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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start: 21 August 2019; experiment end: 12 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

849-166-3

Cas Number:

83841-00-3

Molecular formula:

C4H5NO4

IUPAC Name:

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

Test material form:

solid

Method

Target gene:

Histidine-dependent auxotrophic mutants of S. typhimurium and a tryptophan-dependent mutant of E. coli were used in the test.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction (Sprague-Dawley) was purchased from Moltox and stored at approximately -196 °C in a liquid nitrogen freezer; Lot No. 4123 was used in this study and the protein level was adjusted to 20 mg/mL. For a 10% S9-mix, an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approximately 10% (v/v) in the S9-mix. The S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium orthophosphate buffer (100 mM, pH 7.4) and was prepared using sterilized co-factors immediately prior to use and maintained on ice for the duration of the test.

Test concentrations with justification for top dose:

Plate incorporation test (experiment 1): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-incubation test (experiment 2): 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

Sterile distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

Without S9 mix: 4-nitroquinoline-N-oxide, 9-aminoacridine, N-ethyl-N-nitro-N-nitrosoguanidine
With S9 mix: 2-aminoanthracene, benzo(a)pyrene

Details on test system and experimental conditions:

Bacterial cultures were prepared from frozen stocks by incubating for 10 hours at 37 °C in a shaking incubator. The bacteria were obtained from Trinova Biochem GmbH on 27 June 2017 and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
Media: Top agar was prepared using 0.6% w/v Bacto agar (lot number 8255817 07/2023) and 0.5% w/v sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 51673 09/2019).
Pre-cultures: A culture of each of the bacterial strains was prepared by inoculating nutrient broth with the appropriate coded stock culture and incubated, with shaking, for approximately 10 hours at 37 ± 3 °C. The bacterial cell count for each culture was determined by viable count analysis on nutrient agar plates on the day of test and was as follows
TA100 - 3.1 and 4.0E09/mL (experiment 1 and 2)
TA1535 - 1.3 and 3.5E09/mL (experiment 1 and 2)
WP2 uvrA pKM101 - 2.4 and 4.4E09/mL (experiment 1 and 2)
TA98 - 2.2 and 2.2E09/mL (experiment 1 and 2)
TA1537 - 1.7 and 2.409/mL (experiment 1 and 2)
Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Conditions experiment 1, without S9 mix: 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
Conditions experiment 1, with S9 mix: The procedure was the same as described previously (see 3.5.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and scoring, experiment 1: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Sporadic manual counts were performed due to spreading colonies which prevented an accurate automated count.
Experiment 2: The concentration range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
Conditions experiment 2, without S9 mix: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 30 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
Conditions experiment 2, with S9 mix: The procedure was the same as described previously (see 3.5.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 30 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and scoring experiment 2: All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Rationale for test conditions:

The maximum concentration in experiment 1 was 5000 μg/plate (the maximum recommended concentration). In experiment 2, six test item concentrations per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic concentration levels and the maximum recommended concentration following the change in test methodology from plate incorporation to pre-incubation. No toxicity was observed up to the maximum concentration, with and without S9 mix. No test substance precipitate was observed on the plates at any of the concentrations tested with our without S9 mix in experiments 1 and 2.

Evaluation criteria:

If exposure to a test item produces a reproducible increase, in one or more concentration, in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship in at least one strain with or without metabolic activation system, it will be considered to exhibit mutagenic activity in this test system (Mortelmans and Zeiger 2000). No statistical analysis was performed.
If exposure to a test item does not produce an increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will usually be Dunnett’s test followed, if appropriate, by trend analysis (Mahon et al, 1989). Biological significance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below twice those of the concurrent vehicle controls (as described above) will not be considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director will use his/her scientific judgment.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table: summary of results of plate incorporation test

	Number of revertants (mean) +/- SD
	Base-pair substitution strains						Frameshift strains
Concentration	TA100		TA1535		WP2uvrApKM101		TA98		TA1537
Per Plate	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
Solvent Control	107	144	10	18	146	114	29	38	7	7
(Water)	106	142	7	8	112	108	20	21	12	8
	146	140	10	11	120	124	37	25	14	16
1.5 µg	122	136	14	12	117	136	10	9	11	5
	145	135	8	10	115	106	30	20	15	7
	119	141	11	14	103	143	17	20	10	4
5 µg	111	143	10	11	94	143	17	21	14	9
	112	123	10	14	102	142	27	32	11	4
	135	125	15	14	117	130	18	30	10	8
15 µg	103	151	10	14	117	135	15	18	12	5
	110	147	13	8	112	136	22	30	6	7
	122	122	11	16	120	142	19	20	15	4
50 µg	115	117	12	19	136	130	9	14	17	3
	121	116	7	8	128	155	17	12	15	10
	103	131	10	11	132	128	28	13	13	7
150 µg	130	113	13	10	128	133	21	28	13	9
	129	134	17	23	169	114	20	22	3	12
	105	148	12	9	121	142	20	28	7	9
500 µg	104	147	12	12	125	141	20	16	5	5
	114	126	7	12	144	124	16	20	7	8
	107	124	12	13	137	113	11	14	6	10
1500 µg	103	125	12	15	131	153	15	22	5	19
	97	101	10	14	132	135	27	22	9	12
	118	163	12	11	131	118	25	28	10	2
5000 µg	106	115	7	9	117	153	23	25	5	7
	116	114	8	13	121	121	19	49	4	6
	113	137	17	13	129	138	31	20	7	11
Positive controls	ENNG		ENNG		ENNG		4NQO		9AA
Concentration	3 µg		5 µg		0.5 µg		0.2 µg		80 µg
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
	486		367		910		126		281
	439		546		719		168		380
	408		664		792		228		305
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration		1 µg		2 µg		10 µg		5 µg		2 µg
		1639		295		1143		147		155
		1876		252		1115		130		160
		1869		268		1096		190		179

Table: summary of results of pre-incubation test

	Number of revertants (mean) +/- SD
	Base-pair substitution strains						Frameshift strains
Concentration	TA100		TA1535		WP2uvrApKM101		TA98		TA1537
Per Plate	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
Solvent Control	108	114	9	11	95	77	27	31	12	11
(Water)	129	136	19	13	121	118	18	29	12	16
	134	119	10	8	84	148	29	26	9	14
15 µg	117	131	9	12	100	94	21	44	17	16
	109	111	4	9	98	104	27	29	14	16
	127	145	15	6	87	110	32	34	17	13
50 µg	109	102	9	12	91	119	28	25	12	9
	114	136	11	20	136	124	31	31	18	14
	141	129	13	10	140	104	21	30	11	7
150 µg	111	120	7	5	90	116	17	36	9	8
	107	101	5	10	170	146	26	38	17	14
	101	128	12	11	68	161	30	29	17	13
500 µg	109	129	18	7	106	144	23	28	19	10
	112	131	17	9	106	145	15	26	10	12
	114	133	7	8	107	146	27	45	10	15
1500 µg	132	134	12	9	103	87	22	43	14	14
	117	126	13	10	106	112	19	35	9	10
	120	115	11	8	108	124	18	24	15	16
5000 µg	97	130	11	12	105	124	25	39	9	9
	118	133	8	12	112	125	22	30	16	14
	113	129	15	19	107	127	20	36	12	11
Positive controls	ENNG		ENNG		ENNG		4NQO		9AA
Concentration	3 µg		5 µg		0.5 µg		0.2 µg		80 µg
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
	716		2235		632		205		148
	637		2753		603		195		197
	657		2187		687		209		144
Positive controls		2AA		2AA		2AA		BP		2AA
Concentration		1 µg		2 µg		10 µg		5 µg		2 µg
		1491		322		1035		102		175
		1311		350		1067		154		191
		1283		313		1124		121		222

Applicant's summary and conclusion

Conclusions:

The substance did not induce gene mutations by base pair changes or frameshifts in the genome of S. typhimurium and E. coli strains, and was considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of the substance was studied under GLP to OECD TG 471 with histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrApKM101. The bacteria were exposed to the substance diluted in sterile distilled water. Two independent mutation experiments were performed in the presence and absence of liver preparations (S9-mix) from rats treated with phenobarbital and β-naphthoflavone. Experiment 1 was a standard plate incorporation assay and experiment 2 a pre-incubation test. The maximum concentration of the test item in experiment 1 was 5000 µg/plate, the standard limit concentration recommended in the regulatory guideline that this assay follows. This maximum concentration was also selected for experiment 2. There was no toxicity, evident as a reduction in the number of revertants (below an induction factor of 0.5) or a reduction in the background lawn, in any of the five tester strains either with or without S9 mix following exposure to the substance in either the plate incubation or pre-incubation experiments. No test item precipitate was observed on the plates at any of the concentrations tested in either the presence or absence of metabolic activation (S9-mix) in experiments 1 and 2. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any concentration of the test item, either with or without metabolic activation (S9-mix) in experiments 1 and 2. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.