Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 01, 2011 to June 21, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate
EC Number:
947-751-9
Molecular formula:
C16H34O4P1K1 (representative: mono- C16 PSE, K+) C16H34O1 (representative alcohol: C16 alcohol) C36H72O2 (isostearyl isostearate)
IUPAC Name:
Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate
Test material form:
solid

In vitro test system

Test system:
human skin model
Cell type:
other: three-dimensional reconstructed human epidermis keratinocytes
Cell source:
other: EPISKIN model - reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, France
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test substance was used as supplied. First 5 µL of the distilled water was topically applied to ensure contact between test substance and tissue, and then 10 mg of the test substance was applied on epidermal surface
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Triplicates for the test substance, negative and positive control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minutes exposure
Value:
103
Negative controls validity:
valid
Remarks:
100 % tissue viability
Positive controls validity:
valid
Remarks:
10.9 % tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Direct MTT Reduction test:
The MTT solution containing the test substance did not turn blue which indicated that the test substance did not directly reduce MTT.

Test substance, Positive Control substance and Negative Control substance:
The relative mean viabillity of the test substance treated tissues was 103.0 % after 15 minutes exposure period.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was <40 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was <18 %. The positive control acceptance criterion was therefore satisfied.
The mean OD540 for the negative control treated tissues was >0.6 and the standard deviation value of the percentage viability was >18 %. The negative control acceptance criterion was therefore satisfied.
The standard deviation calculated from individual percentage tissue viabilities of the three identically test substance treated tissues was 18 %. The test substance acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', using Episkin method, according to EU Method B.46, in compliance with GLP. Skin irritation potential was examined using the EpiSkin reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 h. To identify possible interference of the test substance with MTT endpoint, prior to the main test the test substance was checked for the ability to directly reduce MTT. On Day 1 of the main test, triplicate tissues were treated with the undiluted test substance for an exposure period of 15 minutes. First 5 µL of the distilled water was topically applied to ensure contact between test substance and tissue, and then 10 mg of the test substance was applied on epidermal 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. At the end of the exposure period each tissue was rinsed before incubating for 42 h at 37 °C, 5 % CO2 in air. Formazan extraction was performed at Day 3. 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-labeled 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. Absorbance optical density measurements were conducted at Day 6. 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-labeled 96-well plate. The optical density was measured at 540 nm. The relative mean viability of the test substance treated tissues was 103% (which is well above the threshold for classification) after the 15-minute exposure period and 42 h post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied. Under the study conditions, the test substance was determined to be non-irritating to the skin (Harlan, 2012).