[No public or meaningful name is available]

Endpoint summary

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

Reference

Reference 1

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

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.

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-SM^TM)). 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.

Reference 2

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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 Opacity^1	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 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	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 Opacity^2	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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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.