Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 29 November 2016 and 05 Decemebr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

when applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

EC Number:

949-881-1

Molecular formula:

C11H18O2

IUPAC Name:

Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No. NG342-12
- Expiration date of the lot/batch: 01 September 2017
- Purity test date:07 September 2016
- Purity: 96.6%
- Appearance: clear colorless liquid
- Storage condition of test material: Approximately 4”C in the dark

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™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 29 November 2016

EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-048
Maintenance Medium lot number :16-MIAN3-079
Assay Medium lot number : 16-ESSC-051

Test system

Type of coverage:

other: Dissiminated (delete when editing)

Preparation of test site:

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

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Dissiminated see also puzzle pieces (delete when editing)
Test material
- Applied volume: 10 ul
- Concentration (if solution): undiluted

The negative control item, DPBS, was used as supplied.
- Applied volume: 10 ul

The positive control item, SDS, was prepared as a 5% w/v aqueous solution.
- Applied volume: 10 ul

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:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours.
Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarosegel and the insert:

Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2.0 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of three wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

MAIN TEST:
APPLICATION OF TEST ITEM AND RINSING (DAY 1):
2.0 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a
pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. Residual test item remained on the tissues after rinsing. The rinsed tissues were transferred to the second column of 3 wells containing 2.0 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.


MTT LOADING/FORMAZAN EXTRACTION (DAY 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.

2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry.

A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at
562 nm (without a reference filter) using the Anthos 2001 microplate reader.

DATA EVALUATION
Quantitative MTT Assessment (Percentage Tissue Viability)
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562of test item / mean OD562of negative control) x 100

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative mean viability

Value:

ca. 115.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11.4%

Remarks on result:

other:

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was colorless.  It was therefore unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 115.2% after a 15 Minute exposure period and 42 Hour post exposure incubation period.

It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 11.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 6.1 %. The positive control acceptance criterion was therefore satisfied.

The mean OD562for the negative control treated tissues was 0.731 and the standard deviation value of the percentage viability was 5.7 %. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 9.3 %. The test item acceptance criterion was therefore satisfied.

Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.683	0.731	0.041	93.4	100*	5.7
	0.751			102.7
	0.758			103.7
Positive Control Item	0.133	0.083	0.044	18.2	11.4	6.1
	0.050			6.8
	0.065			8.9
Test Item	0.900	0.842	0.068	123.1	115.2	9.3
	0.767			104.9
	0.859			117.5

SD = Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

other: EU CLP Criteria not classified for irritation

Remarks:

UN GHS Not classified for Irritation (category 3 can not be determined).

Conclusions:

The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissue was 115.2%. Since the mean relative tissue viability for the substance was above50% after 15 minutes treatment, the substance is considered to be none irritant.

Executive summary:

The possible skin irritation potential of the substance was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD TG 439. Skin tissue was treated by topical application of 10 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 relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 115.2%.

Since the mean relative tissue viability for Cashmeran was than 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a mean cell viability of 11.4% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.