1-Ethyl-3-methylimidazolium benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-07-09 to 2012-09-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar 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:

2015-07-28, pre-Guideline study

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002-04-24

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-Ethyl-3-methylimidazolium benzoate

EC Number:

695-723-7

Cas Number:

150999-33-0

Molecular formula:

C13H16N2O2

IUPAC Name:

1-Ethyl-3-methylimidazolium benzoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 150999-33-0

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
Basic procedure: Two tissues were treated with the test substance, the PC and NC, respectively. Due to the physical condition of the test substance the protocol for liquids was applied.
Pre-incubation of the tissues: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 - 24 hours (pre-incubation).
Pretreatment of the tissues: After the pre-incubation the tissues were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance: Using a pipette, fifty microliter (50 µL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 µL of sterile de-ionized water (NC) or with 50 µL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
Removal of the test substance and postincubation period: To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).

- RhCE tissue construct used: OCL-200, MatTek Corp., Ashland MA, USA
- Doses of test chemical and control substances used: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min at ~37 °C exposure, 12 min at ~37 °C post-exposure immersion, 2 h at ~37 °C post-exposure incubation

- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device: The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically.
- Description of the method used to quantify MTT formazan: After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
Assay acceptance criterion for the NC:
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is > 1.0. The mean OD570 of the NC should not exceed 2.5.
Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25 %. A viability of < 50 % is acceptable.
Assay acceptance criterion for tissue variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is < 20 %.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
- Reference to historical data of the RhCE tissue construct

- Positive and negative control means and acceptance ranges based on historical data
- Acceptable variability between tissue replicates for positive and negative controls ≤ 20 %
- Acceptable variability between tissue replicates for the test chemica:l ≤ 20 %

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability [%]

Value:

9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Test substance		Tissue 1	Tissue 2	Mean	Inter-tissue variability [%]
NC	mean OD570	1.750	1.681	1.716
	viability [% of NC]	102.0	98.0	100	4.0
12/0365-1	mean OD570	0.153	0.161	0.157
	viability [% of NC]	8.9	9.4	9	0.4
PC	mean OD570	0.424	0.441	0.433
	viability [% of NC]	24.7	25.7	25	1.0

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria