Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Feb 2017 to 06 Mar 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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 of test material (as cited in study report): HEXENYL-3-CIS-ISOBUTYRATE
- Physical state: colourless liquid
- Lot/batch No.: VE00468943
- Expiration date of the lot/batch: 12 November 2017

In vitro test system

Test system:
human skin model
Remarks:
human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: SkinEthic Laboratories, Lyon, France
Details on animal used as source of test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-009.
This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 71 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Amount/concentration applied:
The liquid test item was applied undiluted (25 μl) directly on top of the tissue.^HEXENYL-3-CIS-ISOBUTYRATE was spread to match the size of the tissue.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
91
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

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 HEXENYL-3-CIS-ISOBUTYRATE compared to the negative control tissues was 91%.

Since the mean relative tissue viability for HEXENYL-3-CIS-ISOBUTYRATE was above 50% after 15 ± 0.5 minutes treatment HEXENYL-3-CIS-ISOBUTYRATE is considered to be

non-irritant. The positive control had a mean cell viability of 12% 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 7%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, HEXENYL-3-CIS-ISOBUTYRATE is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate HEXENYL-3-CIS-ISOBUTYRATE for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of HEXENYL-3-CIS-ISOBUTYRATE 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 VE00468943 of HEXENYL-3-CIS-ISOBUTYRATE was a colourless to pale yellow liquid. HEXENYL-3-CIS-ISOBUTYRATE 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 HEXENYL-3-CIS-ISOBUTYRATE compared to the negative control tissues was 91%. Since the mean relative tissue viability for HEXENYL-3-CIS-ISOBUTYRATE was above 50% after 15 ± 0.5 minutes treatment HEXENYL-3-CIS-ISOBUTYRATE is considered to be non-irritant.

The positive control had a mean cell viability of 12% 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 7%, indicating that the test system

functioned properly.

In conclusion, HEXENYL-3-CIS-ISOBUTYRATE is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.