Registration Dossier

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18.05. - 4.07.2017
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
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Entidad Nacional de Acreditación
Test type:
acute toxic class method
Limit test:

Test material

Test material form:

Test animals

Details on test animals and environmental conditions:
- Source: Envigo RMS Spain S.L., Crta. Sant Miquel del Fai, km 3, 08182 Sant Feliú de Codines, Barcelona – Spain
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Group A: males: mean of 224.5 g; females: mean of 193.9 g; Group B: males: mean of 282.9 g; females: mean of 169.0 g
- Fasting period before study: no but animals were deprived during exposure
- Housing: 4/cage (before distribution) and 3/cage (after distribution)
- Diet: Global diet provided ad libitum
- Water: Tap water provided ad libitum
- Acclimation period: 8 days (Group A) or 13 days (Group B) prior exposure

- Temperature (°C): 19.2 - 24.6
- Humidity (%): 25 - 63
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12h light : 12h dark

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Mass median aerodynamic diameter (MMAD):
>= 1 - <= 4 µm
Geometric standard deviation (GSD):
>= 1.5 - <= 3
Remark on MMAD/GSD:
Mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were determined at the target concentration and calculated on the basis of the results from the cascade impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 4 μm for the MMAD and 1.5 to 3 for the GSD. A respirable aerosol (MMAD in the range of 1-4 μm) could be achieved at 3.5 mg/L air. Therefore, starting dose was set at 3.5 mg/L although, as indicated above, the mean actual dose reached during the present study was 3.81 mg/L.
Details on inhalation exposure:
- Exposure apparatus: Exposure chambers type EC-FPC-232
- Exposure chamber volume: approximately 3 L
- Method of holding animals in test chamber: restraint tubes which were positioned radially around the exposure chamber
- Source and rate of air: The flow of air at each tube was approximately 1.1 L/min, which is sufficient to minimize rebreathing of the test aerosol as it is more than twice the respiratory minute volume of rats.
- System of generating particulates/aerosols: A dust aerosol was generated from the desiccated and sieved test item using a Dust Generator SAG 410 (TOPAS GmbH, Germany)
- Method of particle size determination: Mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD)
- Treatment of exhaust air: not reported
- Temperature, humidity, pressure in air chamber: temperature = 19 - 25 ºC; humidity = 30 - 70%;

- Brief description of analytical method used: a stable aerosol of approximately 3.5 mg/L could be achieved with the TOPAS SAG 410 aerosol generator
- Samples taken from breathing zone: yes; test aerosol samples were collected onto a Whatman filter (grade F319.04) using a filter sampling device. The duration of sampling was 5 minutes.

Analytical verification of test atmosphere concentrations:
Duration of exposure:
ca. 4 h
3.81 and 1.02 mg/L
(3.81 mg/L was the highest stable aerosol concentration achievable that could be maintained at least for 4 hours.)
No. of animals per sex per dose:
3 animals/sex/group
Control animals:
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations in response to treatment were performed on all animals hourly during exposure (only grossly abnormal signs), immediately and 1 h after exposure, and once daily thereafter until the end of the observation period. Any visible clinical signs and discomfort were recorded. All animals were weighed once during the acclimatization period, on the day of treatment just before starting exposure, 24 h, 72 h and one week thereafter and also before sacrifice and gross necropsy conducted two weeks after exposure. Nevertheless, animals from Group A exposed to 3.81 mg/L were also weighed 4, 5 and 6 days after exposure for animal welfare reasons due to the mortality recorded and the severe clinical signs present.
- Necropsy of survivors performed: yes; the gross necropsy consisted of the examination of the abdominal and thoracic cavities and contents. Special attention was paid to any change in the respiratory tract. Any organs with gross lesions were collected and preserved in fixation medium (neutral-buffered 4% formaldehyde) for histological evaluation if considered relevant.
- Other examinations performed: Any clinical signs, discomfort and mortality were recorded in accordance with the humane endpoints guidance document of the OECD.
No statistical analysis was required.

Results and discussion

Effect levels
Key result
Dose descriptor:
LC50 cut-off
Effect level:
3.81 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 3.81 mg/L was the highest technically achievable concentration
In the high dose group (A, 3.81 mg/L), two male animals died shortly before finishing exposure and shortly thereafter, respectively, and the third one had to be sacrificed on study day 7 for animal welfare reasons due to the presence of severe labored breathing and continuous body weight loss (35% in total). All 3 females from Group A exposed to the dose of 3.81 mg/L survived the 14-day observation period after exposure.
No mortality was recorded in the low dose group (B, 1.02 mg/L).
Clinical signs:
other: The main clinical signs observed in both groups after finishing exposure were chromodacryorrhea, chromorrhinorrhea, soiled coat and piloerection. All these signs were transient and most of them were not present 24 hours after exposure. Breathing difficult
Body weight:
In all surviving animals, body weight decreased 24 hours after exposure. Afterwards, body weight tended to gradually increase in most animals until the end of the observation period, although in a number of cases slight decreases were also observed until 72-96 hours after exposure (two females from the high dose group and one female from the low dose group).
Gross pathology:
The necropsy conducted in both prematurely dead male animals from the high dose group, revealed a laryngeal obstruction by a white matter compatible with test item rests, whereas in the third male from this group euthanized for humane reasons on study day 7 a nodule of unknown origin was found on the outer tracheal wall. Red spots in the mandibular lymph nodes from both early decedent males and in the lungs from one of them were also observed.

No macroscopical findings were observed either in females from the high dose group or in animals exposed to the low dose, both sexes.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 4 (H302) according to Regulation (EC) No 1272/2008
In an acute inhalation toxicity study conducted according to OECD 436, all male rats in the 3.81 mg/L dose group died. 3.81 mg/L was the highest technically achievable concentration. No mortality was observed in all female rats of the 3.81 mg/L dose group and all animals in the 1.02 mg/L dose group. Thus, the LC50-cut-off was found to be 3.81 mg/L.