Docosyl methacrylate

Eye irritation

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:

2018

Report date:

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Docosyl methacrylate

EC Number:

240-714-1

EC Name:

Docosyl methacrylate

Cas Number:

16669-27-5

Molecular formula:

C26H50O2

IUPAC Name:

docosyl methacrylate

Test material form:

solid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The EpiOcular™ 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. The EpiOcular™ tissues (surface 0.6 cm2) are cultured on cell culture inserts (MILLICELLs®, 10 mm 0) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:

unchanged (no vehicle)

Details on study design:

In order to determine whether the test substance is able to reduce MTT directly, the test substance will be incubated with the substrate in a pretest (experimental conduct in accordance with GLP, but without a GLP status). 50 μL (liquid test substances) or ca. 50 μL bulk volume (solid test substances) are added to 0.9 ml MTT solution. The mixture is incubated in the dark at about 37°C for 3 hours. A negative control (50 μL deionized water) is tested concurrently. When the color of the mixture turns blue/purple, it is assumed that the test substance can reduce MTT directly. This might lead to a false optical signal if after incubation, sufficient amounts of the test substance remain in or on the skin. In case of water-insoluble substances, the coloration will be assessed at the border to the aqueous phase. In case that direct MTT reduction occurs, two freeze-killed control tissues (KC) are treated with each the test article and the negative control in the same way.
Several test substances will be tested in parallel within the present test (test no. 91) using the same control tissues (NC and PC). Two tissues are treated with each test substance, the PC and the NC, respectively. Two additional tissues (KCs) will be used for the test substance and the NC, respectively, if a positive reaction has been observed in the MTT reduction test. There are two separate protocols for liquids and solids differing in exposure time and postincubation period. Due to the physical condition of the test substance, the protocol for solids is applied.
The tissues will be transferred to sterile 6-well plates with 1 ml pre-warmed assay medium and preconditioned in the incubator at 37°C for 1 hour. After 1 hour, the pre-incubation medium is replaced with fresh medium, and preconditioning continues for
16 - 24 hours.
After pre-incubation, the tissues are pretreated with 20 μL PBS to wet the tissue surface. The tissues are incubated at standard culture conditions for 30 minutes.
Due to the physical state of the test substance, application with a pipette or sharp spoon is not possible. Thus, a metal pin is covered with 50 μL undiluted liquid (at ca. 50°C heated) test substance. The pin is applied with direct contact to the tissue, covering the whole tissue surface. Before application, the test substance is cooled down to room temperature. Control tissues are applied concurrently with 50 μL sterile deionized water (NC, NC KC (if applicable)) or with 50 μL methyl acetate (PC) ortest substance (KC, if applicable). After application, the tissues will be placed into the incubator until the total exposure time of 6 hours is completed.
In order to remove the test substance, the tissues are washed with sterile PBS. For this purpose, the tissues are immersed and swiveled thrice in each of three beakers filled with PBS. Washed tissues are immersed immediately into 12-well plates pre-filled with 5 ml/well pre-warmed medium (post-soak immersion) to remove residual test substance. After 25 minutes of post-soak immersion, each tissue is dried on absorbent paper and transferred to fresh 6-well plates filled with 1 ml/well pre-warmed medium.
After the incubation / post-incubation period, the assay medium is replaced with 0.3 ml MTT solution and the tissues are incubated in the incubator for 3 hours. After incubation, the tissues are washed with PBS to stop the MTT incubation.
The formazan that is produced metabolically by the tissues will be extracted by incubation of the tissues in 2 ml isopropanol at room temperature overnight or for at least 2 hours on a plate shaker (ca. 120 rpm). After shaking the isopropanol extract, 2 aliquots of each extract per tissue will be transferred to a 96-well microtiter plate. The optical density (OD570) will be determined spectrophotometrically using a filter with a wavelength of 570 nm.

Results and discussion

In vitro

Results

Remarks on result:

other: EpiOcular: Mean viability of the test-substance treated tissues was 81%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results for EpiOcular Test and applying the evaluation criteria, Docosyl methacrylate does not show an eye irritation potential in the in vitro eye irritation test under the test conditions chosen.