[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2021 - 24 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

EC Number:

275-156-8

EC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Cas Number:

71048-82-3

Molecular formula:

C13H20O

IUPAC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 34746 kit E, 34779 kit F and 33786
- Production date: 7 April 2021, 19 May 2021, 26 April 2021
- Surface: 0.6 cm²
The model 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.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 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 71 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 - 37.3°C). .

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL
- Incubation time: 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.
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- In triplicate

NUMBER OF REPLICATE TISSUES: 4 tissues per test item together with a negative and positive control. 2 replicates per exposure duration (3 minutes and 1 hour) , two positive controls, two negative controls.

In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES: 1

ACCEPTABILITY CRITERIA
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 laboratory historical control data range and the acceptance limits of OECD431 (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 case of color interference and/or MTT interacting test items:
d) The %NSC should be ≤ 30% relative to the negative control OD.
e) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after the 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 mean 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 mean tissue viability after the 1-hour treatment is not decreased below 15%.

INTERPRETATION
See table 1 (Any other information on methods incl. Tables)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL undiluted test item

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water

POSITIVE CONTROL
- Amount applied: 50 μL 8N KOH

Duration of treatment / exposure:

3 minutes or 1 hour

Number of replicates:

2 replicates per exposure duration (4 total), two negative controls, 2 positive controls

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

125

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

113

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0024 and -0.0018, respectively. Therefore it was concluded that the test item did not induce color interference.

In addition, because a color change was observed in the presence of MTT it was concluded that the test item interacted with the MTT endpoint.
Since the test item did interact with MTT, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.5% and 1.0% of the negative control tissues after 3 minutes and 1 hour respectively. The ODs of the test item treated viable tissues were corrected using he OD of the freeze-killed tissues.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. 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 113% and 125% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

ACCEPTANCE OF THE RESULTS
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD TG 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 9.0%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1. Mean Absorption in the in vitro Skin Corrosion Test

	3-minute application			1-hour application
	A (OD570)	B (OD570)	Mean (SD) (OD570)	A (OD570)	B (OD570)	Mean (SD) (OD570)
Negative control	1.960	1.688	1.824 (0.193)	1.707	1.709	1.708 (0.001)
Test item (1)	2.081	2.041	2.061 (0.029)	2.158	2.106	2.132 (0.037)
Positive control	0.104	0.131	0.117 (0.019)	0.148	0.161	0.155 (0.009)

SD = Standard deviation

Duplicate exposures are indicated by A and B.

(1) The test item values are corrected for the non-specific MTT reaction.

 

Table 2. Mean Tissue Viability in the in vitro Skin Corrosion Test

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	113	125
Positive control	6.4	9.0

 

Table 3. Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	14	0.1
Test item	2.0	2.4
Positive control	21	8.0

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The test substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (deionised water) and a positive control (8.0 N KOH) for 3 minutes and 1 hour. The test item did not induce color interference but it did interact with MTT. An additional test was performed with freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were for the cytotoxicity evaluation with MTT at each time point. The ODs of the test item treated viable tissues were corrected using he OD of the freeze-killed tissues Acceptability criteria for the negative control, positive control and variability between measurements were met. The cell viability of the tissues exposed to the substance were 113% and 125% for 3 minutes and 60 minutes exposure, respectively. Both values were not below the threshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.