Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

EC Number:

946-245-5

Molecular formula:

C12H22O

IUPAC Name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

Test material form:

liquid

Details on test material:

According to ECHA communication with reference to Annotation number: SUB-C-2114629169-42-01/F, the substance ID for Koavone has been changed with EC# from 939-627-8 to 946-245-5, and the IUPAC name from Reaction mass of (3R,5R)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (3R,5S)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one to Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one.

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

Preliminary tests
The tissue staining potential of the test substance was investigated by incubating 30 μL of the test substance in 300 μL demineralized water for ca. 1 hour at ca. 37 ºC and 5% CO2. At the end of the exposure period the presence and intensity of the staining (if any) was evaluated visually. The test substance did not significantly change the colour of the solution. Therefore, it was concluded that the test substance did not have the potential to stain the tissue.

Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, prior to the start of the study, 30 μL of the test substance were incubated in 1 μL 1 mg/μL MTT solution for ca. 3 hours at ca. 37 ºC and 5% CO2 and the formation of a blue formazan product was assessed visually. During incubation the test substance solution did not turn blue/purple and therefore it was concluded that the test substance did not reduce MTT.

A nylon mesh was applied to facilitate equal distribution of liquid test substances. To test if the test substance interacts with a nylon mesh, 30 μL of the test substance was applied to a nylon mesh. After ca. 60 min incubation at room temperature possible interaction with the mesh was checked under a microscope. The test substance did not show interaction with a nylon mesh. Therefore, the nylon mesh was used to facilitate equal distribution of the test substance.

EPI-200 skin model
The EpiDerm(TM) (EPI-200) skin model consists of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models were preincubated in 0.9 mL assay medium provided with the kits for 60 min in a humidified incubator (ca. 37 ºC and 5% CO2). At the end of the first pre-incubation period, the tissues were pre-incubated overnight in fresh assay medium for ~22.5 hours in a humidified incubator (ca. 37 ºC and 5% CO2).

Exposure to study substances
Following the second pre-incubation, the skin models were exposed to 30 μL of the negative control, test substance or positive control. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of liquid substances. Exposure was initiated at ambient temperature in the flow cabinet. After dosing the last tissue, all plates were transferred to a humidified incubator (ca. 37 ºC and 5% CO2). After 35 min, the plates were removed from the incubator and placed in the flow cabin until the period of 60 min was completed for the first dosed tissue. When the exposure period was completed, the inserts were removed from the well and the skin surface was carefully washed using excess of phosphate buffered saline.

Subsequently, the insert was blotted dry and the skin membranes were carefully dried using a sterile cotton swab. The inserts were then transferred to a clean 6-well plate containing fresh medium (0.9 mL/well) and incubated in a humidified incubator (ca. 37 ºC and 5% CO2). Medium was refreshed after ~23.5 hours. Following an additional ~18 hours incubation period, viability was determined with MTT assay.

Interpretation of the results
The test is considered valid if the optical density of the negative control is ≥ 1.0 and ≤ 2.5, the optical density of the extractant solvent alone is < 0.1, if skin models treated with the positive control demonstrate a mean tissue viability ≤ 20% compared to the negative control, and if the standard deviation calculated from individual tissue viability percentages of the three replicates is < 18%. The in vitro irritation potential of the test substance is determined from the relative mean tissue viabilities compared to the negative control tissues using the following prediction model:
Mean tissue viability ≤ 50%: irritant, UN GHS category 2
Mean tissue viability > 50%: non-irritant, UN GHS no category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

87

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:

The following results were obtained for the substance and positive and negative controls:

Mean tissue viability ± SD
- Substance: 87 ± 8%
- Phosphate-buffered saline: 100 ± 6%
- 5% sodium dodecyl sulphate: 4 ± 0%

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

In an in vitro skin irritation test using EpiDerm(TM) reconstructed skin membranes (EpiDerm(TM); OECD TG 439), application of the test substance directly to the skin tissue resulted in a mean of 87% cell viability (percentage of control) after an incubation period of 42 hours. The substance is therefore considered to be non-irritating to skin.

Executive summary:

Skin irritating properties of the substance were tested in an in vitro skin irritation test using the EpiDerm(TM) reconstructed skin membranes (OECD TG 439) and in compliance with GLP. Skin tissue was exposed to 30 μL of test material for 60 min, washed, incubated in medium for 42 hours and then examined for viability by the MTT assay. Phosphate-buffered saline and 5% sodium dodecyl sulphate were used as positive and negative control, respectively. Mean tissue viability of the positive control was 4%. Tissue viability following exposure to the substance was 87± 8% (mean ± SD) compared to the negative control. As this value was above the limit value of 50%, the substance was considered to be non-irritating to human skin.