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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
according to
other: OECD Guidance Document No. 39 (2009)
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Strain: Hsd Cpb:WU (SPF) wistar strain
- Source: Harlan-Nederland, AD Horst, Netherlands
- Age at study initiation: approximately 2 months
- Weight at study initiation: males: 178 - 187 g; females: 168 - 183 g
- Housing: singly in conventional Makrolon Type IIIH cages
- Diet: standard fixed-formula diet (KLIBA 3883), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 60
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
- Exposure apparatus: Plexiglas exposure restrainers (TSE, Bad Homburg, Germany)
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. Restrainers were chosen that accomodated the animals' size. Each inhalation chamber segment was suitable to accomodate 20 rats at the perimeter location. The design of the directed-flow inhalation chamber minimizes re-breathing of exhaled test atmosphere. For validation see Pauluhn, Journal of Applied Toxicology 14, 1994, 55-62 and Pauluhn & Thiel, Journal of Applied Toxicology 27, 2007, 160-167.
- Source and rate of air: Dry conditioned air, 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (free from water dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the targeted concentrations were achieved by atomization using the nozzle-baffle system and inhalation chamber. For atomization a binary nozzle (Schlick water jacketed nozzle which was connected to a thermostat, 25°C, using a digitally controlled water bath) and conditioned compressed air was used (15 L/min). The representative dispersion pressure was approximately 600 kPa. The test article was fed into the nozzle system using a digitally controlled infusion pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a glass-pre-separator/baffle system was used.
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (15 L/min x 60 min/(3.8 L) = 237, continuous generation of test atmosphere). Under such test conditions used chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Method of particle size determination: Cascade impactor (Berner critical orifice cascade impactor)
- Treatment of exhaust air: The exhaust air was purified via filter systems.
- Temperature, humidity: Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic). The position of the probe was at the exposure location of rats. Mean temperatures from 20.7 to 22°C, 5% relative humidity.

- The integrity end stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analyzed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 87.5% for 235 mg/m³, 86.9% for 284 mg/m³, 84.6% for 314 mg/m³.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) was 1.8 µm, the average geometric standard deviation (GSD) was approx. 1.6.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
Target concentrations: 200 mg/m³, 275 mg/m³, 300 mg/m³
Analytical concentrations: 235 mg/m³, 284 mg/m³, 314 mg/m³
No. of animals per sex per dose:
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs). Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested (visual placing response, grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflex, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).
Rectal temperatures were determined shortly after cessation of exposure.
Analysis of variance (ANOVA) was used for statistical evaluation.
Calculation of LC50 was performed according to Rosiello et al. (1977; Rosiello, Essigmann and Wogan, Tox and Environ. Health, 3, pp797) as modified by Pauluhn (1983). It is based on the maximum likelihood method of Bliss (1983; Q.J.Pharm.Pharmacol., 11, pp192).

Results and discussion

Effect levelsopen allclose all
Dose descriptor:
Effect level:
264 mg/m³ air
95% CL:
240.53 - 290.7
Exp. duration:
4 h
Dose descriptor:
Effect level:
> 314 mg/m³ air
Exp. duration:
4 h
No animal died in the lowest dose group of 235 mg/m³. Mortality occured at 284 mg/m³ and higher (4 males and 2 females at 284 mg/m³, 5 males and 1 female at 314 mg/m³). Rats succumbed on the exposure day or were found dead in the morning of the first postexposure day.
Clinical signs:
Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation. The following signs were observed (exposure day up to postexposure day 10): bradypnoea, labored breathing patterns, crepitations, irregular breathing patterns, motility reduced, atony, high-legged gait, tremor, gait uncoordinated, hair-coat ungroomed, piloerection, cyanosis, nasal discharge (serous), nose red encrustations, muzzle red encrustrations, stridor, nostrils: red encrustrations, eye lids: red encrustrations, decreased body weights, decreased reflexes, and hypothermia.
Body weight:
Treated animals showed transient reduced body weights as compared to control animals.
Gross pathology:
Necropsy of animals that died during the study revealed nasal/muzzle with red encrustations, nostrils with foamy content/discharge, lung less collapsed with foamy whitish content in trachea, hydrothorax, abdomen bloated and discoloration/bloodless appearance of parenchymatous organs.
Necropsy of surviving animals at the end of the study period did not reveal any abnormal finding compared to control.
Other findings:
All treated animals showed significantly reduced rectal temperatures (hypothermia).
A battery of reflex measurements was made on the first post-exposure day. ln comparison to the rats of the control group, substance treated rats exhibited impaired reflexes.

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of the substance on rats has been conducted in accordance with OECD TG 403. Test procedures were adapted so as to comply also with the EU Directive 92/69/EEC, and especially OECD GD39 (2009). Three groups of rats were nose-only exposed to the liquid aerosol of the test article in actual concentrations of 235, 284 and 314 mg/m³. The aerosol was generated so that it was respirable to rats, i.e. the average mass median aerodynamic diameter (MMAD) was 1.8 µm, the average geometric standard deviation (GSD) was approx. 1.6.

Mortality occured at 284 mg/m³ and higher. Rats succumbed on the exposure day or were found dead in the morning of the first postexposure day. Clinical observation showed evidence of respiratory irritation typical of lower respiratory tract sensory irritation, e.g. bradypnoea, labored and irregular breathing patterns, reduced motility, atony, high-legged and uncoordinated gait, tremor, ungroomed hair-coat, piloerection, cyanosis, serous nasal dischargeand encrustations of nose and eye.

For males the LC50 was determined to be 264 mg/m³. Females were found less susceptible than males, resulting in a LC50 of > 314 mg/m³. The NOAEL for both male and female rats was concluded to be < 235 mg/m³.