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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:
10 Mar - 04 May 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
Adopted in 2009
according to guideline
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
Adopted in 1998
according to guideline
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
Adopted in 2008
GLP compliance:
yes (incl. QA statement)
National Institute of Pharmacy and Nutrition, Budapest, Hungary
Test type:
traditional method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Research Model and Services, Germany GmbH (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Sighting exposure (Group 0.1): 11 weeks; Main Study (Group 1): 8 weeks; Main Study (Group 2): 10 weeks
- Body weight at study initiation: Sighting exposure (Group 0.1): males: 416 g, females: 251 g; Main Study (Group 1): males: 317 - 331 g, females: 213 - 233 g; Main Study (Group 2): males: 361 - 396 g, females: 242 - 276 g
- Housing: sighting study: individually; main study: group housed, 5 animals, by sex per cage in polycarbonate solid floor cages (type II or III) with stainless steel mesh lids, Lignocel® Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH+Co. KG, Rosenberg, Germany), and cotton rolls as nest building material
- Diet: ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: sighting study (Group 0.1): 28 days; main study (Group 1): 8 days; main study (Group 2): 20 days
- Microbiological status: SPF

- Temperature (°C): 21.2 – 25.8
- Humidity (%): 34 – 69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Sighting exposure (Group 0.1): From: 10 Mar 2016 To: 24 Mar 2016; Main Study (Group 1): From: 01 Apr 2016 To: 15 Apr 2016; Main Study (Group 2): From: 20 Apr 2016 To: 04 May 2016

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.39 - <= 2.55 µm
Geometric standard deviation (GSD):
>= 2.81 - <= 2.92
Details on inhalation exposure:
- Exposure apparatus: TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprised of 2 concentric anodised aluminium chambers.
- Exposure chamber volume: 3.85 L
- Method of holding animals in test chamber: polycarbonate restraint tubes located around the chamber
- Method of conditioning air: Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the appropriate dust generator.
- Rate of air (airflow): at least 0.5 L/min at each animal's exposure port (breathing zone)
- System of generating particulates/aerosols: Dust particles were produced in a dust generator according to Wright (TSE GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. In this device a fixed blade scraped continuously the surface of the test substance compacted in a rotating reservoir. Dispersion was carried out by a high velocity air flow inside the outlet nozzle.
- Method of particle size determination: cascade impactor, 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany)
- Temperature and humidity in air chamber: 21.7 - 27 °C, 12 - 17.5%

- Brief description of analytical method and equipment used: Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable volume of test atmosphere through weighed GF10 glass fibre filters (Whatman®, Whatman GmbH, Germany, Ref. no. 10370302). The difference in the pre and post sampling weights and divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration. Filter samples were collected at the breathing zone (approximately every 10 - 20 min) during each 4-h exposure period and analysed. The nominal concentration was calculated by dividing the mass of test substance disseminated into the chamber by the total volume of air that flow through the chamber during the same period.
- Samples taken from breathing zone: yes
- Actual time allowed for equilibrium of exposure concentration before animal exposure : 17 - 26 min

- Particle size distribution: Sighting exposure (Group 0.1): 66.7% <4 µm; Main Study (Group 1): 69.0% <4 µm; Main Study (Group 2): 67.8% <4 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Sighting exposure (Group 0.1): 2.55 / 2.83; Main Study (Group 1): 2.39 / 2.81; Main Study (Group 2): 2.43 / 2.92

- Rationale for the selection of the starting concentration: In a dose-range finding study, sighting exposures using one male and one female animal were performed (Group 0.1). A target concentration of 2.5 mg/L was tested. Animals were exposed for 4 h to the test atmosphere. One female died after the exposure. Based on this lethality the main study was performed at target concentrations of 2.5 and 1.25 mg/L.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
Sighting exposure (Group 0.1): 2.50 mg/L; Main Study (Group 1): 2.50 mg/L; Main Study (Group 2): 1.25 mg/L
No. of animals per sex per dose:
Sighting exposure (Group 0.1): 1 animal/sex/dose; Main Study (Group 1): 5 animals/sex/dose; Main Study (Group 2): 5 animals/sex/dose
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were checked hourly during exposure, 1 h after exposure termination and twice daily during the 14-day observation period for morbidity/ mortality. Animals were observed for clinical signs at hourly intervals during exposure. Following exposure, clinical observations were performed twice on the day of exposure and subsequently once daily for 14 days. Individual body weights were recorded prior to treatment on the day of exposure (Day 0) and on Day 1, 3, 7 and 14, and prior to necropsy.
- Necropsy of survivors performed: yes
- Other examinations performed: Gross necropsy was performed with special attention to the respiratory tract for macroscopic signs of irritancy or local toxicity.
For body weights, mean values were calculated for each sex.

Results and discussion

Preliminary study:
The female animal of Group 0.1 died after the exposure on Day 0.
Following clinical signs of toxicity were observed: laboured respiration (slight to severe), clonic convulsion and decreased activity (slight) were observed in the female animal before its death. Laboured respiration (slight to severe), noisy respiration (moderate to severe), incoordination (slight to moderate), decreased activity (slight to moderate) and irritability were recorded in the surviving male animal. The male animal was symptom free from Day 7.
Slight body weight loss (9.4%) was observed in the surviving male animal after the exposure, which was followed by normal body weight gain.
Effect levels
Key result
Dose descriptor:
Effect level:
> 1.22 - < 2.51 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Group 1: 2/5 male rats died during the exposure, 1 male and 2 females after the exposure on Day 0, 2 female animals were found dead on Day 1, and 1 female on Day 2.
Group 2: 1/5 females died after the exposure on Day 0.
Clinical signs:
other: Laboured respiration was frequently observed. For further details see "Other findings".
Body weight:
Group 1: Slight to moderate body weight losses (7.9 and 10.4%) were recorded in the surviving males after the exposure. All surviving rats regained their initial body weight by Day 7 at the latest.
Group 2: Slight body weight losses (3.9 - 9.9%) were noted post exposure. From Day 3 onwards, all rats showed normal body weight gain.
Gross pathology:
Dark/red discoloration of the non-collapsed/collapsed lungs and white dry/powder material at the perinasal/perioral fur in all found dead animals, enlarged lungs and white mucoid material within trachea in 1/10 animal, and white creamy material observed in digestive content of the stomach in one female were regarded as test substance-related.
In surviving animals no test substance-related changes were noted at the end of the observation period on Day 14.
Other findings:
Clinical signs of toxicity:
Group 1: Laboured respiration (slight to severe) was recorded in the three males before their death. Laboured respiration (slight to severe), noisy respiration (slight), sneezing, activity decreased (slight to moderate), irritability were noted in the surviving two males. Laboured respiration (slight to severe), noisy respiration (slight), activity decreased (slight to moderate), irritability, tonic convulsion were recorded in the females before death. The surviving male animals were symptom free from Day 5 till study termination.
Group 2: Laboured respiration (slight to severe) was recorded in the female before its death. In the surviving animals, laboured respiration (slight to severe), noisy respiration (slight), sneezing, activity decreased (severe), irritability and tremor (intermittent) were observed. The animals were symptom free from Day 4 onwards.
Wet fur, ruffled fur or red-brown staining (as chromodacryorrhea) occurred in study animals from Day 0 up to Day 3. These signs were considered to be related to the restraint and exposure procedures or discomfort of the animals but not to be of toxicological relevance.

Any other information on results incl. tables

Table 2. Summary of acute inhalation toxicity

Number /
Mean Achieved
Onset and Duration
of Toxicological
Clinical Signs (Day)
Occurrence of
Sighting exposure
0.1 / Male 2.53 0/1/1 0 - 6 -
0.1 / Female 2.53 1/1/1 0 0
Main study
1 / Male 2.51 3/5/5 0 - 4 0
1 / Female 2.51 5/5/5 0 - 1 0, 1, 2
2 / Male 1.22 0/5/5 0 - 1 -
2 / Female 1.22 1/5/5 0 - 1 0

* 1st = number of dead animals, 2nd = number of animals with clinical signs, 3rd = number of animals exposed

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Under the conditions of the present study, 8/10 animals (3/5 males and 5/5 females) were found dead after the exposure to 2.51 mg/L of the test substance for 4 h and 1/10 animals was found dead after the exposure to 1.22 mg/L of the test substance. The LC50 is considered to be between 1.22 mg/L and 2.51 mg/L.
Based on these results, the following classification under Regulation (EC) No 1272/2008 is justified: CLP: Acute Tox. 4, Inhalation, H332