Cyclohexyl isocyanate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.11.2022 - 02.12.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

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:

Adopted: 14th June 2021

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: EpiocularTM EIT for the prediction of acute ocular irritation of chemicals, October 2, 2019, 1- 47 – Tests for interference of test item with MTT endpoint

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm, Model EPI-200-SIT, Rev. 08/15/2022, 1-35

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Cyclohexyl isocyanate

EC Number:

221-639-3

EC Name:

Cyclohexyl isocyanate

Cas Number:

3173-53-3

Molecular formula:

C7H11NO

IUPAC Name:

isocyanatocyclohexane

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL of the test item

Duration of treatment / exposure:

60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions)

Number of replicates:

single experiment, composed of three replicate tissues

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

4.11

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

4.05

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

3.87

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Relative cell viability was calculated for each tissue as % of the mean of the negative control tissues. Then the mean relative tissue viability of three individual tissues exposed to the test item was calculated – this value is used for the comparison with limit given in Chapter 3.8.
Under the above-described experimental design of the MTT test, the average viability of tissues treated with the test item Cyclohexyl isocyanate was 4.0±0.1% of the negative control average value i.e., viability was ≤ 50 %. The positive control, SDS, gave the appropriate response.
The test item colour does not influence study results and the test item does not have direct reductive properties, so any correction of MTT test results was not performed.

The test item effect was positive in the EpiDermTM model i.e., the tissue was damaged.
According to the classification criteria given in chapter 3.9 of this report, the test item, Cyclohexyl isocyanate, is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1 - damage to the skin).

Executive summary:

The test item, Cyclohexyl isocyanate, was assayed for in vitro skin irritation in the human epidermal model EpiDerm^TM. The main methodical documents were OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2020), and Protocol for: In Vitro EpiDerm^TM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the main MTT experiment, after pre-incubation of tissues, 30 µL of the test item  was placed directly on the moistened tissue and spread over the entire surface for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used for the test item and for positive and negative controls.

After removal of the test item, tissues were post-incubated in culture medium for approximately 42 hours. After three hours incubation with MTT, samples were extracted with isopropyl alcohol for 2 -3 hours. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

During post-incubation periods, a damage of barrier function was observed in tissues treated by the test item. The appearance of the treated tissues after isopropyl alcohol extraction resembled those affected by positive control.

In the tests for interference of test item with MTT endpoint direct MTT reduction in test tubes was not found. Even the colour of the test item did not interfere with the evaluation of the test. Therefore, the results of the MTT test did not require correction.

Under the above-described experimental design, the average viability of test item-treated tissues was 4.0±0.1%.

The positive control, SDS, gave the appropriate response.

The test item effect was positive in the EpiDerm^TM model i.e., the tissue was damaged.

According to the classification criteria given in chapter 3.9 of this report, the test item, Cyclohexyl isocyanate, is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1 - damage to the skin).