2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-00-22 to 2016-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Reconstructed Human Epidermis (RHE) Test Method, adopted July 28, 2015.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

EC Number:

815-462-6

Cas Number:

124451-79-2

Molecular formula:

C17H26N2O5

IUPAC Name:

2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Test material form:

other: liquid

Details on test material:

2-({[3-(isocyanatomethyl)-3, 5, 5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate of Evonik Degussa GmbH Batch SB 4195 of 28 March 2016

Specific details on test material used for the study:

The test item was applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum.

In vitro test system

Test system:

human skin model

Cell type:

other: three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum

Details on animal used as source of test system:

Human skin model

Justification for test system used:

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.

Vehicle:

unchanged (no vehicle)

Details on test system:

The following Reconstructed Human Epidermis Model was used:
EpiDerm™ (EPI-200-SCT, Lot no. 23344) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.


Cell viability measurements
- MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) reduction, which had been shown
to give accurate and reproducible results, was used to measure cell viability
- Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours
- The precipitated blue formazan product was extracted using the solvent propanol-2, and the concentration of the formazan was measured by
determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer
- Cell viability measurements were carried out at the end of the exposure period (1st period: 3 min; 2nd period: 60 min). The measurements were
made for each of the two tissues in triplicate.
- Previous checks for interference of the test item with the MTT assay, the nylon mesh or the tissues were performed. No discoloration or test item
interference with the vital dye was noted.

ADMINISTRATION
- EpiDerm tissues were conditioned by pre-incubation (1 hour) in Maintenance Medium1 for release of transport stress related compounds and
debris in the incubator (37°C, 5% CO2, 95% humidity).
- After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 1 h,
respectively.
- Two tissues were used per treatment, negative and positive control and exposition time (12 tissues in total).
- 50 µL of the supplied test item were applied to the skin model with a surface area of 0.63 cm2 (corresponding to a minimum of 70 µL/cm2).
- Positive control item was 8 N KOH4, 50 µl
- Negative control was sterile deionised water, 50µl
- After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium2 (final concentration: 1 mg
MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium).
- After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS), blotted and the blue formazan salt was extracted with propanol-2.
- The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as
% of the mean of the negative control tissues.
- Skin corrosion potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure
with the test chemical.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- 50 µL of the supplied test item were applied to the skin model with a surface area of 0.63 cm2 (corresponding to a minimum of 70 µL/cm2).
- Two replicate tissues for each treatment (exposure periods) were employed.
- At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS) .
- Concurrent negative and positive controls were used, each in duplicate.
- The positive control item was 8N KOH and the negative control was sterile deionised water.
- For the controls, a dose volume of 50 µL was used.
- The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

Two tissues were used for each treatment and concurrent control groups.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: cut-off percentage cell viability value

Run / experiment:

The EpiDerm™ model was employed. 3 minute exposure

Value:

108.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value = 108.2 %) or 1 hour (cell viability value = 84.3 %) was non-corrosive to skin.

Results 2

Irritation / corrosion parameter:

other: cut-off percentage cell viability value

Run / experiment:

The EpiDerm™ model was employed, 1-hour exposure

Value:

84.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Under the present test conditions test item tested at two exposure periods of 3 minutes (cell viability value = 108.2 %) or 1 hour (cell viability value = 84.3 %) was non-corrosive to skin.

Other effects / acceptance of results:

no other effects

Any other information on results incl. tables

Assay acceptability criteria

Assay acceptance criterion 1: Negative control

The absolute OD of the negative control (NC) tissues (treated with sterile deionised water) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The tissues treated with the negative control should not be below historically established boundaries.

The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 0.8 and ≤ 2.8.

Assay acceptance criterion 2: Positive control

A8N KOHwas used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.

Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1hour exposure: < 15%).

Assay acceptance criterion 3:variability between tissue replicates

Associated and appropriate measures of variability between tissue replicatesshould not exceed 30% (in the range of 20 – 100% viability).

Interpretation of results

The OD values obtained for each test sample was used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items (or discriminating between different corrosive classes, e.g. subcategories 1A, 1B and 1C, or the statistical procedure(s) used to evaluate the results and identify corrosive materials, is clearly defined and documented, and shown to be appropriate. The criteria of corrosivity associated with the EpiDermTM model are as follows:

- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A8), if the viability after 3

minutes exposure is less than 50%;

- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes

exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;

- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and

the viability after 1 hour exposure is greater than or equal to 15%.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to assess the corrosive properties of test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EpiDerm™ model was employed.

Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

50 µL of the supplied test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. Sterile deionised water was used as the negative control. 8N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 108.2% after a 3-minute exposure period and 84.3% after a 1‑hour exposure. The 3-minute and the 1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥50% and ≥15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean optical density (OD) of the negative control of 2 tissues was 1.536 (3‑minute exposure) or 1.667 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The viability of cells treated with the positive reference item 8N KOH were 9.9% (3-minute exposure) and 6.1% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.

The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

Conclusion

Under the present test conditions test item tested at two exposure periods of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.