[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to the OECD TG 492 and with facility SOPs, but not under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

no

Remarks:

Study performed according to OECD Guideline and testing facility SOPs, but not under GLP.

Test material

Test material information

Constituent 1

Reference substance name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

EC Number:

209-269-0

EC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Cas Number:

564-20-5

Molecular formula:

C16H26O2

IUPAC Name:

[3aR-(3aα,5aβ,9aα,9bβ)]decahydro-3a,6,6,9a-tetramethylnaphth[2,1-b]furan-2(1H)-one

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues (OCL-200)
The EpiOcular™ human cell construct (MatTek Corporation) is composed of stratified human keratinocytes in a threedimensional structure.
Batch No: 27042
Quality control: May 2018

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hrs

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

18 hrs

Number of animals or in vitro replicates:

N/A

Details on study design:

Assessment of Direct Test Article Reduction by MTT
It is necessary to assess this ability for each test article prior to conducting any assays with viable tissues.
1. Approximately 50 mg (for solid test articles) are added to 1 ml of a 1.0 mg/mL MTT solution in a 6-well plate and the mixture is
incubated in the dark at 37°C in a humidified atmosphere of 5 % CO2 for three hours.
2. A negative control (50 μl of sterile deionized water) should be run concurrently.
3. If the MTT solution colour turns blue/purple, the test article is presumed to have reduced the MTT.
4. In cases where the test article is shown to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) should be performed.
5. Blue, dark purple and black test articles should also be tested on killed controlsbecause it may not be possible to assess their potential to directly reduce MTT.

Assessment of Colored or Staining Materials
For non-colored test articles additional tests have to be performed to assess if they become colorants after contact with water or isopropanol. For this purpose 50 mg (for solids) of each test article are added to 1.0 ml of water in a 6-well plate and the mixture is incubated in the dark at 37°C in a humidified atmosphere of 5% CO2 in air for at least one hour. Furthermore, approximately 50 mg of solids are added to 2 ml isopropanol, the same amount as used for MTT extraction, and are incubated in 6-well plates for 2 to 3 hours at room temperature.
If the test article becomes colored either in water or isopropanol, it has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed

Pre-incubation step
For a given chemical a minimum of 2 tissues were used.
An appropriate numbers of 6-well plates were filled with 1 ml of EpiOcular™ Assay Medium. EpiOcular™ EIT test kit was open and tissues were transferred into Assay
medium filled wells, using sterile forceps. Place the tissues at 37°C, 5% CO2 until test substance application (usually 16 – 24 hours).

Test Material Exposure / Rinsing:
Treatment of solid test article
After the overnight incubation, the tissues are pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues are incubated at standard culture conditions for 30 ± 2
minutes.
Each solid test article is tested by applying 50 mg topically on the EpiOcular™ tissues. The tissues are incubated at standard culture conditions for 6 hours ± 15
minutes.
At the end of the treatment time, the test articles are removed by extensively rinsing the tissues with Ca2+Mg2+-free D-PBS, and any remaining liquid should be
decanted onto the absorbent material.
Epitheliums were immersed in a new 12-well plate containing 5 mL of fresh Assay Medium, for 24 ± 2 minutes at room temperature.
Each insert is removed from the Assay Medium, the medium is decanted off the tissue, and the insert is blotted on absorbent material, and transferred to the
appropriate well of the pre-labeled 6-well plate containing 1 ml of warm Assay Medium. The tissues are incubated for 18 ± 0.25 hours at standard culture
conditions.

Viability measurement
24-well plates were filled with 300 μL MTT solution (1 mg/ml).
Treated tissues were transferred in the pre-filled MTT 24-well plates and Incubated for 3 hours (± 10 minutes) at 37°C and 5% CO2.
Treated tissue insert bottom was dried on sterile absorbent paper and transferred in new 6-well plate containing 2 mL of isopropanol so that no isopropanol is
flowing into the insert on the tissue surface.
Plate was protected from evaporation by stretching 3 parafilm layers over the plate and adding the lid on the plate and incubated for 2 hours (± 5 minutes) at
room temperature with gentle agitation (about 150 rpm) for Formazan extraction.
Tissue and polycarbonate filter were not pierced.
Extraction solution was homogenized by pipetting 3 times up and down to complete Formazan crystals solubilization.
2 x 200 μL extraction solution per well (= 2 wells per tissue i.e. 2 replicates per tissue) were transferred into a 96-well plate.
Optical Densities (OD) was read using a 96-well plate spectrophotometer: The concentration of Formazan was measured by determining the OD at 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: tissue viability (%)

Run / experiment:

1

Value:

20.37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cat1/Cat2

Results 2

Irritation parameter:

other: tissue viability (%)

Run / experiment:

2

Value:

22.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cat1/Cat2

Results 3

Irritation parameter:

other: mean tissue viability (%)

Run / experiment:

mean

Value:

21.49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cat1/Cat2

Applicant's summary and conclusion

Interpretation of results:

other: Cat1/Cat2

Conclusions:

According to EpiOcular™ Eye Irritation test, Sclareolide is classified for eye irritation or serious eye damage and defined as UN GHS Cat1/Cat2

Executive summary:

According to EpiOcular™ Eye Irritation test, Sclareolide is classified for eye irritation or serious eye damage and defined as UN GHS Cat1/Cat2