2-Oxazolidinone, 3-ethenyl-5-methyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-01-21 - 2014-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation; EpiOcular TM human cell construct 2010

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

EC Number:

809-852-5

Cas Number:

3395-98-0

Molecular formula:

C6H9NO2

IUPAC Name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

Details on test material:

- Name of test material (as cited in study report): 5-methyl-3-vinyl-oxazolidin-2-on
- Test-substance No.: 14/0031-1
- Physical state: liquid
- Analytical purity: 98.587 area-%
- Lot/batch No.: DEIMLIB 00028 Fr.8
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.
- Homogeneity: The test substance was homogeneous by visual inspection.

Test animals / tissue source

Species:

other: in vitro test

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high speed homogenizer (Ika Tube Drive). The homogeneity of the test-substance preparation during application was
provided by stirring.

Form of application:
BCOP:750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas.
EpiOcular: Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period.

Observation period (in vivo):

not applicable (in vitro test)

Number of animals or in vitro replicates:

not applicable (in vitro test);

Details on study design:

Experimental procedure of the BCOP Test
Preparation of the bovine corneas and measurement of initial corneal opacity Corneas free of defects (opacity, scratches, pigmentation etc.) were
dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both
chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at
least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 530 opacity units1 were
discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.
Application of the test substance and washing
Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette.
Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 100%
dimethylformamider (positive control, PC) using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC
and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing
phenol red) and once with Eagle’s MEM (without phenol red).
Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Post-exposure incubation for liquid test substances and surfactants
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
Measurement of final corneal opacity
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea Determination of permeability
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test
substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well
microtiter plate and the optical density (OD490) was determined.
Histopathology
After determination of opacity and permeability, the corneas were fixed in 4% formaldeyhde for at least 24 h and transferred to the laboratory of
General Pathology for further histotechnical processing and examination by light microscopy.
Histopathological findings were summarized in a histopathological score of irritation (HSI)
as follows:
0 = no findings
I = minimal
II = mild
III = moderate
IV = severe

Experimental procedure of the EpiOcular Test
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized 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 that direct MTT reduction occurred, two freeze-killed control tissues were treated
with, each, the test article and the negative control.
Basic procedure
Two tissues were treated with the test substance, the PC and NC, respectively. In addition two killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. 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 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 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard
culture conditions for 16 – 24 hours.
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) or test substance (killed tissue control, KC).
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 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).
MTT incubation
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

Results

Irritation parameter:

in vitro irritation score

Value:

57.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

In vivo

Results

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Score:

57.7

Reversibility:

other: not applicable

Remarks on result:

other: in vitro test on isolated bovine cornea

Irritant / corrosive response data:

IVIS NC: 1.6
IVIS PS: 102.7

Other effects:

BCOP: Histological evaluation revealed changes indicating severe eye damage.

Any other information on results incl. tables

Findings of the BCOP Test

Mean values for opacity, permeability and IVIS of the test substance, negative control (NC) and positive control (PC)

Test substance	Mean Opacity Value	Mean Permeability Value	Mean  In VitroIrritancy Score
14/0031 -1	53.7	0.254	57.5
NC	1.6	0.002	1.6
PC	102.7	1.218	121.0

Findings of the EpiOcular test

Test Substance		Tissue 1	Tissue 2	Mean KC		Mean	Inter-tissue variability [%]
NC	Mean OD570	1.945	1.924	0.033	-	1.935
	Viability [% of NC]	100.5	99.5	-	-	100	1.1
14/0031 -1	Mean OD570	0.133	0.141	0.047	-	0.137
	Viability [% of NC]	6.9	7.3	-	-	7	0.4
PC	Mean OD570	0.394	0.448	-	-	0.421
	Viability [% of NC]	20.4	23.2	-	-	22	2.8

Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel.

However, the result of the KC did not indicate an increased MTT reduction (difference to KC

of NC is not greater than 0.). Thus the KC was not used for viability calculation.

Decision criteria for the combined assessment

BCOP result	EpiOcular result	Evaluation Test Strategy
IVIS > 55 or HIS = IV	≤ 60% viability	ocular corrosive or severe irritant
IVIS < 55 and HIS < IV	≤ 60% viability	irritant
IVIS < 55 and HIS < IV	> 60% viability	Non-irritant

Applicant's summary and conclusion

Interpretation of results:

other: ocular corrosion or severe irritation in the in vitro eye irritation test strategy

Conclusions:

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria 5-methyl-3-vinyl-oxazolidin-2-on causes ocular corrosion or severe irritation in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

BCOP

The potential of 5-methyl-3-vinyl-oxazolidin-2-on to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 100% dimethylformamide) were applied to three corneas, each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. In addition H&E-stained cross sections were evaluated for the irritation potential of the test substance.Histological evaluation revealed changes indicating severe eye damage.

EpiOcular

The potential of 5-methyl-3-vinyl-oxazolidin-2-on to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control was introduced. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing. The mean viability of the test-substance treated tissues was 7%.