Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2016 to 18 April 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Remarks:
There were no deviations from standard operating procedures that affected the integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: VE00411894
Expiration date: February 11, 2018

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-015,)
Source strain:
other: SkinEthic Laboratories, Lyon, France
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-015, See APPENDIX 4).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Undiluted FRESKOMENTHE was applied (25 μl)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hour
Number of replicates:
1

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean tissue viability (percentage of control)
Value:
8
Negative controls validity:
valid
Remarks:
100% Mean tissue viability (percentage of control)
Positive controls validity:
valid
Remarks:
27% Mean tissue viability (percentage of control)
Other effects / acceptance of results:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 27%.
The absolute mean OD(570) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 19%, indicating that the test system functioned properly.

Any other information on results incl. tables

The mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE was compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE compared to the negative control tissues was 8%. Since the mean relative tissue viability for FRESKOMENTHE was below 50% it is considered to be irritant.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Finally, it is concluded that this test is valid and that FRESKOMENTHE is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In vitro skin irritation test with FRESKOMENTHE using a human skin model.

This report describes the ability of FRESKOMENTHE to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of FRESKOMENTHE was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch VE00411894 of FRESKOMENTHE was a clear colourless liquid. FRESKOMENTHE was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 minutes.

After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with FRESKOMENTHE compared to the negative control tissues was 8%. Since the mean relative tissue viability for FRESKOMENTHE was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

The positive control had a mean cell viability of 27% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 19%, indicating that the test system functioned properly (study plan deviation 1).

Finally, it is concluded that this test is valid and that FRESKOMENTHE is irritant in the in vitro skin irritation test under the experimental conditions described in this report