[No public or meaningful name is available]

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:

2022

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

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

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-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.