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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:
weight of evidence
Study period:
05 Oct 2016 - 16 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(5-Ethyl-tetrahydropyran-2-yl)-phenol
EC Number:
921-114-5
Cas Number:
1173761-32-4
Molecular formula:
C13 H18 O2
IUPAC Name:
4-(5-Ethyl-tetrahydropyran-2-yl)-phenol
Test material form:
solid

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:
SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-114

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Application volume: 16 ± 2 mg per tissue

NEGATIVE CONTROL
- Application volume: 16 ± 5 µL per tissue

POSITIVE CONTROL
- Application volume: 16 ± 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:
The test item as well as the positive and negative control were tested in batch-triplicates.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1
Run / experiment:
Tissue 1
Value:
1.47
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2
Run / experiment:
Tissue 2
Value:
1.54
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 3
Run / experiment:
Tissue 3
Value:
1.28
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
1.43
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The pre-test for direct MTT-reducing capacity of the test item not result in blue colour, i.e. the test item is a direct MTT reducer but the test item has no colorant properties.
Due to the fact that MTT was not reduced after treatment with the test item, no additional killed epidermises were tested to evaluate the extent of a non-specific interaction.

Any other information on results incl. tables

Table 1 Optical density and Tissue viability

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative Control

1.925

98.52%

1.978

101.22%

1.959

100.26%

1.954

100.0%

1.37%

Positive Control

0.029

1.48%

0.028

1.45%

0.030

1.52%

0.029

1.48%

2.30%

Test item

0.029

1.47%

0.030

1.54%

0.025

1.28%

0.028

1.43%

9.28%

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The tissue viability after treatment with the test item was lower than 50 % (men viability: 1.43%). Therefore the test item is considered to possess an irritant potential to skin.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

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 minutes (± 1 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 deionized water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: >1.431). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48% (study acceptance criterion: <3.14%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.43% and, thus, lower than 50%, i.e. according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2).