Ozone

Respiratory sensitisation

Administrative data

Endpoint:

respiratory sensitisation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

No guideline followed, experimental research study

GLP compliance:

no

Test material

Test material information

Constituent 1

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:

O3–oxygen mixture, generated by irradiation of oxygen with ultraviolet (UV) light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Male, specific-pathogen-free Wistar rats (7 wk old, 170–190 g) were obtained from the breeding colony of the RIVM. During the acclimatization period as well as during exposure and recovery experiments the animals were housed individually in 0.2-m3 stainless steel and glass inhalation chambers in the RIVM inhalation facilities (Marra & Rombout, 1990). Each chamber contained one experimental group of animals. Air was purified by an activated charcoal, a permanganate, and a highly efficient particle filter, and was conditioned at a temperature of 22 ± 1°C and 55 ± 5% relative humidity. Airflow of 6 m3/h was maintained through each chamber. The animals were subjected to an acclimatization period of 2 weeks before the onset of exposure and had unlimited access to tap water and pelleted standard laboratory diet (RMH-B, Hope Farms, Woerden, the Netherlands). The animals were maintained on a 12-h, reversed light–dark cycle with lights on from 9 p.m. to 9 a.m.

Test system

Route of induction exposure:

inhalation

Route of challenge exposure:

inhalation

Vehicle:

other: Clean air

Concentration:

0.4 ppm for 12 h per day during the dark phase

No. of animals per dose:

Group size was 5 animals

Details on study design:

For O3 exposure, an O3–oxygen mixture, generated by irradiation of oxygen with ultraviolet (UV) light, was metered into the inlet air stream with stainless-steel mass flow controllers at a rate of approximately 30 ml/ min. The exposures were performed automatically using an exposure control program running on an Altos 1086 microcomputer interfaced to the exposure equipment. Concentrations in the chambers were measured at 2- min intervals with Monitor Labs 8810 O3 analyzers (San Diego, CA), and adjustments of the flow controllers were made to maintain concentrations at the desired levels. The analyzers were checked several times per day against a reference O3–air mixture and zero-air generated by a Monitor Labs 8550 calibrator (San Diego, CA), which was calibrated weekly by means of gas-phase titration.

"Repeatedly exposed" :
Group : 5 consecutive days to 0.4 ppm ozone, autopsy on day 6,
Group : 4 days fresh air and on day 5 0.4 ppm ozone, autopsy day 6,
Group : 5 days fresh air, autopsy on day 6.

"Repeatedly exposed plus challange following recovery" :
Group : 5 consecutive days to 0.4 ppm ozone, followed by recovery and challenge on day 10, autopsy on day 11,
Group : 5 days fresh air, followed by recovery and challenge on day 10, autopsy on day 11,
Group : 10 days fresh air, autopsy on day 11,
Group : 5 consecutive days to 0.4 ppm ozone, followed by recovery and challenge on day 15, autopsy on day 16,
Group : 5 days fresh air, followed by recovery and challenge on day 15, autopsy on day 16,
Group : 15 days fresh air, autopsy on day 16,
Group : 5 consecutive days to 0.4 ppm ozone, followed by recovery and challenge on day 20, autopsy on day 21,
Group : 5 days fresh air, followed by recovery and challenge on day 20, autopsy on day 21,
Group : 20 days fresh air, autopsy on day 21,
Group : 5 consecutive days to 0.4 ppm ozone, followed by recovery and challenge on day 25, autopsy on day 26,
Group : 5 days fresh air, followed by recovery and challenge on day 25, autopsy on day 26,
Group : 25 days fresh air, autopsy on day 26,

Negative control substance(s):

other: concurrent vehicle

Results and discussion

Results:

A single 12-h exposure to 0.4 ppm ozone causes marked increases of alveolar–capillary permeability, inflammatory responses, and cell damage in lower airways of rats, as evidenced by BAL fluid levels of proteins, albumin, inflammatory cell composition, fibronectin, IL-6, and number of dividing cells in terminal bronchiolar epithelium. It also shows that 5 consecutive exposures to 0.4 ppm O3 for 12 h/night results in a very substantial, in some cases almost complete, disappearance of these responses, and the BAL fluid values are not different from or very close to those observed in unexposed controls. Analyses of pulmonary effects in O3-challenged rats demonstrate that for lung tissue to display its full susceptibility again to an O3 challenge following the 5-day preexposure period takes approximately 15–20 days.

Any other information on results incl. tables

Alveolar-Capillary Permeability Indicators in BAL Fluid

Increases in the total protein level in BAL fluid are considered to reflect the transfer of relatively small proteins across the alveolar–capillary barrier, while increases in BAL fluid albumin content have been used as a specific marker of serum transudation accompanying the inflammatory response. Single ozone exposure rats revealed a consistent 3–4 fold increase in the levels of the proteins in BAL fluid. Single O3 challenges in O3-preexposed rats showed at all autopsy dates a significantly decreased protein level in comparison with the single exposure groups, but this value was still significantly higher than the level in unexposed controls. The BAL fluid albumin level at day 6 following 5 days of O3 exposure significantly differed from the single O3 exposure group but did not differ from unexposed controls. Only at day 16 was the albumin  level of the repeated exposure group significantly decreased compared with the single exposure group, but it was still significantly higher compared with the control group. At days 11, 21, and 26 there was no significant difference in the albumin level between the repeated and the single exposure groups, although the albumin level in the repeatedly exposed groups was still lower than in the single exposure groups.

Inflammatory Cell Populations in BAL Fluid

Using the Hatch et al. (1986) lavage protocol, total cell number counted did not significantly change between control and ozone-exposed groups (control level 4.4 × 105 cells). Changes in inflammatory cell populations in BAL fluid were taken as indicators of pulmonary inflammation. Single O3 exposure rats revealed a consistent, approximately 20-fold increase in the percentage of PMNs in BAL fluid. Single O3 challenges in O3-preexposed rats showed distinctive effects depending on the time point of the challenge after preexposure. Following 5 days of O3 exposure, the BAL fluid PMN level at day 6 significantly differed from the single O3 exposure group, but did not differ from clean-air controls. O3 challenge of preexposed groups on day 10, 15, or 20 resulted in significant increases in BAL fluid PMN levels compared to respective unexposed groups. Although the PMN response tended to recover (i.e., getting a normal response following a single ozone exposure) with longer postexposure periods, these values remained different from the PMN increases elicited in the single O3 exposure groups. By day 26, after a 20-day recovery period, PMN response following an O3 challenge in O3 preexposed rats was, although still considerably smaller, not statistically different from the response in the single O3 exposure group. Single O3 exposure rats showed a consistent, approximately 15–20% decrease in the percentage of macrophages in BAL fluid. In parallel with the PMN data, O3 challenges in O3-preexposed rats also showed different effects at various time points of challenge after preexposure. Following an O3 challenge on exposure day 5, 10, 15, or 20, the BAL fluid macrophage levels significantly increased with respect to the level of the single O3 exposure group and differed from unexposed controls (except for day 6). A single O3 challenge on preexposed animals on day 25 resulted in a decrease in BAL fluid macrophage level compared to its respective unexposed group, and this decrease was not different from that elicited in the single O3 exposure group. It should be stressed that the relative decrease in macrophage number in BAL fluid is associated with a relative increase of PMN level.

Inflammatory Proteins and Eicosanoids in BAL Fluid

It could be shown that the BAL fluid data on fibronectin and IL-6, mediators playing a role in inflammation and tissue structuring, as measured in the control and O3-exposed groups. Fibronectin is considered as a mediator facilitating fibroblast recruitment, adhesion, and replication, and is therefore suggested to play an important role in growth, wound healing, and fibrogenic processes in the lungs. Fibronectin levels are also associated with type II alveolar epithelium cell differentiation. Furthermore, bronchiolar epithelial cells can also produce fibronectin. The results show a consistent, approximately 10- to 15- fold increase in BAL fluid fibronectin level following single O3 exposure. O3 challenge in O3-preexposed rats show again distinctive effects depending on the time point of the challenge after preexposure. If the O3 challenge was given on exposure day 5, the BAL fluid fibronectin level at day 6 significantly differed from the single O3 exposure group and did not differ from unexposed controls. O3 challenge of preexposed groups on day 10, 15, 20, or 25 resulted in increases in BAL fluid fibronectin levels compared to respective unexposed groups. Although the fibronectin response tends to recover with longer postexposure periods, these values remained different from, that is, lower than, the fibronectin increases following single O3 exposure groups throughout the 20-day recovery period. IL-6 is a cytokine considered to play an important role in induction and propagation of acute inflammation. In addition, IL-6 is suggested to be involved in the acute-phase response by stimulating the liver to produce and release acute-phase proteins, which are able to limit tissue damage at peripheral sites, such as the lungs. Data show a reproducible, approximately 20- to 25-fold increase in BAL fluid IL-6 level following single O3 exposure. O3 challenges in O3-preexposed rats showed differential effects depending on the time point of the challenge after preexposure. If the O3 challenge was given on exposure day 5 or 15, the BAL fluid IL-6 levels at day 6 and 16 significantly differed from the single O3 exposure group and did not differ from their respective unexposed controls. O3 challenge of preexposed groups on day 10, 20, or 25 resulted in increases in BAL fluid IL-6 levels compared to their time-matched unexposed controls, and, although consistently lower, these levels were not statistically different compared to their single O3 exposure counterparts. Cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism led to biologically active substances thought to play an important role in host defense and inflammatory mechanisms in lung tissue. The BAL fluid levels of the cyclooxygenase pathway metabolite prostglandin E2 (PGE2) and of 15-HETE showed inconsistent responses upon O3 exposure, both after a single exposure and after an O3 challenge of preexposed groups.

Histopathology

No differences in lung weights were seen between any of the groups. No histological changes were detected in any of the clean-air-exposed animals. Some animals of the repeatedly exposed groups followed by an O3 challenge had a slight centriacinar inflammatory reaction consisting mainly of alveolar macrophages. This change was seen in all animals at days 6 and 21, 4 animals at day 11, and 2 animals at day 26.

Morphometry

The number of alveolar macrophages in all single O3 exposure groups was statistically significantly increased compared with the unexposed groups. All groups repeatedly exposed and then challenged with O3 had a statistically significant increase in the numbers of alveolar macrophages compared with the control groups. At days 6, 11, and 21, the numbers of alveolar macrophages in the groups repeatedly exposed and challenged were statistically significantly increased in comparison with the single-exposure groups, but with increasing recovery time this number decreased. In additional groups repeatedly exposed for 5 days and sacrificed after 5, 10, 15, or 20 days of recovery, the number of macrophages decreased to control values within 5 days of recovery.

The number of BrdU-labeled cells per millimeter terminal bronchiolar epithelium was also determined. This number was relative small in all control groups. In all groups exposed a single time to O3 the number of labeled cells increased by a factor of about 20. In the groups repeatedly exposed and challenged, the number of labeled cells was always lower than the number in the matched single exposure groups. This decrease was statistically significant at days 6 and 21. In extra groups repeatedly exposed for 5 days and sacrificed after 5, 10, 15, or 20 days, the number of labeled cells equaled the control value within 5 days of recovery.

Furthermore, the number of BrdU-labeled cells per millimeter bronchiolar epithelium was determined. The number of labeled cells was very small in all control groups and equals the number of labeled cells in terminal bronchioles. A single O3 exposure resulted in a significant decrease in labeling in comparison with the control groups. In the groups repeatedly exposed and challenged, no differences in labeling were seen compared with the values of their respective single-exposed groups except at day 6, when the reeatedly exposed group had a significant increased number of labeled cells versus the single-exposure group.

The number of BrdU-labeled cells in the centriacinar alveolar epithelium was determined as well. In all control groups this number was very small: about 0.1/mm. In all single O3 exposure groups the labeling was largely increased by a factor of about 15. The labeling in the groups repeatedly exposed and challenged was not different from those of the single-exposure groups, except for day 16, when it was statistically significantly decreased. In extra groups repeatedly exposed for 5 days and sacrificed after 5, 10, 15, or 20 days of recovery the number of labeled cells equaled the control value within 5 days.

The data on body weights of all of the experimental groups reveal that body weight was not affected by ozone exposure throughout the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The research study showed that a single 12-h exposure to 0.4 ppm ozone causes marked increases of alveolar–capillary permeability, inflammatory responses, and cell damage in lower airways of rats, as evidenced by BAL fluid levels of proteins, albumin, inflammatory cell composition, fibronectin, IL-6, and number of dividing cells in terminal bronchiolar epithelium. It also shows that 5 consecutive exposures to 0.4 ppm O3 for 12 h/night results in a very substantial, in some cases almost complete, disappearance of these responses, and the BAL fluid values are not different from or very close to those observed in unexposed controls. Analyses of pulmonary effects in O3-challenged rats demonstrate that for lung tissue to display its full susceptibility again to an O3 challenge following the 5-day preexposure period takes approximately 15–20 days.

Executive summary:

The aim of this research study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O3 exposure and to study the time course of attenuation and recovery of these effects using single O3 challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O3 for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O3 after a 5-, 10-, 15-, or 20-day recovery period. Bronchoalveolar lavage (BAL) examination and histopathology were performed 12 h after this O3 challenge. To quantify the magnitude of the O3 response, results were compared with a group exposed only once for 12 h to 0.4 ppm O3 and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O3 causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin ( IL) -6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O3 for 12 h/ night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O3 challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O3 following a 5-day preexposure period is approximately 15–20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O3 challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5–10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O3 may take longer than previously assumed.

This study meets basic scientific pricipals and was therefore used for risk assessment. It provides valuable information on the recovery and attenuation of lung injury following subacute low level ozone exposure. Furthermore it shows, that no hypersensitivity to ozone at the challenge exposure was shown by the test animals. This information is used in a weight of evidence appraoch regarding the respiratory sensitisation properties of ozone.