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

Skin irritation / corrosion

Administrative data

Endpoint:

skin 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:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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

Test guideline 5

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

810-292-9

Cas Number:

1072-70-4

Molecular formula:

C4H7NO2

IUPAC Name:

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 oxazolidin-2-one
- Physical state: liquid, colorless, clear
- Analytical purity: 98.0 area-%
- Lot/batch No.: EWALD-00441

Test animals

Species:

other: in vitro

Strain:

other: in vitro

Test system

Type of coverage:

other: in vitro

Preparation of test site:

other: in vitro

Vehicle:

other: in vitro

Duration of treatment / exposure:

3 min and 1 hour(s)

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. In addition, two killed control tissues per exposure time were treated with the test
substance and NC, respectively, in order to detect direct MTT reduction. Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently treated with 50 μL of de-ionized water (NC, NC KC) or with 50 μL of 8 N potassium hydroxide (PC) or test substance (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, 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.
- Irritation test: Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed control tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards. Control tissues were concurrently treated with 30 μL of sterile PBS (NC, NC KC) or with 30 μL of 5% SDS (PC) or test substance (KC). 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 placed into 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 4 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca.

Vehicle controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Corrosion test

Exposure period: 3 min
Test substance			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.229	2.145	2.187	0.059
		Viability    [% of NC]	101.9	98.1	100.0	2.7	2.7
	KC tissues	mean OD570	0.124	0.193	0.158	0.048
		Viability      [% of NC]	5.7	8.8	7.2	2.2	30.6
15/0112-1	viable tissues	mean OD570	1.928	1.724	1.826	0.145
		Viability    [% of NC]	88.2	78.8	83.5	6.6	7.9
	KC tissues*	mean OD570KC NC corrected	0.000	0.000	0.000	0.000
		Viability     [% of NC]	0.0	0.0	0.0	0.0
	Mean viability of tissues after KC correction [% of NC]:				83.5
PC	viable tissues	mean OD570	0.249	0.261	0.255	0.008
		Viability      [% of NC]	11.4	11.9	11.7	0.4	3.3

 

 

Exposure period: 1 h
Test substance			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.226	2.078	2.152	0.104
		Viability    [% of NC]	103.4	96.6	100.0	4.8	4.8
	KC tissues	mean OD570	0.089	0.081	0.085	0.005
		Viability      [% of NC]	4.1	3.8	3.9	0.2	6.3
15/0112-1	viable tissues	mean OD570	1.836	1.778	1.807	0.041
		Viability    [% of NC]	85.3	82.6	84.0	1.9	2.3
	KC tissues*	mean OD570KC NC corrected	0.000	0.000	0.000	0.000
		Viability     [% of NC]	0.0	0.0	0.0	0.0
	Mean viability of tissues after KC correction [% of NC]:				84.0
PC	viable tissues	mean OD570	0.132	0.132	0.132	0.000
		Viability      [% of NC]	6.1	6.1	6.1	0.0	0.0

* Negative values are set to zero for further calculation

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. However, the results of the KC tissues did not indicate an increased MTT reduction. Thus the KC was not used for viability calculation.

 

  

Irritation test

	Exposure period: 1 h
Test substance				tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC		viable tissues	mean OD570	2.454	2.313	2.320	2.362	0.080
			Viability    [% of NC]	103.9	97.9	98.2	100.0	3.4	3.4
		KC tissues	mean OD570	0.055	0.059	0.061	0.058	0.003
			Viability      [% of NC]	2.3	2.5	2.6	2.5	0.1	5.6
15/0112-1		viable tissues	mean OD570	2.338	2.327	2.393	2.353	0.035
			Viability    [% of NC]	99.0	98.5	101.3	99.6	1.5	1.5
		KC tissues*	mean OD570KC NC corrected	0.010	0.009	0.003	0.007	0.004
			Viability     [% of NC]	0.4	0.4	0.1	0.3	0.2	51.6
		Mean viability of tissues after KC correction [% of NC]:				99.3
PC		viable tissues	mean OD570	0.057	0.068	0.065	0.063	0.006
			Viability      [% of NC]	2.4	2.9	2.7	2.7	0.2	8.9

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.3 % of NC). Thus for the test substance the final mean viability is given after KC correction.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met