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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jan 2014 - 02 Apr 2014

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 640/2012

GLP compliance:

yes

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

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 5-methyl-3-vi nyloxazol id i n-2-one
- Molecular formula (if other than submission substance): C6H9NO2
- Molecular weight (if other than submission substance): 127.14 g/mol
- Physical state: liquid
- Analytical purity: about 99.3 % (as given by the sponsor); characterisation data available under the GLP-study no. 15L00098, from Competence
Center Analytics, BASF SE, D-67056 Ludwigshafen. The characterisation has not been finished to date.
- Lot/batch No.: DEIMLIB-00070
- Storage condition of test material: ambient temperature

Test animals

Species:

other: human epidermis model

Strain:

other: in vitro

Details on test animals or test system and environmental conditions:

Three dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly
differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Test system

Type of coverage:

other: in vitro

Preparation of test site:

other: in vitro test

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro test

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Corrosion test: Fifty microliter (50 μL) of the undiluted liquid test substance was applied
Irritation test: Thirty microliter (30 μL) of the undiluted liquid test substance was applied
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

Corrosion test: 3 min and 1 hour(s)
Irritation test: 1 hour followed by a 42-hours post-incubation period

Details on study design:

TEST SYSTEM
The EpiDerm TM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in viva. The EpiDerm TM tissues (surface 0.6 cm2)
are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm 0) and commercially available as kits (EpiDerm TM 200), containing 24 tissues on shipping agarose.
Skin model: Epi-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEST PROCEDURE

Corrosion test: Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.
Control tissues were concurrently applied with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wave length of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.
- Irritation test: Three tissues were treated with the test substance, the PC and NC, respectively.25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for
24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation 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. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with
isopropanol for each microtiter plate.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation test exposure period 1 hour

Value:

21

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test exposure period 3 minutes

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test exposure period 1 hour

Value:

30

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Corrosion test

		Exposure: 3 min			Exposure: 1 hour
Test substance		tissue 1	tissue 2	mean	tissue 1	tissue 2	mean
NC	mean OD570	1.927	2.168	2.047	1.799	1.762	1.780
	viability [% of NC]	94.1	105.9	100	101.0	99.0	100
14/0031 -1	mean OD570	1.740	1.801	1.770	0.370	0.702	0.536
	viability [% of NC]	85.0	88.0	86	20.8	39.4	30
PC	mean OD570	0.441	0.435	0.438	0.130	0.125	0.127
	viability [% of NC]	21.5	21.2	21	7.3	7.0	7

 

KC:

NC: 3min: 0.113; 1hour: 0.108

Test substance: 3 min: 0.087; 1 hour: 0.194

Irritation test

Test substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.533	2.719	2.389	2.547
	viability [% of NC]	99.4	106.8	93.8	100	6.51
14/0031-1	mean OD570	0.853	0.421	0.328	0.534
	viability [% of NC]	33.5	16.5	12.9	21	11.00
PC	mean OD570	0.075	0.070	0.061	0.068
	viability [% of NC]	2.9	2.7	2.4	3	0.29

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 0.1; Thus the KC was not used for viability calculation.

The value for inter-tissue variability of the test substance for the exposure period of 1 hour is about 0.33 and therefore marginally out of the acceptance range. Since all other quality criteria of the test were met and the viability values of both tissues are well above the cut off for skin corrosion, this deviation is not considered to adversely affect the result of this study.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information

Conclusions:

Based on the observed results and it was concluded, that 5-methyl-3-vinyl-oxazolidin-2-on shows a skin irritation potential in the EpiDerm™ skin
corrosion/irritation test under the test conditions chosen.

Executive summary:

The EpiDerm™ skin corrosion/irritation test showed the following results:

The test substance is able to reduce MTT directly. 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 (performed with corrosion test, only).

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 86%, and it was 30% after an exposure period of 1 hour.

Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 21%.