2,6-dimethyloct-7-en-2-yl formate

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 February 2018 - 07 May 2018

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dimethyloct-7-en-2-yl formate

EC Number:

246-788-1

EC Name:

2,6-dimethyloct-7-en-2-yl formate

Cas Number:

25279-09-8

Molecular formula:

C11H20O2

IUPAC Name:

2,6-dimethyloct-7-en-2-ol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): N°18-RHE-029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test is considered to be irritant to skin, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
The test item is considered as non-irritant to skin, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 ± 2 mg of test item/0.5 cm2

Duration of treatment / exposure:

42 minutes

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:

1 (mean of 3 replicates)

Value:

27.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was not observed between negative control (isopropanol) and dihydromyrcenyl formate. The test item did not form color in isopropanol, therefore results shows no interference in OD due to test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 618-1-06-9641

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 1.2 at 570 ± 30 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18%. The OD values (Corrected ODs) of negative controls in all tissues were between 1.221 to 1.273

- Acceptance criteria met for positive control:
The data met the acceptance criteria since the mean viability, expressed as % of the NC, was < 40 % and the Standard Deviation value is ≤ 18 %. Mean % viability of the positive control was 1.5%

- Acceptance criteria met for variability between replicate measurements:
Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 18%.

- Range of historical values if different from the ones specified in the test guideline:
Optical Density at 570±30 nm
Exposure time: 42-minute exposure time
Negative Control (Dulbecco's phosphate-buffered saline):
Mean: 2.081
Standard deviation: 0.208
Minimum: 1.964
Maximum:2.776
Positive Control (Sodium dodecyl sulfate, 5% aqueous)
Mean: 0.028
Standard deviation: 0.007
Minimum: 0.021
Maximum: 0.053

Any other information on results incl. tables

Data summary of percent viability:

Treatment	Tissue Replicate	O.D. at 570 nm	Blank Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Three Tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	C.V. of % Viability	Corrosivity Class
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS))	1	1.274	1.231	1.238	1.238	100	100	0.01	0.81	NA
		1.282	1.239
		1.287	1.244
	2	1.280	1.237	1.231
		1.264	1.221
		1.277	1.234
	3	1.270	1.227	1.244
		1.276	1.233
		1.316	1.273
Dihydromyrcenyl Formate	1	0.361	0.319	0.336	0.342	27.1	27.6	0.61	2.21	Category 2
		0.384	0.342
		0.388	0.346
	2	0.381	0.339	0.34		27.5
		0.384	0.342
		0.382	0.340
	3	0.395	0.353	0.35		28.3
		0.388	0.346
		0.393	0.351
Positive control (Sodium dodecyl sulphate (5% aq.))	1	0.061	0.018	0.018	0.018	1.5	1.5	0	0.00	Category 2
		0.061	0.018
		0.060	0.017
	2	0.062	0.019	0.019		1.5
		0.062	0.019
		0.061	0.018
	3	0.061	0.018	0.018		1.5
		0.061	0.018
		0.061	0.018

Keys: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable

Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The substance was determined to be irritating to the skin.

Executive summary:

An in-vitro Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 439 (GLP study). Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate, 5% aqueous (SDS)) and test item in triplicate for 42 minutes at room temperature. The mean cell viability in tissues treated with the test item was 27.6% after 42 minutes exposure. A significant reduction in percent cell viability was observed in treated tissues when compared with the concurrent negative control. The Optical density (OD) values for the negative control replicates were between 1.221 to 1.273, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blank was between 0.041 to 0.045 which met the guideline requirement of OD < 0.1. The positive control showed a 1.5% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, compared to concurrent negative control. Variation between tissue replicates (i.e. CV% value) was 4.54% for the test item group, 0.00% for positive control and 0.81% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTM RHE model. All criteria for a valid study were met. Based on these results, the test item was determined to be irritating to the skin (category 2).