6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-10-2019 till 28-10-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

EC Number:

251-717-2

EC Name:

6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-propionaldehyde

Cas Number:

33885-51-7

Molecular formula:

C12H18O

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)propanal

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:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0ºC
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 run (duplicates were used per exposure period)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%. 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%.
- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

3-minute exposure or 1-hour exposure

Duration of post-treatment incubation (if applicable):

Incubation with 300 μL MTT-medium for 3 hours

Number of replicates:

2 replicates of each

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 3-minute exposure

Value:

90

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 1-hour exposure

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two 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 in the initial experiment was 2.6% and 16.7% of the negative control tissues after 3 minutes and 1 hour respectively.
- See "Any other information on results" for the OD570 values.

ACCEPTIBILITY CRITERIA
a) 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
b) The mean relative tissue viability following the 1-hour exposure to the positive control was 6.4%.
c) In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 11%.

Any other information on results incl. tables

Table 1: Mean Absorption in the in vitro Skin Corrosion Test with Pino Acetaldehyde

	3 min			1 hour
	A (OD 570)	B (OD 570)	Mean ± SD	A (OD 570)	B (OD 570)	Mean ± SD
Negative control	1.916	2.058	1.987±0.100	1.941	1.934	1.937±0.005
Test item (1)	1.899	1.697	1.798±0.142	1.873	1.917	1.895±0.031
Positive control	0.161	0.156	0.159±0.004	0.140	0.110	0.125±0.021

SD = Standard deviation

Duplicate exposures are indicated by A and B.

(1) The test item values are corrected for the non-specific MTT reaction (2.6% and 16.7% at the 3 minute and 1 hour treatment, respectively).

In this table the values are corrected for background absorption (0.0456 and 0.0467 for the 3 minute and 1 hour treatment, respectively). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to the skin

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance is not corrosive to the skin.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour in a study performed according to OECD TG 431 and following GLP. The test item was applied undiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 6.4% after the 1-hour exposure. 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. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤11%, indicating that the test system functioned properly. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two 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 2.6% and 16.8% of the negative control tissues after 3 minutes and 1 hour respectively. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 90% and 98%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.