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EC number: 931-597-4 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was carried out between 2 and 4 August 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant guideline study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- The product from the burning of a combination of carbonaceous materials.
- EC Number:
- 931-597-4
- Molecular formula:
- UVCB substance, not available. View remarks field.
- IUPAC Name:
- The product from the burning of a combination of carbonaceous materials.
- Details on test material:
- - Name of test material (as cited in study report): Aqueous extract of Mixed ash
- Composition range of test material (% (w/w)): Aluminium (Al) 13.07, Calcium (Ca) 45.52 , Iron (Fe) 4.15 , Magnesium (Mg) 3.49, Phosphorus (P) 0.79 , Potassium (K) 2.56, Silicon (Si) 28.07, Sodium (Na) 1.08, Sulphur (S) 1.27.
- The critical minor components examined (mg/kg d.w.): Arsenic (As) 18, Barium (Ba) 670 Cadmium (Cd) 3.6, Copper (Cu) 410, Lead (Pb) 180 and Antimony (Sb) 22.
- Purity test date: the substance is UVCB substance
- Lot/batch No.: Mixed ash 1_01032010
- Expiration date of the lot/batch: end of March 2011
- Storage condition of test material: room temperature
- Other: Mixed Ash is named in dossier as Ash. Mixed in the substance name has meant that there has been several (mixed) fuels when producing ash.
- Extraction of the test item: The test item is extracted in culture medium (toxicity assay) or in distilled water (genotoxicity assay) for one night under stirring at +37°C. After centrifugation, the supernatant was filtered and used for dilutions and/or treatments.
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: Human peripheral blood lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: culture medium RPMI 1640
- Collection of lymphocytes: Human lymphocytes were taken from two healthy non-smoker donors, male and female, receiving no medication and who have not suffered any recent viral infection. The blood was drawn onto lithium-heparin in a sterile Venoject tube. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor1254-induced S9 from rat livers (S9-mix)
- Test concentrations with justification for top dose:
- PRELIMINARY CYTOTOXICITY ASSAY
Initial concentrations of Mixed ash expressed as μg/mL:
Without S9-mix : 5000 – 2500 – 1250 – 625 – 312.5 – 156.25 – 78.13 (short and continuous treatments)
With S9-mix : 5000 – 2500 – 1250 – 625 – 312.5 – 156.25 – 78.13 (with either 5 or 10% S9-mix)
GENOTOXICITY ASSAY
Initial concentrations of Mixed ash expressed as μg/mL:
Without S9 mix :
5000 – 2500 – 1250 (assay 1: 4-hour treatment)
5000 – 2500 – 1250 (assay 2: 20-hour treatment)
5000 – 2500 – 1250 (assay 2: 44-hour treatment)
With S9 mix :
5000 – 2500 – 1250 (assay 1: with 5% S9-mix)
5000 – 2500 – 1250 (assay 2: with 10% S9-mix)
METAL CONCENTRATIONS OF THE TEST SOLUTION
The test media (aqueous extract of Ash) contained 548 µg/l of aluminium, 0.89 µg/l of antimony, 0.14 µg/l of arsenic, 4202 µg/l of barium, 0.02 µg/l of cadmium, 2.93 µg/l of copper and 68.7 µg/l of lead. The metal concentrations in the solvent control were 5.45, 0.01, <0.01, 0.23, 0.01, 0.12 and 0.01 µg/l for Al, Sb, As, Ba, Cd, Cu and Pb, respectively. - Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
- RPMI 1640 (Biochrom, batch 1173T), for toxicity assay
- distilled water (Fresenius, batch 13DBP231), for the main genotoxicity assay
Controlsopen allclose all
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without S9-mix; 4 and 20-hour treatments
0.25 µg/mL
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9-mix
10 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
Culture medium
To 100 ml of RPMI 1640 culture medium are added 25 ml of inactivated fetal calf serum, 1ml of 30 mg/ml L-glutamine solution, 1 ml of antibiotic solution (penicillin 50000 IU/ml, streptomycin 25 mg/ml), 0.4 ml of 25000 IU/ml heparin and 2.4 ml of phytohaemagglutinin A solution in water (Wellcome).
- In-life: 03/05/2010 - 13/08/2010
DURATION
- Preincubation period: 48 h
- Exposure duration: Without S9-mix: 4 h + 20 h recovery period (short treatment), 20 and 44 h without recovery period (continuous treatments); With S9-mix: 4 h + 20 h recovery period, with 5% or 10 % S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): in short treatments the cells were harvested 20 h after the beginning of the treatment; in continuous treatments the cells were harvested 20 h and 44 h after the beginning of the treatment.
STAIN (for cytogenetic assays): stained with a 4% dilution of Giemsa reagent in water for 10 minutes.
NUMBER OF REPLICATIONS: 2 assays, 2 replicates per concentration
NUMBER OF CELLS EVALUATED: 100 cells/culture for negative and concentration groups and 50 cells/culture for positive control
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER: Sterility check was valid. - Evaluation criteria:
- ACCEPTANCE CRITERIA FOR THE RESULTS
A study is accepted if:
- In the solvent control group, the mean number of cells carrying structural aberrations (excluding cells presenting gaps only) for the two subjects does not exceed 5%,
- In the positive control group (mitomycin C and cyclophosphamide), the number of breaks per cell and number of cells with aberrations (excluding those with gaps only) is significantly increased. The values observed must be close to those of historical data of reference substances.
- At least 2 of the cultures treated with the test item present a mean mitotic index for the 2 subjects greater than about 50% compared to the reference culture.
INTERPRETATION OF THE RESULTS
A test item is found to demonstrate clastogenicity against cultured human lymphocytes if it results in a statistically significant increase in the number of breaks per cell compared with the control or in the number of cells with aberrations (excluding gaps), if this increase amounts to at least a doubling of the control value and if the genotoxicity detected shows a dose-effect relationship.
A test item is found to have no clastogenic effect on cultured human lymphocytes if it does not comply with any of the 3 criteria listed above. - Statistics:
- Gaps and breaks are analyzed using Student's t-test. The test can be used for large number of samples although the
standard deviations found do not possess normal distribution.
The total number of damaged cells with and without gaps is analyzed statistically using the χ2 test.
All numerical aberrations (aneuploidy and polyploidy) are analyzed statistically using the χ2 test.
Results and discussion
Test results
- Species / strain:
- lymphocytes: Human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was comprised in the acceptable range of 6-8.5 at the highest concentrations tested from 5000 to 1250 μg/mL.
- Effects of osmolality: The initial extract and successive dilutions induced no variation in osmolarity higher than 50 mOsmol/kg when compared to the solvent control.
RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity assay
COMPARISON WITH HISTORICAL CONTROL DATA: Yes
ADDITIONAL INFORMATION ON CYTOTOXICITY: None - Remarks on result:
- other: strain/cell type: Human peripheral blood lymphocytes
Any other information on results incl. tables
Table 1 Osmolarity and pH in the 4 -hour treatment
Compound |
Initial concentration inμg/mL |
pH |
Osmolality (mOsmol/kg) |
Osmolality variation (mOsmol/kg) Compared to solvent control |
Culture medium (RPMI) |
0 |
7.41 |
280 |
- |
Aqueous extract of Mixed ash |
5000 |
8.46 |
277 |
-3 |
2500 |
8.04 |
279 |
-1 |
|
1250 |
7.75 |
279 |
-1 |
Table 2 The pH at the end of the 20 -hour treatment
Compound |
Initial concentration inμg/mL |
pH |
Culture medium (RPMI) |
0 |
7.14 |
Aqueous extract of Mixed ash |
1250 |
7.21 |
2500 |
7.24 |
|
5000 |
7.31 |
Table 3 Summary of the preliminary toxicity assays with or without metabolic activation
COMPOUND |
Assay S9- / 4h |
Assay S9+ / 5% S9-mix |
||
Initial conc. inμg/mL |
% Mitotic index relative to controla |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
|
Solvent control |
0 |
- |
0 |
- |
Aqueous extract of Mixed ash |
5000 |
88.7 |
5000 |
68.0 |
2500 |
76.8 |
2500 |
77.8 |
|
1250 |
69.5 |
1250 |
94.1 |
|
625 |
84.2 |
625 |
100.0 |
|
312.50 |
92.7 |
312.50 |
103.9 |
|
156.25 |
47.5 |
156.25 |
67.3 |
|
78.13 |
59.9 |
78.13 |
62.7 |
COMPOUND |
Assay S9- / 20h |
Assay S9- / 44h |
Assay S9+ / 10% S9-mix |
|||
Initial conc. inμg/mL |
% Mitotic index relative to controla |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
|
Solvent control |
0 |
- |
0 |
- |
0 |
- |
Aqueous extract of Mixed ash |
5000 |
80.8 |
5000 |
158.3 |
5000 |
70.4 |
2500 |
125.6 |
2500 |
175.7 |
2500 |
57.1 |
|
1250 |
89.7 |
1250 |
201.9 |
1250 |
81.5 |
|
625 |
82.7 |
625 |
119.4 |
625 |
83.6 |
|
312.50 |
117.3 |
312.50 |
179.6 |
312.50 |
82.0 |
|
156.25 |
58.3 |
156.25 |
72.8 |
156.25 |
45.5 |
|
78.13 |
75.0 |
78.13 |
51.5 |
78.13 |
49.7 |
a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 1000 cells counted
Table 4 Summary of metaphase analysis assays without metabolic activation
ASSAY 1 SHORT TREATMENT: 4 HOURS |
ASSAY 2 CONTINUOUS TREATMENT: 20 HOURS |
ASSAY 2 CONTINUOUS TREATMENT: 44 HOURS |
||||||||||||||||
COMPOUND |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
NUMERICAL ABERRATIONS /200 CELLSb |
STRUCTURAL ABERRATIONS /200 CELLSb |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
NUMERICAL ABERRATIONS /200 CELLSb |
STRUCTURAL ABERRATIONS /200 CELLSb |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
NUMERICAL ABERRATIONS /200 CELLSb |
STRUCTURAL ABERRATIONS /200 CELLSb |
||||||
Polyploidy |
Break per cell m +/- sd |
Abnormal cells excluding gaps only |
Abnormal cells including gaps only |
Polyploidy |
Break per cell m +/- sd |
Abnormal cells excluding gaps only |
Abnormal cells including gaps only |
Polyploidy |
Break per cell m +/- sd |
Abnormal cells excluding gaps only |
Abnormal cells including gaps only |
|||||||
Solvent control |
0 |
- |
0 |
0.010 ±0.100 |
2 |
4 |
0 |
- |
0 |
0.010±0.100 |
2 |
3 |
0 |
- |
0 |
0.010 ±0.100 |
2 |
2 |
Mitomycin C |
0.25 |
103.7 |
0 * |
0.490 ±0.643 <0.001 |
45 <0.001 |
41 <0.001 |
0.25 |
49.1 |
0 * |
0.620 ±0.814 <0.001 |
46 <0.001 |
47 <0.001 |
|
|
|
|
|
|
Aqueous extract of Mixed ash |
5000 |
108.8 |
0 * |
0.015 ±0.122 N.S. |
3 N.S. |
3 N.S. |
5000 |
124.3 |
0 * |
0.020 ±0.140 N.S. |
4 N.S. |
5 N.S. |
5000 |
109.1 |
0 * |
0.010 ±0.100 N.S. |
2 N.S. |
3 N.S. |
2500 |
98.6 |
0 * |
0.010 ±0.100 N.S. |
2 N.S. |
2 N.S. |
2500 |
134.9 |
0 * |
0.005 ±0.071 N.S. |
1 N.S. |
1 N.S. |
2500 |
112.5 |
0 * |
0.025 ±0.157 N.S. |
5 N.S. |
6 N.S. |
|
1250 |
115.3 |
0 * |
0.025 ±0.186 N.S. |
4 N.S. |
6 N.S. |
1250 |
110.1 |
0 * |
0.020 ±0.172 N.S. |
3 N.S. |
3 N.S. |
1250 |
92.0 |
0 * |
0.010 ±0.100 N.S. |
2 N.S. |
4 N.S. |
m : mean
sd : standard deviation
Student's t-test for breaks per cell
Chi2 test for numerical aberrations, and for structural aberrations (cells with aberrations including or excluding cells with gaps only)
N.S.=not statistically significant at the threshold of p<0.05
a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 2000 cells counted
b : 100 cells for positive control
*: No statistical analysis could be performed
Table 5 Summary of metaphase analysis assays with metabolic activation
|
ASSAY 1 SHORT TREATMENT: 4 HOURS (5% S9-mix) |
ASSAY 2 SHORT TREATMENT: 4 HOURS (10% S9-mix) |
||||||||||
COMPOUND |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
NUMERICAL ABERRATIONS /200 CELLSb |
STRUCTURAL ABERRATIONS /200 CELLSb |
Initial conc. inμg/mL |
% Mitotic index relative to controla |
NUMERICAL ABERRATIONS /200 CELLSb |
STRUCTURAL ABERRATIONS /200 CELLSb |
||||
Polyploidy |
Break per cell m +/- sd |
Abnormal cells excluding gaps only |
Abnormal cells including gaps only |
Polyploidy |
Break per cell m +/- sd |
Abnormal cells excluding gaps only |
Abnormal cells including gaps only |
|||||
Solvent control |
0 |
- |
0 |
0.020 ±0.172 |
3 |
5 |
0 |
- |
0 |
0.005 ±0.071 |
1 |
2 |
Cyclophosphamide |
10 |
45.5 |
0 * |
0.630 ±0.826 <0.001 |
44 <0.001 |
46 <0.001 |
10 |
45.7 |
0 * |
0.700 ±0.759 <0.001 |
54 <0.001 |
54 <0.001 |
Aqueous extract of Mixed ash |
5000 |
97.0 |
0 * |
0.015 ±0.122 N.S. |
3 N.S. |
4 N.S. |
5000 |
111.4 |
1 N.S. |
0.010 ±0.100 N.S. |
2 N.S. |
3 N.S. |
2500 |
105.9 |
0 * |
0.000 ±0.000 N.S. |
0 N.S. |
0 N.S. |
2500 |
100.0 |
0 * |
0.005 ±0.071 N.S. |
1 N.S. |
2 N.S. |
|
1250 |
96.0 |
0 * |
0.010 ±0.141 N.S. |
1 N.S. |
2 N.S. |
1250 |
106.6 |
1 N.S. |
0.005 ±0.071 N.S. |
1 N.S. |
2 N.S. |
m : mean
sd : standard deviation
Student's t-test for breaks per cell
Chi2 test for numerical aberrations, and for structural aberrations (cells with aberrations including or excluding cells with gaps only)
N.S.=not statistically significant at the threshold of p<0.05
a : expressed as percentage of cells undergoing mitosis compared to solvent control, and based on 2000 cells counted
b : 100 cells for positive control
*: No statistical analysis could be performed
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative with metabolic activation
negative without metabolic activation
Not clastogenic in human lymphocytes with or without S9 metabolic activation. - Executive summary:
Clastogenic potential of Ash was assessed in a GLP compliant guideline in vitro study in human lymphocytes in vitro (OECD Guideline 473 and EU Method B.10).
Ash induced neither statistically nor biologically significant increase in the number of breaks per cell or in the frequency of aberrant cells excluding or including gaps in the treatments with or without metabolic activation. Furthermore, no statistically significant increase in the number of cells with numerical aberrations was noted in any treatment. Under experimental conditions, Ash induced no clastogenic activity in human lymphocytes either in presence or in absence of metabolic activation, in the in vitro human lymphocyte metaphase analysis test.
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