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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

809-852-5

Cas Number:

3395-98-0

Molecular formula:

C6H9NO2

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): 5-Methyl-3-vinyloxazolidin-2-on
- Physical state: liquid
- Test article number: 14/0031-1
- Analytical purity: > 98%
- Lot/batch No.: DEIMLIB 00028 Fr.8
- Expiration date of the lot/batch: 12th July 2014
- Storage condition of test material: 2-8°C, under nitrogen

Method

Target gene:

His operon

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.