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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2015 - February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS, CellSystems
- Tissue batch number(s): 100-AE2058-1
- Production date: November 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): ca. 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times with 1mL PBS
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 2 hrs 55 min
- Spectrophotometer: Elx800, BioTek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

3 min and 1 hour

Duration of post-treatment incubation (if applicable):

2 hrs 55 minutes (in MTT solution) + 2 hrs (with isopropanol)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

87.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

109.42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

The SD of the viability of negative control epidermises during 1 hr was 26.2%, instead of <= 18% as initially planned. Considering the results obtained, this deviation is considered as without impact on the study conclusions.

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained under these experimental conditions enable to conclude that the test item Sclareolide is not corrosive for the skin.

Executive summary:

The test item Sclareolide was applied as supplied, at the dose of 25 mg, to 2 Human skin model surfaces during 3 minutes and 1 hour, followed by a rinse with 20 mL of PBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

3 minutes and 1 hour after the test item application, the mean percents viability of the epidermis skins treated with the test item were 87.98% and 109.42% (considered as 100%), versus 3.96% and 0.61 %, respectively, with the positive control item (potassium hydroxide 8N).

The results obtained under these experimental conditions enable to conclude that the test item Sclareolide is not corrosive for the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

no

Remarks:

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

Test system:

other: reconstructed human epidermis model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Details on animal used as source of test system:

N/A

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Ashland Equivalent Epidermis
- Tissue batch number(s): 1806EPID06
- Production date: 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 timesith 1mL DPS
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 3hrs
- Spectrophotometer: yes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be irritant to skin (category 2), if the mean relative viability after 42 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Neat sclareolide, 16 mg

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

117.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

105.57

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

111.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

According to Ashland Equivalent Epidermis skin irritation test performed according to the OECD Guideline 439, Sclareolide is classified as Non irritant.

Executive summary:

According to Ashland Equivalent Epidermis skin irritation test performed according to the OECD Guideline 439, Sclareolide is classified as Non irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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.

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.

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

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.

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

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

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

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

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT, Hungary
- Age of donor animals: approximately 7 weeks

Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

30, 75, 120, 180 and 240 minutes after post-treatment rinse

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head) and avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness were observed in one eye. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole.

REMOVAL OF TEST SUBSTANCE
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3*20 mL saline was performed at each time point when the positive control material remaining on the cornea was observed.

OBSERVATION
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Irritation parameter:

other: maximum corneal swelling at up to 75 min (%)

Run / experiment:

mean

Value:

3.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

other: maximum corneal swelling at up to 240 min (%)

Run / experiment:

mean

Value:

7.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Interpretation of results:

other: Not classified as a severe irritant and not classified as non-irritant.

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with SCLAREOLIDE, the test item is not classified as a severe irritant and not classified as non-irritant.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018).

After the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the study was considered to be valid.

Slight cornea swelling change (mean = 7.5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 1 was on two eyes and severity 2 on one eye) was observed on all three eyes. Slight fluorescein retention change (severity 1 on three eyes) was noted on all three eyes. No other corneal effect was observed.

Based on these in vitro eye irritation assays in isolated chicken eyes with SCLAREOLIDE, the test item is not classified as a severe irritant and not classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available