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:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Batch: CDD-005

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Age at study initiation: Eight to twelve weeks
- Weight at study initiation: 200 to 350g
- Housing: Housed in groups of upto 3 per sex in solid-floor polypropylene cages with stainless steel lids,
- Diet (e.g. ad libitum): Free access except during the exposure period and during acclimitization to the restraining tubes.
- Water (e.g. ad libitum): Free access except during the exposure period and during acclimitization to the restraining tubes.
- Acclimation period: At least 5 days in the detailed housing. Animals were also acclimitized for approximately 2 hours the day prior to exposure to the restraining tubes.

- Temperature (°C): 19°C throughout the exposure period (in the animal room; 19 to 25°C was set to be maintained).
- Humidity (%): 33-34% throughout the exposure period (in the animal room; 30 to 70% was set to be maintained).
- Air changes (per hr): At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: acetone
Details on inhalation exposure:
- Exposure apparatus: Cylindrical exposure chamber.

- Exposure chamber volume: 30L

- Method of holding animals in test chamber: Individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber 'O' ring.

- Source and rate of air: Oil free compressed air provided by a compressor at a rate of 60L/min

- Method of conditioning air: The air was passed though a water trap and respiratory quality filters before being introduced to the nebulizer.

- System of generating aerosols: The test item formulation was aerosolized using a metal concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item formulation under pressure, and to a metered compressed air supply.

- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor. This device consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 μm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.

- Temperature, humidity, pressure in air chamber: The temperature, relative humidity and pressure in the air chamber was determined to be 19°C, 33-34% and negative pressure respectively.

- Brief description of analytical method used: Gravimetric analysis was used. The test atmosphere was sampled at regular intervals during the exposure period. A weighed glass fiber filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump. After sampling, the filter was dried, in a desiccator under reduced pressure between 18 and 20°C, and weighed again 24 hours later. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was the chamber concentration in terms of non-volatile component. The addition of Acetone to improve aerosolization was considered not to affect the gravimetric calculation as all of the filters were dried thoroughly (for 24 hours) prior to weighing.

- Samples taken from breathing zone: yes

- Composition of vehicle (if applicable): Acetone

- Concentration of test material in vehicle (if applicable): A ratio of 20:80 (w/w) test material and acetone.

- Justification of choice of vehicle: The nature of the test item was such that a suitable dust atmosphere could not be generated from the test item as supplied. A formulation was, therefore, prepared with Acetone.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: No toxicity was observed in the preceding acute oral toxicity test.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
A target concentration of 5 mg/L was used for exposure.
No. of animals per sex per dose:
3 males and 3 females per sex per dose
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days

- Frequency of observations and weighing: Clinical signs were observed at hourly intervals during exposure, immeadiately on removal of the restraining tubes at the end of exposure, one hour after the end of exposure, and subsequently once daily for up to 14 days. Bodyweights were were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.

- Necropsy of survivors performed: Yes; all animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Not required in study design.

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
> 5.23 mg/L air
Based on:
test mat.
Exp. duration:
4 h
No deaths occured on study in either sex.
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a
Body weight:
All males and one female animal exhibited body weight losses on the first day post-exposure. With the exception of one female animal which exhibited a slight body weight loss from Days 1 to 3 post-exposure, reasonable body weight gains were noted for all animals during the remainder of the recovery period.
Gross pathology:
With the exception of one instance of dark patches on the lungs, no macroscopic abnormalities were detected amongst animals at necropsy.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.23 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of the test substance, in the RccHanTM : WIST strain rat, was greater than 5.23 mg/L. This results in no classification for acute toxicity via inhalation under CLP regulation.