Registration Dossier

Administrative data

acute toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles.

Data source

Reference Type:
Acute Pulmonary and Systemic Effects of Inhaled Coal Fly Ash in Rats: Comparison to Ambient Environmental Particles
Smith, K.R. et al.
Bibliographic source:

Materials and methods

Principles of method if other than guideline:
Study of pulmonary and systemic effects after inhalation exposure (4 h/day on 3 consecutive days, one dose level) in male rats.
GLP compliance:
not specified
Test type:
other: acute inhalation exposure on 3 consecutive days
Limit test:

Test material

Constituent 1
Reference substance name:
Coal fly ash (respirable fraction)
Coal fly ash (respirable fraction)
Details on test material:
- Name of test material (as cited in study report): Coal fly ash
- Physical state: solid
- Other: Composition and further details on test material see additional information below.

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan, San Diego, CA, USA.
- Age at study initiation: 8 weeks
- Weight at study initiation: 260 - 270 g
- Housing: plastic cages with TEK-Chip pelleted paper bedding
- Diet (e.g. ad libitum): Laboratory Rodent Diet 5001 (LabDiet, Brentwood, MO)
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 days with acclimatisation to nose only exposure tubes.

- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
other: unchanged (no vehicle)
Details on inhalation exposure:
CFA was aerosolized using a belt feeder and fluidized bed system previously described by Teague et al. (2005). PM2.5 (less than 2.5 µm) size-fractionated particles were premixed with 100- to 200 -µm-diameter glass beads and loaded into a dust feed for delivery to a vibrating fluidized bed system for aerosolization of both CFA and glass beads. The aerosol subsequently passed through a cyclone separator to remove the glass beads, while the CFA remained aerosolized passing through a krypton-85 source (to reduce particle agglomeration) to the nose-only inhalation system. Particle concentration during exposure was monitored by a continuous-reading, light-scattering, dust concentration monitor and filter connected to the exposure chamber. A Grimm Series 1.108 Aerosol Spectrometer (GRIMM Aerosol Technik GmbH, Douglasville, GA) extracted a 1.2 Lpm flow, and a proprietary algorithm converted light scattering into particle mass concentration using the default calibration for occupational monitoring (PM10, PM2.5, and PM1). The particle feeder settings of the aerosolization system were adjusted as needed to achieve the target time-averaged concentration for exposure. The aerosol spectrometer was operated in the 16-channel mode to measure particle size distribution. Mass of CFA collected on the filter was used to correct the aerosol spectrometer output to obtain the actual mass concentration during exposures. The filter sample was collected at 3 L/min on a 25-mm Pallflex EMFAB TX40HI20-WW filter using an InTox filter housing connected to a diaphragm pump and a bellows-type dry gas meter.

The actual aerosol particle size generated can be approximated by a count median diameter of 0.3 µm and a geometric standard deviation of 1.4, which corresponds to a mass mode near 1 µm.

Deposition of the CFA aerosol generated was modeled using MPPD V1.0 software developed by Chemical Industry Institute of Toxicology (CIIT Center for Health Research) and Rijksinstituut voor volksgezondheid en milieu (RIVM National Institute for Public Health and the Environment) (Asgharian et al., 2002). The model inputs include measured particle size distribution, time-averaged aerosol concentration during exposure, and observed breathing rate. Based on the MPPD model, the total CFA mass deposited over the 3- day exposure was 32 µg per rat, and the regional deposition was head, 25%; tracheobronchial, 20%; and pulmonary, 55%.
Analytical verification of test atmosphere concentrations:
Duration of exposure:
4 h
Remarks on duration:
4 h/day for 3 days
1400 mg/m³ (= 1.4 mg/L):
The average concentration of CFA particulate matter less than 2.5 µm (PM2.5) was 1400 mg/m³, of which 600 mg/m³ was PM1.
No. of animals per sex per dose:
In total 48 animals were included into the study.
A grouping of 6 animals was reported for tissue sampling.
Control animals:
Details on study design:
- Duration of observation period following administration: Animals were examined 18 and 36 h postexposure.
- Necropsy performed: yes, 18 and 36 hours after last exposure. These necropsy times were selected to measure acute responses and to approximate the 1- to 3-day lag time between increases in particle matter concentration and human effects commonly observed in epidemiology studies.
- Other examinations performed:
Bronchoalveolar lavage and biochemical assay of bronchoalveolar lavage fluid
Blood Serum and hematology parameters (red blood cell count, white blood cell count, and percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils.)
Lung tissues for biochemistry (Cytokines and chemokines, glutathione, total antioxidant potential) and histopathology
All numerical data were calculated as the mean and standard deviation. Comparisons between animals exposed to CFA and filtered air were made by Student’s t-test or, where appropriate, by analysis of variance followed by Fisher’s protected least significant difference posttest.
Comparisons were considered significant if a value of p < 0.05 was observed.
Statistical analysis was performed with StatView 5.0.1 (SAS Institute, Inc., Cary, NC).

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 1 400 mg/m³ air
Based on:
other: test. mat. (respirable fraction of ashes)
Exp. duration:
4 h
Remarks on result:
other: Exposure duration: 4 h/day for 3 days. The average concentration of coal fly ash particulate matter less than 2.5 µm was 1400 mg/m3, of which 600 mg/m3 was less than 1 µm.
No mortalities occured
Other findings:
- Histopathology: Focal alveolar septal thickening and increased cellularity in selected alveoli next to terminal bronchioles. Moderate elevation in the number of alveolar macrophages was noted within the bronchiole-alveolar duct regions of the lungs both 18- and 36-h postexposure. Staining with Prussian blue iron stain demonstrated in a small fraction of alveolar macrophages the presence of iron-positive cytoplasmic inclusions, suggestive of phagocytosized CFA particles.

- Other observations:
Significant increase in neutrophils, both in the lung BALF and in the blood.
Significant increases in cytokines such as macrophage inflammatory protein-2 in BALF and IL-1b in lung tissue.
Significant increased levels of transferrin, lung tissue total antioxidant potential, plasma protein, and blood complement 4.

Applicant's summary and conclusion

Interpretation of results:
practically nontoxic
Migrated information related to total dust Criteria used for interpretation of results: EU
Exposure to 1.4 mg/L of respirable coal fly ash particles derived from a plant burning bituminous coal on three consecutive days did not result in mortalities.
Therefore it can be assumed that the LC50 for male rat is greater than 1.4 mg/L of respirable coal fly ash.
The respirable fraction of Ashes (residues), cenospheres does not exceed 1.5% of the total mass (s. Particle size distribution). Accordingly, the LC50 is expected to exceed 5 mg/L of total Ashes (residues), cenospheres. Therefore, Ashes (residues), cenospheres are considered as non-toxic upon acute inhalation exposure and no classification is needed according to the DSD and CLP criteria for classification and labelling.