Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July/August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD Guideline 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 26 July 2013) and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD 439 adopted 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
The EPISKIN Small ModelTM 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. On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed
Maintenance Medium for 25 hours at 37°C. Maintenance Medium and Assay Medium were supplied by Skinethic Laboratories, Lyon, France. All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 75 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
The skin was moistened with 5 µl Milli-Q water and then the test substance was used as it is and 10 to 10.8 mg of the test itemwas added on top of the tissues.
Duration of treatment / exposure:
exposure period of 15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
skin tissues were incubated for 42 hours at 37°C
Number of replicates:
three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
88
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The positive control had a mean cell viability of 8% after 15 ± 0.5 minutes exposure. The absolute mean absorbance 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 12%, indicating that the test system functioned properly.

Any other information on results incl. tables

Ascorbyl palmitate was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that Ascorbyl palmitate did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Ascorbyl palmitate was -1.5% of the negative control tissues. Since the NSMTT was < 0.0 no correction for the non-specific reduction of MTT was performed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Ascorbyl palmitate compared to the negative control tissues was 88%. Since the mean relative tissue viability for Ascorbyl palmitate was above 50%, Ascorbyl palmitate is considered to be non-irritant.
Executive summary:

This report describes the ability of Ascorbyl palmitate to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of Ascorbyl palmitate was tested through topical application for 15 minutes in triplicate. A positive and negative control were tested concurrently, each on three individual tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Ascorbyl palmitate compared to the negative control tissues was 88%. Since the mean relative tissue viability for Ascorbyl palmitate was above 50% after 15 ± 0.5 minutes treatment Ascorbyl palmitate is considered to be non-irritant.

The positive control had a mean cell viability of 8% after 15 ± 0.5 minutes exposure. The absolute mean absorbance 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 12%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Ascorbyl palmitate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.