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Repeated dose toxicity: inhalation

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Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.4200 (Carcinogenicity)
Principles of method if other than guideline:
The State of California and the Health Effects Institute (HEI) nominated ozone to the National Toxicology Program (NTP) for long-term studies in rats and mice to determine the potential toxicity and carcinogenicity of long-term ozone exposure. The studies reported here include exposures at the current EPA standard (0.12 ppm), the maximum concentration considered to be compatible with long-term survival (1.0 ppm) and an intermediate concentration (0.5 ppm). A second ozone inhalation study was included in which 0.5- and 1.0 ppm exposures were continued to 30 months based on lifetime studies in rats in which the majority of lung tumors developed after 24 months. A third study was included in which male rats were administered 2 concentrations of a known pulmonary carcinogen, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and exposed to 0.5 ppm ozone to determine the co-carcinogenic potential of ozone.
GLP compliance:
yes
Specific details on test material used for the study:
Ozone was generated by corona discharge using an OREC model 03V5-ozonator (Ozone Research and Equipment Corp., Phoenix, AZ) with 100% oxygen. The 03 concentration in each chamber was monitored by a multiplexed Dasibi model 1003-AH or 1003-PC ultraviolet spectrophotometric analyser (Dasibi Environmental Corp., Glendale, CA). The monitor was calibrated by comparing it with a chemical specific, calibrated monitor (neutral-buffered potassium iodide method) simultaneously sampling the exposure chambers.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Simonsen Laboratories (Gilroy, CA). Rats were quarantined for 14 days before the beginning of the study. Five male and five female rats were selected for bacterial culture and selected histopathology prior to the beginning of the study. Approximately 3 weeks after receipt, serology samples were collected for viral screening from up to seven male and seven female rats. Rats were approximately 6 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP sentinel Animal Program.

All animals were housed individually. Water was available ad libitum, and feed (NIH-07 open formula meal diet, Zeigler Brothers, Inc., Gardners, PA; changed weekly) was available ad libitum except during exposure periods. Cage units were rotated vertically (2-year study) or horizontally (life time study) within each chamber weekly.

Environmental conditions:
Average temperature: 23.9 degrees centigrade;
Relative humidity: 40% to 70%;
Fluorescent light: 12 hours/day;
Room air: 12 to 18 changes/hour.

IN-LIFE DATES:
2 Year ozone study
From: 25 January 1990 to 24 January 1992.

Lifetime study
From: 26 October 1989 to 13 March 1992.

2 Year ozone/NKK study
Ozone - From: 28 November 1989 to 27 November 1991.
NKK - From: 27 November 1989 to 13 April 1990.
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Animals were exposed in H-2000 chambers. The animals were hold single in stainless steel, wire bottom cages. The build-up of vapour concentration in the chamber at the beginning of exposure to 90% of its final stable concentration (T90) and the decay of concentration at the end of exposure to 10% (T10) were measured prior to the start of each study in chambers with a full complement of mature F344/N rats. These tests were done in conjunction with the pre-start tests for the ozone study. A T90 value of 30 minutes was used based on the experimental data. The T10 value ranged from 5 to 11 minutes.
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems). Samples were directed to the ozone monitor through computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour. Uniformity of ozone concentration in was measured quarterly during the 2-year and lifetime studies. While the majority of the determinations were within this range, some exceeded this value, and 10.1% was the maximum value found.
The vehicle was conditioned air (single HEPA filters and charcoal).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems) (Glendale, CA). Initially, the UV spectrophotometric analyser (Dasibi Model 1003-AH) was used to monitor the ozone concentration in the exposure chambers, control chamber, room, generator cabinet, and an on-line ozone standard. After approximately 14 months (2-year ozone study), or 16 months (2-year ozone/NNK study and lifetime studies) the Model 1003-AH ozone monitors were replaced with Dasibi Model 1003-PC ozone monitors/generators. This change reduced maintenance and repair costs and maintained an effective system for monitoring ozone.
For both monitoring systems, air sampled at each location was transported to the monitor by transfer lines of Teflon" tubing. Samples were directed to the ozone monitor through a set of eight computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour.
Duration of treatment / exposure:
2 Year ozone study: exposure 6h/d, 5d/w for 105 weeks;
Lifetime study: exposure 6h/d, 5d/w for 125 weeks;
2 Year ozone/NKK study: exposure 6h/d, 5d/w for 105 weeks.
Frequency of treatment:
daily for 5 days/week
Post exposure period:
no post exposure period
Dose / conc.:
0.12 ppm
Dose / conc.:
0.5 ppm
Dose / conc.:
1 ppm
No. of animals per sex per dose:
2 Year ozone study: 50 males and 50 females per dose;
Lifetime study: 50 males and 50 females per dose;
2 Year ozone/NKK study: 48 males
Control animals:
yes, concurrent vehicle
Details on study design:
Concentration levels were based on the National Ambient Air Quality standard (0.12 ppm) and the maximum concentration which the animals would tolerate (1.0 ppm).
Positive control:
n/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, twice daily for mortalities
DETAILED CLINICAL OBSERVATIONS (not in lifetime study): Yes, initially, monthly through week 92 then every 2 weeks until the end of the study.
BODY WEIGHT: Yes, initially, weekly through week 13, monthly through week 92 then every 2 weeks until the end of the study.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes
2 Year ozone study and lifetime study:
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, clitoral gland, esophagus, femur, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (with adjacent skin), nose, ovaries, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland, spleen, stomach, testes (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus
2 Year ozone/NKK study:
Histopathology was performed on all animals. In addition to gross lesions and tissue masses, the tissues examined included lymph nodes (bronchial and mediastinal), lungs, nose, larynx, and trachea.
Other examinations:
n/a
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox's method for testing two groups for equality and Tarone's life table test to identify dose-related trends. P values for the survival analyses are two sided.

Analysis of Neoplasm incidences was by logistic regression analysis. Neoplasm prevalence was modelled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if the fit of the model was not significantly enhanced. The neoplasm incidences of exposed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose (prevalence analysis of Dinse and Lagakos). In addition to logistic regression, other methods of statistical analysis were used. These methods include the life table test (Cox; Tarone), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran Armitage trend test, procedures based on the overall proportion of neoplasm-bearing animals. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall dose-related trend. Continuity corrected tests were used in the analysis of neoplasm incidence, and reported P values are one sided. The procedures described above were also used to evaluate selected non-neoplastic lesions.
All non-neoplastic lesions in this study were considered to be incidental to the cause of death or not rapidly lethal. The primary statistical analysis used was logistic regression analysis. For lesions detected at the interim evaluation, the Fisher exact test was used.
Organ and body weight data were analysed using procedures test of Dunnett or William's, Jonckheere's test and the Mann­ Whitney U test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hypoactivity was observed in male and female rats exposed to ozone. Rats, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Mortality:
mortality observed, treatment-related
Description (incidence):
Hypoactivity was observed in male and female rats exposed to ozone. Rats, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Summary of the 2-Year and Lifetime Carcinogenesis Studies of Ozone

Doses 0, 0.12 (2-year study only), 0.5 and 1.0 ppm ozone by inhalation

2-Year males Lifetime males 2-Year females Lifetime females
Body weights 1.0 ppm group 1.0 ppm group 1.0 ppm group 1.0 ppm group
slightly lower than lower than controls slightly lower than slightly lower than
controls controls controls
Survival rates 8/49, 5/50, 7/50, 7/50 0/50, 0/50, 1/50 28/50, 24/50, 30/50, 6/50, 6/50, 7/50
27/50
Nonneoplastic effects
Nose: goblet cell Nose: goblet cell Nose: goblet cell Nose: goblet cell
hyperplasia (1/50, hyperplasia (1/50, hyperplasia (1/50, hyperplasia (0/50,
4/50, 41/50, 48/50); 46/49, 48/49); lateral 2/50, 45/50, 50/50); 47/49, 50/50); lateral
lateral wall wall hyperplasia lateral wall wall hyperplasia
hyperplasia (0/50, (10/50, 48/49, 47/49); hyperplasia (2/50, (4/50, 49/49, 50/50);
8/50, 50/50, 49/50) squamous metaplasia 8/50, 48/50, 50/50) squamous metaplasia
squamous metaplasia (10/50, 23/49, 40/49) squamous metaplasia (5/50, 25/49, 35/50)
(2/50, 6/50, 36/50, Larynx: squamous (2/50, 11/50, 21!50, Larynx: squamous
46/50) metaplasia (0/50, 45/50) metaplasia (2/49,
Larynx: squamous 20/48, 43/47) Larynx: squamous 16/47, 48/50)
metaplasia (0/50, Lung: metaplasia metaplasia (4/50, Lung: metaplasia
2/50, 16/50, 43/50) (0/50, 45/50, 50/50); 5!50, 9/50, 43/50) (0/50, 44/50, 50/50);
Lung: metaplasia histiocytic infiltration Lung: metaplasia histiocytic infiltration
(0/50, 9/50, 46/50, (0/50, 38/50, 49/50); (0/50, 6/50, 48/50, (0/50, 38/50, 49/50);
47/50); interstitial interstitial fibrosis 48/50); interstitial interstitial fibrosis
fibrosis (0/50, 2/50, (0/50, 44/50, 50/50) fibrosis (0/50, 0/50, (0/50, 41/50, 50/50)
40/50, 44/50) 42/50, 47/50)
Neoplastic effects
None None None None

Level of evidence of carcinogenic activity
No evidence No evidence No evidence No evidence

Ozone/NKK study

Lung: Alveolar epithelial metaplasia and interstitial fibrosis occurred in all groups of rats exposed to ozone (with or without NNK), but were not observed in vehicle controls or in animals exposed to NNK alone. The incidence of alveolar cellular infiltration was greater in males exposed to ozone than in the vehicle control males. There was a dose-related increased incidence of atypical alveolar hyperplasia in groups of rats receiving NNK, and the increase was significant. An increased incidence of alveolar/bronchiolar adenoma or carcinoma (combined) also occurred in rats administered 1.0 mg/kg NNK, with or without ozone. The administration of ozone did not affect the occurrence of pulmonary neoplasms or nonneoplastic lesions in rats administered NNK.

Nose: The incidence of hyperplasia in groups of rats exposed to ozone with and without NNK was greater than the incidence in males not exposed to ozone. Incidences of hyperplasia among groups of rats exposed only to NNK were low and similar to that of the controls. The nasal lesions were similar to those seen in rats exposed to ozone by inhalation for 2 years.





Conclusions:
The results of the large "NTP study" (2-year and lifetime inhalation studies) on rats showed that there was no evidence of carcinogenic activity of ozone in male or female F344/N rats exposed to 0.12, 0.5, or 1.0 ppm.
Executive summary:

The toxicity and carcinogenicity of ozone was evaluated in Fischer 344/N rats and B6C3Fl mice exposed 6 hr/day, 5 days/week, to 0.12 (2 years only), 0.5 or 1.0 ppm ozone by inhalation for 2-year and lifetime exposures. A 2-year cocarcinogenicity study (male rats only) included the subcutaneous administration of 0, 0.1 or 1.0 mg/kg body weight. of 4-(N-methyl-Nnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) for the first 20 weeks along with inhalation exposure to 0 or 0.5 ppm ozone followed by additional 84 weeks of ozone exposure alone.

Ozone exposure in rats did not cause an increased incidence of lung neoplasms. In the co-carcinogenicity study, ozone exposure did not have an additive carcinogenic effect. We conclude, that under the conditions of these studies: (a) ozone exposure is not carcinogenic to either male or female F-344/N rats, (b) ozone does not enhance the incidence of pulmonary neoplasms in F-344/N rats exposed to a known pulmonary carcinogen (NNK), and (c) mild site-specific toxic lesions characteristic of ozone exposure persist in the nasal passage and lung throughout the lifetime of the rat with continued ozone exposure

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Research study, effects on lung morphology were investigated in subgroups of rats from a long-term inhalation study.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ozone
EC Number:
233-069-2
EC Name:
Ozone
Cas Number:
10028-15-6
Molecular formula:
O3
IUPAC Name:
trioxygen
Test material form:
gas
Specific details on test material used for the study:
Ozone was generated by corona discharge using an OREC Model 03V5-0 ozonator (Ozone Research and Equipment Corporation, Phoenix, AZ) with 100% oxygen.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were purchased from Simonsen Laboratories (Gilroy, CA) at three weeks of age. After a quarantine period of 14 days, rats were randomly assigned to air or ozone exposure groups, and individually housed in stainless-steel wire-bottom cages. Rats were given NIH-07 open formula pellets (Zeigler Bros., Gardner, PA) and softened tap water ad libitum, except during exposure periods. Relative humidity (55°/rJ ± 15%}, temperature (24° ± 0.7°C), and lighting (12 hours light/12 hours dark) were maintained aulomatically.

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: n.a.
Details on inhalation exposure:
Ozone was generated from 100% oxygen corona discharge (OREC Model 03V5-0, Ozone Research and Equipment Corp., Phoenix, AZ). Ozone concentrations were measured with multiplexed ultraviolet spectrophotometric analyzers (Model 1003-AH, Dasibi Environmental Corp., Glendale, calibrated by a chemical method using neutral-buffered potassium iodide. Ozone in the control atmosphere was below the limit of detection (0.002 ppm). All the rats from the NTP/HEI collaborative study were exposed to 0, 0.12, 0.5, or 1.0 ppm ozone for 6 hours/day, 5 days/week for 20 months. . Animals in both studies were killed one week after the end of the exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Ozone concentration in each chamber was monitored by a multiplexed Dasibi Model 1003-AH (Dasibi Environmental Corporation, Glendale, CA) ultraviolet spectrophotometric analyzer.
Duration of treatment / exposure:
The ozone exposure was 6 hours/day, 5 days per week
Frequency of treatment:
Exposure took place for 20 months
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
0.12 ppm (nominal)
Dose / conc.:
0.5 ppm (nominal)
Dose / conc.:
1 ppm (nominal)
No. of animals per sex per dose:
A total of 38 rats, equally divided between males and females, were used for this study; the group size from each ozone exposure concentration was
10 from 0.0 ppm,
12 from 0.12 ppm,
8 from 0.5 ppm, and
8 from 1.0 ppm.
Control animals:
yes, concurrent vehicle
Details on study design:
Male and female rats were exposed to filtered air or to 1.0, 0.5, or 0.12 ppm ozone for 6 h/day, 5 days/week for 20 months. Animals were killed one week after the end of the exposure. Immediately after death, lungs were fixed, removed from the carcass and stained. Microdissection was done to prepare blocks for further processing for electron microscopic morphometric analysis.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
Not reported
Sacrifice and pathology:
Animals were killed by anaesthesia with sodium pentobarbital, and the tracheas were cannulated. The diaphragms were punctured to deflate the lungs, and the lungs were fixed by instillation at 30 cm of water pressure with 2% glutaraldehyde in 0.85 M sodium cacodylate buffer (350 mOsm; pH 7.4). After fixing the lungs in the chest for 15 minutes, they were removed and stored in fixative until processed. Lung volumes were measured by fluid displacement. Three 2-mm slices of the left lung were cubed into 4 mm x 4 mm pieces. To enhance the visibility of the interstitial matrix components, tissues were stained en bloc in 1% osmium tetroxide for four hours, 1 (%) tannic acid for 2.5 hours, and in 2% uranyl acetate for 2.5 hours (Mercer et al., 1991). The tissue was extensively washed in 8% sucrose after each step, dehydrated in an ethanol series, and embedded in epoxy resin with standard procedures. To assure adequate infiltration of the resin, the tissue blocks were allowed to incubate a long time (approximately 1 hour) in the propylene oxide-resin solution.

MICRODISSECTION
The microdissection technique used to isolate terminal bronchioles and proximal alveolar regions is described before(Chang et al. 1988). Tissue blocks selected arbitrarily were cut in a random orientation into slices 0.5 mm thick. They were examined sequentially with a dissecting microscope. Small airways, identified by their smooth circumference and thicker epithelium, were followed to the bronchiole-alveolarduct junction.
Terminal bronchioles were cut in cross sections to facilitate morphometric analysis of their tubular and oriented structure. The alveolar tissue surrounding the first alveolar duct bifurcation, designated as the proximal alveolar region, also was studied in cross section on sections that presented a distinct alveolar duct at each side of the first alveolar duct bifurcation. Three blocks of each anatomic location were selected randomly, and mounted on blank epoxy blocks. Since the orientation of the terminal bronchioles or proximal alveolar regions in the selected blocks were predetermined, thin sections of the blocks were cut by aligning the diamond knife to tho block faces. Sections were placed on 200-mesh copper grids, poststained in uranyl acetate and lead, and examined on a Zeiss lOC electron microscop (Carl Zeiss Inc., Germany).
Other examinations:
ELECTRON MICROSCOPIC MORPHOMETRY
Electron microscopic morphometric analysis was used to determine tissue volume, surface area, and cell characteristics regions, and terminal bronchioles. A total of 177 morphologic parameters were calculated for each animal (2 related to the whole animal, 94 in the proximal alveolar region, 47 in the random alveolar regions, and 34 in the terminal bronchioles).

Proximal Alveolar Regions
Three sites in the proximal alveolar region were randomly selected from each animal. Thin sections from each of these sites were stained and examined with an electron microscope. Two photomicrographs were taken of each grid square, one in the upper left corner and one in the lower right corner. All nuclear profiles in each grid square used for photography were counted on the electron microscope using Gundersen's rule of forbidden lines (Gundersen 1977). Micrographs were enlarged to x 8500 on 11- by 19-inch photographic paper that previously had been printed with a point-counting lattice of 448 lines, each 1.37 cm long. Points, intercepts, and nuclear profiles were counted to determine cell volume and cell surface densities. The formula used in the morphometric analysis has been described in detail {Barry and Crapo 1965; Chang and Crapo 1990).

Epithelium.
The epithelium is subdivided into alveolar (type I and type II) and bronchiolar-like (ciliated, Clara, and other) cells. The volume of total epithelium and of each of the components of the epithelium were determined. The cell area (numerical density), mean cell volume, and, for epithelial and endothelial cells only, mean cell surface area.

Interstitial Matrix.
Morphometric analysis of the interstitial matrix followed procedures designed by Vincent and associates (1992). The interstitial matrix was subdivided into four compartments: collagen fibers, elastin, basement membranes, and cellular space. Collagen fibers were defined as any arrangement of fibrils that can be delineated by an estimated perimeter and distinguished, according to the density of fibrils in the fibers, from other neighboring components.
Elastin was recognized as amorphous material uniformly stained by tannic acid and uranyl acetate. A basement membrane was juxta~posed to both epithelial and endothelial cells and was easily recognized. The volume of each interstitial matrix component was calculated and normalized to basement membrane surface area in the same manner described for tissues and cells in the proximal alveolar regions.

Endotbelium and Capillaries.
The volume of capillary endothelium normalized to the surface area of the basement cells were measured. The volume of the capillary bed was subdivided into red blood cells and plasma. The plasma component included all white blood cells. Because the results of numerous earlier studies of ozone exposure fail to show changes in white blood cells in the pulmonary vasculature, volume and cell characteristics of white blood cells were
not measured as a separate category.
Evidence of Inflammation.
Inflammatory cells (macro phages and neutrophils) in the alveolar spaces and in the interstitium were used as indicators of inflammation. The volumes and the cell characteristics of both were measured.

Random Alveolar Regions
Tissue and cell volumes were measured morphometrically as indices of the responses to ozone exposure in the total gas exchange region, referred to as the random alveolar region. Three blocks of tissue from each animal were randomly selected from the embedded tissue blocks without prior examination under the dissection microscope and without knowledge of the presence or absence of bronchiolealveolarduct junctions. Fifteen micrographs, taken from the upper left corner of 15 consecutive grid squares, were obtained. Photographs were printed and analyzed in a manner similar to those from the proximal alveolar regions. Only total cell volume, matrix volume, and surface area were measured. Cell characteristics and matrix components were not analyzed.

Terminal bronchioles
The terminal bronchioles were examined using morphometric techniques described by Barry and associates (1988) and Chang and associates (1 988). The complete epithelium of each terminal bronchiole examined was photographed by 25 to 30 overlapping micrographs taken at a magnification of x 2000. Pictures were enlarged to x 8500 and printed on 11- by a 14-inch photographic paper. A montage of each terminal bronchiole was constructed and the portion contributed by each micrograph was marked on the composite. The pictures were then placed under a Merz overlay sheet marked with 224 points. Points falling on each cell type and intercept between test lines and a luminal surface, a basal surface, or a cilium were counted. The number of nuclei of each type of cell in the montage was recorded. The area of the bronchiolar epithelium and the lengths of the luminal surface and the basement membrane were measured by a digitizer. The thickness of the epithelium was derived from these measurerments by assuming that the cross section of a bronchiole was a circle. The volume density of each cell type and the surface densities of the luminal and basement membrane surface areas for each type of cell in relation to the total volume of the terminal bronchiolar epithelium (the reference space) were calculated using point and intercept counts. For each terminal bronchiole, the number of cells is expressed in relation to the surface area of the bronchiolar epithelial basement membrane.

Statistics:
For the purposes of statistical analysis, we developed an analytical approach that would use all information most efficiently. Five categories of injury were established and one or two of the 177 measured parameters identified as the most sensitive indicators for each kind of injury. These we called primary variables. First, a multivariate analysis of variance (MANOVA) was done to test for statistical significance in this vector of primary variables. When the MANOVA revealed a significant relationship, univariate analysis of variance (ANOVA) was performed. If the MANOVA did not demonstrate significance, the variable was not tested further. In this study, six primary variables were established in the proximal and randomly selected alveolar regions, and seven primary variables in the terminal bronchioles were established.
If a primary variable showed statistical significance in the first MANOVA, then a second MANOV A was performed. The second vector for multivariate analysis consisted of 1 to 5 key variables that provided more information than the primary variable alone about the category of injury.
In this study, 15 key variables were identified in the proximal alveolar region and 11 in the random alveolar regions. No key variables were counted in the terminal bronchioles. As for the primary variables, multivariate significance was required for each of the five (corresponding to the five injury categories) key variable MANOVAs before examining univariate significance and comparisons among exposure concentrations. This analysis provides the statistical basis for the statements of significant effects given in this report.

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
lung (see "Details on results"). Other information in this context is presented in the NTP study.
Histopathological findings: neoplastic:
not examined
Details on results:
EFFECTS OF OZONE CONCENTRATION AND GENDER ON THE PROXIMAL ALVEOLAR REGION
The MANOVA applied to the primary variables in the first stage of the statistical analysis revealed an effect due to ozone exposure concentration in the following injury classes: bronchiolarization, interstitial, vascular, and inflammation. No significant effects were attributable either to gender or to an interaction between gender and concentration. Therefore, effects from gender were not considered in subsequent stages of the statistical analysis. Significant effects due to ozone concentration were indicated by changes in the following primary variables: the percentage of bronchiolarization, the volume of interstitium, the volume of alveolar macrophages, and the surface area of capillaries. The volume of type I epithelium did not change following ozone exposure.
An uncontrolled ANOVA indicated that ozone concentration had an effect on a confirmatory variable, the total volume of tissue (epithelium, interstitium, and endothelium) due to increases in the volumes of both epithelium and interstitium.

Epithelium
The epithelium in the proximal alveolar regions was not significantly altered by exposure to 0.12 ppm ozone, but major changes were found in the epithelium of rats exposed to either 0.5 or 1.0 ppm ozone. The higher concentrations of ozone induced epithelial metaplasia (a change of the squamnous alveolar epithelium to cuboidal bronchiolar epithelium, also referred to as bronchiolarization) in the proximal alveolar regions. Normally, only a small number of bronchiolar epithelial cells, if any, can be found extending from terminal bronchioles into the proximal alveolar region.
In control rats, less than 2 % of the surface area in the proximal alveolar region was covered by bronchiolar cells. After exposure to either 0.5 or 1.0 ppm ozone, 7% or 12%, respectively, of basement membrane surface was covered by bronchiolar epithelium, and the volume of bronchiolar epithelium in the proximal alveolar region increased 2.5- fold or 4.5-fold, respectively. The extent of bronchiolarization induced by ozone, therefore, is dependent on dose, although only the effect at 1.0 ppm ozone was statistically significant. The bronchiolar cells lining the alveolar ducts and the alveoli consisted mainly of fully differentiated ciliated cells and Clara cells that were structurally identical to those found in terminal bronchioles. Both types of cells increased in number and volume by the prolonged ozone exposures. Unlike the terminal bronchiolar epithelium in normal proximal alveolar regions, in which the volume of ciliated cells is approximately twice as large as the volume of Clara cells, the metaplastic epithelium in the proximal alveolar regions contained approximately equal volumes of ciliated and Clara cells. There was also a large increase in the volume of unidentified cells (referred to as "other cuboidal cells"). These cells were observed only in the metaplastic proximal alveolar region, and typically contained differentiated features of both ciliated cells and Clara cells, including fiber bundles, secretory granules, basal bodies, and glycogen granules. Preciliated cells and brush cells that are normally found in the terminal bronchioles were not observed in the metaplastic cuboidal epithelium in the proximal alveolar regions. Morphometric analysis of the characteristics of the ciliated and Clara cells in the metaplastic epithelium indicated that there was no effect of ozone exposure on mean cell size or on mean cell surface area.
Although the total epithelial volume increased, duo to the metaplasia of the normal squamous epithelium to a cuboidal type of epithelium, in rats exposed to 0.5 or 1.0 ppm ozone, the total volumes of type I and type II epithelium were not changed by the exposures. Univariate ANOVA, however, showed an effect of ozone concentration on the characteristics of type I epithelial cells. The number of type I cells increased 64% to 74% after exposure to 0.5 or 1.0 ppm ozone, respectively. The size of the type I cells decreased approximately 40%, and the luminal and basal cell surface areas of alveolar type I cells decreased to approximately 50% of the control values. Few structural abnormalities wore noted, except for focal thickening of type I epithelium and small areas of cell necrosis. The characteristics of alveolar type II cells were not altered by the ozone exposures.

Interstitium
The volume of the interstitium increased as a function of ozone concentration. No difference in interstitial volume was noted between control rats and those exposed to 0.12 ppm ozone; however, the volume of interstitium was significantly increased after exposure to 0.5 or 1.0 ppm ozone. The vector of key variables for interstitial injury consisted of the cellular components and the compartments making up the interstitial matrix: acellular space, basement membrane, collagen, and elastin. Exposure to ozone had a significant effect on the volume of each of these variables. Elastin and acellular space were significantly elevated after exposure to 1.0 ppm ozone, and the volumes of cellular interstitial components, collagen, and the basement membrane were significantly increased after exposure to 0.5 or 1.0 ppm ozone.
The total interstitial volume increased 5:3 % after exposure to 0.5 ppm ozone, with a 40% increase in the cellular component and a 60% increase of the noncellular component. After 1.0 ppm exposure to ozone, total interstitial volume increased 71 % with a 44% increase of the cellular component and an 84% increase of the noncellular components. The increase in the volume of cellular interstitium was due mainly to an increase in interstitial fibroblasts that constituted approximately 80% of all interstitial cells. The increase of interstitial volume after exposure to 0.5 ppm ozone arises from small increases of both the number of fibroblasts and their mean cell size. Exposure to 1.0 ppm ozone, on the other hand, resulted in a significant increase in the number, but no increase in the mean cell volume, of interstitial fibroblasts. Furthermore, neither the number nor volume of interstitial cells was changed after ozone exposure.
Of the total noncellular interstitium, 40% to 50% is occupied by collagen. After 20 months of exposure to 0.5 ppm ozone, the volume of collagen increased 64%, and after exposure to 1.0 ppm ozone, the volume increased 78%. Basement membrane accounts for 22% to 26% of the noncellular interstitium. Exposure to 0.5 or 1.0 ppm ozone induced thickening of the basement membrane. The magnitude of changes were similar to those observed with collagen. The thickened basement membranes contained inclusion bodies the origin of which is not known. Elastin makes up only 2% of the total noncellular interstitium, and is mainly localized at septal tips. Exposure to 1.0 ppm ozone for 20 months caused an 80% increase in the volume of elastin. Acellular space made up 22% to 27% of the noncellular interstitium, and its volume increased 11% by exposure to 1.0 ppm ozone.

Endothelium and Capillaries
The vectors of primary variables for vascular injury were the volume of endothelium and the surface area of capillaries. No significant change in the volume of endothelium was observed in this study. The mean cell volume and the mean cell surface area of capillary endothelial cells remained unaltered after 20 months of exposure to either 0.12, 0.5, or 1.0 ppm ozone. The number of endothelial cells increased slightly after exposure to 1.0 ppm ozone. A concentration effect was observed and found to be due to a significant increase in the capillary surface area after exposure to 0.5 ppm ozone. However, exposure to 0.12 or 1.0 ppm ozone did not cause significant change in the parameter of capillary surfaces. The inconsistency of the trend of ozone effect on capillary surface suggests that the change found after 0.5 ppm ozone may not be biologically significant.

Evidence of Inflammation
The primary variable used for analyzing inflammation was the volume of the inflammatory cells (the sum of the alveolar and interstitial macrophages). An ozone concentration effect was found for inflammatory cells by MANOVA. Exposure to 0.12 or 0.5 ppm ozone for 20 months did not change either the number or the size of alveolar macrophages. However, rats exposed to 1.0 ppm ozone exhibited a 113% increase in alveolar macrophages in the proximal alveolar region. Interstitial macrophages were rare in all exposure groups, and no significant changes in the volume of interstitial macrophages were noted after ozone exposure. Other inflammatory cells such as neutrophils and monocytes were not included in the quantitative analysis because previous studies with subchronic and prolonged exposures to ozone had shown little or no involvement of these cells. Qualitative examination of the sections in this study confirmed the near absence of neutrophils and monocytes in the proximal alveolar regions of rats exposed to 0.12, 0.5 or 1.0 ppm ozone for 20 months.

EFFECTS OF OZONE EXPOSURE ON RANDOM ALVEOLAR REGIONS
Because previous studies have shown that the effects of 0.12 ppm ozone are strictly confined to the proximal alveolar regions, the morphometric study of random alveolar regions was carried out only with animals exposed to 0.5 or 1.0 ppm ozone. The set of primary variables was assessed with MANOVA. No statistically significant ozone concentration effect was found. Because the epithelium of the distal alveolar regions, which constitute the great majority of the gas exchange region from which the alveolar region blocks were randomly selected, is composed completely of alveolar type I and type II epithelial cells, the percentage of bronchiolarization measured in all exposure groups was not significantly different from the control group. Type I and type II epithelial volumes were not altered by the exposures. The volumes of cellular interstitium and interstitial matrix and the volumes of the components of the interstitial matrix also were not changed by exposure to any concentration of ozone. The ultrastructure of the alveolar epithelial cells, interstitial fibroblasts, and capillary endothelial cells was normal. No significant increase of inflammatory cells was noted in either the alveolar spaces or in the interstitium. Further studies of the group exposed to 0.12 ppm ozone were not performed because no effect was found for the higher ozone doses.

EFFECTS OF OZONE EXPOSURE ON TERMINAL BRONCHIOLES
Multivariate analysis was carried out using the primary variables listed. Statistically significant effects of exposure to 1.0 ppm ozone were observed in the number of ciliated cells, the number of Clara cells, and the mean cell volume of Clara cells. In addition, no effect was found for the thickness of the terminal bronchiole epithelium, the mean cell volume of ciliated cells, the mean luminal surface areas of both ciliated and Clara cells, and the average diameter of terminal bronchioles.
Five types of cells were studied from the terminal bronchioles, ciliated cells, Clara cells, brush cells, preciliated cells, and unidentified cells. Exposure to 1.0 ppm ozone for 20 months was found to cause a 13% reduction in the total number of cells per unit of basement membrane (mm2) in terminal bronchioles. This reflects the combined result of a cell population shift with loss of ciliated cells but an increase in Clara cells. The total number of ciliated cells reduced 37% from 11,508 cells/mm2 to 7,255 cells/mm2 of basement membrane surface area. The percentage of ciliated cells in terminal bronchioles decreased from 71% in control rats to 51% in rats exposed to 1.0 ppm ozone. Despite the loss of ciliated cells from terminal bronchioles, the characteristics of those cells remained normal except for a 20% increase of their basement membrane surface area. The surface area of cilia per cell was not changed. However, due to the reduced number of ciliated cells, the total density of ciliated surface (or cilia) in terminal bronchioles was reduced. In contrast to ciliated cells, the number of Clara cells in terminal bronchioles increased 54% after exposure to 1.0 ppm ozone, and the volume fraction of Clara cells increased from 21% to 36% of terminal bronchiolar cells. The size of the dome or luminal surface of Clara cells was not affected, but the average size of Clara cells decreased 13%. Aside from swollen cilia, the ultrastructure of ciliated cells and Clara cells was normal. Brush cells were identified by their brush Larders and filament bundles. They consisted of only 2% (by number) of the terminal bronchiolar cells in rats exposed to 0.0 ppm, 0.12 ppm, and 0.5 ppm ozone. Exposure to 1.0 ppm ozone increased the percentage of brush cells in terminal bronchioles to approximately 3%. Preciliated cells, a precursor of ciliated cells containing basal bodies and fibrinogen granules, were found to become smaller after exposure to 0.5 ppm ozone, and had a reduced basal surface area. These minor changes did not follow dose patterns and are probably random variability resulting from the small number of both cell types found in the terminal bronchioles.

Effect levels

Dose descriptor:
NOAEC
Effect level:
ca. 0.12 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
0.5 ppm
System:
respiratory system: lower respiratory tract
Organ:
alveolar duct
alveoli
bronchi
bronchioles
lungs
trachea
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
The study provides valuable supportive information from morphometric studies of rats exposed to 0.0, 0.12, 0.5, or 1.0 ppm ozone for 20 months. The major finding was that exposure to 0.5 or 1.0 ppm ozone caused significant effects. Changes in the proximal alveolar region consisted of epithelial metaplasia, with replacement of squamous (type I) alveolar epithelial cell types by a cuboidal (type II) bronchiolar epithelium, and a thickening of the interstitium due to increases in both cells and extracellular matrix components. In the terminal bronchioles after exposure to 1.0 ppm ozone, decreases in cilia and in the volume and number of ciliated cells were noted, with a proportional increase in Clara cells. The number of alveolar macrophages examined also increased at this exposure level. No morphometric effects of exposure to 0.12 ppm ozone were observed in the proximal alveolar region or the terminal bronchioles, indicating no effects of a 20-month exposure to 0.12 ppm ozone in these lung locations in the animals. Suggested NOAEC chronic = 0.12 ppm = 0.24 mg/m³.
Executive summary:

This research study is part of the NTP/HEI Collaborative Ozone Project published in a peer-reviewed publication. The study provides valuable supportive information from morphometric studies of rats exposed to 0.0, 0.12, 0.5, or 1.0 ppm ozone for 20 months. Morphometric techniques were used to examine cellular and tissue changes occurring in male and female rat lungs exposed to ozone for a prolonged time.

F344/N rats were exposed to ozone concentrations of 0.0 (control) 0.12, 0.5, or 1.0 parts per million (ppm), six hours per day, five days per week for 20 months.

The major finding was that exposure to 0.5 or 1.0 ppm ozone caused significant effects. Changes in the proximal alveolar region consisted of epithelial metaplasia, with replacement of squamous (type I) alveolar epithelial cell types by a cuboidal (type II) bronchiolar epithelium, and a thickening of the interstitium due to increases in both cells and extracellular matrix components. In the terminal bronchioles after exposure to 1.0 ppm ozone, decreases in cilia and in the volume and number of ciliated cells were noted, with a proportional increase in Clara cells. The number of alveolar macrophages examined also increased at this exposure level. No morphometric effects of exposure to 0.12 ppm ozone were observed in the proximal alveolar region or the terminal bronchioles, indicating no effects of a 20-months exposure to 0.12 ppm ozone in these lung locations in the animals.

Suggested NOAEC chronic = 0.12 ppm = 0.24 mg/m³

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