2-phenylpropionaldehyde-dimethyl acetal

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylpropionaldehyde-dimethyl acetal

EC Number:

202-022-8

EC Name:

2-phenylpropionaldehyde-dimethyl acetal

Cas Number:

90-87-9

Molecular formula:

C11H16O2

IUPAC Name:

(1,1-dimethoxypropan-2-yl)benzene

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

PRE-TESTS
- Direct MTT Reduction: 10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
- Assessment of Color Interference with the MTT endpoint: 10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

MAIN TEST
- Day 0: 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.
- Day 1: 2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.
- Day 3: Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
- Day 6: At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
- For the test item the relative mean tissue viabilities obtained were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way: Relative viability (%) = [mean OD (test item / positive control ) / mean OD negative control] * 100.
- Classification of irritation potential is based upon relative mean tissue viability according to the following criteria: The test chemical is considered to be irritant to skin if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%. The test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL (26.3 μL/cm2)

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

Value:

67.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Direct MTT Reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Assessment of Color Interference with the MTT endpoint: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.
- Quality Criteria: The relative mean tissue viability for the positive control treated tissues was 3.4% 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 OD570 for the negative control treated tissues was 0.892 and the standard deviation value of the viability was 2.5%. 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 12.3%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: Not a skin irritant

Remarks:

according to EU CLP (EC 1272/2008 and its amendments)

Conclusions:

Under the conditions of this study the test item was considered to be not irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.

Executive summary:

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL undiluted test item for an exposure period of 15 minutes. Concurrent positive (5% SDS solution in aqueous solution) and negative (Dulbecco’s Phosphate Buffered Saline) controls were included. The optical density was measured at 570 nm. Tests for direct MTT reduction and colour interference were negative. The relative mean viability of the test item treated tissues was 67.2%. Under the conditions of this study the test item was considered to be not irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.