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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Oct 04, 2017 - Jan 09, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
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 material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

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-118
- Expires: Nov 27, 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

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1 (3 min)
Value:
94.3
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:
97.2
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:
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 1.826 to 1.879

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

Acceptance Criterion Result
Mean OD negative control ≥ 0.8 and ≤ 3.0 1.879 (3 min)
1.826 (1 hour)

Mean viability positive control < 15% after 1-hour exposure 0.5%

Range between identically treated < 30% 14.4% (3 min)
tissues with test item 18.3% (1 hour)

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.568 (3 min) 1.902 (3 min)
≥ 1.434 (1 hour) 1.973 (1 hour)

Mean viability positive control ≤ 0.95% 0.58%

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% 2.6% (3 min)
treated tissues 8.2% (1 hour)
Positive control < 30% 16.7% (1 hour)
Test substance < 30% 8.4% (3 min)
0.7% (1 hour)


The study met all acceptance criteria.





Any other information on results incl. tables

 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 3 1.879 100.0 
Negative Control 60

1.826

100.0

 Positive Control

60

0.010

0.5

 Test Material 3 1.772 94.3
 Test Material 60 1.775 97.2

Applicant's summary and conclusion

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:

Objective

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in anin vitrohuman skin model.

Study Design

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 corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for
1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

Results

After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.7% and, thus, lower than the historically established threshold of 0.95%. After treatment with the negative control (deionised water) the mean ODs were 1.879 (3 minutes exposure) and 1.826 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.568 and 1.434, respectively. Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was ≥50% after 3 minutes exposure (mean viability: 94.3%) and ≥15% after 1 hour exposure (mean viability: 97.2%),i.e.according to OECD 431 the test item is not considered as corrosive to skin.

Conclusion

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.