[1aR-(1aα,4aα,7α,8aS*)]-octahydro-4,4,8,8-tetramethyl-4a,7-methano-4aH-naphth[1,8a-b]oxirene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2014 - 31 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2013)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

(2012)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[1aR-(1aα,4aα,7α,8aS*)]-octahydro-4,4,8,8-tetramethyl-4a,7-methano-4aH-naphth[1,8a-b]oxirene

EC Number:

247-851-6

EC Name:

[1aR-(1aα,4aα,7α,8aS*)]-octahydro-4,4,8,8-tetramethyl-4a,7-methano-4aH-naphth[1,8a-b]oxirene

Cas Number:

26619-69-2

Molecular formula:

C15H24O

IUPAC Name:

[1aR-(1aα,4aα,7α,8aS*)]-octahydro-4,4,8,8-tetramethyl-4a,7-methano-4aH-naphth[1,8a-b]oxirene

impurity 1

Reference substance name:

7,7,8,8-tetramethyloctahydro-2,3b-methanocyclopenta[1,3]cyclopropa[1,2]benzen-4-ol

Cas Number:

29461-20-9

Molecular formula:

C15H24O

IUPAC Name:

7,7,8,8-tetramethyloctahydro-2,3b-methanocyclopenta[1,3]cyclopropa[1,2]benzen-4-ol

Test material form:

liquid

Details on test material:

- Name of test material (as cited in the expert statement): Folenox
- Chemical name: 4,4,8,8-Tetramethyloctahydro-4a,7-methanonaphtho[1,8a-b]oxirene (IUPAC)
- Empirical formula: C15H24O
- Structural formula: See IUCLID section 1

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 35.3 - 36.9
- Humidity (%): 76 - 89

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 25 μl of the undiluted test substance

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

15 minutes

Number of animals:

3 tissues / substance or control

Details on study design:

TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 14-EKIN-010).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates 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.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Folenox was checked for possible direct 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction before the study was started. To assess the ability of the test substance to reduce MTT, 25 μl of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.
- Cytotoxicity (irritancy) is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability

Value:

16

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 5%. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean tissue viability obtained after 15 minutes treatment with Folenox compared to the negative control tissues was 16%. Since the mean relative tissue viability for Folenox was below 50% after 15 minutes treatment it is considered to be irritant.

Any other information on results incl. tables

Folenox was checked for possible direct MTT reduction by adding the test substance to MTT medium.

Because no colour change was observed it was concluded that Folenox did not interact with MTT.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
Based onthe results it is concluded that Folenox is irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model), the influence of the test substance on the viability of human skin was tested. The test substance (25 μl) was applied directly to 0.38 cm²cultured skin.

After 15 minutes, the substance was removed and cells were cultured for 42 hours.

Folenox was checked for possible direct MTT reduction by adding the test substance to MTT medium.

Because no colour change was observed it was concluded that Folenox did not interact with MTT.

The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 5% whereas the test substance showed cell viability of 16%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that Folenox is irritating in the in vitro skin irritation test.