(4R)-2-oxo-1,3-oxazolidine-4-carboxylic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: 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:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4R)-2-oxo-1,3-oxazolidine-4-carboxylic acid

EC Number:

849-166-3

Cas Number:

83841-00-3

Molecular formula:

C4H5NO4

IUPAC Name:

(4R)-2-oxo-1,3-oxazolidine-4-carboxylic acid

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiSkin supplied by SkinEthic, France, batch 19-EKIN-031, expiry date 05 August 2019

Justification for test system used:

The Episkin model has been validated for corrosivity testing in an international study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 431)

Vehicle:

unchanged (no vehicle)

Details on test system:

Preparation: adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Units: EpiSkinTM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” (Batch No.: 19-MAIN3-034; Exp. Date: 07 August 2019)
A flask of sterile “Assay Medium” (Batch No.: 19-ESSC-032; Exp. Date: 07 August 2019)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test substance: 20 mg (plus 100 μL physiological saline)
Positive and negative controls: 50 μL

Duration of treatment / exposure:

4 hours

Number of replicates:

2 (substance, positive and negative controls), additional 2 with test substance for non-specific optical density evaluation

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.
The mean OD value of the two negative control tissues was 0.717, which satisfied the acceptability criterion of being in the range of 0.6-1.5.
The positive control treated tissue showed 0.5% cell viability demonstrating the proper performance of the assay.
The difference of cell viability between the two test item-treated tissue samples in the MTT assay was 4.2%.
The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 7.5%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.

Any other information on results incl. tables

The test item did not react with MTT as no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

Table 1: Optical density measurements and calculated relative viability of the samples

	Optical density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative control: physiological saline	1	0.791	0.743	103.7
	2	0.737	0.690	96.3
	mean	-	0.717	100.0
Positive control: glacial acetic acid	1	0.051	0.003	0.5
	2*	0.047	-0.001	-0.1
	mean	-	0.003	0.5
Test substance	1	0.071	0.023	3.3
	2	0.072	0.024	3.4
	mean	-	0.024	3.3

Mean blank was 0.047; optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places); * negative values of positive control were excluded from the calculation

Validity of the test

After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.

The mean OD value of the two negative control tissues was 0.717, which satisfied the acceptability criterion of being in the range of 0.6-1.5.

The positive control treated tissue showed 0.5% cell viability demonstrating the proper performance of the assay.

assay was 4.2%.

The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 7.5%.

The mean OD value of the blank samples (acidified isopropanol) was 0.047.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The substance was assessed to be corrosive to the skin.

Executive summary:

The skin corrosion potential of the substance was studied under GLP in accordance with OECD TG 431 using a reconstructed human epidermis model (EpiSkin). This model is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (Thiazolyl blue, CAS number 298-93-1) assay. Disks of the dermal model (two units) were treated with the powdered test substance and incubated for 4 hours at room temperature. Exposure of the skin model surface to the test substance was terminated by rinsing the units with phosphate buffered saline solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm.

The negative control epidermis units were treated with physiological saline (0.9% w/v NaCl solution), and the positive control units were treated with glacial acetic acid (two units per control). Two additional disks were used to provide an estimate of colour contribution from the coloured test item. For each treated tissue, viability was expressed as a percentage relative to the negative control. All the validity criteria were fulfilled in this study. As the mean cell viability of the test substance was 3.3% compared to the negative control after 4 hours, and this value is clearly less than the cut-off of 35%, the test substance was considered to be corrosive to skin.