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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initation: April 13, 2018 - Experimental Start: April 17, 2018 - Experimantal Termination: April 19,2018 - Study Termination (Draft Report): April 26, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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)
Version / remarks:
OECD Guideline for testing of chemicals: Reconstructed Human Cornea-like
Epithelium (RhCE) Test Method for Identifying Chemicals not Requiring
Classification and Labelling for Eye Irritation or Serious Eye Damage OECD 492, last
version from 9 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-ethyl-3-methylimidazol-3-ium;acetate
EC Number:
604-344-8
Cas Number:
143314-17-4
Molecular formula:
C8H14N2O2
IUPAC Name:
1-ethyl-3-methylimidazol-3-ium;acetate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: proionic GmbH, 25PI195_3
- Expiration date of the lot/batch: 02.2019
- Purity test date: 99.45 +/- 0.12 wt%


Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™ human cell construct for eye irritation testing (OCL-212-EIT)
Details on test animals or tissues and environmental conditions:
The EpiOcular™ human cell construct for eye irritation testing (OCL-212-EIT) (Lot No. 27033) was obtained from MatTek In Vitro Life Science Laboratories, SR.
Standard Assay Kit Components (OCL-200-EIT)
Half kit (12 cultures instead of standard 24) was used.

Amount Reagent Description
1 Sealed 24-well plate of EpiOcularTM tissues (OCL-200) Contains 12 tissues of cell culture inserts, package on agarose
2 6-Well Plates (Falcon) Used for maintaining tissues during assay protocol
1 12- Well Plate (Falcon) Used during assay protocol
2 24-Well Plate (Falcon) Used to perform MTT assay
1 bottle, 200 mL EpiOcularTM Assay medium (OCL-200-ASY) DMEM based medium
1 bottle, 100 mL DPBS Rinse Solution (TC-PBS) Used for wetting and rinsing the inserts


Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl


Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
two tissues
Details on study design:
After the overnight incubation, the tissues were pre-wetted with 20 L of DPBS. The tissues were incubated at standard culture conditions for 30 minutes. After pre-wetting, the negative and positive controls were tested by applying 50 L topically on the tissues. The test item was applied topically onto the tissue surface at amount of 50 L. Two tissues were used per treatment, negative and positive controls. The cultures were returned to the incubator for 30 minutes.
After treatment time, tissues were rinsed with DPBS (in three glass beakers) to remove any residual test material. After rinsing, the tissue was immediately transferred to and immersed in 5 mL of previously-warmed assay medium in 12-well plate for a 12-minute immersion
incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, each insert was removed from the medium and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm medium. The tissues were post-incubated for an additional 2 hours. Then, the cultures were transferred to 24-well plate containing 0.3 mL/well of MTT reagent (1 mg/mL) and incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 3 hours.
After incubation, the cultures were blotted on absorbent paper and transferred to new 24-well plate and extracted in 2 mL of isopropanol overnight without shaking at 2 – 8°C in the dark.
Volume of 2x 200 μL of each extraction solution were transferred to a 96-well plate and the absorbances (ODs) were recorded.

Results and discussion

In vitro

Results
Irritation parameter:
other: tissue viability
Value:
77.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: viability mean 100 %
- Acceptance criteria met for positive control: viability mean 32.0 %

Any other information on results incl. tables

Table 1.  Eye irritation potentialof1-Ethyl-3-methylimidazolium acetateafter 30-min exposure in human model EpiOcularTM

 

 Test item

OD

SD

Viability

SD of

in vivo

 

mean

of OD

mean (%)

viabilities

prediction

 Negative controla

1.800

 0.001

100.0

0.06

NI

 Positive controlb

0.576

0.110

 32.0

6.08

I

 1-Ethyl-methylimidazolium                 acetate

1.388

0.233

 77.1

12.91

NI

a  H2O

b  methyl acetate

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item 1-Ethyl-3-methylimidazolium acetate was examined for in vitro eye irritation in human model EpiOcularTM. The magnitude of viability was quantified by using MTT test.

Validity of the test method was ascertained by positive control methyl acetate. Two tissue replicates were used for each treatment (exposure time 30 minutes), including negative and positive controls.
The tissue viability met the acceptance criterion (mean OD of negative control was 1.800). The viability of culture treated by positive control methyl acetate was 32.0%. The positive control met the acceptance criterion: mean tissue viability less than 50%.

Determined viability of culture treated by 1-Ethyl-3-methylimidazolium acetate (77.1%) fulfilled the criteria for irritancy.
Therefore, the test item 1-Ethyl-3-methylimidazolium acetate is considered to be non irritant to the eye.
Executive summary:

The test item 1-Ethyl-3-methylimidazolium acetatewas examined for eye irritation potential in EpiOcularTMEye Irritation Test (OCL-200-EIT). 

The irritationpotential of the test item was assessed in compliance with:

OECD Guideline for testingof chemicals: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals not Requiring Classification and Labelling for Eye Irritation or Serious EyeDamage OECD 492 [2]

and

Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) -For the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcularTMModel[1].

Two tissue replicates were used for each treatment (exposure time of 30 min), including negative and positive controls. The magnitude of viability was quantified by MTT test.

Validity of the test method was ascertained by a positive control.

The tissue viability met the acceptance criterion (mean OD of negative control was 1.800). The viability of culture treated by positive control methyl acetate was 32%. The positive control met the acceptance criterion: mean tissue viability less than 50%.

The viability of culture treated by1-Ethyl-3-methylimidazolium acetatewas 77.1%.

 

Based on the results of the study, the test item1-Ethyl-3-methylimidazolium acetateaccording to Evaluation criteria and Acceptance criteria is considered to be non-irritant (NI).