Registration Dossier

Administrative data

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

Test material

Test material form:
liquid: viscous

Test animals

Details on test animals and environmental conditions:
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland, AD Horst, The Netherlands
- Age at study initiation: approximately 2 months
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days, during this period, rats were also acclimatized to the restraining tubes

- Temperature (°C): 22 ± 3 °C
- 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
other: test article contains 20 % butyl acetate
Details on inhalation exposure:
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. The design of the directed-flow inhalation chamber prevents rebreathing of the test atmosphere.
- Generation of atmosphere: Atmospheres were generated under dynamic conditions using a digitally controlled Harvard PHD 2000 infusion pump and a binary nozzle.
- Generation of aerosol: The test substance was nebulized neat using conditioned compressed air (dispersion pressure approximately 600 kPa, 15 L air/min. The targeted concentration was achieved by using constant concentrations of the test article in butyl acetate as vehicle.
- Inhalation chamber: internal volume = about 3.8 L.
- 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.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- 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(t95 % = 3 x chamber volume/chamber airflow). At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Exhaust air treatment: The exhaust air was purified via filter systems.
- 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.

- 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).
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 75.6 % for 398 mg/m³, 72.4 % for 1337 mg/m³, 69.3 % for 3379 mg/m³, 63.7 % for 3967 mg/m³.
- MMAD (Mass median aerodynamic diameter): The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was smaller than 2.5 µm, the geometric standard deviation (GSD) was 1.7-2.4.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
Target Concentrations: 500, 1500, 3000 and 5000 mg/m³
Actual Concentrations (test substance 80 % in butyl acetate): 460, 1546, 3906 and 4586 mg/m³
Gravimetric Concentration (non-volatile fraction of the test article): 398, 1337, 3379, 3967 mg/m³
No. of animals per sex per dose:
Control animals:
other: yes, concurrent solvent (butyl acetate)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: 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).
Weighing: Body weights were measured before exposure, on days 1, 3 and 7, and weekly thereafter. Individual weights are also recorded at death, if applicable.
- Necropsy of survivors performed: yes
- Other examinations performed: Rectal temperatures were measured shortly after cessation of exposure using a digital thermometer with a rectal probe for rats.
For necropsy findings: pair-wise Fisher test after the R x C chi-squared test in accordance with Gad and Weil (Statistics for Toxicologists. Principles and Methods of Toxicology, ed. AW. Hayes, Raven Press, New York, 280, 1982). For statistical evaluation of the body weight gain for each group a one-way ANOVA (vide infra) is used. Data of rectaI temperature measurements are statistically evaluated using the ANOVA procedure (vide infra). For LC50 calculation: method of Rosiello et al. (J. Tox. and Environ. Health, 3, 797-809, 1977) as modified by Pauluhn (Computer-Aided Estimation of the LD50/LC50 BAYER AG Report No. 11835, dated May 18, 1983).

Results and discussion

Effect levelsopen allclose all
Dose descriptor:
Effect level:
4 090 other: mg/m³ air (test substance 80 % in butyl acetate)
Exp. duration:
4 h
Dose descriptor:
Effect level:
3 537.5 other: mg/m³ air (non-volatile fraction of the test article)
95% CL:
3 364.9 - 3 718.9
Exp. duration:
4 h
Mortality did not occur up to 1546 mg/m³ (actual concentration of the test substance 80 % in butylacetate) whereas at higher concentration rats succumbed up to the second postexposure day.
Animals that died [actual concentration (onset of mortality)]:
- males: 0/5 at 460 mg/m³; 0/5 at 1546 mg/m³; 1/5 at 3906 mg/m³ (1d); 5/5 at 4586 mg/m³ (1d-2d).
- females: 0/5 at 460 mg/m³; 0/5 at 1546 mg/m³; 2/5 at 3906 mg/m³ (2d); 5/5 at 4586 mg/m³ (1d-2d).
Clinical signs:
Rats exposed at higher concentrations showed evidence of respiratory irritation with mortality patterns typical of an irritation-related acute lung edema. The following signs were observed (exposure day up to second postexposure week): bradypnea, dyspnea, labored breathing patterns, breathing sounds, irregular breathing patterns, motility reduced, limp, high-legged gait, prostration, tremor, squatted posture, piloerection, hair-coat ungroomed, cyanosis, nose: red encrustations, muzzle: red encrustations, nostrils: red encrustations, emaciation, decreased body weights, decreased reflexes, and hypothermia.
Body weight:
Transient changes in body weights of toxicological significance at 460 mg/m³ (actual concentration) and above.
Gross pathology:
Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable amongst exposure and control groups. One rat at 460 mg/m³ (actual concentration of the test substance 80 % in butylacetate) showed light colorized lungs (bloodless).
Animals succumbing within the observation period: Foamy discharge from nose, trachea with foamy content, lung edema and hydrothorax. Lung less collapsed, with dark-red discolorations (marbled) and gray aereas. Bloated gastrointestinal tract.
Other findings:
Rectal temperature: Significant changes in body temperature (increased for concentration group 460 mg/m³ - actual concentration, decreased for high concentrations).

Applicant's summary and conclusion

Executive summary:

An acute inhalation toxicity study of the substance (80 % in butyl acetate) was conducted in rats in accordance with OECD TG 403 and OECD GD 39. Rats were nose-only exposed to the liquid aerosol of the substance at 0 (butyl acetate control), 460, 1546, 3906, and 4586 mg/m³ (actual concentrations of test substance 80 % in butyl acetate). The respirability of the aerosol was adequate, i.e. the mass median aerodynamic diameter (MMAD) was smaller than 2.5 µm and the geometric standard deviation (GSD) 1.7-2.4.

Mortality did not occur up to 1546 mg/m³. Rats exposed at higher concentrations showed evidence of respiratory irritation with mortality patterns typical of an irritation-related acute lung edema. The LC50 determined was 4090 mg/m³ (actual concentration of the substance 80 % in butyl acetate); LC50 for the non-volatile fractions of the test article (based on gravimetric concentrations) = 3537.5 mg/m³.