Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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)
Deviations:
yes
Remarks:
The Negative Control narrowly failed the required acceptance criterion in the first run of the assay. Therefore the test was repeated with a fresh batch of EPISKINTM tissues
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
The test substance is identified as 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate. The purity is 100%. The physical state/appearance of material is clear colorless liquid. The expiry date of material is 01 February 2019. The substance can be stored at 4 oC in dark conditions.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Cell source:
other: A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum from an adult human.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface
Duration of treatment / exposure:
Tissues were treated with the test item for an exposure period of 15 minutes
Duration of post-treatment incubation (if applicable):
42-Hour post-exposure incubation period
Number of replicates:
Triplicate tissues were treated with the test item.

Test system

Details on study design:
Pre-Test Procedure
To identify this possible interference, the test item is checked for the ability to directly reduce MTT.

Pre-incubation (Day 0: Tissue Arrival)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main test

Application of Test Item and Rinsing (Day 1)

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis 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 removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
98.6
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: The test item was classified as non-irritant
Conclusions:
The test item was classified as non-irritant.EU CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

The skin irritation potential of the 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate was evaluated using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the post-exposure incubation period tissues were taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made for extraction of formazan crystals. At the end of the formazan extraction period the optical density was measured at 570 nm. The percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) was calculated. The relative mean viability of the test item treated tissues was 98.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The relative mean viability of negative control was 100% and for positive control was 12.8%.

The test item was classified as non-irritant.The test item is not requiring classification for skin irritancy under either UN GHS or EU CLP.