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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:
04 Oct 2005 - 07 Nov 2005
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)
according to guideline
EPA OPPTS 870.1300 (Acute inhalation toxicity)
according to guideline
other: EU Guideline 92/69/EEC and 93/21/EEC
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Molecular formula:
Reaction mass of Bisbenzimidazo[2,1-a:1',2'-b']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-6,11-dione and Bisbenzimidazo[2,1-a:2',1'-a']anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-10,21-dione
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation: solid, powder
- Particle size distribution (TEM): 30.3 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles: 16.8 m²/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1515 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable
Specific details on test material used for the study:
- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
- Homogeneous

Test animals

Details on test animals or test system and environmental conditions:
- Source: Laboratory Animal Services; Wölfersraße 4, 4414 Füllinsdorf, Switzerland
- Age at study initiation: 9 weeks for males and approx. 11 weeks for females.
- Weight at study initiation: males: 260,5 g (mean); females: 205,3 g (mean)
- Housing: singly in cages type DK III (Becker, Germany) without bedding
- Diet: KLIBA mouse / rat laboratory diet 10 mm pellets "GLP", Provimi Kliba SA, Kaiseraugst, Basel Switzerland, ad libitum
- Water: drinking water ad libitum
- Acclimation period: at least 1 week in which they were adapted to the surroundings

- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): dark cycle of 12 hours

Administration / exposure

Route of administration:
other: dust aerosol
Type of inhalation exposure:
nose/head only
other: 2% (w/w) of Aerosil 200
Mass median aerodynamic diameter (MMAD):
>= 3 - <= 3.1 µm
Geometric standard deviation (GSD):
>= 3.1 - <= 3.3
Details on inhalation exposure:
- The test substance was stirred in its container before a sample for dust generation was taken. The test substance was desagglomerated in a mixer under addition of 2% (w/w) of Aerosil 200 before introduction into the dust generator, in order to improve dust aerosol formation. The dust was produced inside the inhalation system with a dosing-wheel dust generator and compressed air.

- The exposure system was located inside an exhaust cabin in an air-conditioned laboratory. A supply air flow (compressed air) of 1.5 m³/h was used for the exposure. The exhaust airflow was set to 1.35 m³/h. An air change of about 27 times per hour can be calculated by dividing the supply air flow by the volume of the inhalation system.
- The lower amount of exhaust air, which was adjusted by means of a separate exhaust air system, achieved a positive pressure inside the exposure system . This ensured that the mixture of test substance and air was not diluted with laboratory air in the breathing zones of the animals. The animals were exposed to the inhalation atmosphere for 4 hours plus equilibration time of the inhalation systems (t99 about 10 min).

Measurement of operation conditions
- The flows of supply and exhaust air were adjusted and continuously measured with flowmeters. They were recorded four times in about 1-hour intervals. The temperature in the inhalation system was measured continuously with a digital thermometer and recorded four times in about 1-hour intervals. The humidity in the inhalation system was measured with a dielectric probe four times in about 1-hour intervals.
- No surveillance of the oxygen content in the inhalation system was performed. The air change was judged to be sufficient to prevent oxygen depletion by the breathing of the animals, and the concentration of the test substance used could not have a substantial influence on oxygen partial pressure.
Gravimetric determination of the inhalation atmosphere concentration:
Equipment: balance Mettler AT 250. Preweighed filters were placed into the filtration equipment. By means of the vacuum pump metered volumes of the dust were drawn through the filter. For each sample the dust aerosol concentration in mg/I was calculated from the difference between the preweight of the filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmospheres. Mean and standard deviation were calculated for the concentration from the results of the individual measurements. The mean concentration was corrected for the amount of additive used.
Particle size analysis:
• EACD 50% (effective aerodynamic cutoff diameter 50%) defines the separation characteristic of each impactor stage. 50% of particles with the EACD given are deposited in the pertinent impactor stage; the remainder has reached one of the following stages.
• MMAD (mass median aerodynamic diameter) is the calculated aerodynamic diameter which divides the size distribution in half when measured by mass.
• Geometrical standard deviation (GSD) is the ratio of the estimated 84 percentile to the 50 percentile and indicates the slope of the cumulative particle size distribution curve.
• Inspirable aerosol
Inspirable aerosols can enter the respiratory system. Aerosols with MMAD <100 Nm are considered inspirable (MAK Commission of the DFG (German Research Society, MAK list 1997).
• Respirable aerosol
Respirable aerosols can enter the alveolar region of the lung. Aerosols with MMAD < 4 Nm are considered respirable (MAK Commission of the DFG (German Research Society, MAK list 1997).

Before sampling, the impactor was assembled with preweighed glass-fiber collecting discs, and a backup particle filter. The impactor was connected to the vacuum pump and two samples were taken from the breathing zone of the animals starting not earlier than 30 minutes after the beginning of the exposure. The sample volume was 6 L. After sampling the impactor was taken apart. The collecting discs and the backup particle filter were re-weighed. The amounts of material adsorbed to the walls of the impactor and in the sampling probe (wall losses) were also determined quantitatively. The results from the particle size analysis were not corrected for the additive.

- Exposure conditions:
Supply air (m³/h): 1.5
Exhaust air (m³/h): 1.35
Substance flow (g/h): 36.1
Temperature (°C): 21.8±0.3
Relative humidity (%): 47.7±2.2

- Analytical concentration:
Mean mg/l: 5.2
Standard deviation: 1.0
Nominal concentration mg/l: 24.1

- Particle size analysis:
MMAD (µm): 3.1 and 3.0
Geometrical standard deviation: 3.1 and 3.3
Analytical verification of test atmosphere concentrations:
Gravimetric determination
Duration of exposure:
4 h
5.2 mg/l (nominal concentration: 24.1 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: A check for overt clinical signs of toxicity or mortality as well as a check for the presence of feed and drinking water was made twice a day on workdays and once daily on weekends and public holidays. Detailed clinical observations were recorded for each animal separately several times during exposure and at least once on each workday of the observation period.
- Body weights: The body weight of the animals was determined just prior to exposure (day 0), weekly thereafter and at the end of the observation period.
- Necropsy of survivors performed: yes. At the end of the observation period the animals were sacrificed with C02 and were subjected to gross-pathological examination

A binomial test was used for statistical evaluation.

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 5.2 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
not determinable
(no mortality observed)
No lethality occurred at the tested concentration of 5.2 mg/I during the study period of 14 days.
Clinical signs:
other: Accelerated respiration, pulmonary respiration e sounds, squatting posture, piloerection and smeared fur. Findings were observed from hour 0 of exposure until including study day 3.
Body weight:
The mean body weights of the animals increased throughout the study period.
Gross pathology:
No pathological abnormalities of the organs were observed in all animals at termination of the study.
Other findings:
Contaminated fur was noted during necropsy in all animals.

Any other information on results incl. tables

Analytical concentration:

Mean (mg/l) standard deviation nominal concentration (mg/l)
5.2 1 24.1

Particle Size Analysis:

Sample MMAD (µm) geometrical standard deviation
1 3.1 3.3
2 3 3.1

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the conditions of this study the LC50 for male and female rats after dust inhalation was determined to exceed 5 .2 mg/I.
Executive summary:

For determination of the acute inhalation toxicity (single 4-hour-exposure) of the test substance as dust, a GLP compliant study in male and female Wistar rats was performed according to OECD-Guideline method 403, as well as EEC and EPA guidelines. A measured concentration of 5 .2 mg/I was tested (Limit test). Cascade impactor measurements resulted in particle size distributions with mass median aerodynamic diameters (MMADs) of 3.0 and 3.1 µm, which are well within the respirable range. No mortality occurred at the tested concentration. Clinical signs of toxicity comprised visually accelerated respiration, pulmonary respiration sounds, squatting posture, piloerection and smeared fur. Findings were observed from hour 0 of exposure until including study day 3. Moreover, contaminated fur was observed in all animals from day 0 onward until termination of the study. The mean body weights of the animals increased throughout the study period. No pathological abnormalities of the organs were observed in all animals at termination of the study. Contaminated fur was noted during necropsy in all animals. Under the conditions of this study the LC50 for male and female rats after dust inhalation was > 5 .2 mg/l.