Reaction mass of 2-[7-isopropyl-5-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane and 2-[8-isopropyl-6-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane

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 - 05 May 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:

Reaction mass of 2-[7-isopropyl-5-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane and 2-[8-isopropyl-6-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane

EC Number:

916-737-4

Molecular formula:

Reaction mass of 2-[7-isopropyl-5-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane and 2-[8-isopropyl-6-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane

IUPAC Name:

Reaction mass of 2-[7-isopropyl-5-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane and 2-[8-isopropyl-6-methylbicyclo[2.2.2]oct-5-en-2-yl]-1,3-dioxolane

Test material form:

other: liquid

Details on test material:

Please note that although the substance is stated as the mono-constituent substance 2-(6-methyl-8-(-1-methylethyl)bicyclo(2.2.2)oct-5-en-2(3)-yl-1,3-dioxolane in the report, analytical data on the same batch shows that the test substance is in fact the multi-constituent substance Reaction mass of 2-(7-isopropyl-5(6)-methylbicyclo[2.2.2]oct-5-en-2-yl)-1,3-dioxolane and 2-(8-isopropyl-5(6)-methylbicyclo[2.2.2]oct-5-en-2-yl)-1,3-dioxolane.

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 34.5 - 36.8
- Humidity (%): 80 - 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, Batch no.: 14-EKIN-015). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded 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:
- Glycolierral 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:

27

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 8%. (migrated information)

In vivo

Irritant / corrosive response data:

The relative mean tissue viability obtained after 15 minutes treatment with Glycolierral compared to the negative control tissues was 27%. Since the mean relative tissue viability for Glycolierral was below 50%, it is considered to be irritant.

Other effects:

Glycolierral 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 Glycolierral 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.
It is concluded that this test is valid and that Glycolierral 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 cm² cultured skin. After 15 minutes, the substance was removed and cells were cultured for 42 hours. Glycolierral 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 Glycolierral 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 8% whereas the test substance showed cell viability of 27%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.