Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
EpiOcular
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs®, 10 mm ø) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23769
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Details on study design:
In order to assess the ability of the test material to directly reduce MTT, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the MTT solution color or in case of water-insoluble test substances the border to the water-phase turned blue / purple the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case of direct MTT reduction, two freeze-killed control tissues were treated with the test article and the negative control each in the same way as described in the following section.
Several test substances were tested in parallel within the present test (test no. 84) by using the same control tissues (NC and PC). Two tissues were treated with each the test substance, the PC and the NC. There are two separate protocols for liquids and solids differing in exposure time and postincubation period. Due to the physical state of the test substance, the protocol for liquids was applied.
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 37°C. After 1 hour, the pre-incubation medium was replaced by fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. After pre-incubation, the tissues were pretreated with 20 μL PBS to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. By using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed. By using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed. In order 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) 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 overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Negative control (NC): Deionized water, sterile
Positive control (PC): Neat methyl acetate (CAS No.: 79-20-9)

Results and discussion

In vitro

Results
Remarks on result:
other: Mean viability of the tissues treated with the test substance was 105.3%.

Any other information on results incl. tables

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance is not able to directly reduce MTT.

The final mean viability of the tissues treated with the test substance was 105.3%.

Test substance

 

tissue 1

tissue 2

mean

inter-tissue variability [%]

NC

mean OD570

1.651

1.614

1.632

 

viability

[% of NC]

101.1

98.9

100.0

2.3

16/0068-1

mean OD570

1.798

1.639

1.719

 

viability

[% of NC]

110.2

100.4

105.3

9.8

PC

mean OD570

0.325

0.438

0.382

 

viability

[% of NC]

19.9

26.8

23.4

6.9

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that C9-11 Methacrylate does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.