Fatty acids, tall-oil, polymers with glycidyl neodecanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 22, 2015 to June 26, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

EC Number:

617-001-2

Cas Number:

80207-00-7

Molecular formula:

not applicable

IUPAC Name:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

Test material form:

liquid

Remarks:

Brown

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:

Human three dimensional epidermal model (EpiDerm (EPI-200)). Obtained from MatTek In Vitro Life Science Laboratories, Bratislava.

Justification for test system used:

Guideline recommended.

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 μL

Duration of treatment / exposure:

60 min

Details on study design:

PERFORMANCE OF THE STUDY:

Additional tests:
1. Nylon mesh compatibility: The test substance was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 μL test substance were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 h exposure.
2. Assessment of coloured or staining test substance: It was tested whether the test substance develops a colour without MTT addition. 30 μL test substance were given in a test tube with 0.3 mL H2O demineralised and incubated at 37±1°C and 5±0.5% CO2 for 60 min. The resulting solution was colourless, therefore no binding capacity had to be tested.
3. Assessment of direct reduction of MTT by the test substance: The test substance was tested for the ability of direct MTT reduction. To test for this ability, 30 μL test substance were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37±1°C and 5±0.5% CO2 for 60 min. Untreated MTT solution was used as control. The MTT solution did not change its colour within 1 h, therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Pre-Incubation of tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (3 per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37±1°C and 5±0.5% CO2 for 1 h. After the pre-incubation (1 h), the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37±1°C and 5±0.5% CO2 for 18 h.

Treatment:
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only. One plate (three tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 μL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used for treatment with the test substance: 30 μL test substance was applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface. Tissues were dosed in 1 min. intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. 37±1°C and 5±0.5% CO2 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-min.-intervals. After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 24 h.

Medium renewal:
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h for post-incubation.

MTT assay:
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 μL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h. After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature. After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well plate which was read in a plate spectral photometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative absorbance (%)

Value:

97.8

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Any other information on results incl. tables

- Comparison of formazan production:

For the test substance and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table: Formazan production

Designation	Test substance	Positive control
% Formazan production (tissue 1)	93.7%	4.5%
% Formazan production (tissue 2)	95.7%	3.7%
% Formazan production (tissue 3)	103.9%	4.5%
% Formazan production (mean)	97.8%	4.2%

 

The relative absorbance values were reduced to 97.8% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test substance was considered as not irritant to skin.

 

- Validity and acceptability:

All validity criteria were met. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment was considered valid.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Conclusions:

The test substance is non irritating to skin.

Executive summary:

A study was conducted to evaluate the skin irritation potential of the test substance in an in vitro human skin model according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. The test consisted of a topical exposure to neat test substance (30 µl) of a human reconstructed epidermis model followed by a cell viability assay. After treatment, the relative absorbance values were reduced to 97.8%. This value was well above the threshold for skin irritation (50%). The optical density (OD) of the negative control was well within the required acceptability criteria. The positive control induced a decrease in the relative absorbance as compared to the negative control down to 4.2%. Variation within replicates was within the accepted range for negative and positive controls, and test substance. Under the study conditions, the test substance was found to be not irritating to skin (Andres, 2015).