Registration Dossier

Administrative data

Description of key information

Skin irritation in vitro (OECD 439): not irritating, reference 7.3.1 -1

Eye irritation in vitro (OECD 405): not irritating, reference 7.3.2 -1

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 6, 2011 - October 25, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
standard model
Vehicle:
unchanged (no vehicle)
Remarks:
No vehicle used in this study; The test item was applied neat to the tissues.
Details on test system:
CELL CULTURE
- Supplier: SkinEthic Laboratories (Nice, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 11 022A 0809


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL PBS using a pipette. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1
Value:
100.34
Vehicle controls validity:
not applicable
Remarks:
The test item was applied neat to the tissues
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: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
After treatment with the negative control the absorbance values reached a mean OD of 2.11 (SD 5.96 %). Therefore, the negative control fulfilled the validity criteria.
Treatment with the positive control revealed a mean relative viability of 1.36 % (SD 4.03 %), thus the positive control reached the validity criteria.



 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  2.111 100 
 Positive Control 42

0.029

1.36

 Test Material

42

2.118

100.34

Interpretation of results:
GHS criteria not met
Conclusions:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the experimental conditions reported, the test material is not irritating to the skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test and in accordance with OECD TG 439. The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential. Triplicates of the human skin model RHE (Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute).
16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue.
Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) ≥ 1.2 and ≤ 2.5 for the treatment interval thus showing the quality of the tissues.

The tissue viability after treatment with the test material was higher than 50 % (mean viability: 100.34 %). Therefore, the test material is not considered to possess an irritant potential to the skin. In conclusion, under the experimental conditions reported, the test material is not irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
For this endpoint information from structural similar compounds is available. The studies for these similar compounds were performed according to GLP and the methods applied are fully compliant with OECD TG 405. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
No signs of irritation were observed at the cornea or iris after instillation of the test item. Two animals showed redness of the conjunctivae (score 1) 1 hour after treatment, that recovered completely 6 hours after administration. No abnormalities were detected in the untreated right eyes.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation, in vitro

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test and in accordance with OECD TG 439. The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential. Triplicates of the human skin model RHE (Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the treatment interval thus ensuring the validity of the test system. After treatment with the negative control the absorbance values reached the required acceptability criterion of a mean optical density (OD) ≥ 1.2 and ≤ 2.5 for the treatment interval thus showing the quality of the tissues. The tissue viability after treatment with the test material was higher than 50 % (mean viability: 100.34 %). Therefore, the test material is not considered to possess an irritant potential to the skin. In conclusion, under the experimental conditions reported, the test material is not irritating to the skin.

Eye irritation, in vivo, read-across

There are no experimental data available on eye irritation with the registered substance. However, data with a structural analogue substance are available which are considered suitable and sufficient to conclude on this endpoint. For detailed read-across justification please refer to IUCLID section 13.

The objective of the present study was to investigate the eye irritating potential of the test item in rabbits. For this purpose, the test item was applied in a single dose to the left eye of rabbits, the untreated right eye served as control. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris at specific intervals. The first examinations of the eyes followed 1 and 6 hours after treatment. Subsequently, the eyes were investigated twice daily for a further 4 days. In addition, potential systemic effects and the body weight development was determined. The study was performed initially with one animal, followed by the confirmatory test with two further animals. No mortality occurred and no clinical symptoms were seen during the experimental phase of the study. The body weight development of the rabbits was inconspicuous. No signs of irritation were observed at the cornea or iris after instillation of the test item. Two animals showed redness of the conjunctivae (score 1) 1 hour after treatment, that recovered completely 6 hours after administration. No abnormalities were detected in the untreated eyes. In conclusion, under the conditions of the present study, no eye irritating potential was detected for the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance does not require classification for eye or skin irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.