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Administrative data
Description of key information
The skin and eye irritation/corrosion potential for the susbtance was assesed follwoing in vitro guidine methods OECD 439, 431 and 437. Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres was found to be non-corrosive (OECD 431 test) and non-irritant (OECD 439) in the in vitro experimental conditions. Therefore, the susbtance is not classified as skin irritant/corrosive as per the CLP criteria.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- 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
- 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)
- 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 - 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
- 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 - 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.
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 Apr 2022 to 10 Jun 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- 2019
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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 - Test system:
- other: reconstructed human epidermis (RHE)
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: not specified
- Justification for test system used:
- Recommended test system in international guidelines (OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Source
MatTek Corporation, Ashland MA, U.S.A.
batch
EpiDerm Skin Model (EPI-200, Lot no.: 36937 kit L + M; 36983 kit M + G
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 (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES:
duplicate
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze killed
- N. of replicates : duplicate
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
two
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test material is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test material considered non-corrosive (viability 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15%.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%. - Control samples:
- yes, concurrent no treatment
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
25.0 to 48.6 mg of the solid test material was added into the 6-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
50 µL Milli-Q water
POSITIVE CONTROL
50 µL 8N KOH - Duration of treatment / exposure:
- 3 minutes (experiment 1)
1 hour (experiment 1) - Duration of post-treatment incubation (if applicable):
- Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.
- Number of replicates:
- duplicate
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Experiment 2 / run 1 (1 hour) (the first experiment was rejected and a repeat experiment was performed. Results of the first experiment are not reported but are retained within the raw data).
- Value:
- 100
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Experiment 2 / run 1 (3 minute) (the first experiment was rejected and a repeat experiment was performed. Results of the first experiment are not reported but are retained within the raw data).
- Value:
- 106
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: no
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 - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
- 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 corrosion on a human three-dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour.
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 25 µL of Milli-Q water and at least 25 mg of the test material was applied directly on top of the skin tissue.
The positive control had a mean relative tissue viability of 8.9% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit £2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was £ 7.5%, indicating that the test system functioned properly.
Since a color change in presence of MTT could not be determined, the test material was considered as a MTT reducer.
In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test material and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test material was 0.06% and -1.61% of the negative control tissues after 3 minutes and
1 hour respectively. Since %NSMTT was below 0.0%, no correction was MTT interference was applied for the 1-hour exposure. For the 3-minute exposure, the ODs of the test material treated viable tissues was corrected using the OD of the freeze-killed tissues.Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 106% and 100%, respectively. Because the mean relative tissue viability for the test material was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test material is considered to be not corrosive.
In conclusion, Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report
Referenceopen allclose all
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.
Table 1
Mean Absorption in the in vitro Skin Corrosion Test with Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres
| 3-minute application | 1-hour application | ||||||||
A (OD570) | B (OD570) | Mean (OD570) | SD | A (OD570) | B (OD570) | Mean (OD570) | SD | |||
Negative control | 1.800 | 1.819 | 1.809 | ± | 0.013 | 1.823 | 1.784 | 1.803 | ± | 0.027 |
Test material (1) | 1.840 | 1.990 | 1.915 | ± | 0.106 | 1.797 | 1.825 | 1.811 | ± | 0.020 |
Positive control | 0.227 | 0.487 | 0.357 | ± | 0.184 | 0.183 | 0.138 | 0.161 | ± | 0.031 |
Table 2
Mean Tissue Viability in the in vitro Skin Corrosion Test with Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres
| 3-minute application viability (percentage of control) | 1-hour application viability (percentage of control) |
Negative control | 100 | 100 |
Test material | 106 | 100 |
Positive control | 20 | 8.9 |
Table 3
Coefficient of Variation between Tissue Replicates
| 3-minute | 1-hour |
Negative control | 1.0 | 2.1 |
Test material | 7.5 | 1.5 |
Positive control | 53 | 24 |
Table 4
Historical Control Data for In Vitro Skin Corrosion Studies
| Negative control | Positive control | ||
| 3-minute treatment (OD570) | 1-hour treatment (OD570) | 3-minute treatment (OD570) | 1-hour treatment (OD570) |
Min | 1.427 | 0.994 | 0.080 | 0.022 |
Max | 2.264 | 2.321 | 0.444 | 0.319 |
Mean | 1.740 | 1.772 | 0.168 | 0.137 |
SD | 0.171 | 0.180 | 0.073 | 0.048 |
n | 132 | 132 | 132 | 134 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13-20 April 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Version / remarks:
- 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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 - Vehicle:
- unchanged (no vehicle)
- Remarks:
- Since no workable suspension of the test material in physiological saline could be obtained, the test material was used as delivered by the sponsor and added pure on top of the corneas.
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 322.43 to 359.14 mg
VEHICLE
No vehicle - Duration of treatment / exposure:
- 240 +- 10 minutes
- Number of animals or in vitro replicates:
- triplicate
- Details on study design:
- NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: physiological saline
SOLVENT CONTROL USED (if applicable): none
POSITIVE CONTROL USED: 20% (w/v) Imidazole
APPLICATION DOSE AND EXPOSURE TIME: applied directly on the corneas in such a way that the cornea was completely covered (322.43 to 359.14 mg). Corneas were incubated in a horizontal position for 240 10 minutes at 32 +- 1 degrees C.
TREATMENT METHOD: cMEM filled chamber
POST-INCUBATION PERIOD: No
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: thrice
METHODS FOR MEASURED ENDPOINTS:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance by a light meter. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each cornea treated with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 L of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 1
- Value:
- >= 0.2 - <= 3.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- Mean in vitro irritancy score of 1.6 after 240 minutes of treatment.
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, since Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
- Executive summary:
The objective of this study was to evaluate the eye hazard potential of Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).
This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage potential of the test material was tested through topical application for approximately 240 minutes.
The study procedures described in this report were based on the most recent OECD guideline.
Batch 2021/3/17/1 of the test material was a fine black powder. Since no workable suspension in physiological saline could be obtained, the test material was used as delivered and added pure on top of the corneas.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 127 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.6 after 240 minutes of treatment.
In conclusion, since Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Reference
Table 1: Summary of Opacity, Permeability and In Vitro Scores
Treatment | Mean Opacity | Mean Permeability | Mean In vitro Irritation Score 1, 2 |
Negative control | 1.7 | -0.007 | 1.6 |
Positive control | 88 | 2.595 | 127 |
Test material | 1.2 | 0.023 | 1.6 |
1 Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test material.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Table 2: Opacity Score
Treatment | Opacity before treatment | Opacity after treatment | Final Opacity1 | Negative control corrected Final Opacity 2 | Mean Final Opacity |
|
|
|
|
|
|
Negative control | 3.2 | 7.3 | 4.2 |
| 1.7 |
2.3 | 3.1 | 0.8 | |||
2.8 | 3.1 | 0.3 | |||
|
|
|
|
|
|
Positive control | 0.7 | 91 | 91 | 89 | 88 |
1.8 | 83 | 81 | 79 | ||
0.7 | 99 | 98 | 97 | ||
|
|
|
|
|
|
Test material | 0.3 | 2.0 | 1.6 | -0.1 | 1.2 |
1.2 | 5.9 | 4.7 | 3.0 | ||
0.8 | 3.3 | 2.5 | 0.8 |
Calculations are made without rounding off.
1 Final Opacity = Opacity after treatment – Opacity before treatment.
2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control
Table 3: Permeability Score Individual Values (Uncorrected)
Treatment | Dilution factor | OD490 | OD490 | OD490 | Average OD | Final OD | Mean final negative control |
1 | 2 | 3 | |||||
|
|
|
|
|
|
|
|
Negative control | 1 | -0.005 | -0.005 | -0.005 | -0.005 | -0.005 | -0.007 |
1 | -0.010 | -0.009 | -0.008 | -0.009 | -0.009 | ||
1 | -0.007 | -0.006 | -0.006 | -0.006 | -0.006 | ||
|
|
|
|
|
|
|
|
Positive control | 6 | 0.548 | 0.552 | 0.551 | 0.550 | 3.302 |
|
6 | 0.382 | 0.401 | 0.404 | 0.396 | 2.374 |
| |
6 | 0.349 | 0.348 | 0.358 | 0.352 | 2.110 |
| |
|
|
|
|
|
|
|
|
Test material | 1 | 0.021 | 0.018 | 0.019 | 0.019 | 0.019 |
|
1 | 0.015 | 0.018 | 0.015 | 0.016 | 0.016 |
| |
1 | 0.034 | 0.034 | 0.033 | 0.034 | 0.034 |
|
Calculations are made without rounding off.
Table 4: Permeability Score Individual Values (Corrected)
Treatment | Dilution factor | Negative control corrected OD490 11 | Negative control corrected OD490 21 | Negative control corrected OD490 31 | Negative control corrected OD490 Average | Negative control corrected final OD490 | Average OD |
|
|
|
|
|
|
|
|
Positive control | 6 | 0.548 | 0.552 | 0.551 | 0.550 | 3.302 | 2.595 |
6 | 0.382 | 0.401 | 0.404 | 0.396 | 2.374 | ||
6 | 0.349 | 0.348 | 0.358 | 0.352 | 2.110 | ||
|
|
|
|
|
|
|
|
Test material | 1 | 0.021 | 0.018 | 0.019 | 0.019 | 0.019 | 0.023 |
1 | 0.015 | 0.018 | 0.015 | 0.016 | 0.016 | ||
1 | 0.034 | 0.034 | 0.033 | 0.034 | 0.034 |
Calculations are made without rounding off.
1 Since the mean final OD490 value of the negative control is below zero, no correction was made.
Table 5: In Vitro Irritancy Score
Treatment | Final Opacity2 | Final OD490 | In vitro Irritancy Score 1 |
|
|
|
|
Negative control | 4.2 | -0.005 | 4.1 |
0.8 | -0.009 | 0.6 | |
0.3 | -0.006 | 0.2 | |
|
|
|
|
Positive control | 89 | 3.302 | 138 |
79 | 2.374 | 115 | |
97 | 2.110 | 128 | |
|
|
|
|
Test material | -0.1 | 0.019 | 0.2 |
3.0 | 0.016 | 3.2 | |
0.8 | 0.034 | 1.3 |
1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).
2 Positive control and test material are corrected for the negative control.
Table 6: Historical Control Data for the BCOP Studies
| Negative control | Positive control | ||
| Opacity | Permeability | In vitro Irritancy Score | In vitro Irritancy Score |
Min | -2.42 | -0.009 | -2.34 | 69 |
Max | 5.80 | 0.202 | 5.90 | 280 |
Mean | 1.00 | 0.012 | 1.18 | 147 |
SD | 1.60 | 0.019 | 1.61 | 32 |
n | 193 | 193 | 193 | 200 |
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 February 2019 to February 2022.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Additional information
Justification for classification or non-classification
Amorphous carbon and silicon dioxide recovered from two-stage pyrolysis of spent tyres was found to be non-corrosive (OECD 431 test) and non-irritant (OECD 439) in the in vitro experimental conditions. Therefore, the susbtance is not classified as skin irritant/corrosive as per the CLP criteria.
Similarly, based on the the results of OECD 437 test, the substance is not classified for eye corrosion/irritation.
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