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Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2016 to 15 February 2017
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 403 (Acute Inhalation Toxicity)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C12 H24 O
Test material form:
Clear, colorless

Test animals

Details on test animals or test system and environmental conditions:
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 to 12 weeks
- Weight at study initiation: within the range of 200 g to 350 g
- Fasting period before study: Not fasted
- Housing: The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet: free access, with the exception of the exposure period
- Water: free access, with the exception of the exposure period
- Acclimation period: at least 5 days
- Method of randomisation in assigning animals to test and control groups: On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card.

- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): at least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Mass median aerodynamic diameter (MMAD):
3.09 µm
Geometric standard deviation (GSD):
Details on inhalation exposure:
- Exposure chamber volume: approximately 30 liters (dimensions: 28 cm diameter x 50 cm high)
- Method of holding animals in test chamber: each rat was 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. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air (airflow): 50 L/minute
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) 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 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 (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 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.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (percentage) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 μm was calculated.
The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (percentage) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature, humidity, pressure in air chamber: 20 ºC, 38-41%, negative pressure

- Brief description of analytical method and equipment used: GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: ZB-5 (30 m x 0.53 mm id x 5 μm film)
- Samples taken from breathing zone: yes
- Time needed for equilibrium of exposure concentration before animal exposure: The theoretical chamber equilibration time (T99) was 3 minutes (Silver, 1946). The test atmosphere was generated for a total of 16 minutes prior to animal insertion to ensure the target test item concentration was being achieved.

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.09 um/ 2.42
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
4.97 mg/L
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: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes

Results and discussion

Effect levels
Key result
Dose descriptor:
Effect level:
> 4.97 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Clinical signs:
bodyweight loss
Body weight:
All animals exhibited body weight losses on the first day post-exposure. With the exception of two male and two female animals which exhibited body weight losses or showed no body weight gain from Days 1 to 3 post-exposure and one female which showed no body weight gain from Days 7 to 14 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period.
Gross pathology:
With the exception of four instances of dark and/or pale patches on the lungs, no macroscopic abnormalities were detected amongst animals at necropsy.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Executive summary:

A study was performed to assess the acute inhalation toxicity of the test item. The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals No. 403 “Acute Inhalation Toxicity” (2009), Method B.2. (Inhalation) of Commission Regulation (EC) No. 440/2008.

No deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 4.97 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 4.97 mg/L.