3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 July 2018 (Experimental Start) to 6 July 2018 (Experimental Completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

EC Number:

230-512-1

EC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Cas Number:

7158-25-0

Molecular formula:

C15H18

IUPAC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia Inc.
- Lot/batch No.of test material: RR229-0714
- Expiration date of the lot/batch: 6 June 2020
- Purity test date: 28 March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%), under inert (Nitrogen) gas
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: The test item was applied as supplied, no formulation required.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, administered to the test system as supplied

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not specified, test item applied to the test system as supplied.

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN TM(SM)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult human derived epidermal keratinocytes (EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-027, Expiry Date: 09 July 2018

Source strain:

other: Strain No's: 10-KERA-003 & 10-KERA-004

Justification for test system used:

The EPISKIN TM (SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439). It was therefore considered as suitable for use in this study.

Vehicle:

unchanged (no vehicle)

Remarks:

Test item was applied as supplied, no formulation required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM (SM) Model
- Tissue batch number: EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-027, Expiry Date: 09 July 2018)
- Production date: 3 July 2018
- Expiry date: 9 July 2019
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 4 July 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Disks of EPISKIN TM(SM) were treated with the test item and incubated for 4 hours (corrosivity testing) and for 15 minutes (irritation testing) at room temperature.
- Temperature of post-treatment incubation (if applicable): After 15 minutes incubation time (irritation test) or 4 hours incubation time (corrosivity test), the EPISKIN TM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

For the irritation test, after rinsing, the units were placed into the plate wells with fresh pre-warmedMaintenance Medium (2 mL/well) below them and then incubated for 42 hours (±1 hour) at 37 °C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 15 minutes incubation time (irritation test) or 4 hours incubation time (corrosivity test), the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- 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: In both the irritation and corrosion tests, MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except for the two living colour control units). The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours (±5 minutes), protected from light, in a > 95% humidified atmosphere.
- Incubation time: 37 °C in 5% CO2 for 3 hours (±5 minutes), protected from light, in a > 95% humidified atmosphere.
- Spectrophotometer: Not specified
- Wavelength: The OD (optical density or absorbance) of samples was measured using a plate reader at 570 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: In the irritation test, the mean cell viability was 105.6% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as non-irritant to skin.
- Viability: In the corrosivity test, the mean cell viability was 109.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.
- Barrier function: Appropriate cell morphology. The EPISKIN TM(SM) kits are manufactured according to ISO 9001. All biological components of the epidermis and the
kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell
viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the
EPISKIN TM(SM) Test Kits used in this study.
- Morphology: A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994)
- Contamination: None
- Reproducibility: All parameters were within acceptable limits and therefore the study was considered as valid.

NUMBER OF REPLICATE TISSUES:
In the corrosivity test, two replicates per time point were used for the test item accompanied by two negative controls and two positive controls.
In the irration test, three replicates per time point were used for the test item accompanied by three negative controls and three positive controls.
For both tests, two additional test item-treated living tissues were used for the non-specific OD evaluation.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : For both tests, two additional test item-treated living tissues were used for the non-specific OD evaluation. No killed tissues were used.
- N. of replicates: two
After three hours of incubation, a yellow colour of the mixture was detected in the test tube. The test item did not therefore react with MTT and thus the use of additional controls was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if following exposure with Distilled Tricyclopentadiene, the mean cell viability was above the threshold of 35%. As the mean cell viability was 109.6% following exposure to TCPD when compared to the negative control, the test item was therefore considered as non-corrosive.

- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative Control

Corrosivity testing:
Physiological saline (0.9% (w/v) NaCl solution):

Manufacturer: B. Braun Pharmaceuticals SA
Batch No.: 80353Y05-1
Expiry Date: December 2020
Grade: Sterile


Irritation testing:

Phosphate Buffered Saline:
Abbreviation: PBS
Supplier: Life Technologies Corporation
Batch number: 1930056
Expiry date: November 2019


Positive Control

Corrosivity testing:
Glacial acetic acid:
Supplier: VWR International Ltd.
Batch No.: 17H074111
Expiry date: 05 July 2020


Irritation testing:

5% (w/v) Sodium Dodecyl Sulphate solution:
The positive control solution (abbreviated as SDS in the raw data and report) was prepared freshly in the testing laboratory. The following chemicals were used for the preparation of the positive control solution:

Sodium Dodecyl Sulphate:

Supplier: REANAL
Batch number: PP/2016/11637
Expiry date: 30 June 2021

Distilled water:
Supplier: B. Braun Pharmaceuticals SA
Batch number: 73751Y25-2
Expiry date: August 2020

Duration of treatment / exposure:

In case of the corrosivity testing, 30 mg of test item was applied evenly to each of two test units and each additional control skin units and then 100 µL physiological saline was added to the test item to ensure good contact with the epidermis in each case. In case of the irritation testing, first an appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 30 mg of test item were applied evenly to each of three test units and each additional control skin units. The amounts were sufficient to cover the epidermal surface.

Duration of post-treatment incubation (if applicable):

After 15 minutes incubation time (in the irritation test) or 4 hours incubation time (in the corrosivity test), the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

Number of replicates:

Two replicates per time point were used for test item (corrosivity test). Two negative controls and two positive controls were also run in the corrosivity test.
Three replicates per time point were used for test item (irritation test). Three negative controls and three positive controls were also run in irritation test.
In both tests, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Corrosivity test

Run / experiment:

1 (Negative control) Physiological saline

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No indication of corrosivity

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (Test item - TCPD)

Value:

109.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No indication of corrosivity

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (Positive control) Glacial acetic acid

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No indication of corrosivity

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: After three hours of incubation the test item did not react with MTT.
- Colour interference with MTT: No colour change was observed after three hours incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility chosen to conduct these tests (OECD 431 and OECD 439) is technically proficient in these in vitro tests, is GLP compliant and has appropriate historical data on which to compare the test item againsts to define a study outcome of non-corrosive and non-irritant to skin.

ACCEPTANCE OF RESULTS:
General validity criteria: The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5. The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.

- Acceptance criteria met for negative control:
In the corrosivity test, the mean OD value of the two negative control tissues was in the recommended range (0.830).
In the irritation test, the mean OD value of the three negative control tissues was in the recommended range (0.876).

- Acceptance criteria met for positive control:
In the corrosivity test, the two positive control treated tissues showed 0.6% viability demonstrating the proper performance of the assay.
In the irritation test, the positive control treated tissues showed 1.6% viability demonstrating the proper performance of the assay.

- Acceptance criteria met for variability between replicate measurements:
In the corrosivity test, the difference of viability between the two test item-treated tissue samples in the MTT assay was 4.6%. The difference of viability between the two negative control tissue samples in the MTT assay was 3.8%.

In the irritation test, the standard deviation of the viability results for negative control samples was 3.8%. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 7.5%.

All parameters were within acceptable limits and therefore the studies considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN TM (SM) model test with Distilled Tricyclopentadiene (TCPD), the results indicate that the test item is non-corrosive to the skin, UN GHS Classification: No Category.