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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Ozone uptake in healthy adult males during quiet breathing
Wiester M.J. et al.
Bibliographic source:
Fundamental and Applied Toxicology 29, 102-109

Materials and methods

Objective of study:
Test guideline
no guideline followed
Principles of method if other than guideline:
Research study
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Ozone was generated in-situ using an uv ozone generator

Test animals

Details on test animals or test system and environmental conditions:
Ten male subjects, 19-32 years of age, were recruited from the community in and around Chapel Hill, North Carolina. Excluded from participation in the study was anyone who smoked, had a history of asthma, allergic rhinitis, cardiac disease, acute respiratory disease within the previous 4 weeks. Screening procedures included a medical history, a physical examination, and a complete blood count plus differential white cell count. Accepted subjects were informed of the purpose of the study, the experimental methods, and the potential risks of participation before signing a statement of informed consent. This study was approved by the Committee on the Protection of the Rights of Human Subjects of the University of North Carolina School of Medicine.

Administration / exposure

Route of administration:
inhalation: gas
other: air
Details on exposure:
The exposure apparatus was located in a 4 x 6 x 3.2-m stainless-steel walk-in environmental chamber. The chamber was maintained at 22°C and 40% relative humidity. Ozone was produced for the chamber by passing incoming air through a series of ultraviolet light O3 generator tubes. Ozone concentration was monitored by chemiluminescence detection using Bendix Model 8002 analyzers which were calibrated against an ultraviolet O3 photometer (Dasibi Model 1003AH). A detailed description of the environmental chamber operation has been published.
The subject exposure apparatus consisted of 4-in. stainless-steel pipe which opened to the environmental chamber at one end and attached to a fan/damper assembly at the other end. Chamber air was drawn down through the apparatus by the fan/damper assembly. Ports were cut into the apparatus for sampling of O3, O2, and CO2. A face port was also cut into the pipe for the subject to insert his nose and mouth and breathe from the airstream flowing through the apparatus. The exposure apparatus flow rate was approximately 40 L/min. At this rate, the downstream (relative to the face port) dilution of exhaled air with the airstream was similar to that used in the rat and guinea pig studies. Testing indicated that this rate was sufficient to prevent the rebreathing of downstream air by the subject and to supply an adequate volume of upstream air during maximum inspiratory flow. Flow measurements were determined using a Roots meter (Model 5M). Additionally, flows were checked for accuracy with a Gilibrator Primary Flow Calibrator (Gilian Instrument Corp., West Caldwell, NJ).
Duration and frequency of treatment / exposure:
Single 10 min exposure, followed by a 2nd 10 min exposure two weeks later
Doses / concentrations
Dose / conc.:
0.3 ppm
No. of animals per sex per dose / concentration:
n=10 subjects
Control animals:
Positive control reference chemical:
not applicable
Details on study design:
Subjects were measured, using the same experimental protocol, on two visits. These visits were separated by at least 14 days.
On the day of an exposure, the subject completed a symptom questionnaire and performed forced expiratory maneuvers. These consisted of three forced vital capacity (FVC) maneuvers performed on a 12-1 dry seal spirometer (CPI model 220). Peak expiratory flow rate (PEF) and forced expiratory volume at 1 sec (FEV, 0) were calculated for each maneuver. The subject was then fitted with a telemetered ECG monitor and the Respitrace bands and exposed to clean air in the exposure chamber. The first 5 min for system equilibration followed by 10 min sampling to collect air exposure data. Similar runs were performed for nose and mouth breathing. Subsequently the whole procedure was repated, but with exposure to ozone. The percentage of O3 uptake was calculated using O3 upstream (µg/L); O3 downstream (µg/L); O3 background loss (µg/L); SAF is the system airflow (L/min); and VE (L/min).

Details on dosing and sampling:
Breathing parameters.
Subject chest wall movements caused by breathing efforts were measured using Respitrace inductance plethysmography chest bands (Respitrace, Ambulatory Monitoring, Inc., Ardsley, NY). The fitted Respitrace was calibrated by having the subject quietly breathe into a rolling seal spirometer that had been previously calibrated with a 1-liter syringe.
Tidal breathing measurements were obtained dunng the experiment by sampling the Respitrace signal at 12 msec intervals for 20 sec each minute. Breathing parameters reported represent averaged values from all breaths completed during this 20-sec sampling period (usually four to six breaths). These include tidal volume (VT), breathing frequency (f) expiratory minute volume (VE); maximum inspiratory and expiratory flows (Vimax. Vemax), inspiratory and expiratory times (Ti, Te), oxygen consumption and carbon dioxide production and the respiratory quotient (VO2, VCO2, RQ).
All breathing parameters and the percentage of O3 uptake were examined for time-related trends over the 10-min exposure period using linear regressions. These were calculated for each subject and breathing mode during each exposure. Because no trends were found, the individual minute by minute data were averaged for each subject dunng each exposure for each breathing mode. These means were used as the unit of observation for the subsequent analyses.
A two-way multivariate analysis of variance (MANOVA) was used to examine the effects of "route" of exposure (nasal or oral), "visit" (first or second), and the interaction between these two factors on the percentage of O3 uptake. Significance of an effect was determined using the Hotelling- Lawley trace. Because there were only two categories for each main effect companson (i.e., nasal versus oral or visit 1 versus visit 2), no further subtesting was required for significant main effects. Interactions between route and visit were tested using paired / tests. Three-way MANOVAs were used to examine the effects of route, visit, and exposure (air versus O3) and all possible interactions in the breathing data.
Pearson correlation coefficients were calculated to test for linear relationships between the percentage of O3 uptake and the breathing parameters, spirometric measurements of lung function, and subject weight and height.

Results and discussion

Preliminary studies:

Toxicokinetic / pharmacokinetic studies

Details on absorption:
There was no significant effect of visit on the percentage of O3 uptake, demonstrating the reproducibility of the measurement. There was a slight but significant route effect such that the mean percentage of O3 uptake when breathing orally was 76.5% ± 3.3 SE compared to 73.1% ± 3.0 when breathing nasally. There was no interaction between visit and route.
The relative extent of intra- and intersubject variability was assessed. The variability between subjects was substantial. The lowest percentage of O3 uptake was observed in one subject, ranging from about 50 to 60% whether breathing orally or nasally. Among the nine other subjects, the percentage of O3 uptake ranged between about 65 and 95%, a difference of about 30%. In general, the variability in percentage of O3 uptake for an individual subject was less than this although in some cases (e.g., oral breathing), there was a difference of approximately 25% between the first and second visits. The average difference in percentage of uptake between the first and second visits for oral breathing was -3.1% ± 3.7 SE and for nasal breathing was -0.2% ± 3.9. Neither of these values are statistically significantly different from zero, suggesting that the percentage of uptake is generally not influenced between visits.
No statistically significant differences were found between spirometric measurements made on the first and second visits. The absence of statistically significance changes in breathing parameters among the exposure periods indicates that the subjects were breathing at steady state and that the O3 exposures were low enough not to affect ventilation.
Details on distribution in tissues:
Details on excretion:

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Applicant's summary and conclusion

In conclusion, the study provides information on the absorption of ozone in healthy adult male subjects during normal quiet breathing by mouth and by nose under steady-state exposure (0.3 ppm for 10 min) and breathing conditions. Among the 10 subjects, the average percentage of O3 uptake was slightly, but statistically significantly greater, with oral breathing (76%) than with nasal breathing (73%). The percentage of O3 uptake ranged from approximately 50 to over 95% in 10 subject indicating the difference between subjects. This study also illustrated that the percentage of O3 uptake is reproducible within subjects, varying by about 25% or less.

Executive summary:

In this study, 10 healthy adult male subjects were exposed to 0.3 ppm O3 while seated and breathing naturally through the nose or mouth. Total respiratory tract O3 uptake, spontaneous breathing parameters, and respiratory gas exchange were measured for 10 min under steady-state conditions. The exposure protocol was replicated in each subject approximately 2 weeks after the first visit. On each visit, health exams were performed and spirometric lung measurements were obtained. The experimental design provided comparisons of total O3 uptake during nasal and oral breathing, differences in uptake in an individual at two time points, and an examination of between-subject variability in O3 uptake. Exposure to O3 had no effect on the breathing parameters or gas exchange. Oral and nasal breathing frequency averaged 16.2 ± 1.1 (SE) and 16.0 ± 1.2 breaths per minute with tidal volumes averaging 651 ± 46 and 669 ± 67 mL, respectively. A significant correlation (p < 0.01) was found for the minute volume during resting breathing with the percentage of uptake. The percentage of O3 uptake was consistently higher (p = 0.02) during oral breathing (76.5% ± 3.3) than during nasal breathing (73.1% ± 3.0) although this difference may not be biologically significant. The variability in percentage of uptake between subjects was substantial with calculated uptakes ranging from 51 to 96%, a difference of about 45%. Variability in percentage of uptake for an individual was less with the maximal difference between the first and second visits being about 20%; the average difference, however, was only about 3%. It is concluded that total percentage of O3 uptake is approximately 75% in adult males during resting breathing. It is slightly greater during oral than during nasal breathing, will vary considerably among subjects, and is moderately reproducible within a subject.