyperplasia lesions decreased dramatically in NA-exposed males and females after the 4-week recovery period (Figure 7 and Table 2 for male data). There were no olfactory epithelial effects seen in male or female rats exposed at 0.1 or 1.0ppm NA (Table 1 for male data). Also observed, and possibly related to NA exposure at the highest concentrations, was goblet cell hypertrophy/hyperplasia of the nasopharyngeal duct (Tables 1 and 2 for male data).”

Male Fischer Rats