2-benzyl-4,4,6-trimethyl-1,3-dioxane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-30 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-benzyl-4,4,6-trimethyl-1,3-dioxane

EC Number:

266-795-3

EC Name:

2-benzyl-4,4,6-trimethyl-1,3-dioxane

Cas Number:

67633-94-7

Molecular formula:

C14H20O2

IUPAC Name:

2-benzyl-4,4,6-trimethyl-1,3-dioxane

In vitro test system

Test system:

human skin model

Source species:

other: human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): 16-EKIN-021
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

PRE-TEST PROCEDURE
- Pre-incubation: On day of receipt the tissues were transferred into a 12-wells plate filled with maintenance medium. A different 12-wells plate was used for the test and each control item were. The tissue were incubated at 37°C, 5% CO2 in air overnight.
- Test item colour interference: To assess colour interference, 10 μL of Peomosa (mono-constituent) was added to 90 μL sterile water. The mixture was mixed for approximately 15 minutes. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 10 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C, 5% CO2 in air. Untreateed MTT solution was used as a control. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure/ post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: not specified
- Wavelength: 562 nm (without reference fildeter)

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubat
ion is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of
post incubation is > 50% of the mean viability of the negative controls.

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 OD562 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% (w/v)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

26.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.9

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
-The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
-The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
-The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.0%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD562 Values and 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	1.265 / 1.345 /1.269	1.293	0.045	97.8 / 104.0 / 98.1	100∗	3.5
Positive Control Item	0.076 / 0.058 / 0.055	0.063	0.011	5.9 / 4.5 /4.3	4.9	0.9
Test Item	0.365 / 0.333 / 0.355	0.348	0.013	27.5 / 25.8 / 27.5	26.9	1.0

OD = Optical Density

SD = Standard deviation

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

⊕ = The control groups were shared with Envigo - Shardlow study number TW67BS, MS48XJ, FC59GT, LL32QY, RS46VL, JD61DM and NL15CD.

Applicant's summary and conclusion

Interpretation of results:

other: Category 2 (skin irritant)

Remarks:

based on CLP criteria (EC 1272/2008 and its amendments)

Conclusions:

Under the conditions of this test, the relative mean tissue viability for the test item determined to be 26.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Reseda body is an irritant to skin (Category 2 according to the CLP Regulation).

Executive summary:

The possible skin irritation potential of Reseda body was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. 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. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.9% 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. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 26.9%. This value is below the threshold for irritancy of ≤50%.