4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April, 2015 - 22 May, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

1

Reference substance name:

4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

EC Number:

238-620-0

EC Name:

4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Cas Number:

14576-08-0

Molecular formula:

C11H20O

IUPAC Name:

4-(2-methoxypropan-2-yl)-1-methylcyclohex-1-ene

Test material form:

liquid

In vitro test system

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: EPISKIN Small ModelTM
- Supplier : SkinEthic Laboratories, Lyon, France
- EpiSkinTM Tissues (0.38cm2) lot number: 15-EKIN-020

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 25 μL into 12-well plates on top of the skin tissues

Duration of treatment / exposure:

15 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

Run / experiment:

mean compared to the negative control

Value:

7.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Orange Flower Ether compared to the negative control tissues was 7.2%. Since the mean relative tissue viability for Orange Flower Ether was below 50% it is considered to be irritant.

Any other information on results incl. tables

Direct MTT Reduction

Orange Flower Ether was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that Orange Flower Ether did not interact with the MTT endpoint.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 7.2 % after a 15 -minute exposure period and 42 hours post-exposure incubation period.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 24% relative to the negative control treated tissues.The positive control acceptance criterion was therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.957 and the standard deviation was 0.023. The negative control acceptance criterion was therefore satisfied. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD562 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	0.983	0.957	0.023		100
	0.946
	0.940
Positive Control Item	0.271	0.226	0.057	28	24
	0.245			26
	0.162			17
Test Item	0.060	0.069	0.010	6.1	7.2
	0.066			7.0
	0.080			8.5

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Applicant's summary and conclusion

Interpretation of results:

other: Criteria for Cat 2 are met

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

The relative mean tissue viability after 15 minutes treatment with Orange Flower Ether compared to the negative control tissue was 7.2%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be irritant. The substance needs to be classified as Cat 2 and Cat 2A for EU CLP and GHS, respectively.

Executive summary:

The skin irritation potential of the test substance was tested through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean cell viability of 24% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 7.2%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment the substance is considered to be irritant. Based on these results the substance needs to be classified as Cat 2 and Cat 2A for EU CLP and GHS, respectively.