4-tert-pentylcyclohexanone

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan 2017 to 10 Feb 2017

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:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Test material

Test material information

1

Reference substance name:

4-tert-pentylcyclohexanone

EC Number:

240-642-0

EC Name:

4-tert-pentylcyclohexanone

Cas Number:

16587-71-6

Molecular formula:

C11H20O

IUPAC Name:

4-tert-pentylcyclohexanone

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
- Tissue batch number(s): 17-EKIN-006
- 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 in a 12-well plate
- Delivery date: 07 February 2017.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
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
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Wavelength: 570 nm
- 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL AND CONTROLS
10 µL

Duration of treatment / exposure:

15 min.

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:

15 min exposure with 10 µL undiluted substance

Value:

9.4

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 its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

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™ 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% 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. Data are presented in the form of 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 9.4%. 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.