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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative

MNT (in vitro): negative

HPRT: negative

In vitro mouse lymphoma assay: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2014 - 25 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 (Arochlor 1254)

Test concentrations with justification for top dose:

1st Experiment: 5, 16, 50, 160, 500, 1600, 5000 µg/plate
2nd Experiment: 160, 300, 625, 1250, 2500, 5000µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

5-Methyl-3-vinyloxazolidin-2-on was tested for mutation (and toxicity) in four strainsof Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments at the concentrations detailed previously, using triplicate plates without and with S-9. Vehicle controls were included in quintuplicate and positive controls were included in triplicate in both assays without and with S-9. These platings were achieved by the following sequence of additions to molten agar at 46±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from
light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant
colonies were counted .
As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test
article or control solution, bacteria and S-9 mix detailed above (except positive control volume reduced
to 0.05 mL) were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2.5 mL molten agar at 46±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic
chemicals that could be detected in the assay.
Volume additions for the positive controls in the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) used in the formulation of these chemicals. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in
this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise
or eliminate any toxic effects of the vehicle that may have otherwise occurred.
Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction (to ≤0.5-fold) in revertants compared to the concurrent vehicle controls and/or a reduction in
mutagenic response. A minimum of five treatment concentrations were evaluated for mutation in each strain in the absence and presence of S-9.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535, TA1537 or WP2 uvrA) the concurrent vehicle control values
2. The positive trends/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

1st Experiment:

Compound	Concentration (µg/plate)	Revertant numbers/plate
		TA 98 (-S9)	TA 98 (+S9)	TA 100 (-S9)	TA 100 (+S9)	TA 1535  (-S9)	TA 1535 (+S9)	TA 1537 (-S9)	TA 1537 (+S9)	WP2 uvrA (-S9)	WP2 uvrA (+S9)
Purified Water		28.6	37.4	114.6	128.2	27.6	19.2	10.4	14.0	12.0	17.4
Test item	5	24.0	43.0	129.7	120.7	31.3	18.7	6.0	19.0	11.3	18.3
	16	23.3	46.7	113.0	119.3	29.3	23.0	8.3	18.7	14.3	14.3
	50	26.3	42.3	112.7	125.3	30.3	25.0	7.3	14.7	16.7	16.7
	160	25.3	34.3	102.0	129.3	30.3	18.3	9.3	19.0	9.0	19.3
	500	24.3	45.3	106.3	128.0	31.7	20.3	9.0	20.3	17.3	14.0
	1600	27.7	43.0	122.3	138.7	23.0	12.7	7.7	19.0	8.7	14.0
	5000	24.3	44.0	109.7	137.0	31.7	26.0	9.0	18.7	8.3	14.3
Positive Control	-	725.3	470.7	660.3	1612.7	692.3	310.7	296.0	166.3	902.0	402.3

2 nd Experiment:

Compound	Concentration (µg/plate)	Revertant numbers/plate
		TA 98 (-S9)	TA 98 (+S9)	TA 100 (-S9)	TA 100 (+S9)	TA 1535  (-S9)	TA 1535 (+S9)	TA 1537 (-S9)	TA 1537 (+S9)	WP2 uvrA (-S9)	WP2 uvrA (+S9)
Purified Water		17.8	35.8	101.4	124.2	16.4	19.6	11.8	20.6	13.4	19.8
Test item	160	13.3	35.3	89.0	127.3	17.7	24.3	14.3	17.3	15.7	21.3
	300	12.7	30.7	80.0	144.0	18.0	22.7	13.3	13.3	15.0	15.3
	625	12.7	33.0	93.3	112.7	17.7	20.3	6.3	15.7	14.3	19.0
	1250	8.0	44.7	103.3	128.0	18.0	19.0	9.3	11.7	14.3	20.0
	2500	13.3	27.0	98.7	117.3	22.0	21.0	11.3	15.7	19.0	16.7
	5000	10.7	34.3	99.3	125.0	17.7	26.0	8.7	9.7	18.7	20.3
Positive Control	-	748.0	490.7	778.7	1290.0	614.3	188.7	127.0	80.7	1151.3	211.0

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that 5-Methyl-3-vinyloxazolidin-2-on did not induce mutation in four histidine-requiring strains (TA98, TA100, TA1535 and
TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli when tested under the conditions of this
study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration
according to current regulatory guidelines) in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

5-Methyl-3-vinyloxazolidin-2-on was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli, both in the absence and

presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All 5-Methyl-3-vinyloxazolidin-2-on treatments in this study were performed using formulations prepared in water for irrigation (purified water).

Experiment 1 (plate incorporation test) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 5-Methyl-3-vinyloxazolidin-2-on at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity was observed at 5000 μg/plate in strain WP2 uvrA in the absence of S-9 only.

Experiment 2 (plate incorporation test without S-9; pre-incubation test with S-9) treatments of all the tester strains were performed. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 μg/plate, in order to examine more closely those concentrations of 5-Methyl-3-vinyloxazolidin-2-on approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments evidence of toxicity was observed at 5000 μg/plate in strain TA1537 in the presence of S-9. A marked reduction in revertant colony numbers was also observed at 1250 μg/plate in strain TA98 in the absence of S-9 but as this was observed only at one intermediate concentration, it was not considered clear evidence of toxicity.

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following 5-Methyl-3-vinyloxazolidin-2-on treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535, TA1537

and WP2 uvrA) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any 5-Methyl-3-vinyloxazolidin-2-on mutagenic activity in this assay system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

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:

other: OECD Guideline for the Testing of Chemicals No. 487, 26 Sep 2014, “In vitro Mammalian Cell Micronucleus Test”

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital i.p. and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1: 40.6, 81.2, 162.5, 325.0, 650.0, 1300.0 µg/mL (with and without S9 mix / 4 h exposur + 24 h preparation interval)
Experimant 2: 162.5, 325.0, 650.0, 1300.0 µg/mL (with and without S9 mix / 24 h exposur + 24 h preparation interval)
162.5, 325.0, 650.0, 1300.0 µg/mL (with and without S9 mix / 4 h exposur + 44 h preparation interval)

Vehicle / solvent:

culture medium (MEM)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: ethyl methanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 24h
- Exposure duration: 4h or 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): At the end of the 4-hour exposure period, the medium was removed and the cultures were
rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) supplemented with CytB (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO; AppliChem, Cat.No. A7657) was added and the cultures were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for the respective recovery time. In the case of 24-hour continuous exposure, CytB was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9 mix CytB was added 24 hours before preparation of the cultures.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B
STAIN (for cytogenetic assays): Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation index
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in at least 1000 cells per culture (at least 2000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerisation inhibitor cytochalasin B (17, 18). The number of mononucleated, binucleated and multinucleated cells was recorded.

OTHER EXAMINATIONS:
The cytospin slides were scored by fluorescence microscopy (Axio Imager.Z2, Zeiss, Göttingen, Germany).
As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle (3):
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.
Slides were coded before microscopic analysis with an appropriate computer program. Cultures with only few isolated cells were not analysed for micronuclei.
Since the absolute values shown were rounded but the calculations were made using the unedited values, there may be deviations in the given relative values.

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) have been printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no distinct cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest required test substance concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Exp.	Exposure / Preparation interval	Test groups	S9 mix	Prec.*	Genotoxicity Mico- nucleated cells** [%]	Cytotoxicity
						Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD)  [%]
1	4/24 hrs	Negative control	-	n.d.	0.4	0.0	100.0
		40.6 µg/mL	-	-	n.d.	n.d.	107.2
		81.3 µg/mL	-	-	n.d.	n.d.	109.1
		162.5 µg/mL	-	-	n.d.	n.d.	105.2
		325.0 µg/mL	-	-	0.3	-3.1	108.5
		650.0 µg/mL	-	-	0.4	-1.3	104.8
		1300.0 µg/mL	-	-	0.3	3.8	99.8
		Positive control^1	-	-	1.4^s	2.7	103.4
		Positive conrtol^2	-	n.d.	2.3^s	4.2	103.3
2	24/24 hrs	Negative control	-	n.d.	0.7	0.0	100.0
		162.5 µg/mL	-	-	n.d.	n.d.	81.4
		325.0 µg/mL	-	-	0.3	16.7	89.9
		650.0 µg/mL	-	-	0.6	26.5	61.2
		1300.0 µg/mL	-	-	0.3	45.2	60.5
		Positive control^1	-	n.d.	2.6^s	31.2	93.9
1	4/24 hrs	Negative control	+	n.d.	0.1	0.0	100.0
		40.6 µg/mL	+	-	n.d.	n.d.	83.4
		81.3 µg/mL	+	-	n.d.	n.d.	89.7
		162.5 µg/mL	+	-	n.d.	n.d.	84.2
		325.0 µg/mL	+	-	0.3	-2.1	86.2
		650.0 µg/mL	+	-	0.3	-0.2	85.9
		1300.0 µg/mL	+	-	0.3	8.0	76.5
		Positive control^3	+	n.d.	2.4^s	28.6	76.3
2	4/44 hrs	Negative control	+	n.d.	0.6	0.0	100.0
		162.5 µg/mL	+	-	n.d.	n.d.	102.0
		325.0 µg/mL	+	-	0.5	0.4	96.6
		650.0 µg/mL	+	-	0.4	2.1	102.6
		1300.0 µg/mL	+	-	1.2	1.8	94.1
		Positive control^3	+	n.d.	5.3^s	-10.3	74.0

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

SFrequency statistically significant higher than corresponding control values

n.d. Not determined

1EMS 400 μg/mL2EMS 500 μg/mL3CPP 0.5 μg/mL

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions described, 5-Methyl-3-vinyloxazolidin-2-on is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Executive summary:

The substance 5-Methyl-3-vinyloxazolidin-2-on was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested. The test groups printed in bold type were evaluated.

1st Experiment: 4 hours exposure, 24 hours harvest time, without S9 mix, 0; 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 μg/mL and 4 hours exposure, 24 hours harvest time, with S9 mix, 0; 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 μg/mL μg/mL.

2nd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix, 0; 162.5; 325.0; 650.0; 1300.0 μg/mL and 4 hours exposure, 44 hours harvest time, with S9 mix, 0; 162.5; 325.0; 650.0; 1300.0 μg/mL.

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

In this study, no distinct cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest required test substance concentration.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, 5-Methyl-3-vinyloxazolidin-2-on is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from male Wistar rats received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.

Test concentrations with justification for top dose:

Experiment 1: 81.3; 162.5; 325.0; 650.0; 1300.0 µg/mL (4h, without S9 mix); 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 µg/mL (4h, with S9 mix)
Experiment 2 +3: 250.0; 500.0; 1000.0; 1300.0 µg/mL (4h, with and without S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6 - 8 d
- Selection time (if incubation with a selection agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 15 d

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL); 10% (v/v) FCS

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criterion:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
No cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in both evaluated experiments tested for gene mutations in the presence and absence of S9 mix.
CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations.
TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment. In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest required test substance concentration.
MUTANT FREQUENCY
The 2nd Experiment was discontinued due to microbial contamination. Thus, no data for the mutation frequencies were obtained. In the 1st and 3rd Experiment, no increase in the number of mutant colonies was observed in the presence and absence of S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.68 – 3.58 per 106 cells) were close to the respective vehicle control values (MFcorr.: 2.13 – 5.70 per 106 cells) and clearly within the range of our historical negative control data (without S9 mix:
MFcorr.: 0.00 – 16.43 per 106 cells; with S9 mix: MFcorr.: 0.00 – 16.12 per 106 cells). In all evaluated experimental parts, no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 138.14 – 202.22 per 106 cells; with S9 mix: MFcorr.: 239.41 – 356.65 per 106 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 47.35 – 383.57 per 106 cells; with S9 mix: MFcorr.: 41.99 – 812.14 per 106 cells).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of results

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr.         [per 10^6cells]	Cytotoxicity***
						CH1 [%]	CH2 [%]
1	4	Negative control	-	n.d.	2.13	100.0	100.0
		81.3	-	-	n.c.	97.3	n.c.
		162.5	-	-	3.54	95.7	108.0
		325.0	-	-	3.58	96.9	89.4
		650.0	-	-	1.79	96.3	93.6
		1300.0	-	-	1.35	100.4	102.8
		Positive control^1	-	n.d.	138.14	99.4	95.5
3	4	Negative control	-	n.d.	3.43	100.0	100.0
		250.0	-	-	2.86	108.1	101.9
		500.0	-	-	1.61	95.2	99.0
		1000.0	-	-	1.36	98.9	99.1
		1300.0	-	-	0.68	91.4	98.7
		Positive control^1	-	n.d.	202.22	95.3	89.5
1	4	Negative control	+	n.d.	3.22	100.0	100.0
		40.6	+	-	n.c.	104.1	n.c.
		81.3	+	-	n.c.	99.1	n.c.
		162.5	+	-	3.25	110.8	96.2
		325.0	+	-	2.08	105.0	104.3
		650.0	+	-	3.61	105.3	94.6
		1300.0	+	-	2.30	98.1	112.2
		Positive control^2	+	n.d.	356.65	90.3	88.4
3	4	Negative control	+	n.d.	5.70	100.0	100.0
		250.0	+	-	3.54	109.7	111.9
		500.0	+	-	1.62	103.6	106.6
		1000.0	+	-	0.72	95.3	104.0
		1300.0	+	-	2.52	91.9	108.3
		Positive control^1	+	n.d.	239.41	100.8	83.3

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106cells corrected with the CE2value

*** Cloning efficiency related to the respective vehicle control

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.d. Not determined

¹EMS 400μg/mL     

²DMBA 1.25μg/mL

Conclusions:

Interpretation of results (migrated information):
negative

Thus, in the absence and the presence of metabolic activation, 5-Methyl-3-vinyloxazolidin-2-on is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The substance 5-Methyl-3-vinyloxazolidin-2-on was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment: without S9 mix 0; 81.3; 162.5; 325.0; 650.0; 1300.0 μg/mL and with S9 mix 0; 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 μg/mL. 2nd Experiment (discontinued due to technical reasons), without S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL and

with S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL. 3rd Experiment, without S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL and

with S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL.

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant

increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance 5-Methyl-3-vinyloxazolidin-2-on is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Feb 2019 - 12 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 Jul 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 ;May 2008

Deviations:

yes

Remarks:

Due to some differences between the above cited guidelines at diverging points (e.g. dose selection) the recommendations of the most recent OECD Guideline (490) were followed

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

aug 1998

Deviations:

yes

Remarks:

Due to some differences between the above cited guidelines at diverging points (e.g. dose selection) the recommendations of the most recent OECD Guideline (490) were followed

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

- Name of test material (as cited in study report): 5-methyl-3-vinyloxazolidin-2-one
- Molecular formula (if other than submission substance): C6H9NO2
- Molecular weight (if other than submission substance): 127.14 g/mol
- Physical state, appearance: liquid, yellow
- Homogeneity: The homogeneity of the test substance was ensured by mixing before
preparation of the test substance preparations.
- Expiration date of the lot/batch: The stability of the test substance under storage was
guaranteed until 09 Feb 2020 as indicated by the sponsor, and the sponsor holds this responsibility.
- Content: about 95.9g/100g (1H-nmr)%
- Lot/batch No.: Z019-2018
- Storage condition of test material: room temperature

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The mouse lymphoma L5178Y TK+/- cell line (clone 3.7.2c [7]) is derived from a permanent cell
line of 3-methylcholanthrene induced tumors (thymus) in DBA/2 mice and has a
- high proliferation rate (doubling time of about 9 - 10 hours)
- high plating efficiency (about 90%)
- stable karyotype with a near diploid number of 40 ± 1 chromosomes.
Stocks of the mouse lymphoma L5178Y TK+/- cell line (1-mL portions) will be maintained at
- 196°C in the gas phase above the liquified nitrogen using 7% (v/v) DMSO in FCS as a
cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma
contamination previously.
Culture media
RPMI 1640 medium including stable glutamine supplemented with:
1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
1% (v/v) sodium pyruvate (10 mM) (= RPMI-0)
For treatment medium (with S9 mix):
RPMI-0 supplemented with 5% (v/v) fetal calf serum (= RPMI-5)
For treatment medium (without S9 mix) and sub culturing cells:
RPMI-0 is supplemented with 10% (v/v) fetal calf serum (= RPMI-10)
For cloning efficiency and selection medium:
RPMI-0 supplemented with 20% (v/v) fetal calf serum (= RPMI-20)
Pretreatment medium A ("THMG" medium)
RPMI-10 supplemented with: thymidine 3.0 μg/mL; thymidine 3.0 μg/mL;
methotrexate 0.1 μg/mL; glycine 7.5 μg/mL.
Pretreatment medium B ("THG" medium)
RPMI-10 supplemented with: thymidine 3.0 μg/mL; hypoxanthine 5.0μg/mL; glycine 7.5 μg/mL
Selection medium ("TFT" medium)
RPMI-20 supplemented with: trifluorothymidine (TFT) 4.0 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital and β-naphthoflavone induced rats

Test concentrations with justification for top dose:

Pretest:
In the pretest for toxicity based on the purity and the molecular weight of the test substance
1400.0 μg/mL (approx. 10 mM) 5-Methyl-3-vinyloxazolidin-2-on was used as top
concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-
hour exposure time.

1st Experiment:
With and without S9 mix (4-hour exposure period): 0, 87.5, 175.0, 350.0, 700.0 , 1400.0 μg/mL
2nd Experiment
Without S9 mix (24-hour exposure period): 87.5, 175.0, 350.0, 700.0 , 1400.0 μg/mL
With S9 mix (4-hour exposure period): 100.0, 200.0, 400.0, 700.0, 1400.0 μg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in culture medium, culture medium (RPMI 1640)
was selected as vehicle.
After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by
reduced relative suspension growth of about or below 20% was observed. After 24 hours
treatment in the absence of S9 mix slightly reduced relative suspension growth was observed
after treatment with 1400.0 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Pretest
The pretest was performed following the method described for the main experiment. The
relative suspension growth (RSG) was determined as toxicity indicator for dose selection and
various parameters were checked for all or at least for some selected doses.
In the pretest the parameters pH value and osmolality were not relevantly influenced by the
addition of the test substance preparation to the culture medium at the concentrations
measured.
In culture medium no test substance precipitation occurred up to the highest applied at the end
of treatment in the absence and the presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by
reduced relative suspension growth of about or below 20% was observed. After 24 hours
treatment in the absence of S9 mix slightly reduced relative suspension growth was observed
after treatment with 1400.0 μg/mL.

Preparation of test cultures
Logarithmically growing cells in suspension culture (3 x 105 cells per 75 cm² flask in a total
volume of 30 mL in exponential growth per treatment group required) were incubated 4 - 5 days
prior to the start of the experiment.
3.7.4. Pretreatment of cells
During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated
from the stock cultures by incubating 3 x 105 cells per 75 cm² flask in a total volume of 30 mL
for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in
“THG" medium (pretreatment medium B).
3.7.5. Treatment of test cultures and expression period
Following centrifugation and resuspension the cells were dispensed into 75 cm² flasks
(4-hour exposure: 1.5 x 107 cells per culture; 24-hour exposure: 1 x 107 cells per culture). Two
cultures were treated in parallel for each test group. Subsequently the treatment medium was
added The cultures were incubated for the respective exposure period.
At the end of the exposure period, the cells were transferred in tubes, centrifuged for 5 minutes
at 800 rpm (134 g) and were resuspended in RPMI-5 medium. The washing of the cells was
repeated at least once. Then the cells were centrifuged at 800 rpm (134 g, 5 min) and
resuspended in RPMI-10 medium. From each culture a sample of treated cells
(2 x 105 cells/mL or 6 x 106 cells/flask) was pipetted in 75 cm² flasks and incubated for a 2-day
expression period.
To maintain exponential growth during this phase, each culture was counted daily and the cell
numbers were adjusted at each day to 2 x 105 cells/mL in 30 mL RPMI-10 medium. The cell
numbers were determined using a cell counter.
Selection period
For the selection of the mutants, the cells were centrifuged (134 g, 5 min) and 5 x 105 cells
from each culture were resuspended in 50 mL selection medium (“TFT" medium;
1 x 104 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates
(2000 cells/well). After incubation for at least 9 days, both the number of negative wells and
the number of wells containing small or large colonies were scored for calculation of the mutant
frequency (MF).
Cytotoxicity determination
Cloning efficiency 1 (survival)
At the end of the exposure period, the cells were centrifuged (134 g, 5 min) and 400 cells from
each test group were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture 200 μL
were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for
9 - 11 days the plates were scored for empty wells.
Cloning efficiency 2 (viability)
After the expression period, 2 days after end of exposure, the cells were centrifuged (134 g,
5 min) and 400 cells from each culture were resuspended in 50 mL RPMI-20 medium
(8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates
(1.6 cells/well). After incubation for at least 9 days the plates were scored for empty wells.
Relative suspension growth and relative total growth
For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the
cell counts determined within the expression period at 2nd and 3rd passage after exposure in
the case of 4-hour exposure and 1st, 2nd and 3rd passage after exposure in the case of 24-
hour exposure were used.
In the pretest single cultures per test group were conducted only.
Other:
The pH was measured at least for the top concentration and for the negative controls with and
without S9 mix at the beginning of treatment.
Osmolality was measured at least for the top concentration and for the negative controls with
and without S9 mix at the beginning of treatment.
Test substance precipitation was checked immediately after treatment of the test cultures and
at the end of treatment with the unaided eye.

Rationale for test conditions:

Following the requirements of the current OECD Guideline 490 a test substance with defined
composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM,
whichever is the lowest. When the test substance is not of defined composition, e.g. substance
of unknown or variable composition, complex reaction products or biological materials (socalled
UVCBs), or environmental extracts, the top concentration should be higher to increase
the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top
concentration should result in approximately 10 - 20% relative suspension growth (RSG,
pretest) or relative total growth (RTG, main experiments), but not less than 10%. For relatively
insoluble test substances only one concentration should be tested showing turbidity or
precipitation in culture medium at the end of exposure period determined with the unaided eye.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After 24 hours continuous treatment without S9 mix slightly reduced relative total growth of 56.6% was observed in the test group with the highest concentration (1400.0 μg/mL).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Treatment conditions
Osmolality and pH values were not relevantly influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix no precipitation in culture medium
was observed up to the highest applied test substance concentration.
Cytotoxicity
After four hours treatment in the absence and presence of metabolic activation relevant
cytotoxic effects indicated by reduced relative total growth of below 20% of control were not
observed.
After 24 hours continuous treatment without S9 mix slightly reduced relative total growth of
56.6% was observed in the test group with the highest concentration (1400.0 μg/mL).
Mutant frequency
In the absence of S9 mix, no dose-related increases in the corrected mutation frequency were
observed as determined by testing for linear trend. In the 1st Experiment, the values of all test
groups (MFcorr.: 48.8 – 80.0 per 106 cells) were below the respective calculated
threshold for a mutagenic effect based on the GEF (194 mutant colonies per 106 cells). After
24 hours treatment without S9 mix in the 2nd Experiment the corrected mutation frequencies
ranged between 78.5 and 100.1 colonies per 106 cells and were below the calculated threshold
of 209 mutant colonies per 106 cells.
In the presence of S9 mix the statistical analyses by testing for linear trend led to clearly
negative findings. The values after 4 hours treatment with S9 mix (MFcorr.:
64.4 – 80.0 per 106 cells) were lower than the respective threshold of 194 mutant colonies per
106 cells.
Both the positive control substances known to induce gene mutations, MMS (without S9 mix;
5 and 15 μg/mL; MFcorr.: 883.7 and 864.8 per 106 cells, respectively) and DMBA (with S9 mix;
1.0 μg/mL; MFcorr.: 748.6 per 106 cells), and the well-known clastogen CPP (with S9 mix;
2.5 μg/mL; MFcorr.: 409.0 per 106 cells) led to clearly increased mutant frequencies as expected.
The values exceeded the respective calculated thresholds for a mutagenic effect based on the
GEF (126 plus the mutant frequency of the respective negative control). In addition, the
corrected mutant frequencies were comparable with the historical positive control data range.

Selection period

For the selection of the mutants, the cells were centrifuged (134 g, 5 min) and 5 x 105 cells

from each culture were resuspended in 50 mL selection medium (“TFT" medium;

1 x 104 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates

(2000 cells/well). After incubation for at least 9 days, both the number of negative wells and

the number of wells containing small or large colonies were scored for calculation of the mutant

frequency (MF).

In the pretest for toxicity based on the purity and the molecular weight of the test substance

1400.0 μg/mL (approx. 10 mM) 5-Methyl-3-vinyloxazolidin-2-on was used as top

concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-

hour exposure time.

The pretest was performed following the method described for the main experiment. The

relative suspension growth (RSG) was determined as toxicity indicator for dose selection and

various parameters were checked for all or at least for some selected doses.

In the pretest the parameters pH value and osmolality were not relevantly influenced by the

addition of the test substance preparation to the culture medium at the concentrations

measured.

In culture medium no test substance precipitation occurred up to the highest applied at the end

of treatment in the absence and the presence of S9 mix.

After 4 hours treatment in the absence and presence of S9 mix no cytotoxicity indicated by

reduced relative suspension growth of about or below 20% was observed. After 24 hours

treatment in the absence of S9 mix slightly reduced relative suspension growth was observed

after treatment with 1400.0 μg/mL

MUTANT FREQUENCY

In the absence of S9 mix, no dose-related increases in the corrected mutation frequency were

observed as determined by testing for linear trend (cf. attached background material). In the 1st Experiment, the

values of all test groups (MFcorr.: 48.8 – 80.0 per 106 cells) were below the respective calculated

threshold for a mutagenic effect based on the GEF (194 mutant colonies per 106 cells). After

24 hours treatment without S9 mix in the 2nd Experiment the corrected mutation frequencies

ranged between 78.5 and 100.1 colonies per 106 cells and were below the calculated threshold

of 209 mutant colonies per 106 cells.

In the presence of S9 mix the statistical analyses by testing for linear trend led to clearly

negative findings. The values after 4 hours treatment with S9 mix (MFcorr.:

64.4 – 80.0 per 106 cells) were lower than the respective threshold of 194 mutant colonies per

106 cells.

Both the positive control substances known to induce gene mutations, MMS (without S9 mix;

5 and 15 μg/mL; MFcorr.: 883.7 and 864.8 per 106 cells, respectively) and DMBA (with S9 mix;

1.0 μg/mL; MFcorr.: 748.6 per 106 cells), and the well-known clastogen CPP (with S9 mix;

2.5 μg/mL; MFcorr.: 409.0 per 106 cells) led to clearly increased mutant frequencies as expected.

The values exceeded the respective calculated thresholds for a mutagenic effect based on the

GEF (126 plus the mutant frequency of the respective negative control). In addition, the

corrected mutant frequencies were comparable with the historical positive control data range.

Besides, the obtained values fulfilled the criteria for positive controls as

mentioned in the current OECD Guideline 490.

Cytotoxicity determination

Cloning efficiency 1 (survival)

At the end of the exposure period, the cells were centrifuged (134 g, 5 min) and 400 cells from

each test group were resuspended in 50 mL RPMI-20 medium (8 cells/mL). Per culture 200 μL

were dispensed in each well of two 96-well plates (1.6 cells/well). After incubation for

9 - 11 days the plates were scored for empty wells.

Cloning efficiency 2 (viability)

After the expression period, 2 days after end of exposure, the cells were centrifuged (134 g,

5 min) and 400 cells from each culture were resuspended in 50 mL RPMI-20 medium

(8 cells/mL). Per culture 200 μL were dispensed in each well of two 96-well plates

(1.6 cells/well). After incubation for at least 9 days the plates were scored for empty wells.

Relative suspension growth and relative total growth

For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the

cell counts determined within the expression period at 2nd and 3rd passage after exposure in

the case of 4-hour exposure and 1st, 2nd and 3rd passage after exposure in the case of 24-

hour exposure were used.

In the pretest single cultures per test group were conducted only.

Conclusions:

The test substance 5-Methyl-3-vinyloxazolidin-2-on was tested for its ability to induce gene
mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at
chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method.
Two independent experiments were carried out with and/or without the addition of liver S9 mix
from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding cytotoxicity test for the determination of the experimental
doses and the molecular weight of the test substance, the following concentrations were tested,
whereby the top concentration corresponds to approx. 10 mM. The test groups printed in bold
type were evaluated for gene mutations.
1st Experiment
without S9 mix (4-hour exposure period)
0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL
with S9 mix (4-hour exposure period)
0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL
Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic
activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were
cultured for an expression period of about 48 hours and then cultured in selection medium for
another approx. 10 days. Finally, the number of large and small colonies was determined.
The negative controls gave mutant frequencies within the range expected for the
L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene
mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and
7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of
forward mutations.
No relevant cytotoxicity indicated by reduced relative total growth (RTG) below 20% of control
was observed in both experiments up to the highest tested concentration.
Based on the results of the present study, the test substance did not cause any biologically
relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after
adding a metabolizing system in two experiments performed independently of each other.
According to the results of the present in vitro study, the test substance 5-Methyl-3-
vinyloxazolidin-2-on did not lead to a dose-dependent increase in the number of mutant
colonies either without S9 mix or after the addition of a metabolizing system in two experiments
performed independently of each other. The mutant frequencies at any concentration were
below the respective thresholds of 126 colonies per 106 cells (GEF) and in the range of the
concurrent negative control values.
The mutation frequencies of the negative control groups were within our historical negative
control data range including all vehicles used and, thus, fulfilled the acceptance criteria of this
study.
The proficiency of the laboratory to perform the mouse lymphoma assay with L5178Y TK+/-
cells was demonstrated by the laboratory’s historical control database on vehicle and positive
controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of mutant colonies induced by the positive control substances
MMS, DMBA and CPP clearly demonstrated the sensitivity of the test method and/or the
metabolic activity of the S9 mix employed. The values were within the range of the historical
positive control data.
Thus, under the experimental conditions described, 5-Methyl-3-vinyloxazolidin-2-on did not
induce forward mutations or structural chromosome aberrations in vitro in the mouse
lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic
activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

5-Methyl-3-vinyloxazolidin-2-one was assayed for mutation in four histidine-requiring strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium and one tryptophan-requiring strain (WP2 uvrA) of Escherichia coli, both in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments (BASF SE, 2014).

Experiment 1 (plate incorporation test) treatments of all the tester strains were performed in the absence and presence of S-9, using final concentrations of 5-Methyl-3-vinyloxazolidin-2-on at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments evidence of toxicity was observed at 5000 μg/plate in strain WP2 uvrA in the absence of S-9 only.

Experiment 2 (plate incorporation test without S-9; pre-incubation test with S-9) treatments of all the tester strains were performed. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 μg/plate, in order to examine more closely those concentrations of 5-Methyl-3-vinyloxazolidin-2-on approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments evidence of toxicity was observed at 5000 μg/plate in strain TA1537 in the presence of S-9. A marked reduction in revertant colony numbers was also observed at 1250 μg/plate in strain TA98 in the absence of S-9 but as this was observed only at one intermediate concentration, it was not considered clear evidence of toxicity. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. Following 5-Methyl-3-vinyloxazolidin-2-on treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535, TA1537 and WP2 uvrA) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any 5-Methyl-3-vinyloxazolidin-2-on mutagenic activity in this assay system.

The substance 5-Methyl-3-vinyloxazolidin-2-one was assessed for its potential to induce micronuclei in V79 cells in vitro (BASF SE, 2016). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations and the toxicity data of a previously performed pretest for a HPRT study the following concentrations were tested:

1st Experiment: 4 hours exposure, 24 hours harvest time, without S9 mix, 0; 325.0; 650.0; 1300.0 μg/mL and 4 hours exposure, 24 hours harvest time, with S9 mix, 0; 325.0; 650.0; 1300.0 μg/mL.

2nd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix, 0; 325.0; 650.0; 1300.0 μg/mL and 4 hours exposure, 44 hours harvest time, with S9 mix, 0; 325.0; 650.0; 1300.0 μg/mL.

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

In this study, no distinct cytotoxicity indicated by clearly reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest required test substance concentration.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, 5-Methyl-3-vinyloxazolidin-2-one is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence metabolic activation.

The substance 5-Methyl-3-vinyloxazolidin-2-one was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested:

1st Experiment: without S9 mix 0; 162.5; 325.0; 650.0; 1300.0 μg/mL and with S9 mix 0; 162.5; 325.0; 650.0; 1300.0 μg/mL. 2nd Experiment (discontinued due to technical reasons), 3rd Experiment without S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL and with S9 mix 0; 250.0; 500.0; 1000.0; 1300.0 μg/mL.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance 5-Methyl-3-vinyloxazolidin-2-on is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

The test substance 5-Methyl-3-vinyloxazolidin-2-on was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at

chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method. Two independent experiments were carried out with and/or without the addition of liver S9 mix

from phenobarbital and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental

doses and the molecular weight of the test substance, the following concentrations were tested, whereby the top concentration corresponds to approx. 10 mM.

1st Experiment: without S9 mix (4-hour exposure period) 0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL; with S9 mix (4-hour exposure period) 0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL

2nd Experiment without S9 mix (24-hour exposure period) 0; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL

Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were

cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined.

The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and 7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of forward mutations. No relevant cytotoxicity indicated

by reduced relative total growth (RTG) below 20% of control was observed in both experiments up to the highest tested concentration.

Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions described, 5-Methyl-3-vinyloxazolidin-2-on did not induce forward mutations or structural chromosome aberrations in vitro in the mouse

lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the results of the genetic toxicity testing, the classification criteria according to Regulation (EC) No 1272/2008 (CLP) are not met.