3-(p-methoxyphenyl)-2-methylpropionaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Nov 2016 to 14 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Test material

Test material information

Constituent 1

Reference substance name:

3-(p-methoxyphenyl)-2-methylpropionaldehyde

EC Number:

226-749-5

EC Name:

3-(p-methoxyphenyl)-2-methylpropionaldehyde

Cas Number:

5462-06-6

Molecular formula:

C11H14O2

IUPAC Name:

3-(4-methoxyphenyl)-2-methylpropanal

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Tissue batch number(s): 16-EKIN-045
- Supplier: SkinEthic Laboratories (Lyon, France)
- The EpiSkin™ tissues surface: 0.38 cm²

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: approximately 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 2 mL assay medium containing 0.3 mg/mL MTT per well. 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.
- Incubation time: 3 hours at 37°C
- 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.
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

TEST FOR SUBSTANCE SPECIFIC INTERFERENCE IN MTT ASSAY
- Test for 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. If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
- 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL AND CONTROLS
10 µL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min exposure with 10 µL undiluted substance

Value:

76.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also the substance is colorless.

Applicant's summary and conclusion

Interpretation of results:

other: Not skin irritating

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

Under the conditions of this study the test item was not considered to be 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™ after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study 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% aqueous SDS) and negative (DPBS) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 562 nm. Data are presented as relative viability (%) (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 76.4%. Under the conditions of this study the test item was not considered to be irritating to the skin, because the relative mean tissue viability was above 50% after 15 min exposure.