Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March, 2016 - 21 March, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

(2015)

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:

Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

EC Number:

904-551-6

Molecular formula:

C16H24O

IUPAC Name:

Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Test material form:

liquid

In vitro test system

Test system:

human skin model

Test animals

Species:

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

EPISKIN Small ModelTM
Supplier : SkinEthic Laboratories, Lyon, France
EpiSkin-SMTM Tissues (0.38cm^2) batch number : 16-EKIN-011

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.3 - 37.3
- Humidity (%): 67 - 87

Test system

Type of coverage:

other: None; Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.

Preparation of test site:

other: The test item was applied topically to the corresponding tissues ensuring uniform covering.

Vehicle:

unchanged (no vehicle)

Controls:

other: 3 Tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively.

Amount / concentration applied:

Test material
- Applied volume: 25 μL

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of animals:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay

The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction and colour interference:
The test substance (multi-constituent) was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of the test substance (multi-constituent) was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
To assess the ability of the test item to reduce MTT, 25 μL of the test item 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. At the end of the incubation period a colour check was performed.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Run / experiment:

Time point: 15 minutes exposure.

Value:

97

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The relative mean tissue viability compared to the negative control tissues (100%).

In vivo

Irritant / corrosive response data:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance (multi-constituent) compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test substance (multi-constituent) was above 50% the test substance (multi-constituent) is considered to be non-irritant.

Any other information on results incl. tables

Direct MTT Reduction

The test substance (multi-constituent) was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that the test substance (multi-constituent) did not interact with the MTT endpoint.

Test Item, Positive Control Item and Negative Control Item

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance (multi-constituent) compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test substance (multi-constituent) was above 50% the test substance (multi-constituent) is considered to be non-irritant.

Quality Criteria

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

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	1.066	1.047	0.022		100
	1.052
	1.024
Positive Control Item	0.398	0.462	0.078	37	44
	0.549			52
	0.437			43
Test Item	0.815	1.018	0.176	76	97
	1.116			106
	1.123			110

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

other: Not a skin irritant

Remarks:

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

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance (multi-constituent) compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test substance (multi-constituent) was above 50% the test substance (multi-constituent) is considered to be non-skin irritant.

Executive summary:

The possible skin irritation potential of the test substance (multi-constituent) was tested in vitro using a human skin model 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. Reliable negative and positive controls were included. The positive control had a mean cell viability of 44% 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 97%. Since the mean relative tissue viability for the test substance (multi-constituent) was above 50% after 15 minutes treatment the substance is considered to be not a skin irritant.