Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
Only males were studied; 2 concentrations were used for the ALC exposures and 4 lower concentrations were used to examine respiratory tract pathology; 5 animals/group were used for the ALC exposures; 15 animals/group were used for the pathology exposures.
GLP compliance:
yes
Test type:
standard acute method
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)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 220 - 315 g
- Fasting period before study: No
- Housing: Except during exposure, animals were housed individually in solid bottom caging with bedding or stainless steel, wire mesh cages suspended above cage boards.
- Diet (e.g. ad libitum): Ad libitum except during exposure
- Water (e.g. ad libitum): Ad libitum except during exposure
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Approximate 12-hour light/dark cycle.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chamber was constructed of glass (cylindrical). A polycarbonate baffle at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Exposure chamber volume: 34L
- Method of holding animals in test chamber: During exposure, animals were individually restrained in perforated stainless steel cylinders with conical nose pieces. The restrainers were inserted into a polymethylmethacrylate faceplate attached to the exposure chamber so that the nose of each animal extended into the exposure chamber.
- Source and rate of air: Chamber airflow was 15 L/min which provided at least 26 air changes per hour
- Method of conditioning air: Chamber oxygen concentration was 21%.
- System of generating particulates/aerosols: Chamber atmospheres were generated by aerosolization of the test substance in air with a nebulizer. The test substance was metered into the nebulizer with a syringe infusion pump. Since the test substance was a viscous material, it was necessary to dilute one part of the test substance with one part distilled water so that the nebulizer would not plug. Dilution of the test substance with water had a very small effect on the percent weight loss of the gravimetric filters, i.e., the weight of filters immediately after sampling relative to the weight after 24 hours in a dessicator. High-pressure air, metered into the nebulizer by a flow controller, carried the resulting atmosphere into the exposure chamber. The mass flow controller and the syringe infusion pump were controlled and monitored by an automated data system. Chamber concentrations of test substance were controlled by varying the syringe infusion pump rate to the nebulizer.
- Method of particle size determination: A sample to determine particle size distribution (mass median aerodynamic diameter, geometric standard deviation, and percent particles less than 1, 3, and 10 μm diameter) was taken during each exposure with a cyclone preseparator/cascade impactor and a constant flow air sampler.
- Treatment of exhaust air: Test atmospheres were exhausted through a dry-ice cold trap prior to discharge into the fume hood.
- Temperature, humidity, pressure in air chamber: 20-24°C, 38 - 56%, pressure not reported.

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration of the test substance in the test chamber was determined by gravimetric analysis at approximately 30-minute intervals during the first ALC exposure and at approximately 60 minute intervals for the other test substance exposures. Only one 30-minute chamber sample was taken for the air-only exposure. Known volumes of chamber atmosphere were drawn from the sampling port through a 25 mm filter cassette containing a pre-weighed glass fiber (Type A/E) 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 between 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 an automated data system. Upon completion of the exposures, sample results were transferred to a reporting and analysis system, which collated sample calculations. Because of the chemical composition of the test substance, it was decided to sample for ammonia in the exposure chamber during the 47 mg/m³ aerosol exposure. Using a Draeger-Tube apparatus specific for ammonia, a concentration of 3 ppm of ammonia was measured in the exposure chamber. Because of the low concentration, ammonia was not measured in subsequent exposures at lower chamber aerosol concentrations.
- Samples taken from breathing zone: yes/no

VEHICLE
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable):
- Justification of choice of vehicle:

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: A sample to determine particle size distribution (mass median aerodynamic diameter, geometric standard deviation, and percent particles less than 1, 3, and 10 μm diameter) was taken during each exposure with a cyclone preseparator/cascade impactor and constant flow air sampler.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The average mass median aerodynamic diameter (MMAD) for the ALC exposures was 2.1 μm ± 2.0 (MMAD ± GSD) with 69 to 70% of the particles less than 3 μm MMAD. The MMAD for the pathology exposure atmospheres ranged from 0.68 to 2.4 μm.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Animals were exposed to the test substance at mean aerosol concentrations of 47 ± 3.9 and 20 ± 2.3 mg/m³ (mean ± SD) for the ALC exposures and to 0, 0.12, 1.0, 8.0, and 19 mg/m³ for the pathology exposures.
No. of animals per sex per dose:
Two groups of 5 male rats each to determine an inhalation approximate lethal concentration (ALC).
Four groups of 15 male rats each to examine respiratory tract pathology.
Control animals:
other: 15 male rats for the pathology evaluation.
Details on study design:
- Duration of observation period following administration: 14 days following ALC exposures. Five rats in each group exposed for respiratory tract histopathology evaluation were sacrificed on post-exposure day 1, 7, or 14.
- Frequency of observations: Animals were observed for mortality and response to alerting stimuli 3 times during each exposure and observed for mortality and clinical signs of toxicity immediately after they were removed from the restrainers following exposure. During the recovery period, all rats were observed each day for mortality. Rats were observed for clinical signs of toxicity on the day following exposure and at least twice more during the recovery period, except for the rats that were sacrificed at one day post-exposure.
- Frequency of weighing: Rats were weighed on the day following exposure and at least twice more during the recovery period, except for the rats that were sacrificed at one day post-exposure.
- Necropsy of survivors performed: Yes for respiratory tract pathology animals; gross pathology and histopathology of lung, larynx/pharynx, trachea, nose, and gross lesions.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: approximate lethal concentration (ALC)
Effect level:
47 mg/m³ air
Exp. duration:
4 h
Remarks on result:
other: a.i. TNS (gravimetric measurement)
Sex:
male
Dose descriptor:
other: NOAEL for the anatomic pathology evaluation
Effect level:
1 mg/m³ air
Exp. duration:
4 h
Remarks on result:
other: a.i. TNS (gravimetric measurement)
Mortality:
Three of the 5 rats exposed to 47 mg/m³ died. One was found dead the morning after the exposure, one was found dead that afternoon, and one found dead the next day. All other animals survived.
Clinical signs:
other: One of the 5 rats exposed to 47 mg/m³ exhibited nasal discharge immediately after exposure. Labored breathing was observed in the rats that died. No adverse clinical observations were observed in any of the other animals.
Body weight:
Weight loss was observed in the rats that died. The 2 rats that survived exposure to 47 mg/m³ did not lose weight at any time after exposure. None of the 5 rats exposed to 20 mg/m³ had body weight losses throughout the 14-day recovery period. Following the pathology exposures, 3/15, 0/15, 5/15, 2/15 and 6/15 rats from the 0, 0.12, 1.0, 8.0, and 19 mg/m³ exposure groups, respectively, displayed body weight losses of 1 to 8 grams one day post-exposure. All rats gained weight for the remainder of the recovery period.
Gross pathology:
There were no test substance-related gross observations.
Other findings:
- Organ weights: No data
- Histopathology: Microscopic examination of the respiratory tissues of rats exposed to 8.0 and 19 mg/m³ revealed changes in the ventral larynx characterized by mucosal erosion and ulceration, inflammation, and cartilage necrosis. Following 7 or 14 days of recovery, changes were mostly limited to the submucosa and were characterized by mineralization of the laryngeal u-cartilage and microgranulomas, usually in association with mineralized debris.

Applicant's summary and conclusion

Conclusions:
ALC (male rats) = 47 mg/m³
Test substance-related mucosal erosion and ulceration, inflammation, and cartilage necrosis of the ventral larynx in the respiratory tract of rats exposed to 8.0 and 19 mg/m³. The no-observable-adverse-effect-level (NOAEL) for the anatomic pathology evaluation is 1.0 mg/m³.
Executive summary:

Two groups of 5 male rats each were exposed nose-only for a single 4-hour period to the test substance in air to determine an inhalation approximate lethal concentration (ALC). Four groups of 15 male rats each were exposed nose-only for a single 4-hour period to the test substance in air to examine respiratory tract pathology. As a control group for the pathology evaluation, an additional group of 15 male rats was exposed nose-only for a single 4-hour period to air only. The test atmosphere was generated by aerosolization and the aerosol concentration was determined by gravimetric analysis. There was no significant vapor component present in the exposure atmospheres. A Draeger-Tube, specific for NH3, measured 3 ppm of ammonia in the chamber during the 47 mg/m³ exposure.

For the ALC exposures, rats were exposed to mean atmospheric aerosol concentrations of 47 ± 3.9 mg/m³ and 20 ± 2.3 mg/m³. The fractional mortality (number dead/number exposed) for these exposures was 3/5 and 0/5, respectively. The average mass median aerodynamic diameter (MMAD) measured for the test substance atmospheres was 2.1 μm with 69 to 70% of the particles less than 3 μm MMAD. Three of the 5 rats exposed to 47 mg/m³ died. One was found dead the morning after the exposure, one was found dead that afternoon, and one found dead the next day. All other animals survived. One of the 5 rats exposed to 47 mg/m³ exhibited nasal discharge immediately after exposure. Labored breathing was observed in the rats that died. No adverse clinical observations were observed in any of the other animals.

Weight loss was observed in the rats that died. The 2 rats that survived exposure to 47 mg/m³ did not lose weight at any time after exposure. None of the 5 rats exposed to 20 mg/m³ had body weight losses throughout the 14-day recovery period. Six of 15 rats exposed to 19 mg/m³ lost minimal weight (1-8 grams) one day post-exposure and all gained weight for the remainder of the recovery period. Four of 15 rats exposed to 8.0 mg/m³ lost minimal weight (3-7 grams) one day post-exposure and all gained weight for the remainder of the recovery period. Two of 15 air control rats (0.0 mg/m³) lost minimal weight (2 grams or less) one day post-exposure and all gained weight for the remainder of the recovery period.

Four groups of 15 male rats each were exposed nose-only for a single 4-hour period to the test substance in air to examine respiratory tract pathology. Exposure concentrations were 0, 0.12, 1.0, 8.0, and 19 mg/m³. The MMAD for the pathology exposure atmospheres ranged from 0.68 to 2.4 μm. Microscopic examination of the respiratory tissues of rats exposed to 8.0 and 19 mg/m³ revealed changes in the ventral larynx characterized by mucosal erosion and ulceration, inflammation, and cartilage necrosis. Following 7 or 14 days of recovery, changes were mostly limited to the submucosa and were characterized by mineralization of the laryngeal u-cartilage and microgranulomas, usually in association with mineralized debris.

Under the conditions of this study, the approximate lethal concentration (ALC) for the test substance is 47 mg/m³ and the no-observable-adverse-effect-level (NOAEL) for the anatomic pathology evaluation is 1.0 mg/m³.