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Diss Factsheets

Administrative data

Description of key information

Under the experimental conditions reported, the test material is not irritating to the skin.


Under the conditions of the present study, the test item did not show an eye hazard potential.

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:
Dec 05, 2018 - Feb 25, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
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 (Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 19-RHE-021

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:
111.7
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 1.778 (SD 1.1 %). Therefore, the negative control fulfilled the validity criteria.
Treatment with the positive control revealed a mean relative viability of 1.4 % (SD 7.1 %), thus the positive control reached the validity criteria.

 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.778 100 
 Positive Control 42

0.025

1.4

 Test Material

42

1.986

111.7
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions reported, the test material is not irritating to the skin.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.


The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.
Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.


All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met.
Following treatment with the test item, the tissue viability was 111.7 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).


Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

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:
Dec 05, 2018 - Feb 25, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
October 9, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,
Version / remarks:
September 14, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation
Version / remarks:
June 29, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue
Sterile deionized water was used as negative control.

POSITIVE CONTROL: 50 µL per tissue
Synonym: Methyl acetate
Supplier: Merck KGaA
CAS No.: 79-20-9
Batch No.: S7451611
Appearance: Liquid
Content (GC): 99.6 % (a/a)
Expiry Date: June 30, 2022
Storage conditions: Tightly closed, dark, at room temperature (15 – 25 °C)
Duration of treatment / exposure:
30 ± 2 minutes
Number of animals or in vitro replicates:
in vitro: duplicate design
Irritation parameter:
mean percent tissue viability 
Run / experiment:
Run 1 / Experiment 1
Value:
115.4
Vehicle controls validity:
valid
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 observations

After treatment with the negative control (sterile deionized water) the mean OD was 2.014 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met.

 

























  Mean OD Mean Viability
 Negative Control2.014100.0 % 
 Positive Control0.59929.7 %
 Test Item2.323115.4 %
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The study was conducted according to OECD 492.


The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.


After treatment with the negative control (sterile deionized water) the mean OD was 2.014 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met.


Following treatment with the test item the test material, the tissue viability was 115.4 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).


Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation


This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.


The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.
Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5 % aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.


All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5 % aqueous solution of sodium dodecyl sulfate) were met.
Following treatment with the test item, the tissue viability was 111.7 % and, thus, higher than 50 %, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).


Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).


 


Eye irritation


The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The study was conducted according to OECD 492.


The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.


After treatment with the negative control (sterile deionized water) the mean OD was 2.014 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.7 % (study acceptance criterion: <50 %). Thus, the acceptance criteria were met.


Following treatment with the test item the test material, the tissue viability was 115.4 % and, thus, higher than 60 %, i.e. according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).


Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.


Based on available data on skin irritation/corrosion, the test item does not require classification for causing skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP).


Based on available data on eye irritation/corrosion, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).