1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2020 to 27 July 2020

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

EC Number:

214-946-9

EC Name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Cas Number:

1222-05-5

Molecular formula:

C18H26O

IUPAC Name:

4,6,6,7,8,8-hexamethyl-1H,3H,4H,6H,7H,8H-indeno[5,6-c]pyran

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 20-EKIN-030
- Production date: 21 July 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- Incubation time: 3 h at 37°C
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

ACCEPTANCE CRITERIA
-Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
-Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
-Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is <=50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is >50% of the mean viability of the negative controls

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
Amount(s) applied (volume): 10 μL
NEGATIVE CONTROL
Amount(s) applied (volume): 10 μL PBS
POSITIVE CONTROL
Amount(s) applied (volume or weight): 10 μL (5% solution)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

76.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

6.8%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

84.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

78.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY AND ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.712 and the standard deviation value of the viability was 13.7%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.8% relative to the negative control treated tissues and the standard deviation value of the viability was 2.5%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 8.4%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.608			85.4
	0.729	0.712	0.097	102.4	100	13.7
	0.800			112.4
Positive Control Item	0.067			9.4
	0.048	0.049	0.018	6.7	6.8	2.5
	0.031			4.4
Test Item	0.603			84.7
	0.556	0.548	0.060	78.1	76.9	8.4
	0.484			68.0

OD = Optical Density

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 skin irritant

Remarks:

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

Conclusions:

The substance does not cause skin irritation in the in vitro dermal irritation test (OECD guideline 439).

Executive summary:

The skin irritation potential of test material was tested in accordance to OECD TG 439 in accordance with GLP. Undiluted test item was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. The tissue viability obtained after 15 minutes treatment with test material compared to the negative control tissues was 76.9% (84.7, 78.1 and 68.0%). The standard deviation value of the percentage viability of three tissues treated with positive or negative control was resp. 2.5 and 13.7%. Both the positive (6.8% viability) and the negative control were within the historical control data range for both assays and therefore considered valid, indicating that the test system functioned properly. The relative mean tissue viability of the test item is >50% and therefore, under the experimental conditions reported in this study, the test item was classified as non-irritant.