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Administrative data

Description of key information

Oral: LD50 (rat) > 2000 mg/kg bw
Inhalation: LD50 (rat) > 1400 mg/m³ (respirable fraction), > 5000 mg/m³ (total ashes)
Dermal: LD50 (rat) > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jan 2008 - 22 Jan 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeding farm BioTest s.r.o., Konárovice, 281 25, CZ, RČH CZ 21760152
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 158-170 g (see table No. 1)
- Fasting period before study: About 20 hours prior to oral administration the animals were not fed, water was given ad libitum
- Housing: 3 animals of one sex in one plastic breeding cage Velaz T4
- Diet (e.g. ad libitum): ST 1 BERGMAN, standard pelleted diet ad libitum (producer: Mill Kocanda, Jesenice u Prahy)
- Water (e.g. ad libitum): Drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law)
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%):30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 07 Jan 2008 To: 22 Jan 2008
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose in water
Details on oral exposure:
VEHICLE
- 0.5% methyl cellulose in water
- Justification for choice of vehicle: It is not toxic. Is it possible to prepare a homogeneous suspension of the test substance in this vehicle.
- Lot/batch no.: DT157078

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg bw

DOSAGE PREPARATION:
Immediately before administration, the test substance was weighed, mixed in vehicle (0.5% methyl cellulose in water) and the resulting suspension was administered by gavage. The single volume of administered suspension was 1mL/100 g body weight.

CLASS METHOD
- Rationale for the selection of the starting dose: according to the methology
Testing schedule (according to EU Method B.1 tris Annex 1D):
Step No. 1: 3 females dosed 2000 mg/kg. Result: no deaths. Therefore, Step No. 2: 3 females dosed 2000 mg/kg. Result: no deaths. Thus, no further testing necessary.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 females (Step No.1: 3 females, Step No.2: 3 females)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: After administration, the animals were observed individually. Twice on the first day (30 minutes and 3 hours after application), twice on the second day (in the morning and in the afternoon), and daily thereafter up to the end of the study. Observations included changes in skin and fur, eyes, visible mucous membranes, behaviour, somatomotor activity, reactions to stimuli, presence of lacrimation, salivation and discharge from nostrils, function of respiratory, digestive and urogenital system.
Animals were weighed before administration, on day 8 of the study and on day 15 prior to sacrifice. Body weight gain was calculated from body weight at the start and at the end of the study.
- Necropsy of survivors performed: yes
- Other examinations performed: All test animals survived. Animals were sacrificed on day 15 by injection of a veterinary preparation T61 (1 mL iv.) and gross necropsy was carried out. Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortalities occurred
Clinical signs:
No clinical sings of intoxication were observed.
Body weight:
No effects on body weight (gain) observed.
Gross pathology:
No macroscopic changes were observed.
Interpretation of results:
other: The test substance does not fufil the requirements to be classified according to CLP (EU-GHS) criteria.
Conclusions:
The test substance administered orally at 2000 mg/kg bw caused no death of animals. No clinical signs of intoxication were observed. No pathologic macroscopic changes were diagnosed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

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Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles. Fly-ash from bituminous coal.
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:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- 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.

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
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:
yes
Duration of exposure:
4 h
Remarks on duration:
4 h/day for 3 days
Concentrations:
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:
yes
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
Statistics:
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).
Sex:
male
Dose descriptor:
LC50
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.
Mortality:
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.

Interpretation of results:
other: The test substance does not fufil the requirements to be classified according to CLP (EU-GHS) criteria.
Conclusions:
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) does not exceed 20% of the total mass (s. Particle size distribution). Accordingly, the LC50 is expected to exceed 5 mg/L of total Ashes (residues). Therefore, Ashes (residues) is considered as non-toxic upon acute inhalation exposure and no classification is needed according to the CLP (EU-GHS) criteria for classification and labelling.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jan 2008 - 29 Jan 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
adopted 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted February 24, 1987
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeding farm BioTest s.r.o., Konárovice, 281 25 CZ, RČH CZ 21760152
- Weight at study initiation: 264-287 g (males), 207-242 g (females)
- Housing: single
- Diet (e.g. ad libitum): ST 1 BERGMAN complete pelleted diet ad libitum, (producer: Mill Kocanda, Jesenice u Prahy) ad libitum
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14 Jan 2008 To: 29 Jan 2008
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: 6x6 cm
- % coverage: 10
- Type of wrap if used: The application site was covered by mull, plastic foil and held in contact by plaster (strapping).


REMOVAL OF TEST SUBSTANCE
- Washing (if done): After 24 hours the occlusive dressing and remains of the test substance were removed with water.
- Time after start of exposure: 24 h


TEST MATERIAL
- Amount(s) applied: 2000 mg/kg bw
- For solids, paste formed: yes. The test substance was moistened with a small amount of water and applied onto the depilated area of the skin.
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were weighed on day 1 at the start of the study (prior to application), on day 8 and on day 15 at the end of the study. After application, animals were observed individually: twice on day 1 (30 min and 3 h post-application), twice on day 2 (in the morning and in the afternoon) and daily thereafter up to day 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross necropsy
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
No mortalities occurred.
Clinical signs:
No clinical signs of toxicity were observed.
Body weight:
In two females, a decrease of weight was observed on the day 8 of the study. This was not considered treatment-related as no clinical signs of toxicity and no pathological changes were observed.
Gross pathology:
No macroscopic changes were observed.
Interpretation of results:
other: The test substance does not fufil the requirements to be classified according to CLP (EU-GHS) criteria.
Conclusions:
The test substance applied to the rat skin at 2000 mg/kg bw caused no death of animals. No clinical signs of intoxication were observed. No pathologic macroscopic changes were diagnosed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Oral

The acute toxicity of Ashes (residues) after oral administration to Wistar rats was investigated according to EU Method B.1 tris under GLP conditions. Two groups of 3 female rats each were given the test material by gavage at 2000 mg/kg bw as a suspension in 0.5% methyl cellulose in water. No mortalities occurred and no clinical or pathological signs of toxicity were noted during the observation period of 14 days. The oral LD50 value for female rats was therefore considered to be greater than 2000 mg/kg bw (ČEZ Energetické produkty, 2008, key).

Oral LD50 values greater than 2000 mg/kg bw have also been reported in studies carried out in mice using bottom ash derived from coal combustion (Iberdrola S.A., 2009), as well as in rats given coal fly ash derived from a burning plant using different black coals mixed with food (Institut für Umwelthygiene und Umweltmedizin, 1995).

 

Inhalation

Male Sprague-Dawley rats were exposed to 1400 mg/m³ coal fly ash derived from a 400- MW power plant burning bituminous coal from multiple mines in the Wasatch Plateau, UT, for 4 h on three consecutive 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. The observation period was 18 and 36 hours. Bronchoalveolar lavage, lung tissue and blood samples were analysed. No mortalities occured. Histopathologic changes in the lung (focal alveolar septal thickening, increased cellularity, elevation in the number of alveolar macrophages with iron-positive cytoplasmic inclusions) and significant increase in neutrophils, both in the lung BALF and in the blood were noted (Smith, 2006). Based on these results, the LC50 value for male rats was assumed to be greater than 1400 mg/m³ of respirable coal fly ash particles.

The respirable fraction of Ashes (residues) typically accounts for approximately 10% of the total mass and does not exceed 20% (s. Particle size distribution). Accordingly, the LC50 of total Ashes (residues) is expected to exceed 5000 mg/m³ (5 mg/L).

 

Dermal

The acute dermal toxicity of ashes (residues) was tested in accordance with EU Method B.3 and in compliance with GLP. The study was performed as a limit test in two groups of Wistar rats (5 males and 5 females) at a dose of 2000 mg/kg. The test substance was applied unchanged on the shaved skin of the test animals (moistened with a small amount of water) for 24 hours under occlusive conditions. The test animals were observed for 14 days after application, and sacrificed thereafter for gross pathological examinations. No mortalities occurred and no clinical signs of toxicity were observed during the study in all animals. Macroscopic changes were not diagnosed during pathological examination in all animals.

According to the results of this study, the dermal LD50 value of ashes (residues) for rats was greater than 2000 mg/kgbw (ČEZ Energetické produkty, 2008, key).

In another study, 5 male and 5 female New Zealand White rabbits received single topical applications of 2000 mg/kg of the test substance according to OECD guideline 402. The animals were observed for 14 days after administration. No mortalities occured and no clinical signs of toxicity were observed (Iberdrola S.A., 2009). Under the conditions of this study the acute dermal LD50 value for rabbits was found to be greater than 2000 mg/kg bw.

 

Other routes

The respiratory tract response to two types of dust, fly ash “Maasvlakte” and lytag powder, was investigated in rats in compliance with GLP and following a non-guideline protocol (Kema, 1993). Groups of 16 male Wistar rats per dose level were administered one of the test materials suspended in saline via intratracheal instillation at 0, 2, 10 or 50 mg/kg bw. In addition, groups of 16 male Wistar rats per dose level were instilled with silicon dioxide (alpha-quartz, positive control) and titanium dioxide (negative control) at 10 and 50 mg/kg, respectively. On days 7, 14 and 28 post-instillation, 4 animals of each dose group were sacrificed for bronchoalveolar lavage analysis. The lavage fluids were analysed for selected biochemical and cellular markers of toxicity. After the last lavage procedure, the remaining animals of each dose group were sacrificed for histopathological examination of the lower respiratory tract.

In general, condition, behaviour and survival were not adversely affected. Compared with saline controls, animals given 50 mg/kg bw and the 10 mg/kg bw lytag powder group showed significantly decreased body weight gains during the first week, mainly resulting in decreased body weights lasting for 14 days (lytag powder-treated groups) and 28 days (fly ash “Maasvlakte”-treated group).

Effects on cellular and biochemical markers for toxicity such as increased total cell count , decreased relative number of monocytes/macrophages, increased relative numbers of granulocytes and lymphocytes, high protein content and increased beta-glucuronidase and LDH activities were mostly seen in rats treated with 50 mg/kg bw fly ash “Maasvlakte” or lytag powder. To a lesser extent, effects were also noted at 10 mg/kg bw. Doses of 2 mg/kg bw showed no significant changes at all.

In a similar way, histopathological changes were mainly observed in rats given 50 mg/kg bw of either substance. These changes included: spotted, discoloured, swollen and/or nodular lungs and discoloured and/or enlarged mediastinal lymph nodes; multifocal accumulation of particluate material in the alveoli; perivascular lymphoid aggregates and increased septal cellularity (also seen at 10 mg/kg bw); and development of multiple granulomas in alveolar tissue. Treatment with 2 mg/kg bw fly ash “Maasvlakte” or lytag powder showed an insignificant number of rats with very slight pulmonary changes or no changes at all, respectively. In the mediastinal lymph nodes, accumulation of material was observed in almost all animals treated with 50 mg/kg bw fly ash “Maasvlakte” or lytag powder; lymph nodes showed an activated appearance.

Justification for classification or non-classification

The available data on both the acute oral, dermal and inhalation toxicity of ashes (residues) is conclusive but not sufficient for classification.