Imidazolium compounds, 2-(C9-19 and C9-19-unsatd. alkyl)-1-[(C10-20 and C10-20-unsatd. amido)ethyl]-4,5-dihydro-1-Me, Me sulfates

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 20, 2017 to May 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-(C9-19 and C9-19-unsatd. alkyl)-1-[(C10-20 and C10-20-unsatd. amido)ethyl]-4,5-dihydro-1-Me, Me sulfates

EC Number:

296-019-9

EC Name:

Imidazolium compounds, 2-(C9-19 and C9-19-unsatd. alkyl)-1-[(C10-20 and C10-20-unsatd. amido)ethyl]-4,5-dihydro-1-Me, Me sulfates

Cas Number:

92201-88-2

Molecular formula:

Molecular formula of major constituents: C38H77N3O5S1 (C16 carbon chain) C42H81N3O5S1 (C18:1 carbon chain)

IUPAC Name:

Imidazolium compounds, 2-(C9-19 and C9-19-unsatd. alkyl)-1-[(C10-20 and C10-20-unsatd. amido)ethyl]-4,5-dihydro-1-Me, Me sulfates

Test material form:

solid

Test animals / tissue source

Species:

other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)

Strain:

other: Keratinocyte 4F1188

Details on test animals or tissues and environmental conditions:

Test system:
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium (for information see www.mattek.com). QC results for the specific lot of models received (Lot# 23779) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS

Test system

Vehicle:

other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 µl of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.

Duration of treatment / exposure:

6h ± 15 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation.

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes

Number of animals or in vitro replicates:

Three tissues per condition (n=3).

Details on study design:

Preliminary test:
The test substance was first checked for its potential for MTT interference and solvent interference.

Main test overview:
Day 0: On the day of receipt, EpiOcularTM tissues were pre-incubated overnight at 37 °C, 5 % CO2, 95 % relative humidity.
Day 1: Exposure to and removal of test and reference substances (50 mg of test substance or 50 µl of reference substances for 6h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation). 
Day 2: End of MTT viability test, readings at 570 nm without reference filter.

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

6 h exposure

Value:

15.6

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with sterile water) tissues is ≥ 0.8 and ≤ 2.5.
Result for the test: 1.334
- The mean of the positive control relative percentage viability is below 50% of negative control viability after 6 hours exposure.
Result for the test: 47.2
- The SD between three tissues replicates should not exceed 18 % in the same run (for negative and positive control tissues and tissues of test substances).
Results for the test:
NC: 3.88
PC: 4.976
Test substance: 4.582

Applicant's summary and conclusion

Interpretation of results:

other: Inconclusive for the classification

Conclusions:

Under the study conditions, the percentage viability obtained was 15.6% and therefore, the test substance was classified as irritant to the human eye. However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage).

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16 and C18-unsatd. AAEMIM-MS', using Reconstructed human Cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. After pre-wetting the tissues with 20µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, test system was exposed to 50 mg of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation. After post-treatment incubation period, MTT test was performed with readings at 570 nm without reference filter, and percentage viability value for EpiOcularTM models exposed to the test substance relative to the negative control was calculated. The percentage viability obtained for negative control, positive control and test substance were calculated be 100%, 47.2% and 15.6% respectively. The test was considered to have met all the validity criteria. Since the percentage viability of the test substance was much below the threshold (>60%) indicating no irritation potential, the test substance was classified as irritant to the human eye by the study authors (XCellR8, 2017). However, based on the viability percentage in the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.