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Description of key information

For the assessment of irritation / corrosion in vitro methods have been applied. The following results have been obtained:

OECD 439: negative

OECD 437: negative

OECD 492: no prediction possible

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:
Jun 13 - Aug 23, 2018
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)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied undiluted.
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 (Nice, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 18-RHE-086


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:
104.2
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 1.851 (SD 4.2%). 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  1.851 100 
 Positive Control 42

0.024

1.4

 Test Material

42

1.929

104.2

Interpretation of results:
GHS criteria not met
Remarks:
UN GHS: No Caterory (according to OECD TG 439)
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 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.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 16 - Oct 19, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OCD 437
Version / remarks:
09 October 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) No. 1152/2010 amending, for the purpose of its adaption to technical progress, Regulation (EC) No. 440/2008 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
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20% (w/v) suspension in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Preparation

The test item was prepared as a 20% (w/v) solution in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

TEST MATERIAL: 750 µL (i.e. 150mg/750µL)
NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 17361013
Storage: 2 to 8°C
Released until: August 2020

POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S5798623
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: April 30, 2023



Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Value:
7.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.5 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.1). After treatment with the positive control (20% Imidazole) the calculated IVIS was 99.6 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.3 – 132.6). Therefore, the study fulfilled the acceptance criteria.
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
-0.5
0.002
-0.470
0.5
0.9
0.7
0.004
0.760
1.2
0.003
1.245
Positive control
20% Imidazole solution
62.6
2.727
96.680
99.6
4.5
59.2
2.536
97.240
67.0
2.516
104.740
Test item
Test Item
5.3
0.060
6.200
7.5
2.3
8.7
0.103
10.245
6.0
0.013
6.195


Interpretation of results:
other: inconclusive
Conclusions:
Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 08, 2018 - Aug 09, 2018
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 µL per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Art. 8.09711
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:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
1.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability
3. The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.


   Mean OD  Mean Viability
 Negative Control 2.220 100.0% 
 Positive Control 0.673 30.3%
 Test Item 0.041 1.9%
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the provided information there is need for classification for eye irritation class 2 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures. No further classification is warranted.