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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 18 October 2017. Experimental completion date 30 October 2017
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Due to a technical error in the initial run it was necessary to repeat the test in one further run. This deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Due to a technical error in the initial run it was necessary to repeat the test in one further run. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
Batch: 52611021
CAS Number: 68475-94-5
EC Number: 500-215-4
Purity: 95-100%
Physical state / Appearance: Clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Batch: 52611021
Purity: 95 – 100%
Physical state/Appearance: Clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 24 October 2017
EpiSkinTM Tissues (0.38cm2) lot number: 17-EKIN-043
Maintenance Medium lot number: 17-MAIN3-045
Assay Medium lot number: 17-ESSC-042
Vehicle:
unchanged (no vehicle)
Details on test system:
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (OD570) (without a reference filter) using the Labtech LT 4500 microplate reader and LT-com analysis software.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. The test item was used as supplied.
10 μL of DPBS served as the negative controls. The negative control item, DPBS, was used as supplied.
10 μL of SDS 5% w/v served as the positive controls. The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution and was used within 2 hours of preparation.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
93.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 93.3% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 10.5% relative to the negative control treated tissues and the standard deviation value of the viability was 7.8%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.819 and the standard deviation value of the viability was 1.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 8.2%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD570of tissues

Mean OD570of triplicate tissues

±SDof OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.831

0.819

0.012

101.5

100*

1.5

0.820

100.1

0.807

98.5

Positive Control Item

0.040

0.086

0.064

4.9

10.5

7.8

0.058

7.1

0.159

19.4

Test Item

0.842

0.764

0.068

102.8

93.3

8.2

0.732

89.4

0.719

87.8

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
other: EU CLP Not classified for Irritation.
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 cannot be determined).
Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 93.3% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 cannot be determined).