3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers reaction product with 3-(cyclohexylamino)propane-1-sulfonic acid and N,N-dimethylcyclohexanamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid - solid: mixture of

Specific details on test material used for the study:

- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Test system epiCS has been validated for skin corrosion in a multicenter validation study and is commercially available.

Vehicle:

unchanged (no vehicle)

Details on test system:

DETAILS OF THE TEST PROCEDURE USED
The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item. All tests were performed in triplets for each time point. The test item was applied unchanged, i.e. 50 µl per insert for 3 min. (room temperature) and 60 min (incubator conditions). Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density compared to negative control).

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature) for 3 min.-exposure and 37 ± 2° C (incubator conditions) for 60 min.-exposure

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Incubation conditions: 37 ± 2° C, 5 % CO2, maximum humidity
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
3 for test substance and negative control (for determination of cell viability the absorption of the isopropanol-extracts were measured in duplicates = 6 OD values for each)

DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Classification according to UN GHS: The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (Category 1A), or 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% (Category 1B/1C). The test substance 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%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

50 μL per insert

Duration of treatment / exposure:

3 min. (room temperature) and 60 min. (37 °C)

Duration of post-treatment incubation (if applicable):

post-exposure incubation: none

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of exposure period 3 min.

Value:

76.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: no indication of corrosive potential

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of exposure period 60 min.

Value:

79.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: no indication of corrosive potential

Other effects / acceptance of results:

- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The reliability of the test conduction was previously confirmed in an interlaboratory validation study.

Any other information on results incl. tables

Summary of results

Sample No.	Test item	Time [min.]	OD mean*	StdDev	% Viability
1 -3	Negative control (NaCl 0.9 %)	60	2.51	0.02	100
4 -6	Test item, undiluted	60	2.00	0.09	79.8
10 -12	Negative control (NaCl 0.9 %)	3	2.612	0.14	100
13 -15	Test item, undiluted	3	2.00	0.15	76.5

*: 6 values

Applicant's summary and conclusion

Interpretation of results:

other: negative

Executive summary:

An in vitro study was performed for the assessment of skin corrosion. The experiment was carried out in accordance to OECD TG 431 using a commercially available reconstructed human epidermis test (RhE; epiCS®). In that study a test item with the substance at approx. 70 % (= "active ingredient") in approx. 30 % solvent was used. 50 µL of this test item was applied topically on the RhE. After an exposure period of 3 minutes (room temperature) and 60 minutes (37 °C; incubator conditions), the cell viability was 76.5 % and 79.8 %, respectively, as measured by a MTT conversion assay. The test item was thus not considered to have a corrosive potential to the skin.