[No public or meaningful name is available]

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Unknown

Cas Number:

Unknown

IUPAC Name:

Unknown

Constituent 2

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

Constituent 3

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

Constituent 4

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

Constituent 5

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

Constituent 6

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

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

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)

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.