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Acute Toxicity: inhalation

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acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 March to 16 April 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
Test type:
acute toxic class method

Test material

Details on test material:
Batch No.: 0953-1Purity: Not indicated by the sponsor; treated as 100% pure

Test animals

Details on test animals and environmental conditions:
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: Young adult animals (approximately 12 weeks old)
- Housing: Group housing of five animals per sex per cage in labelled Macrolon cages (type IV; height 18 cm) containing sterilised sawdust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet except during exposure to the test substance.
- Water (e.g. ad libitum): Free access to tap water except during exposure to the test substance.
- Acclimation period: at least 5 days

- Temperature (°C): 21.0 ± 3.0 ℃ (actual range: 20.0 – 21.7 ℃)
- Humidity (%): 40-70% (actual range: 34 - 57%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: 31 March 2010 To: 16 April 2010

No additional data

Administration / exposure

Route of administration:
other: aerosol/vapor mixture
Type of inhalation exposure:
nose only
Details on inhalation exposure:
- Exposure apparatus: The design of the exposure chamber is based on the flow past nose-only inhalation chamber. The chamber consisted of three animal sections with eight animal ports each.
- Method of holding animals in test chamber: The animals were placed in restraining tubes and connected to the animal ports.
- Source and rate of air: The mean total airflow was 29 and 54 L/min. for the 5 and 1 mg/L exposure groups respectively.
- System of generating particulates/aerosols: An aerosol/vapor mixture was generated by nebulization of the test substance by means of a nebulizer (type 950, Hospitak Inc., Lindenhurst, NY, USA). The primary aerosol/vapor mixture was diluted with pressurized air before it entered the exposure chamber.
- Method of particle size determination: For 5 mg/L, two representative droplet size distribution characterizations were conducted for the aerosol during exposure. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters and a fiber glass back-up filter. Amounts of test substance collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined.
For 1 mg/L, the test atmosphere almost completely consisted of a vapor and therefore no droplet size characterization was conducted.
- Treatment of exhaust air: From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.
- Temperature, humidity, pressure in air chamber: The temperature of the atmosphere was between 19.9 and 21.7 ℃ and relative humidity was between 22.4 and 25.5%.

- Brief description of analytical method used: A small stream of the test atmosphere was drawn from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The tube was heated to at least 200 ℃ to avoid condensation of the test atmosphere. The tube was connected to a gas cell, which was heated to 200 ℃ and mounted in a FTIR spectrophotometer (method developed under NOTOX project 493452). Both phases of the aerosol/vapor mixture were measured completely by the FTIR.
- Samples taken from breathing zone: no

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): For 5 mg/L, the Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice at 4.7 μm (gsd 1.8) and 4.5 μm (gsd 1.5), respectively. For 1 mg/L, the test atmosphere almost completely consisted of a vapor and therefore no droplet size characterization was conducted.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: The target concentration was based on the hazard categories for dust and mists specified in the UN and EC classification guidelines. The upper limit of the guideline concentration was increased with 10% in order to avoid the mean actual concentration to fall below the cut off value due to experimental variation. Three animals of each sex were exposed in a limit test for 4 hours to a target concentration of the test substance of 5.5 mg/L. Based on mortality at 5.5 mg/L, an additional group of 6 females (most susceptible sex) was exposed to 1.1 mg/L.

No additional data
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
The mean actual measured concentration was 5.39 ± 0.96 mg/L at 5 mg/L and 1.18 ± 0.42 mg/L at 1 mg/L.
No. of animals per sex per dose:
Three animals per sex were exposed in a limit test; 6 females were exposed to an additional dose
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights: On Days 1 (pre-treatment), 2, 4, 8 and 15 and at death (if found dead or sacrificed after day 1); Clinical signs: Three times during exposure for mortality, behavioural signs of distress and effects on respiration, and twice (at 1 and at 3 hours after exposure) on the day of dosing (Day 1) and once daily thereafter, until Day 15.
- Necropsy of survivors performed: yes
- Other examinations performed: Mortality/Viability
No statistical analysis was performed (the method used was not intended to calculate a LC50 value).

Results and discussion

Effect levels
Dose descriptor:
Effect level:
1 - 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
All females and one male were found dead or sacrificed moribund within approximately 2 and 24 hours after exposure to 5 mg/L. No mortality occurred at 1 mg/L.
Clinical signs:
other: Clinical signs noted among most animals after exposure at 5 mg/L included lethargy, flat/hunched posture, ventro-lateral recumbency, laboured/shallow respiration, rales, piloerection, ptosis and/or chromodacryorrhoea. The surviving animals had recovered f
Body weight:
Mean body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study.
Gross pathology:
Isolated dark red foci were noted in the lungs of the two females found dead after exposure at 5 mg/L. One male sacrificed at 5 mg/L showed small intestines distended with gas.
No abnormalities were found at macroscopic post mortem examination of other animals at 5 mg/L and animals at 1 mg/L.
Other findings:
No information provided

Any other information on results incl. tables

The MMAD at 5 mg/L exceeded the recommended range of 1 - 4 μm, possibly due to agglomeration of aerosol particles at this high concentration. The deposition in the lungs was however sufficient considering the mortality that occurred at this concentration.

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category IV
Migrated information Criteria used for interpretation of results: EU
The inhalatory LC50 value of the test substance in Wistar rats was considered to be within the range of 1.0 – 5 mg/L under the conditions of this study.
Executive summary:

The study was carried out with Wistar rats based on OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.436, "Acute Inhalation Toxicity-Acute Toxic Class Method", September 2009. Mortality, clinical signs, body weight, gross pathology were observed.

The inhalatory LC50 value of the test substance in Wistar rats was considered to be within the range of 1-5 mg/L under the conditions of this study.