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EC number: - | 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June 21, 2022 - July 04, 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 2021
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Unknown
- Cas Number:
- Unknown
- IUPAC Name:
- Unknown
- Reference substance name:
- Calcium oxide
- EC Number:
- 215-138-9
- EC Name:
- Calcium oxide
- Cas Number:
- 1305-78-8
- Molecular formula:
- CaO
- IUPAC Name:
- oxocalcium
- Reference substance name:
- Quartz (SiO2)
- EC Number:
- 238-878-4
- EC Name:
- Quartz (SiO2)
- Cas Number:
- 14808-60-7
- Molecular formula:
- SiO2
- IUPAC Name:
- Quartz
- Reference substance name:
- Zinc sulphide
- EC Number:
- 215-251-3
- EC Name:
- Zinc sulphide
- Cas Number:
- 1314-98-3
- Molecular formula:
- SZn
- IUPAC Name:
- thioxozinc
- Reference substance name:
- Silicon dioxide
- EC Number:
- 231-545-4
- EC Name:
- Silicon dioxide
- Cas Number:
- 7631-86-9
- Molecular formula:
- O2Si
- IUPAC Name:
- Silicon dioxide
- Reference substance name:
- Carbon
- EC Number:
- 231-153-3
- EC Name:
- Carbon
- Cas Number:
- 7440-44-0
- Molecular formula:
- C
- IUPAC Name:
- Carbon
- Test material form:
- solid: nanoform
- Remarks:
- Fine black powder
- Details on test material:
- - State of aggregation:
spherical particles clustered together to form loose aggregates in organic media.
- Particle size distribution:
median (d(50)) size of 27.51 nm at the major axis and 23.62 nm at the minor axis.
- Shape of particles:
Spheroid
- Surface area of particles:
136.368 m2 cm-3
- Coating:
none
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Constituent 6
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Sponsor; Lot: 2021/3/17/1
- Purity, including information on contaminants, isomers, etc.:
UVCB
RADIOLABELLING INFORMATION (if applicable)
none
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in a closed container
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: Not specified. origin: 'adult donors'
- Justification for test system used:
- In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- three-dimensional human epidermis model
Source
EPISKIN-SM
batch
0.38 cm2, Batch no.: 22 EKIN 026
TEMPERATURE USED FOR TEST SYSTEM
37 degree C
REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands)
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 1 mg/mL in PBS) diluted (3x) in Assay medium (final concentration 0.3 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES:
triplicate
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze killed
- N. of replicates : triplicate
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
one
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test material is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test material is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50% of the mean viability of the negative controls. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
21.16 to 26.98 mg of the solid test material was added into the 12-well plates on top of the skin tissues
VEHICLE
The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test material to the tissue
NEGATIVE CONTROL
25 µL PBS
POSITIVE CONTROL
25 µL 5% SDS - Duration of treatment / exposure:
- 15 ± 0.5 min
- Duration of post-treatment incubation (if applicable):
- After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 ± 1 hours at 37°C.
- Number of replicates:
- Triplicate
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Single experiment
- Value:
- 104
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Any other information on results incl. tables
Table 1
Mean Absorption in the In Vitro Skin Irritation Test with Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres
| A (OD570) | B (OD570) | C (OD570) | Mean (OD570) |
| SD |
Negative control | 1.001 | 1.050 | 1.066 | 1.039 | ± | 0.034 |
Test material | 1.056 | 1.047 | 1.127 | 1.076 | ± | 0.044 |
Positive control | 0.063 | 0.126 | 0.088 | 0.092 | ± | 0.032 |
OD = optical density
SD = Standard deviation
Triplicate exposures are indicated by A, B and C.
In this table the values are corrected for background absorption (0.044). Isopropanol was used to measure the background absorption.
Table 2
Mean Tissue Viability in the In Vitro Skin Irritation Test with Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres
| Mean tissue viability (percentage of control) | Standard deviation (percentage) |
Negative control | 100 | 3.3 |
Test material | 104 | 4.2 |
Positive control | 8.9 | 3.1 |
Table 3
Individual OD Measurements at 570 nm
| A | B | C |
(OD570) | (OD570) | (OD570) | |
Negative control |
|
|
|
OD570 measurement 1 | 1.0495 | 1.0960 | 1.1197 |
OD570 measurement 2 | 1.0406 | 1.0909 | 1.1005 |
Test material on viable tissue |
|
|
|
OD570 measurement 1 | 1.0971 | 1.0922 | 1.1786 |
OD570 measurement 2 | 1.1022 | 1.0886 | 1.1631 |
Test material on killed tissue |
|
|
|
OD570 measurement 1 | 0.1124 | 0.1221 | 0.0948 |
OD570 measurement 2 | 0.1169 | 0.1233 | 0.0942 |
Non treated killed tissue |
|
|
|
OD570 measurement 1 | 0.3111 | 0.1151 | 0.0877 |
OD570 measurement 2 | 0.3296 | 0.1199 | 0.0890 |
Positive control |
|
|
|
OD570 measurement 1 | 0.1066 | 0.1711 | 0.1325 |
OD570 measurement 2 | 0.1067 | 0.1695 | 0.1309 |
OD = Optical density
Triplicate exposures are indicated by A, B and C.
Table 4
Historical Control Data for In Vitro Skin Irritation Studies
| Negative control (absorption; OD570) | Positive control (absorption; OD570) |
Min | 0.507 | 0.021 |
Max | 1.478 | 0.549 |
Mean | 1.090 | 0.097 |
SD | 0.164 | 0.077 |
n | 177 | 177 |
SD = Standard deviation
n = Number of observations
The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2019 to May 2022.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
- Executive summary:
The objective of this study was to evaluate Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of the test material was tested through topical application for 15 minutes.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch 2021/3/17/1 of the test material was a fine black powder. Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test material was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
The test material did possibly interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test material and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was -6.2% of the negative control tissues. Since the %NSMTT was below 0%, there was no need to correct for the MTT reduction
Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 104%. Since the mean relative tissue viability for the test material was above 50% after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.
The positive control had a mean cell viability of 8.9% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit £1.5). The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.2%, indicating that the test system functioned properly.
In conclusion, Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
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