Cyclohexyl isocyanate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.11.2022 - 2.12.2022

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted: 18 June 2019

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OCL-200-EIT-Eye-Irritation-Test-Protocol-MK-24-007-0055_02_02_2021

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

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Test System - the reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0). The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

- immersion incubation (post-soak) at room temperature - 12 ±2 min
- post-treatment incubation at standard culture conditions - 2 h and 3 min
- incubation with MTT medium at standard culture conditions - 180 min

Number of animals or in vitro replicates:

A single experiment has been composed of two replicate tissues for the test item, two for negative and two for positive controls.

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1 and 2

Value:

7.8

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The average viability of tissues treated by the test item Cyclohexyl isocyanate was 7.8 % of negative control average value, i.e. viability was < 60 %.
The effect of the test item was positive in EpiOcularTM model (tissues were damaged).
If the test item-treated tissue viability is ≤ 60.0% relative to negative control-treated tissue viability,.

Executive summary:

The test item, Cyclohexyl isocyanate, was assayed for the in vitro eye irritation in human epidermal model EpiOcular^TM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek).

MTT test was performed as follows: After pre-incubation and wetting of tissues, 50 µL of the test item was placed directly atop to the moistened tissue and it was spread on the entire tissue surface. Length of exposition was 30 minutes at 37±1°C in humidified CO₂ incubator (5±1% CO₂). Two tissues were used for the test item and every control.

After removal of the test item, tissues were post-soaked in medium for approximately 12 minutes and post-incubated for 2 hours at culture conditions. Three hours incubation with MTT and 2-3 hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

In the complementary experiments, 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 average viability of treated tissues was 7.8 % i.e., viability was ≤ 60 %.

The effect of the test item was positive in EpiOcular^TM model (tissues were damaged).