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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 February 2017 - 17 March 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-ethynylcyclohexyl acetate
EC Number:
EC Name:
1-ethynylcyclohexyl acetate
Cas Number:
Molecular formula:
1-ethynylcyclohexyl acetate
Test material form:

In vitro test system

Test system:
human skin model
Source species:
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable.
unchanged (no vehicle)
Details on test system:
- Model used: EPISKIN Small Model, 0.38 cm^2
- Tissue batch number: 17-EKIN-011
- Delivery date: 14 March 2017

- Temperature used during incubation: 37 ± 1.5°C

Assessment of Color Interference with the MTT endpoint:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the 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.
The test item did not dye water and the colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. An additional test with viable tissues (without MTT addition) to determine a correction factor for calculating the true viability in the main experiment did not have to be performed.
Test for Direct MTT Reduction:
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.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment did not have to be performed.

Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for about 22.5 hours.

The negative control, positive control and the test item were added into the inserts atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were treated for 15 minutes.
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. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42.5 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials.
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 3.5 hours while shaking at room temperature.
Per tissue sample 2 x 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were calculated from the 2 wells per tissue sample.

For the current test, a test item is considered irritant if the mean relative tissue viability of three individual tissues is reduced to <= 50% of the negative control.
For the current test, a test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material:
- Applied volume: 10 μL
Duration of treatment / exposure:
15-Minute exposure period.
Duration of post-treatment incubation (if applicable):
Approximately 42.5 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: mean relative viability (%)
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: The mean relative tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
Colour interference and Direct MTT Reduction:
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.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

Test item:
The mean relative absorbance value of the test item, corresponding to the cell viability, was reduced to 6.4% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Quality Criteria:
After treatment with the negative control the absorbance values (OD 0.760 - 0.888) were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% (criterion: ≤ 40%) thus ensuring the validity of the test system.
The rel. standard deviations between the % variability values of the test item, the positive and negative controls were below 9% (criterion: ≤ 18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Mean Absorbance (OD570) Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Dose Group

Absorbance 570 nm
Tissue 1**

Absorbance 570 nm
Tissue 2**

Absorbance 570 nm
Tissue 3**

Mean Absorbance of 3 Tissues

Relative Absorbance [%] Tissue 1, 2 + 3***

Standard Deviation

Relative Standard Deviation [%]

Rel. Absorbance

[% of Negative Control]****

Negative Control









Positive Control









Test Item









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

** = Mean of two replicate wells after blank correction

*** = Relative absorbance per tissue: 100 x (absorbance tissue / mean absorbance negative control)

**** = Relative absorbance per treatment group: 100 x (mean absorbance test item / mean absorbance negative control)

Since the results derived from the MTT assay were not unclear or borderline, the IL-1 α concentration in the medium was not determined.

Applicant's summary and conclusion

Interpretation of results:
other: Skin irritant, Category 2
According to EU CLP Regulation (EC) No. 1272/2008 and its amendments.
After treatment with the test item the mean relative tissue viability decreased to 6.4%. Since this value is below the threshold for irritancy of ≤ 50% the substance is considered to be irritant to skin.
Executive summary:

The possible skin irritation potential of the substance was tested in vitro, in compliance with OECD TG 439 and GLP principles. Each three tissues of the human skin model EpiSkin™ were treated by topical application of 10 μL of the undiluted test item, the negative control (deionised water), or the positive control (5% sodium lauryl sulfate) for 15 minutes. After approximately 42.5 hours post-exposure incubation period, determination of the cell viability was performed. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison with untreated negative controls is used to predict skin irritation potential. After treatment with the test item the mean relative tissue viability decreased to 6.4%. This value is below the threshold for irritancy of <= 50%. Therefore, the test item is considered to be a skin irritant. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD >= 0.6 till <= 1.5 (actual values 0.760 – 0.888), the mean relative tissue viability of the positive control was <= 40% (3.9%) and the rel. standard deviations in between tissues of the same treatment group were <= 18% (< 9%), indicating that the test system functioned properly.