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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
according to
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/InterLaboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diethyl-1H-imidazol-3-ium acetate
EC Number:
Cas Number:
Molecular formula:
C7 H13 N2 . C2 H3 O2
1,3-diethyl-1H-imidazol-3-ium acetate
Details on test material:
Name of the test substance used in the study report: EEIM Acetate
Purity: 97.4 ± 0.1 g/ 100 g
pH value: ca. 6 (undiluted test substance)

Test animals / tissue source

other: Three dimensional human cornea model

Test system

unchanged (no vehicle)
Amount / concentration applied:
Single topical application of 50 uL
Duration of treatment / exposure:
30 minutes
Observation period (in vivo):
2 hours post-incubation period
Number of animals or in vitro replicates:
2 samples
Details on study design:
The present test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short-term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcular. After application of the test material to the surface of the EpiOcular tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of testsubstance treated tissues is compared to negative control values from tissues and expressed as relative tissue viability.
Using a pipette, fifty microliter (50 uL) of the undiluted liquid test substance was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 uL of sterile de-ionized water (NC) or with 50 uL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
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 prewarmed 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).
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.

Results and discussion

In vitro

Irritation parameter:
percent tissue viability 

Any other information on results incl. tables

Mean tissue viability    (% of negative control)

<= 50irritant
> 50 <= 60no prediction
> 60non-irritant

The test substance is not able to reduce MTT directly.
The mean viability of the test-substance treated tissues was 71%.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Based on the observed results and the evaluation criteria it was concluded, that EEIM Acetate does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.