Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 to 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.431 and under GLP compliance.

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Dated to 29 July 2016.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

EC Number:

252-335-9

EC Name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

Cas Number:

35044-59-8

Molecular formula:

C12H18O2

IUPAC Name:

ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Physical appearance: colourless to pale yellow liquid
- Stored in a dry, well ventilated and dark location at room
temperature (i.e. 10–30°C)

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis (EpiDerm Skin Model)

Cell type:

other: EpiDerm Skin Model

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
- The EpiDerm Skin Model (EPI-200, Lot no.: 24940 kits J and K) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, U.S.A.
- Rationale: Recommended test system in international guidelines (OECD and EC).

CELL CULTURE
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium (Supplemented DMEM medium, serum-free supplied by MatTek Corporation). The plates were incubated for approximately 1.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 47 - 82%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EVALUATION OF DIRECT INTERACTION WITH MTT
- Colour interference by the test item in aqueous conditions: To assess the colour interference, 50 μl of the test item or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Reduction of MTT by the test item: To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.
> Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

TREATMENT
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively.

REMOVAL OF TEST MATERIAL
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM), both
supplied by MatTek Corporation, was prepared.
The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

PREDICTION MODEL / DECISION CRITERIA:

- Acceptability of the assay:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

In addition to the quality criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.

- Data evaluation and statistical procedures:
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute
treatment is considered corrosive if the relative tissue viability after 1-hour treatment with
the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the
negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below
15%.

The following information presents the data interpretation and optional sub-categorisation in case a test item will be corrosive.

Viability measured after 3-minutes and 1 hour

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 25% after 3 min exposure ==> Optional Sub-category 1A
≥ 25% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

During 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C± 1°C, 5% CO2.

Number of replicates:

2 living human skin models for each time

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 3 minutes

Results 2

Irritation / corrosion parameter:

% tissue viability

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 60 minutes

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 95%, versus 7% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 112 %, versus 14% in the positive control.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	± SD	Mean viability %	Viability difference between replicates %
Negative control	A	1.7327 1.7449 1.7441	1.698	1.658	± 0.056	100.00	4.7
	B	1.6579 1.6502 1.6745	1.6118
Positive control	A	0.1701 0.1674 0.1657	0.125	0.120	± 0.008	7	9.0
	B	0.1577 0.1562 0.1553	0.114
Test item	A	1.6523 1.7420 1.6830	1.650	1.567	± 0.117	95	10
	B	1.5378 1.5326 1.5101	1.484

 

 

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	± SD	Mean viability %	Viability difference between replicates %
Negative control	A	1.4840 1.4800 1.4840	1.440	1.406	± 0.049	100.00	4.8
	B	1.4386 1.3726 1.4303	1.371
Positive control	A	0.2751 0.2888 0.2664	0.234	0.202	± 0.046	14	28
	B	0.2121 0.2125 0.2126	0.170
Test item	A	1.6631 1.6308 1.5944	1.587	1.578	± 0.013	112	1.2
	B	1.6585 1.5719 1.6022	1.568

SD: standard deviation

 

In these tables the values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.

 

Acceptability criteria:

- The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. - The mean relative tissue viability following the 1-hour exposure to the positive control was 14% ( < 15%).
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 10%, indicating that the test system functioned properly (≤ 30%).

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

Executive summary:

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item Ethyl Safranate was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (EpiDerm (EPI-200)) for 3 minutes and 1 hour. The application was followed by a rinse with PBS to remove residual test item. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, the remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control).

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 95% and 112% versus 7% and 14% respectively, with the positive control.

Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

 

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required.