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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 25.1% solids in water

Test animals

Species:
rat
Strain:
other: RccHan®:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 to 12 weeks old
- Weight at study initiation: 369 g and 210 g for males and females of group 1; 302 g and 209 g for males and females of group 2
- Fasting period before study: no
- Housing: animals were housed in groups of three, separated by sex, in macrolon cages (type 4S) with a bedding of wood shavings and a wooden block and strips of paper as environmental enrichment.
- Diet: rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England) ad libitum
- Water: domestic mains tap-water ad libitum
- Acclimation period: 28 days group 1; 8 days group 2


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): about 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: humidified air during exposure of the first group or with a stream of compressed dry air for the second group
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical stainless steel column, surrounded by a transparent cylinder
- Exposure chamber volume: about 50 litres
- Method of holding animals in test chamber: secured in plastic animal holders
- Source and rate of air: at least 1 litre/min for each rat
- Method of conditioning air: The air entering the unit was controlled at 22 ± 3˚C. The target range for the relative humidity was 30% - 70%.
- System of generating particulates/aerosols: The test material was diluted in demineralized water (10% w/w) and continuously stirred by a magnetic stirrer. An amount of test material, controlled by a peristaltic pump (Peristaltic pump Minipulse, Gilson, Villiers le Bel, France), was nebulized using an air-driven atomizer (Schlick type 970/S, Coburg, Germany). The atomizer was supplied with a stream of humidified air during exposure of the first group or with a stream of compressed dry air for the second group. The air pressure on the atomizer was regulated by a reducing valve and the airflow was measured with a mass view meter (MASSVIEW, Bronkhorst Hi Tec, Ruurlo, The Netherlands). The test atmosphere was introduced at the top of the exposure chamber, directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted.
- Method of particle size determination: Particle size distribution measurements were carried out by means of a 10-stage cascade impactor (2110k cascade impactor, Sierra instruments, Carmel Valley, California, USA).
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test material in the test atmosphere was measured by gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing approximately 50 L (group 1) or 25 L (group 2) test atmosphere at 5 L/min through fibre glass filters (Sartorius, 13400- 47). Filters, contained in a drying cup, were weighed before sampling, loaded with a sample of test atmosphere, weighed, dried for at least 15 minutes but not longer than about half an hour (which was sufficient to obtain a stable filter weight) and weighed again. The loaded filters were dried by means of a stream of compressed dry air controlled by a mass flow controller (0.125 normal litre/min/filter). A total number of 8 samples were taken during exposure of each group of animals. The actual concentration was calculated by dividing the amount of test material present on the filter after drying by the volume of the sample taken (the dry mass was converted to ‘test material’ as such using a solids content of 25.1%). The atmospheric concentration of total solids was also calculated by dividing the dried mass on the filter by the volume of the sample taken.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
- Group 1:
- preliminary test atmosphere generation: MMAD 2.23 μm, GSD 1.94
- during exposure, 1st measurement: MMAD 2.19 μm, GSD 2.01
- during exposure, 2nd measurement: MMAD 2.18 μm, GSD 1.99
Group 2:
- preliminary test atmosphere generation: MMAD 2.33 μm, GSD 2.13
- during exposure, 1st measurement: MMAD 2.42 μm, GSD 1.91
- during exposure, 2nd measurement: MMAD 2.26 μm, GSD 2.12

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Group 1 = 126 (± 9) mg total solids/m3 (504 mg test material/m3, corrected for solids)
Group 2 = ± 19 mg total solids/m3 (1046 mg test material/m3, corrected for solids)
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: On the exposure day, the animals were observed for clinical signs just before exposure, four times during exposure (about once per hour), and twice after exposure. During the observation period, each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon. In
weekends only one check per day was carried out. During exposure, when observation was hindered by the restraining tubes, attention was
directed to breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment and mortality were recorded.
- Frequency of weighing: The body weight of each (surviving) animal was recorded on days -1, 0 (just before exposure), 1, 3, 7 and 14 (prior to necropsy).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
>= 126 - <= 263 other: mg total solids/m3
Exp. duration:
4 h
Remarks on result:
other: gravimetric measurement
Mortality:
Group 1, 126 mg total solids/m3: 0 males died and 1 of 3 females died
Group 2, 263 mg total solids/m3: 2 of 3 males and 2 of 3 females died
Clinical signs:
126 mg total solids/m3: During exposure, treatment-related clinical signs were seen in all animals (restless and altered breathing frequency). After exposure, treatment-related clinical signs were seen in two female animals (hunched posture, blepharospasm, dyspnea) and in one male animal (blepharospasm). One female was found dead on the day after exposure

263 mg total solids/m3: During exposure, treatment-related clinical signs were seen in all animals (altered breathing rate, mostly increased, and shallow breathing). After exposure, all animals showed irregular breathing, piloerection, blepharospasm and lethargy. On the day after exposure, two male animals and two female animals were found dead. The surviving animals showed no abnormalities from the day after exposure until scheduled sacrifice.
Body weight:
126 mg total solids/m3: All male and one female animal had lost weight the day after exposure and gained weight thereafter. One female animal had gained a little weight on the day after exposure, lost weight in the period day 1-3 and gained weight thereafter.

263 mg total solids/m3: The surviving male and female animal had lost weight the day of exposure and showed catch up growth thereafter.
Gross pathology:
126 mg total solids/m3: The female that was found dead on the day after exposure had blood at the nose and dark red and poorly collapsed lungs.
One surviving female showed a swollen uterus at scheduled necropsy. The latter finding was not ascribed to treatment because it is a common background finding in female rats. Necropsy of the other survivors showed no abnormalities.

263 mg total solids/m3: The two male and two female animals that were found dead on the day after exposure had blood around their noses and dark red lungs. The surviving male and female animal had grey discoloured lungs.

Applicant's summary and conclusion

Interpretation of results:
Category 2 based on GHS criteria
Conclusions:
LC50 is between 126 mg total solids/m3 and 263 mg total solids/m3.
Executive summary:

The aim of the study was to determine the acute inhalation toxicity of the test substance in rats.

A group of three males and three females was exposed to 126 mg total solids/m3 for four hours. Mass median aerodynamic diameter was 2.19 – 2.18 µm and geometric standard deviation was 2.01 – 1.99. During exposure, treatment-related clinical signs were seen in all animals (restless and altered breathing frequency). After exposure, treatment-related clinical signs were seen in two female animals (hunched posture, blepharospasm, dyspnea) and in one male animal (blepharospasm). One female was found dead on the day after exposure. Following some initial weight loss, body weight development was considered to be in the normal range for rats of this strain and age. At necropsy of survivors, no treatment related abnormalities were seen.

A second group of three males and three females was exposed to 263 mg total solids/m3 for four hours. Mass median aerodynamic diameter was 2.42 – 2.26 µm and geometric standard deviation was 1.91 -2.12. During exposure, treatment-related clinical signs were seen in all animals (altered breathing rate, mostly increased, and shallow breathing). After exposure, all animals showed irregular breathing, piloerection, blepharospasm and lethargy. On the day after exposure, two male animals and two female animals were found dead. The surviving animals showed no abnormalities from the day after exposure until scheduled sacrifice. These animals had lost weight on the day of exposure and grew normally thereafter. At necropsy, the surviving animals had grey discoloured lungs. This finding was considered to be related to treatment.

As one animal died following exposure to 126 mg total solids/m3 and two male and two female animals died at 263 mg total solids/m3, it was concluded that the 4-hour LC50 of the test substance in rats was between 126 and 263 mg total solids/m3.