Esterification products of Grape seeds, Vitis vinifera L. (Vitaceae), extract with hexadecanoyl chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: BIO-HC internal procedures

Deviations:

no

Principles of method if other than guideline:

The eye irritant potential of the test material is assessed by measurement of its cytotoxic effect on a 3-dimensional reconstituted human corneal epithelial model (HCE SkinEthic Model).
The test material is applied as supplied, topically to the tissues for 60 minutes. After a 16h post-treatment incubation, the viability of tissues is evaluated by a MTT test. The results of the MTT assay allows to classify the test material.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Esterification products of Grape seeds, Vitis vinifera L. (Vitaceae), extract with hexadecanoyl chloride

EC Number:

941-319-3

Molecular formula:

Unknown (Substance of Unknown or Variable Composition)

IUPAC Name:

Esterification products of Grape seeds, Vitis vinifera L. (Vitaceae), extract with hexadecanoyl chloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Palmitoyl grape seed extract is the chemical name of BERKEMYOL PEPIN DE RAISIN (the trade name).

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The study is performed on the reconstituted human corneal epithelial model from SkinEthic laboratories (HCE model). This model consists of an epithelium derived from immortalized human corneal epithelial cells (HCE) cultured in a defined medium at the air-liquid interface.

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test material

Duration of treatment / exposure:

60 minutes

Duration of post- treatment incubation (in vitro):

16 hours

Number of animals or in vitro replicates:

0.5 cm² of the tissue surface

Details on study design:

Test material application:
Prior to the application of the test substance, epithelia are transferred into 24-well plates filled with SkinEthic maintenance medium.
30mg of the test material are topically applied on the 0.5cm² of the tissue surface. The treated tissues are incubated at room temperature for 60 minutes.
In parallel, 3 untreated tissues (nagative control) and 3 tissues treated with ethanol (positive control) are placed in cluster plates containing maintenance medium and incubated for 60 minutes.

Post treatment incubation:
After 60 minutes exposure with the test material, the treated tissues are rinsed thoroughly with PBS to remove all residual test material from the surface. After rinsing, the tissue units are incubated with new maintenace medium at 37°C in 5% CO2 humidified atmosphere for additional 16 hour post-treatment incubation period.

Tissue viability - MTT test:
At the end of the assay, tissues are transferred in a 24-wel culture dish containing MTT solution. After 3hr incubation at 37°C, the epithelia are transferred in a 24-wel culture dish containing acidified isopropanol. The plates are stored 2 hours at room temperature, under gentle shaking, protected from light. At the end of the extraction period, 3 x 200 µl aliquots of extracts are transferred to a 96-well flat bottom plate before measuring optical density (O.D.) at 570 nm with isopropanol as a blank, using a microtiter plate reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: Tissue viability

Vehicle controls validity:

valid

Remarks:

Viability : 106%

Negative controls validity:

valid

Remarks:

Viability : 100%

Positive controls validity:

valid

Remarks:

Viability : 2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The viability of 2% for the positive control validates the assay.
No deviation to the experimental protocol was reported and not any amendment was made on the protocol.

Applicant's summary and conclusion

Interpretation of results:

other: No significant cytotoxicity was recorded. The test material is not irritant

Conclusions:

No significant cyototoxic effect was observed in the experimental conditions described in attached study report.
The results of the MTT assay indicate: Tissue Viability > 50%. So the test material is not irritant to eyes

Executive summary:

The assay was performed on reconstituted human corneal epithelia SkinEthic (RHC / S /5 batch 09 022D 0703) of 0.5 cm² surface.

30 mg of the test material as supplied were applied on the epithelia surface. Viability of epidermis was assessed by a MTT test.

Tissue viability results are summarized in the following table:

	Negative control (PBS)	Positive control	Test article
Tissue viability	100%	2%	106%

No significant cyototoxicity was recorded. Taking into account the results obtained (viability more than 50%) the test article can be classed as Not irritant.

Under the same experimental conditions, ethanol induced an important cyototoxic effect (Viability = 2%). This result (viability<50%) validates the assay.

No deviation to the experimental protocol was reported.