Registration Dossier

Toxicological information

Acute Toxicity: inhalation

Currently viewing:

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:
solid: compact

Test animals

Details on test animals and environmental conditions:
- Strain: HsdRCCHan: Wist
- 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 (based on A. Spiegel and R. Gonnert, Zschr. Versuchstierkunde, 1. 38 (1961) and G. Meister, Zschr. Versuchstierkunde, I. 144-153 (1965)
- 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
- 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: ethyl acetate
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 accommodated the animals' size. Each inhalation chamber segment was suitable to accommodate 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 (freed 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, 10 °C, using a digitally controlled cryostat) 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 pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles through evaporation of the vehicle and to prevent larger particles from entering the chamber a 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 chamber equilibrium is attained in less than one minute of exposure. 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.
- 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.

- 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 analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 70.7 % for 2379 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 1.96 µm, the average geometric standard deviation (GSD) was 2.2.

VEHICLE: ethyl acetate
- Justification of choice of vehicle: The vehicle ethyl acetate allows the aerosolisation of the resin-like solid test item. The vehicle (ethyl acetate) evaporates instantly after aerosolisation of the liquid test article
Analytical verification of test atmosphere concentrations:
gravimetric analysis
Duration of exposure:
4 h
Target concentrations: 5000 mg/m³
Actual concentrations: 2379 mg/m³ (maximum technical achievable concentration)
No. of animals per sex per dose:
5/sex for vehicle control group, 3/sex for test substance group
Control animals:
other: yes, concurrent vehicle (ethyl acetate)
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: The appearance and behaviour 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. Individual weights are also recorded at death, if applicable.
- 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 behavioural changes stimulated by sounds (finger snapping) and touch (back)).
Rectal temperatures were measured shortly after cessation of exposure using a digital thermometer with a rectal probe for rats.
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 levels
Dose descriptor:
Effect level:
> 2 379 mg/m³ air
Based on:
act. ingr.
Exp. duration:
4 h
Remarks on result:
other: maximum technically achievable concentration
Mortality did not occur up to the maximum technically achievable concentration of 2379 mg/m³.
Clinical signs:
other: The following clinical signs were observed at 6/6 rats: laboured breathing patterns, irregular breathing patterns, hair coat partially pasted, alopecia (pasted areas of hairs). The lead pathodiagnostic effects suggestive of portal of entry were borderline
Body weight:
Comparisons between control and exposure group revealed transient decrease in body weights.
Gross pathology:
Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable amongst the groups. In rats of test substance group lungs were pallid and increased lung-associated lymph node weights were observed.
Other findings:
Reflexes: No differences between vehicle control and exposure group observed.
Rectal temperature: Statistical comparisons between control and exposure groups did not reveal statistical significant changes in body temperature.

Any other information on results incl. tables

Of note was that the test substance as a resin-like solid molten-mass of no dustiness had to be dissolved in ethyl acetate to allow the aerosolisation. As a result of this process of dissolution, the test article became sticky and glue-like. Most of the observations made appeared to be related to the physical glue-like properties of this dissolution. Due to the lack of any apparent test article specific toxicity, apart from its gluing characteristics, higher concentrations were considered to be scientifically not justified.

Applicant's summary and conclusion

Executive summary:

A study on the acute inhalation toxicity of the substance was conducted in rats in accordance with OECD TG 403. Two groups of rats were nose-only exposed to the liquid aerosol of the substance at 0 (vehicle control) and 2379 mg/m³ (maximum technically achievable concentration of test substance). The aerosol was generated using the solvent ethyl acetate as vehicle. As the vehicle evaporates instantly after aerosolisation of the liquid test article, rats were in fact exposed to a dry powder aerosol. The respirability of the aerosol was adequate, i.e. the average MMAD was 1.96 µm (GSD 2.2).

Mortality did not occur up to the maximum tested concentration of 2379 mg/m³, thus the LC50 was >2379 mg/m³. The lead pathodiagnostic effects were borderline irregular and laboured breathing patterns which lasted up to postexposure day 7. No substance specific toxicity and particular no indications of systemic toxicity were observed. Of note is that the test substance as a resin-like solid molten-mass of no dustiness had to be dissolved in ethyl acetate to allow the aerosolization. As a result of this process of dissolution, the test article became sticky and glue-like. Most of the observations made appeared to be related to the physical glue-like properties of this dissolution. Due to the lack of any apparent test article specific toxicity, apart from its gluing characteristics, higher concentrations were considered to be scientifically not justified.