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Direct observations: clinical cases, poisoning incidents and other

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Administrative data

Endpoint:
direct observations: clinical cases, poisoning incidents and other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and according to GLP
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Nasal inflammatory and respiratory parameters in human volunteers during and after repeated exposure to chlorine
Author:
Schins, R.P.F.; Emmen, H.; Hoogendijk, L.; Borm, P.J.A.
Year:
2000
Bibliographic source:
Eur Respir J 2000; 16: 626-632

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
acute toxicity: inhalation
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline followed, but very good documentation. For details see materials and methods in IUCLID5 dossier (Lung function measurements).
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorine
EC Number:
231-959-5
EC Name:
Chlorine
Cas Number:
7782-50-5
Molecular formula:
Cl2
IUPAC Name:
Active chlorine generated from chloride of ambient water by electrolysis
Details on test material:
not indicated

Method

Type of population:
general
Subjects:
Testing was conducted in 8 subjects using a repeated measures design.Inclusion and exclusion of subjectsFor inclusion in the study, each volunteer had to meet the following criteria: 1) be male and Caucasian, 2) 20±50 yrs of age, 3) healthy as determined by medical and laboratory examination, 4) have normal lung function (i.e. FEV1>90%; FVC>80%), 5) an interleukin-6 (IL-6) concentration in NALF under detection limit (<20 pg. mL-1), 6) skin calliper: body fat volume <30%, 7) Dutch as their native language, and 8) have given written informed consent.Subjects were excluded when one of the following exclusion criteria was met: 1) a history of medical/surgical disease that may significantly affect the outcome, 2) hay-fever or rhinitis based on anamnesis and NALF, 3) abnormal respiratory impedance values (i.e. frequency dependence of resistance<0; resonant frequency >15Hz), 4) a history of alcohol, amphetamine, cocaine, barbiturate, or other drug abuse, 5) participated in a clinical study within 3 months of present study, 6) presently using any chronic medication, 7) using more than 28 alcoholic beverages a week, 8) show evidence of liver or kidney dysfunction, 9) an employee of the TNO research institute or their first or second removed relatives, 10) be claustrophobic, 11) be a smoker, 12) having a cold, the week before or during the study, 13) be a regular swimmer, 14) regularly in contact with chlorine, bleaching agent or known respiratory irritants.
Ethical approval:
not specified
Route of exposure:
inhalation
Reason of exposure:
intentional
Exposure assessment:
measured
Details on exposure:
The subjects were exposed to the test material for 6 h. day-1 (9:00±12:00 h and 12:30±15:30 h), on 3 consecutive days/week over alternate weeks for a total period of eight weeks. Subject exposures were carried out in an air-conditioned exposure chamber (13.6 m exp(-3)). The number of air changes was slightly more than 2 h exp(-1). The temperature range was 21.5-23.4°C, and the relative humidity range was 37-50%. The inhalation equipment was designed to expose the subjects to a continuous supply of fresh test atmosphere. To generate the test atmosphere, the test material was passed from the gas cylinder, which was kept just outside the exposure room, via a special reducing valve, stainless tubing and a mass flow controller to a mixing chamber where it was mixed with nitrogen. The nitrogen was also delivered via a reducing valve and a mass flow controller. Using a third mass flow controller, a small, filtered part of the generated mixture was passed to the exposure room where it was mixed with clean air. The main part of the mixture was passed to the exhaust using a constant pressure controller.
Examinations:
Nasal lavageFive mL of sterile phosphate buffered saline solution (37°C) was delivered into each nostril and allowed to remain there for 10 s, during which the volunteers should hold their breath. During the morning sessions and during the 1-day and 4-days after exposure the left nostril was used for lavage, while in the afternoon sessions the right nostril was chosen to avoid possible wash-out effects due to repetitive washing of one nostril within a single day. The lavage fluid was processed as described previously. Albumin was measured in NALF aliquots by an automated micro-assay using immunoturbidometry with Beckman reagents (Array, Beckman Inc. Mijdrecht, the Netherlands). IL-8 and IL-6 (used an exclusion criterion) were determined with specific enzyme-linked immunosorbent assays (ELISA) as described previously, with a detection limit of 20 pg.mL-1 for both assays.Cytospin preparations were scored by two cytologists: if present, 300 cells were counted and damaged cells excluded. If less than 20 cells were present, no differential cell count was performed. Total cell number was determined by using a haemocytometer.Lung function measurementsLung function was evaluated using both the effortdependent (FEV1,FVC,FEV1/FVC)andeffort-independent (MMEF) parameters from forced-expiratory manoeuvres recorded using a portable Jaeger Masterscope (Breda, the Netherlands) equipped with a pneumatograph. All flowvolume values were related to European community for Steel and Coal (ECSC) reference values for individual diagnosis. All subjects were measured by the same operator (except the check-out measurement) and using the same apparatus. The spirometer was calibrated at 4 h intervals on ambient conditions (pressure, temperature) and volume. Data were evaluated according to the ECSC and American Thoracic Society (ATS) criteria.Statistical evaluationsFor all effect parameters, the distribution characteristics were examined using the K-S- Lilliefors and Shapiro- Wilks tests. Since the effect parameters lacked normal distribution, non-parametric tests were used. First, the preexposure baseline values "over-time" (i.e. the mean scores of the Tuesday morning measurements grouped by study period) were tested using the Friedman's test. In addition, data were evaluated with respect to the mean scores of post-exposure (Monday-morning) measurements. A p-value of less than 0.05 was considered to be statistically significant.Exposure related data were evaluated using the Wilcoxon matched-pairs tests. Control values (i.e. data from the zero exposure condition) were compared with mean values of the three exposure conditions (0.1, 0.3, and 0.5 ppm) combined. This test was applied as a first indication of a possible difference between exposure to Cl2 and control (0 ppm). To identify differences between all four exposure conditions, the Friedman's test was applied. Correcting for the number of planned post-hoc comparisons (0.5 versus 0 ppm; 0.3 versus 0 ppm; 0.1 versus 0 ppm), a value of p<0.017 was considered to be statistically significant. In case of a significant finding, additional Wilcoxon-tests (p<0.05) were carried out to compare all exposure group data to the control condition. Finally, the Cochran-MantelHaenzel (non-zero) statistics were calculated to examine the association between scores on the effect parameters and the Cl2 exposure levels. It is noted that a significant result in this case, does not necessarily indicate a linear trend. For statistical analyses SPSS (version 6.0 and 7.0) and the SAS software package (version 6.12; Heidelberg, Germany) were used.
Medical treatment:
not applicable

Results and discussion

Clinical signs:
Adverse events/respiratory symptomsAt the end of each session the well-being of all subjects was checked by a physician. None of the registered adverse events were judged to be treatment-related per se. In 6 cases the relation to exposure was considered as "not", 18 cases were judged as "unlikely" (i.e. relation to exposure is unlikely, but not impossible), while in 9 cases the relation to Cl2 exposure was judged "possible" (i.e. relation is not likely, but may exist). None of these adverse events was reason to undertake action with regard to the protocol. However, by the end of the study it appeared that one of the subjects (NR. 8) had a thyroid carcinoma (follicular) which was judged as not treatment related. The subject decided not to participate any further in the study and although he completed nearly all the tests, this subject was excluded from further statistical evaluations.
Results of examinations:
Effects on nasal lavage parameters No differences were observed in baseline values of any of the nasal lavage parameters over time. This testing was done by comparing the mean scores of the Tuesday morning measurements grouped by study period (Friedman's test, p<0.05). In addition no significant time-effect was noted with respect to the mean scores of the Monday morning (post-exposure) nasal lavage parameters.Albumin and IL-8 values (table 4) were all within range of previous studies in the laboratory. Significant differences were found between the control group and the combined exposure conditions (0.1, 0.3 and 0.5 ppm) for albumin over the time interval 2±1 (i.e. Tuesday postexposure minus pre-exposure), and for IL-8 over the time interval 3-1 (i.e.Wednesday pre-exposure minus Tuesday pre-exposure) (not shown). For IL-8 levels, the Friedman's analyses revealed no significant differences between all four exposure conditions and no association between exposure levels and IL-8. On the other hand, albumin data revealed significant differences (p<0.017) for two time points. However, these are explained by a difference between 0 and 0.1 ppm Cl2 (post-hoc Wilcoxon testing). Also correlation analysis (Cochrane- Mantel-Haenzel) indicated that albumin levels were related to exposure at several time points, but these findings were not consistent with the results of the post-hoc tests.For the total cell counts a significant difference was found between the control conditions and the combined exposure conditions at time interval 4-1. Friedman analysis revealed a significant difference in total cells between all exposure conditions for the time interval 7-1. At this time interval, however, the "post hoc" Wilcoxon tests only indicated a significance between the 0.1 ppm and control conditions and not at higher exposure . Moreover, no significant correlations were observed between total cell counts and exposure levels.Differential cell counts revealed several significant differences, that should be interpreted with caution. With regard to neutrophil percentages, significant differences between control and combined exposure conditions were found at several time points/intervals. However, Friedman analysis revealed no difference between all exposure conditions that would forward additional post-hoc testing (i.e. p<0.017). Cochran-Mantel-Haenzel analysis suggests a decreasing trend for the percentage neutrophils along with increasing Cl2 exposure at time points/intervals 4, 4-1, 4-3, and (2, 4, 6)-1. However, it should be emphasized that changes in neutrophil percentage are interdependent with changes in other cell types, and that neutrophil percentages were found not to be significantly different at any of the exposure levels versus the control (zero) condition. With the exception of one time point (i.e. 4) no significant difference in the percentage of epithelial cells was found in control versus combined exposure conditions. Although the Cochran-Mantel-Haenzel statistic indicated an association between exposure level and percentage epithelial cells at 5 different occasions, the required statistical significance (p<0.017) was not attained in a Friedman's test comparing all four exposure conditions. Monocyte percentages were not significantly different between the various exposure periods. Further detailed statistical analysis provides no indication for a positive or negative dose-response association with the Cl2 exposure level. Although a significantly higher percentage of monocytes was found at pre-exposure timepoints at 0.3 ppm compared to zero exposure, a biological explanation for such a finding is not present. Since the absolute monocyte counts are rather low, this difference is considered to be caused by chance or minor variability in visual cytological scoring. Although eosinophils and lymphocytes were well detectable in nasal lavage, there was no or little statistical proof that these cell types are affected by Cl2 exposure. No significant differences between different study periods were observed. Although an association between cell percentage and exposure was found at an incidental test point, comparison to findings with other cell types suggests that this is merely a matter of chance.Effects on lung function parametersWith the exception of MMEF no differences were observed in baseline (i.e. Tuesday morning) values of lung function parameters over time. No significant time-effect was noted with respect to the mean scores of the Monday morning (post-exposure) lung function parameters, i.e. this time also including MMEF values.Several significant differences for FVC (e.g. time point 2, time interval 2-1), FEV1 (e.g. time point 2, intervals 4- 1, 7-1) and FVC/FEV1 (e.g. time point 1, intervals 4-1, 6-1) were observed comparing different time-points or time intervals using the Wilcoxon matched pairs test. However, none of these differences "survived" more rigid statistical testing comparing the four exposure conditions using Friedman's test. The differences inMMEF, however, remained statistically significant (e.g. time interval 6-1) although changes are smaller than individual physiological variations. As already stated, comparison (Tuesday) preexposure MMEF revealed a statistically significant difference between the four study periods. Also post-hoc Wilcoxon analyses indicated a difference between various periods, and was caused by the fact that MMEF values in the third period (zero exposure in both protocols) were significantly lower (4.29 L) than in other periods (0.1 ppm: 4.59 L; 0.3 ppm: 4.79 L; 0.5 ppm: 4.79 L). To further interpret this finding, one subjects (NR 04) with a high intra-subject variation in MMEF and clearly deviant from others was excluded from statistical analysis and testing was done using repeated measures ANOVA. This analysis confirmed the baseline drift as a cause of the observed difference in MMEF.
Effectivity of medical treatment:
not applicable
Outcome of incidence:
The study concluded that nasal lavage measurements did not support an inflammatory response or irritant effects on the nasal epithelium. Furthermore no significant effect on lung function parameters was found. The study did not support an inflammatory effect in the nose nor shows changes in the respiratory function at repeated exposure up to 0.5 ppm (1.5 mg/m3).

Applicant's summary and conclusion

Conclusions:
The study concluded that nasal lavage measurements did not support an inflammatory response or irritant effects on the nasal epithelium. Furthermore no significant effect on lung function parameters was found. The study did not support an inflammatory effect in the nose nor shows changes in the respiratory function at repeated exposure up to 0.5 ppm (1.5 mg/m3).
Executive summary:

The study was well documented and was done according to Good Clinical Practice. The objectives of this study were:

1) to determine if chlorine exposure at low levels induces nasal effects in humans as it does in rodents; and

2) to establish a possible occurrence of respiratory effects in human volunteers exposed to chlorine vapour at concentrations of 0, 0.1, 0.3 and 0.5 ppm (0, 0.3, 0.9 and 1.5 mg/m3). The 8 male volunteers were exposed for 6 hour per day on 3 consecutive days to each of the 4 exposure conditions. Data analysis was limited to 7 subjects since one volunteer decided to stop participating for reasons not related to the study.

Some adverse effects were reported by the volunteers and registered by the physician. Most of them were classified as “impossible” or “unlikely” to be treatment related. The following effects were judged as “possible” to be treatment related: sinus tension (1 case), eye irritation (5 cases), coughing (2 cases), nose congestion (2 cases), dry throat (1 case), dry mouth (1 case), throat irritation (1 case), expiratory wheeze (1 case), mucus production in nasal cavity (1 case).

The study concluded that nasal lavage measurements did not support an inflammatory response or irritant effects on the nasal epithelium. Furthermore no significant effect on lung function parameters was found. The study did not support an inflammatory effect in the nose nor shows changes in the respiratory function at repeated exposure up to 0.5 ppm (1.5 mg/m3).

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