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Description of key information

Skin irritation in vitro

OECD 439: UN GHS Cat 2 or Cat 1

OECD 431: not corrosive

According to the result of the OECD Guideline study 439, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). To decide whether the test item is irritating or corrosive to skin, a further in vitro study according to OECD 431 was performed. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin. Based on this weight of evidence approach, the test item is considered to be irritating to skin.

Eye irritation in vitro

OECD 492: GHS 1 or GHS 2

OECD 437: no prediction can be made

A weight of evidence approach was conducted to determine the eye irritation/corrosive potential of the test item.

Based on the result of the OECD Guideline study 492, the test item is considered to be UN GHS category 1 (cause serious eye damage) or category 2 (eye irritation). Since this study cannot resolve between UN GHS 2 or serious damage UN GHS 1, a further study was performed.

A study according to OECD 437 was performed to identify whether the test item induced serious eye damage (UN GHS 1) or not requiring classification for eye irritation or serious eye damage (No UN GHS category). The test item is not predicted to cause serious eye damage (UN GHS 1).

Based on the results of the weight of evidence approach, it can be concluded that the test item is irritating to eye.

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:
September 26, 2017 -November 13, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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
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
Guideline:
other: INVITTOX Protocol SkinEthic Skin Corrosivity Test, April 2012.
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: 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: 17-RHE-108
- Expires: October 30, 2017

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:
98.6
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:
99.6
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:
The negative control OD values were 1.719, 1.745, 1.743 and 1.661 and, thus, in the range of ≥0.8 and ≤3.0.

Acceptability of the Positive and Negative Control Data:
After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.5% and, thus, lower than the historically established threshold of 0.97%.
After treatment with the negative control (deionised water) the mean ODs were 1.731 (3 minute exposure) and 1.703 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.590 and 1.468, respectively.

Negative Control, Positive Control and Test Substance Data Acceptance Criteria:
The range between identically treated tissues with the negative control was less than 30% after 3 minutes exposure (1.4%) and after 1 hour exposure (5.1%).
The range between identically treated tissues with the positive control was higher than 30% after 1 hour exposure (50.0%), but the optical densities measured were <0.3.
The range between identically treated tissues with the test item was less than 30% after 3 minutes exposure (0.3%) and after 1 hour exposure (7.2%).


The study met all acceptance criteria.




 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 3 1.731 100.0 
Negative Control 60

1.703

100.0

 Positive Control

60

0.009

0.5

 Test Material 3 1.706 98.6
 Test Material 60 1.696 99.6
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 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:
July 17, 2017 - September 4, 2017
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:
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
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: 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: 17-RHE-085
- Expires: August 14, 2017

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 of solid test material
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:
6.8
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:
positive 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 1.754 to 1.943

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.827
Mean viability positive control < 40% 1.3%
SD of group-mean value ≤ 18% 7.7% (positive control)
5.5% (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 1.827
Mean viability positive control ≤ 2.97% 1.3%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 2.9%

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.827 100 
 Positive Control 42

0.024

1.3

 Test Material

42

0.125

6.8

Interpretation of results:
other: UN GHS: Category 2 or Category 1
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 identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).
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 identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
weight of evidence
Study period:
August 1, 2017 - October 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
2013
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) 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. QA statement)
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

Synonym: Imidazole

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:
13.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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 0.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 112.1 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS: 80.9 – 133.0).

Therefore, the study fulfilled the acceptance criteria.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.3 -0.005
0.225
0.1
0.6
-0.4
-0.006
-0.490
0.7
-0.005
0.625
Positive control
20% Imidazole solution
77 .0
2.077
108.155
112.1
9.4
65.0
2.689
105.335
73.4
3.295
122.825
Test item

12.9
0.015
13.125
13.3
0.7
12.6
0.009
12.735
14.6
0.005
14.175


Interpretation of results:
other:
Remarks:
The IVIS obtained after treatment with the test item was 13.3 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.
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. The IVIS obtained after treatment with the test item was 13.3 and, thus higher than 3 and lower than 55, i.e. according to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 27, 2017 - February 6, 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 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30°C
Duration of treatment / exposure:
6 hours
Number of animals or in vitro replicates:
in vitro: duplicate design
Irritation parameter:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
7.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.403 and 1.615).
2. TThe mean relative viability of the positive control is below 50% of the negative control viability (47.0%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.3% to 14.0%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

   Mean OD  Mean Viability
 Negative Control  1.509 100.0% 
 Positive Control  0.709 47.0%
 Test Item  0.112 7.5%
Interpretation of results:
other: UN GHS Category 1 or Category 2
Conclusions:
Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).
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 test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

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 evidence for classification for skin and eye irritation Cat 2 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.