3-(1-ethoxyethyl)-5-methyl-oxazolidin-2-one

Endpoint summary

Description of key information

The objective was to assess the skin sensitizing potential of 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:protein reactivity (DPRA), activation of keratinocytes (LuSens), and activation of dendritic cells (h-CLAT).

 

However, in the current case for 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

 

DPRA:The reactivity of 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and Uvdetection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Two test runs were performed. Per test run, three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. The test substance was dissolved in acetonitrile at a concentration of 100 mM. In the 1st test run a purity of 97.8% was considered for the test substance (information of the sponsor, which was available at the time of the 1st test run, only). A 2nd test run was performed as a lower purity of 94.3% was determined analytically. In both test runs the samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present.

1st test run:

The mean C-peptide depletion, caused by the test substance was determined to be -2.57%. The mean K-peptide depletion, caused by the test substance was determined to be 4.21%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 2.11%.

2nd test run:

The mean C-peptide depletion, caused by the test substance was determined to be 2.04%. The mean K-peptide depletion, caused by the test substance was determined to be 0.06%. Thus, the mean peptide depletion was calculated to be 1.05%. The 2nd test run confirms the result of the 1st test run. Hence, based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

 

LuSens:The keratinocyte activating potential of test substance 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. No relative viability below 70% was observed. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

 

In summary, after 48 hours of exposure to test substance 3-(1-Ethoxyethyl)-5- methyloxazolidin-2-on luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on does not have a keratinocyte activating potential.

 

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	2.11% (1st test run) or 1.05% (2nd test run) mean peptide depletion	Negative
Kertinocyte Activation Assay - LuSens	In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (H-CLAT)	Not determined	Not determined

Based on the results and applying the evaluation criteria described, 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on is not peptide reactive and does not activate keratinocytes. Applying the evaluation criteria 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on is predicted not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Experimental procedure of the DPRA:
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Test substance solubility:
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Preparation of peptide stock solutions:
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

Preparation of the test-substance samples:
The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Preparation of the vehicle controls:
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (acetonitrile) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Preparation of the co-elution control:
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Controls (DPRA):
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Synthetic peptides (DPRA):
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Remarks on result:

other: 2.11% (1st test run) or 1.05% (2nd test run) mean peptide depletion

The test substance was dissolved in acetonitrile at a concentration of 100 mM. In the 1st test run a purity of 97.8% was considered for the test substance (information of the sponsor, which was available at the time of the 1st test run, only). A 2nd test run was performed as a lower purity of 94.3% was determined analytically.

In both test runs the samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates

in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

1st test run:

The mean C-peptide depletion, caused by the test substance was determined to be -2.57%.

The mean K-peptide depletion, caused by the test substance was determined to be 4.21%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 2.11%.

2nd test run:

The mean C-peptide depletion, caused by the test substance was determined to be 2.04%.

The mean K-peptide depletion, caused by the test substance was determined to be 0.06%.

Thus, the mean peptide depletion was calculated to be 1.05%.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Preparation of the cells:
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing.
Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay:
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. The test substance was prepared as described. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

Visual inspections:
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

Luciferase assay:
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT:
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Cell line (LuSens):
Human transgenic keratinocyte cell line derived from HaCaT cells,
prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Controls (LuSens):
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
Positive control (PC): ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

Test substance preparation for the LuSens:
The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). The test-substance preparations were prepared by stirring.
Reason for the vehicle: The test substance was soluble in 4% DMSO in culture medium 3. Form of application: Visual inspection of each dilution step was performed.

Remarks on result:

other: does not have a keratinocyte activating potential

Summary of LuSens Main Experiments. Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

Concentration (test substance) [µM]	1st experiment				Concentration (test substance) [µM]	2nd experiment
	Fold induction rel. Viability [%]		t-test			Fold induction rel. Viability [%]		t-test
	mean	mean	p-vale	markers		mean	mean	p-vale	markers
592	1.14	93	0.168	n.s.	592	1.23	91	0.115	n.s.
710	1.11	90	0.004	**	710	1.26	91	0.000	**
852	1.12	89	0.215	n.s.	852	1.20	93	0.000	**
1023	1.12	92	0.012	*	1023	1.14	91	0.067	n.s.
1227	1.18	88	0.093	n.s.	1227	1.17	88	0.009	**
1473	1.16	92	0.185	n.s.	1473	1.22	87	0.000	**
1768	1.14	87	0.090	n.s.	1768	1.16	87	0.011	*
2121	1.36	79	0.024	*	2121	1.21	90	0.076	n.s.
VC	1.00	100	-	-	VC	1.00	100	-	-
EGDMA 90.8 µM	3.75	74	0.000	**	EGDMA 90.8 µM	4.14	75	0.000	**
LA 5000 µM	1.12	99	0.005	**	LA 5000 µM	0.92	104	0.119	n.s.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

 

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. The following results were observed: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

In summary, after 48 hours of exposure to test substance 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on does not have a keratinocyte activating potential.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the evaluation criteria, 3-(1-Ethoxyethyl)-5-methyloxazolidin-2-on does not have a kerationocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone in-house validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of the in house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90% and an accuracy of 90%.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

 

Each individual assay was performed under GLPand the cell based assays LuSens consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays. The DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). An OECD testing guideline for the dentritic cell activation assay is under preparation.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

 

Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).