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

Endpoint summary

Description of key information

BCOP (in vitro test): corrosive (BASF SE, 2015)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 640/2012

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

other: in vitro

Strain:

other: in vitro

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.

Irritation / corrosion parameter:

% tissue viability

Value:

ca.

Vehicle controls validity:

valid

Remarks on result:

no indication of irritation

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.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: 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)

Species:

other: in vitro test

Strain:

other: in vitro test

Vehicle:

unchanged (no vehicle)

Details on study design:

BCOP Test:
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 < 545 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment 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. For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 100% ethanol (positive control, PC), were applied into the anterior chamber 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 with an opacitometer.
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. An aliquot was diluted 1:5 in Eagle’MEM (without phenol red) and measured analogously (test substance, only).

Irritation parameter:

in vitro irritation score

Value:

143.4

Remarks on result:

positive indication of irritation

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Score:

143.4

Remarks on result:

other: in vitro test on isolated bovine cornea

Mean values for opacity, permeability and IVIS of the test substance, NC and PC

Test substance	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritancy Score
15/0112-1	99.2	2.953	143.4
NC	0.0	0.002	0.0
PC	32.2	1.084	48.4

5-methyl oxazolidin-2-one the results derived with BCOP alone were sufficient for a final assessment. Therefore further testing in EpiOcular was waived.

Based on the observed results for the BCOP Test alone and applying the evaluation criteria it was concluded, that 5-methyl oxazolidin-2-one causes ocular corrosion or severe irritation under the test conditions chosen

IVIS	Prediction
≤3	no classification for eye irritation^1
> 3;≤55	no prediction can be made for eyeirritation, further testing with another suitable method is required^2
> 55	ocular corrosive or severe irritant

¹According to the current OECD Guideline 437 (adopted July 2013), this prediction is possible, however, not recommended by the test facility. If the IVIS obtained for the substance tested in this study fell within this range, this aspect is discussed in section 5.

²The test method according to the OECD test guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation. I.e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

The objective was to assess the potential for skin corrosion and skin irritation of 5-methyl oxazolidin-2-one. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential including transport classification.

Therefore, three in vitro assays were proposed for this in vitro skin irritation and corrosion test strategy including transport classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane Barrier Test (Corrositex®). However, in the current case for 5-methyl oxazolidin-2-one the results derived with SCT and SIT were sufficient for a final assessment. Therefore further testing in Corrositex® was waived.

The potential of 5-methyl oxazolidin-2-one to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model

(EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-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 EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced. Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 83.5%, and it was 84.0% 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 99.3%.

Based on the observed results and applying the evaluation criteria cited in chapter 3.8 it was concluded, that 5-methyl oxazolidin-2-one does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen. Hence, 5-methyl oxazolidin-2-one is not assigned to a transport category.

Eye irritation/corrosion

The objective of the present study was the determination of a possible eye irritating potential of 5-methyl oxazolidin-2-one. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test. However, in the current case for 5-methyl oxazolidin-2-one the results derived with BCOP alone were sufficient for a final assessment. Therefore further testing in EpiOcular was waived. The potential of 5-methyl oxazolidin-2-one 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% ethanol) 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. The mean ICVS for the test substance was 143.4. Based on the observed results for the BCOP Test alone and applying the evaluation criteria it was concluded, that 5-methyl oxazolidin-2-one causes ocular corrosion or severe irritation under the test conditions chosen.

Justification for selection of skin irritation / corrosion endpoint:
only available study

Justification for selection of eye irritation endpoint:
only available study

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based on the results of the available data, the test item is classified as Xi, R41 (according to Directive 67/548/EEC (DSD)) and eye irritation cat. 1 (H318) according to Regulation (EC) No 1272/2008 (CLP).