Registration Dossier

Administrative data

acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 8 to 28, 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD test guidelines and GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
Test type:
standard acute method

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): E-BK105
- Physical state: Black powder (Solid)
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: June 7, 2016
- Storage condition of test material: Room temperature, dark place, tight container

Test animals

other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 265-276 g for males, 210-224 g for females
- Fasting period before study: From the evening on the day before the administration (approx 18 hours predose) to about 3 hours postdose.
- Housing: Polycarbonate cages (W265xD426xH200 mm), stainless steel racks and feeders, autoclaved bedding was used, two animals same sex per cage
- Diet (ad libitum): Pellet diet (MF, Oriental Yeast Co., Ltd.) (each lot analysed: contaminants confirmed to be within acceptable limits of testing facility)
- Water (ad libitum): Tap water filtered through a 5 microm filter and irradiated by UV light (analysed every 6 months: contaminants confirmed to be within acceptable limits of testing facility)
- Acclimation period: at least 5 days

- Temperature (°C): 21.9-22.4
- Humidity (%): 47.8-58.7
- Air changes (per hr): 6 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Details on inhalation exposure:
- Exposure apparatus: a flow-past nose-only inhalation exposure chamber
- Method of holding animals in test chamber: Animals were restrained in restraining tubes for nose-only inhalation exposure. The chamber has 16 exposure ports per tier, and a one tier chamber was used.
- Source and rate of air: The flow rate air supply was 16 L/min
- Method of particle size determination: Test atmosphere collected in 8 stages using a cascade impactor at 1 and 3 hr after the start of the exposure. A glass fiber filter was used as the collection substrate. Air flow ratio for the collection was set at 500 ml/min, duration was set at 1.5 min. Glass fibers were weighed before and after collection, the test substance weights in each stage being the weight difference. The particle size distribution was calculated with the effective cut-off diameter of each stage of the cascade impactor and the amount collected in each stage. The following parameters were calculated: MMAD, GSD, weight ratio of particles with <4µm to total particles.
- Treatment of exhaust air: Flow rate set at 14 L/min
- Temperature, humidity, pressure in air chamber: Pressure of chamber positive. Temperature and relative humidity measured at exposure port with digital thermo-hygrometer: Temperature: 23.0-23.3°C and humidity: 52-53% throughout exposure.

- Brief description of analytical method used: The measurement of the exposure concentration before the start of exposure was conducted after a particle count concentration (relative concentration), which was measured at the exposure port, reached equilibrium.


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: At 1 and 3 hrs after the start of the exposure, MMAD of the dust measured was 4.7 and 6.2 µm and GSD at each measuring oint was 1.9 and 1.6,. respectively. The ratio of the particles with a diameter less than 4 µm, considered to be the inhalable particle size for rats, were 40.0 and 16.7%, respectively.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 4.7 - 6.1 µm / 1.6-1.9 µm

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: The exposure concentration was set at 2 mg/L which is permissible concentration as maximum concentration according to the applied guideline. Since the MMAD of the dust obtained in the preliminary dust generation trial was larger than the recommended range in the guideline, the target concentration was set at 2 mg/L to achieve the particle size of the recommended range.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
2.8 mg/l mean actual exposure concentration; nominal concentration 4 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: Mortality and clinical signs were observed immediately after the end of exposure (on Day 1, day of exposure), 1, 2 hours postdose on Day 1 and once daily for 14 days thereafter. Body weight was measured just before the administration (Day 1) and on Days 2, 4, 8, and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: none
none required.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 2.8 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
No animals died.
Clinical signs:
other: Soiled fur on the whole body derived from the test substance was observed in males and females immediately after exposure to day 2. Soiled fur on the tail was observed in males and females from day 2. Chromaturia (reddish brown) was observed in males and
Body weight:
Body weight losses or depression of body weight gain was noted in males and females on day 2. Their body weight increased in all males and females on day 4 and thereafter.
Gross pathology:
There were no abnormalities in any males or females.

Applicant's summary and conclusion

Interpretation of results:
not classified
Migrated information Criteria used for interpretation of results: EU
The LC50 of E-BK105 was determined to be more than 2.8 mg/L (dust) under the conditions of this study (OECD 403).
Executive summary:

A single exposure to 2.8 mg/L dust of E-BK105 (nose only) was conducted to male and female SD (Crl:CD(SD)) rats aged 8 weeks to evaluate its acute inhalation toxicity in accordance with the OECD test guideline 403. The MMAD of the dust was 4.7 and 6.2 µm at 1 and 3 hours, respectively, after start of exposure. The ratios of particles with a diameter < 4 µm, were 40.0% and 16.7% at 1 and 3 hours after start of exposure, respectively. As a result, there was no mortality nor necropsy findings. Decreases in body weight or suppression of body weight gain were noted in females or males, respectively, on day 2. In addition, soiled fur, chromaturia

(reddish brown) and compound colored stool was noted. In conclusion, the LC50 of E-BK105 in rats is determined to be more than 2.8 mg/L under the conditions of this study.