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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: K2 since being used for read-across

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Exposures for 9 days; only males were tested; food consumption was not collected; 5 males/concentration were sacrificed at end of exposures, rather than 10; hematology, clinical chemistry, & pathology deviations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: not reported as such

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 231 - 276 g
- Fasting period before study: No
- Housing: stainless steel, wire-mesh cages suspended above cage boards
- Diet (e.g. ad libitum): ad libitum except during exposure and fasting periods prior to necropsy
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: Approximately 14 days, with 7-day quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle); air
Remarks on MMAD:
MMAD / GSD: MMAD ranged from 0.90 to 2.1 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style) with a nominal internal volume of 150 L. A polycarbonate baffle at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: individually restrained in perforated stainless steel cylinders with conical nose pieces; the restrainers were inserted into a polymethylmethacrylate faceplate which was attached to the exposure chamber so that the nose of each animal protruded into the exposure chamber.
- Source and rate of air: Filtered, high-pressure air, metered into the nebulizer by a mass flow controller, atomized the test substance and carried the resulting atmosphere through a cyclone, through a glass flow-splitter, and into the exposure chamber.
- Method of conditioning air: Not Reported
- System of generating particulates/aerosols: Chamber atmospheres were generated by nebulization of the test substance in air with a Spraying Systems Nebulizer. The test substance was metered into the nebulizer with an infusion pump. Filtered, high-pressure air, metered into the nebulizer by a mass flow controller, atomized the test substance and carried the resulting atmosphere through a cyclone, through a glass flow-splitter, and into the exposure chamber. The flow splitter diverted aerosol to the hood exhaust to reduce the amount of test material reaching the exposure chamber. Dilution air, metered into the chamber by a Brooks model 5878 Mass Flow Controller, helped to evenly distribute the test substance throughout the exposure chamber. The infusion pump and mass flow controllers were controlled and monitored by the automated data system. Fine control of the chamber concentration was accomplished by varying the test substance feed rate to the nebulizer
- Temperature, humidity, pressure in air chamber: Mean temperature ranged from 19 to 25°C (targeted at 21 to 25°C). Daily mean relative humidities ranged from 36 to 67% (targeted at 40 to 60%). In some cases the temperature and relative humidity measurements were slightly out of range; these deviations were minor and were not expected to affect the study outcome.
Chamber oxygen concentration was 21% (targeted to be at least 19%). Pressure was not reported.
- Mean airflow rate: 0 mg/m³: 35 L/min; 0.2 mg/m³: 65 L/min; 2 mg/m³: 65 L/min; 20 mg/m³: 50 L/min.
- Air change rate: At least 12 air changes per hour
- Method of particle size determination: once per week in the 2 and 20 mg/m³ chambers with a cyclone preseparator/cascade impactor and a constant flow air sampler. On the last exposure day of the study, the aerosol particle size in all 3 chambers was measured in duplicate with an impactor.
- Treatment of exhaust air: Test atmospheres were exhausted through an MSA filter cartridge prior to discharge into the fume hood.

TEST ATMOSPHERE
- Brief description of analytical method used: atmospheric concentration of test substance was determined by gravimetric analysis at approximately 60-minute intervals during each exposure. The control chamber was sampled once during the study. Known volumes of chamber atmosphere were drawn from the breathing zone of the animals through a 25 mm filter cassette that contained a pre-weighed glass fiber filter. The filters were weighed on a microbalance. The filter weights were automatically transferred to an automated data system which calculated the chamber concentrations based on the difference in pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled. Gravimetric sample start- and stop-times for each sample were controlled and recorded by the automated data system.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of Analysis: GC
Concentrations Analyzed: 0, 0.2, 2, 20 mg/m³
Results: Mean concentrations were 0, 0.22 mg/m³ (range: 0.15-0.26); 1.9 mg/m³ (range: 1.2 - 2.6); 20 mg/m³ (range: 18 - 22) for design concentrations of 0, 0.2, 2, 20 mg/m³, respectively.
Duration of treatment / exposure:
6 hours per day for a total of 9 exposures in 14 days with a recovery period of 15 days
Frequency of treatment:
6 hours/day, 9 days in 2 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.2, 2, 20 mg/m³
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 0.22, 1.9, 20 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
15 males per concentration.
The first 10 rats per group were designated for standard toxicity testing. The remaining 5 rats per group were designated for total fluorine analysis sampling.
Control animals:
yes, sham-exposed
Details on study design:
The purpose of this 2-week inhalation study was to investigate the effects of repeated inhalation exposure in rats to airborne concentrations of test substance aerosol. Inhalation is an expected route of human exposure. The data will be used to provide information for safe handling of the test substance.
- Dose selection rationale:
In a previous study with the same substance, the 4-hour approximate lethal concentration (ALC) in nose-only exposed rats was 57 mg/m³; no rats died after exposure to 23 mg/m³. Immediately prior to the present study, a range-finding experiment was conducted in groups of rats exposed nose-only for approximately 5 ½-to-6 hours/day for 6 days, within an 8-day period, to a mean concentration of approximately 18 mg/m³ of the test substance. No significant clinical signs of toxicity were observed. Following a 3-day period without exposure, the same rats were exposed for 5 hours to a mean concentration of approximately 44 mg/m³ followed by a final 4 hour and 20 minute exposure to a mean concentration of 69 mg/m³. Five of 6 rats died within 2 days of the last exposure. Clinical signs of toxicity observed immediately following the last exposure included: red nasal discharge, irregular respiration, and lethargy. The day following the last exposure lung noise and severe weight loss were also observed.
- Rationale for animal assignment (if not random): Prior to the end of the quarantine period, rats were assigned to 4 groups of 15 male rats each. Rats were divided into groups with the aid of a computerized, stratified, randomization program, so that mean body weights for each group were similar.
The first 10 rats per group were designated for standard toxicity testing. Immediately following the last exposure, collection of an overnight urine specimen was initiated for each rat. On the day following the last exposure, blood samples were collected for clinical analyses from each standard toxicity rat, and 5 rats per group were sacrificed for anatomic pathology examination.
- Rationale for selecting satellite groups: The remaining 5 rats per group were designated for total fluorine analysis sampling. Blood for total fluorine analysis was collected from each rat during the exposure phase on test days 0, 3, and 9, and during the recovery period on test days 14 and 24. Selected blood samples were subsequently analyzed for fluorine, and the remaining samples were saved for possible future analysis. At the end of the recovery period, all rats were sacrificed without further examination.
- Post-exposure recovery period in satellite groups: All remaining rats were allowed to recover for a 15-day period. At the end of the recovery period, standard toxicity rats were fasted overnight, blood and urine samples were collected for clinical analyses from each rat, and all surviving standard toxicity rats were sacrificed for anatomic pathology examination.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
GENERAL CLINICAL OBSERVATIONS (Individually examined for clinical signs of toxicity): Yes
- Time schedule: Before and after each exposure; during recovery, twice per week when weighed

GROUP CLINICAL OBSERVATIONS: Yes
- Time schedule: During exposures

DETAILED CLINICAL OBSERVATIONS (Alerting response to an auditory stimulus): Yes
- Time schedule: Prior to each exposure, approximately every 2 hours during each exposure, and after each exposure

BODY WEIGHT: Yes
- Time schedule for examinations: On exposure days; during recovery, twice per week

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 10 days after initiation of the study
- Anaesthetic used for blood collection: Yes, light carbon dioxide anesthesia
- Animals fasted: Approximately 16 hours
- How many animals: 10
- See Table 1.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 10 days after initiation of the study:
- Animals fasted: Approximately 16 hours
- How many animals: 10
- See Table 2.

URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 10 days after initiation of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Approximately 16 hours
- See Table 2.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to each exposure, approximately every 2 hours during each exposure, and after each exposure
- Dose groups that were examined: All groups
- Battery of functions tested: Other, Alerting response to an auditory stimulus

BLOOD FLUORINE ANALYSIS: Yes
- Time schedule for collection of blood: Afternoons of test days 0, 3, and 9 and during recovery on test days 14 and 24.
- How many animals: 5 rats per group
- Type of analysis: Total fluorine content of blood samples.
- See Table 2.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 3)
Five animals per group were sacrificed and necropsied following the last exposure (test day 10 sacrifice).
Another 5 animals per group were allowed to recover and were sacrificed and necropsied on the 15th day of recovery (test day 24 sacrifice).
Rats were euthanatized by carbon dioxide anesthesia and exsanguination.
Gross examinations were performed on all rats.

ORGAN WEIGHTS: Yes (See Table 3)

HISTOPATHOLOGY: Yes (see Table 3)
Statistics:
See Table 4.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL CHEMISTRY
There were no adverse changes in clinical chemistry parameters in male rats exposed to the test substance.
There was a statistically significant and/or treatment-related change in urea nitrogen which was considered to be non-adverse or not related to exposure to the test compound. Urea nitrogen was minimally decreased in male rats exposed to 20 mg/m³ after 10 days of treatment and at the end of the recovery period (test days 10 and 24). At both time-points, urea nitrogen concentrations were generally within the range of those in other groups and similar to historical control values. There were no alterations in other related clinical pathology parameters (urine volume, creatinine, phosphorus), and no alterations in renal histopathology. Albumin was minimally increased in males exposed to 0.2 mg/m³ on test day 10.

HISTOPATHOLOGY:
Compound-related microscopic observations were present in the lungs and larynx of rats exposed to 2 and 20 mg/m³ test substance sacrificed on test day 10. Inflammation in the lungs, characterized by mixed inflammatory cells within scattered alveolar lumina, was observed at 2 mg/m³ in one of 5 rats and at 20 mg/m³ in 3 of 5 rats, and was considered compound related and adverse. Minimal to mild squamous metaplasia of the mucosa lining the ventral floor of the larynx was observed at 2 mg/m³ in 4 of 5 rats and at 20 mg/m³ in 5 of 5 rats, and was considered compound-related and adverse. See Table 5.
There were no compound-related microscopic lesions observed in rats sacrificed on test day 24, indicating recovery of compound-related lesions in the lung and larynx.

BLOOD FLUORINE ANALYSIS
Small amounts of fluorine (approximately 2 ppm), slightly above background (approximately 1.3 ppm in controls at test day 9), were detected in the blood of rats in the 20 mg/m³ group on test days 0, 3, and 9 (compared to test day 9 control data), indicating the presence of the test compound or its metabolites. No above-background fluorine was detected in rats exposed at this concentration after the 2-week recovery period (test day 24) compared to the test day 24 control group. No fluorine was detected above background levels in the 0.2 and the 2 mg/m³ groups on test day 9 (the last day of the exposure period).

Effect levels

Dose descriptor:
NOEL
Remarks:
Repeated Inhalation Exposure
Effect level:
0.2 mg/m³ air
Sex:
male
Basis for effect level:
other: Based on histological findings in the lungs and larynx of rats exposed to 2 and 20 mg/m³ test substance sacrificed on test day 10.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 5

Anatomical Pathology
Incidences and Lesion Grades of
Compound-Related Microscopic Observations In Male Rats
at Day 10 Sacrifice

 

Lesion Incidence (Numeric)

Group

I

III

V

VII

Exposure (mg/m3)

0

0.2

2

20

Lesions

 

 

 

 

LUNGS

(5)

(5)

(5)

(5)

No Abnormality Detected

5

5

4

2

Inflammation, Subacute/Chronic.

 

 

 

 

Minimal

 

 

1

3

Total observations/Lesion

 

 

1

3

PHARYNX/LARYNX

(5)

(5)

(5)

(5)

No Abnormality Detected

5

5

1

 

Squamous Metaplasia.

 

 

 

 

Minimal

 

 

4

1

Mild

 

 

 

4

Total Observations/Lesion

 

 

4

5

Number in parenthesis indicates number of animals examined.

Absence of a number indicates the lesion specified was not identified.

Applicant's summary and conclusion

Conclusions:
Inhalation exposure to 2 and 20 mg/m³ of test substance produced adverse changes in the lung and larynx. Based on adverse microscopic findings in the respiratory tract of rats exposed to test substance for 2 weeks, the No-Observed-Effect Level (NOEL) for repeated inhalation exposure was 0.2 mg/m³. Absence of lesions in the lung and larynx of recovery animals indicated complete lesion reversal within the 2-week recovery period.
Executive summary:

Four groups of 15 male rats each were exposed to concentrations of test substance aerosol targeted to 0.2, 2, or 20 mg/m³ (actual mean concentrations were 0.22, 1.9, and 20 mg/m³). The mass median aerodynamic equivalent diameter of the aerosols ranged from 0.90 to 2.1 µm. All rats were exposed nose-only for 6 hours per day for a total of 9 exposures over a two-week period. A similarly comprised control group of 15 male rats was simultaneously exposed to air only.
The first 10 rats per group were designated for standard toxicity testing. The remaining 5 rats were designated for blood fluoride analysis sampling. On the day of the last exposure, collection of an overnight urine specimen was initiated for each standard toxicity rat. On the day following the last exposure, blood samples were collected for clinical analyses from each standard toxicity rat, and 5 rats per group were sacrificed for anatomic pathology examination. All remaining rats were allowed to recover for a 15-day period. At the end of the recovery period, blood and urine samples were collected for clinical analyses from each standard toxicity rat and all surviving rats were sacrificed. Only the standard toxicity rats were given an anatomic pathology examination.
No deleterious effects were observed in clinical observations during the exposure or recovery periods and body weights were similar in test and control rats. Clinical pathology evaluations, including fluoride measurements, showed no effects in urine, hematology, or blood chemistry parameters as a result of exposure to test substance.
Small amounts of fluorine (approximately 2 ppm), slightly above background, were detected in the blood of rats in the 20 mg/m³ group on test days 0, 3, and 9 (compared to test day 9 control data) indicating the presence of the test compound or its metabolites. No above-background fluorine was detected in rats exposed at this concentration after the 2‑week recovery period (test day 24) compared to the test day 24 control group. No fluorine was detected above background levels in the 0.2 and the 2 mg/m³ groups on test day 9 (the last day of the exposure period).
Weight analysis of the major organs showed no effects attributable to the test substance. Histological findings were confined to the respiratory tract. There were no adverse effects in any other evaluated organs. Compound-related microscopic observations were present in the lungs and larynx of test day 10 sacrifice rats at concentrations of 2 and 20 mg/m³. Inflammation in the lungs, characterized by mixed inflammatory cells within scattered alveolar lumina, was observed at 2 mg/m³ in 1 of 5 rats and at 20 mg/m³ in 3 of 5 rats. Minimal to mild squamous metaplasia of the mucosa lining the ventral floor of the larynx was observed at 2 mg/m³ in 4 of 5 rats and at 20 mg/m³ in 5 of 5 rats.
There were no compound-related microscopic lesions observed in test day 24 sacrifice rats, indicating the reversibility of compound-related lesions in the lung and larynx.
The no-observed-effect level (NOEL) for this study is defined as the highest concentration at which no toxicologically important effects attributable to the test substance were detected. The NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985) and to the no-observed-adverse-effect level (NOAEL) as defined by the European Union (1994).
Based on the above findings in the respiratory tract of rats exposed to test substance for 2 weeks, the NOEL for repeated inhalation exposure was 0.2 mg/m³.