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Description of key information

OECD 437: serious eye damage (Cat 1)

OECD 439: non-irritatin to skin (No GHS category)

OECD 431: non-corrosive to skin

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 2, 2018 - June 28, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006. B.40.bis. In vitro skin corrosion: human skin model test
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 29, 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol SkinEthic Skin Corrosivity Test, April 2012.
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 18-RHE-060
- Expires: June 11, 2018

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
At the end of the exposure periods, the test item, positive and negative control was removed immediately by gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg ± 3 mg of solid test material
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL ± 3 µl (deionised water )
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL ± 3 µl
- Concentration (if solution): An 8N potassium hydroxide solution dissolved deionised water pure was used as positive control.
Duration of treatment / exposure:
3 min & 1 hour
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1 (3 min)
Value:
100.4
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:
other: Non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1 (1 hour)
Value:
23.5
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:
other: Non-corrosive
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:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.563 to 1.703

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 0.8 and ≤ 3.0 1.694 (3 min)
1.608 (1 hour)

Mean viability positive control < 15% after 1-hour exposure 0.9%

Range between identically treated < 30% 1.4% (3 min)
tissues with test item 17.6% (1 hour)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.596 (3 min) 1.694 (3 min)
≥ 1.467 (1 hour) 1.608 (1 hour)

Mean viability positive control ≤ 0.94% 0.9%

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:

Group Acceptance Criterion Result
Range between identically Negative control < 30% 1.0% (3 min)
treated tissues 5.8% (1 hour)
Positive control < 30% 83.3% (1 hour)
Test substance < 30% 1.4% (3 min)
17.6% (1 hour)

The range between identically treated tissues with the positive control was higher than 30% after 1 hour exposure (83.3%), but the optical densities measured were <0.3.
The study met all acceptance criteria.





 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 3 1.694 100.0 
Negative Control 60

1.608

100.0

 Positive Control

60

0.014

0.9

 Test Material 3 1.701 100.4
 Test Material 60 0.379 23.5
Interpretation of results:
GHS criteria not met
Remarks:
Non-corrosive
Conclusions:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 18, 2018 - July 2, 2018
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:
Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; The test item was applied neat to the tissues.
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: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 18-RHE-068
- Expires: June 25, 2018

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 DPBS using a pipette. Excess DPBS 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 ± 2 mg
NEGATIVE CONTROL: 16 µL (Dulbecco`s 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:
97.8
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:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.594 to 1.794

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.665
Mean viability positive control < 40% 1.4%
SD of group-mean value ≤ 18% 7.1% (positive control)
6.7% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.665
Mean viability positive control ≤ 2.97% 1.4%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 6.5%

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.665 100 
 Positive Control 42

0.022

1.4

 Test Material

42

1.629

97.8

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

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. 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:
March 28, 2018 - May 24, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to 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. certificate)
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:
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: 16455011
Storage: 2 to 8°C
Released until: October 2019


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S6746923
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: August 31, 2018

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:
64
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
According to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1)
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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: -1.5 – 3.1).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 118.9 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 82.4 – 132.9).

Therefore, the study fulfilled the acceptance criteria.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.6 0.001
0.615
0.5
0.2
0.3
0.000
0.300
0.5
-0.001
0.485
Positive control
20% Imidazole solution
83.8
2.301
118.315
118.9
8.8
56.2
3.619
110.485
85.4
2.842
128.030
Test item
Test item
75.7
0.012
75.880
64.0
10.4
54.7
0.106
56.290
59.8
0.010
59.950


Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the conditions of the present study, the test item did show an eye hazard potential. The test item is inducing serious eye damage (UN GHS: Category 1).
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 test item did show an eye hazard potential. The test item is inducing serious eye damage (UN GHS: Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

A Weight of Evidence approach was applied to investigate the potential of the test item to induce skin irritation/corrosion. The corrosive potential of the test item was examined according to OECD Guideline 431.

Following treatment with the test item, the tissue viability was ≥50% after 3 minutes exposure (mean viability: 100.4%) and ≥15% after 1 hour exposure (mean viability: 23.5%),i.e.according to OECD 431 the test item is not considered as corrosive to skin.

To exclude that the test item exhibits an irritating potential to skin, a study according to OECD 439 was performed. Following treatment with the test item, the tissue viability was 97.8% 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).

In conclusion, the test item is not considered to be irritating or corrosive to skin.

Eye irritation/corrosion

The potential of the test item to induce serious eye damage was investigated in the BCOP assay according to OECD Guideline 437.

The IVIS obtained after treatment with the test item was 64.0 and, thus, higher than 55, i.e.according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1).

Justification for classification or non-classification

Based on the provided information there is need for classification for eye irritation cat.1 (H318) according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776. The test item is not considered to be classified for skin irritation/corrosion accordint to EU Regulation (EC) No 1272/2008.