Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 20, 2018 -June 22,2018
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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM, Performance Standards for In-Vitro Skin Irritation Test Methods based on Reconstructed Human Epidermis (RHE)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
other: SkinEthic Skin Irritation Test-42bis Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX Version 2.1
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17 obtained from Episkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number: 18-RHE-068

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C and 5% CO2.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL DPBS
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes) at 37°C and 5% CO2 protected from light
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD =1.2 (CV= 3.1 %)
- Barrier function: 4.5 h
- Morphology: well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum, absence of significant histological abnormalities
- Contamination: none

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is ≥ 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is ≤ 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

ACCEPTABILITY CRITERIA
The OD values for the negative control shall be in the range of ≥ 0.8 and ≤ 3.0 as given in OECD Guideline 439.
Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered valid if ≤ 18% of group mean-value.
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%. The standard deviation value is considered valid if ≤ 18% of group mean-value.
Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (1.450).
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (2.87%).
Test Item Data Acceptance Criteria:
The standard deviation of the three tissues treated with the test item should be ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied (volume or weight with unit): 16 mg ± 2 mg per tissue
Before adding 16 mg of the test item, 10 µL of deionised water were spread to the epidermis surface to improve further contact between the test item and the epidermis.

NEGATIVE CONTROL
- Concentration: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Concentration: 16 µL ± 0.5 µL per tissue
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st experiment
Value:
107.5
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
- Colour interference with MTT: The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Results

Group

Tissue 1

 

Tissue 2

 

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative
Control

1.794

107.7%

1.607

96.5%

1.594

95.7%

1.665

100.00

6.7%

Positive
Control

0.025

1.5%

0.021

1.3%

0.021

1.3%

0.022

1.4%

7.1%

Test item

1.993

119.7%

1.682

101.0%

1.693

101.7%

1.789

107.5%

9.9%

Table 2: 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

Table 3: 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)

 

Table 4: Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory

 

Acceptance Criterion

Result

Mean OD negative control

≥ 1.450

1.665

Mean viability positive control

≤ 2.87%

1.4%

Table 5: Test Item Data Acceptance Criteria:

 

Acceptance Criterion

Result

SD of group-mean value

≤ 18%

9.9%

 

 

Table 6: Historical Data

The mean of negative control and positive control of all performed experiments in the testing laboratory is given in the following table.

Positive Control

Negative Control

Mean Viability [%]

142

Mean Absorption [OD570]

1.987

Standard Deviation

0.48

Standard Deviation

0.268

Based on the historical data, acceptability ranges for the positive and negative control were stated.

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following treatment with the test item, the tissue viability was 107.5% 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).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in anin vitro human skin model according to OECD TG 439. 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 minutes1 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 107.5% 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).