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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Oct 2016 to 25 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 23, 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
ENVIGO CRS GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf

Test material

1
Chemical structure
Reference substance name:
3-(p-cumenyl)propionaldehyde
EC Number:
231-885-3
EC Name:
3-(p-cumenyl)propionaldehyde
Cas Number:
7775-00-0
Molecular formula:
C12H16O
IUPAC Name:
3-[4-(propan-2-yl)phenyl]propanal
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:
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Supplier: SkinEthic Laboratories (69007 Lyon, France)
- The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
- EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels.
- Delivery date: 22 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37±1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were immediately removed from the 12- well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 2 mL assay medium containing 0.3 mg/mL MTT per well.
- Incubation time: 3hrs at 37±1.5°C
- Extraction of formazan: The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for nearly 3 hours while shaking at room temperature.
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Wavelength: 570 nm

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
- Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the colourless test item (10 µL) was mixed with 90 µL of deionised water in a pre-experiment. The test item/water mixture was gently shaken for 15 minutes at room temperature.
- For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item was added to 2 mL of MTT solution (0.3 mg/mL) and the mixture will be incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution with 10 µL of DMEM was used as the control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42.25 hrs
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:
23.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 (0.612-0.617) for the 15 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% (< 40%) thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 13% (≤ 18%), thus ensuring the validity of the study.

Applicant's summary and conclusion

Interpretation of results:
other: Skin irritant category 2
Remarks:
in accordance with CLP (1272/2008 and its updates)
Conclusions:
Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 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 42.25 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% SLS solution in deionized water) and negative (deionized water) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm.Tests for direct MTT reduction and colour interference were negative.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 23.8%. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.