1-[5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]ethanone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Japan Test Guidelines for Agricultural Chemicals 12-Nousan-8147: Guideline Number 2-1-19-1 (2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Purity: 99.9%

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 from male Sprague-Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

toxicity-mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg per plate
mutagenicity test: 333, 667, 1000, 3333, and 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

triethylenemelamine

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, Acridine mutagen ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: agar plate incorporation

DURATION
- All samples were vortexed and overlaid onto the surface of minimum glucose agar plates. After the overlay solidified, the plates were inverted and incubated for approximately 50-54 hours at 37 ± 2ºC. Plates that were not evaluated immediately following the incubation period were stored at approximately 4ºC. All toxicity-mutation test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in duplicate. All mutagenicity test dose preparations of negative (vehicle) controls, test substance, and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered as signs of toxicity. A minimum of 3 non-toxic scorable dose levels are required to validate the study. A dose level is considered toxic if it causes:
• A > 50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibits a dose-dependent drop in the revertant count, or
• A reduction in the background lawn.

Evaluation criteria:

Strains TA1535 and TA1537: Data will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

Strains TA98, TA100 and WP2uvrA: Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

Statistics:

For each tester strain, the mean of the number of revertants and the standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Toxicity was observed as a >50% reduction in revertant colonies with tester strain TA1537 starting at 3333 μg per plate in the absence of S9 activation. Toxicity was observed as a reduction in background lawn with tester strain TA100 starting at 3333 μg per plate in the presence of S9 activation and at 5000 μg per plate in the absence of S9 activation. No other appreciable toxicity was observed. No test substance precipitation was observed.

No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

Summary of the toxicity-mutation test without rat liver S9
Dose (µg/plate)	Number of Revertants Per Plate
	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Vehicle	18	2	95	2	14	10	10	5	28	9
Positive control	169	9	1311	43	1164	31	1628	171	448	23
33.3	16	4	109	13	14	3	8	3	33	6
66.7	21	5	91	8	11	6	9	5	36	1
100	25	2	103	8	12	1	7	1	30	3
333	22	1	102	3	15	4	8	3	23	1
667	28	1	97	4	12	0	8	4	28	1
1000	21	8	87	9	14	7	8	3	27	0
3333	13	4	81	2	11	0	4	0	27	4
5000	15	6	59	1	12	1	3	1	22	4
Experiment No: T-1 Plate Aliquot: 100 µL

 

Summary of the toxicity-mutation test with rat liver S9
Dose (µg/plate)	Number of Revertants Per Plate
	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Vehicle	27	11	111	5	12	1	6	1	35	6
Positive control	489	28	2323	53	186	11	107	6	214	20
33.3	30	6	111	3	15	2	7	4	37	6
66.7	30	0	132	11	10	1	8	6	34	0
100	28	7	118	6	14	2	8	2	36	4
333	29	1	122	23	12	10	8	4	32	2
667	28	1	103	19	9	0	9	2	35	3
1000	31	2	127	4	8	3	10	1	34	1
3333	30	4	134	33	10	1	10	1	28	13
5000	25	12	113	13	9	5	5	1	32	4
Experiment No: T-1 Plate Aliquot: 100 µL

 

Summary of the mutagenicity test without rat liver S9
Dose (µg/plate)	Number of Revertants Per Plate
	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Vehicle	16	4	82	9	12	2	9	3	30	5
Positive control	189	47	1065	112	948	19	1149	34	529	66
333	15	4	81	20	8	4	8	3	23	8
667	18	4	71	12	12	1	5	2	25	4
1000	16	6	83	20	9	2	7	2	18	5
3333	13	3	67	5	7	3	2	1	31	7
5000	14	3	53	8	9	3	3	2	23	1
Experiment No: E-1 Plate Aliquot: 100 µL

 

Summary of the mutagenicity test with rat liver S9
Dose (µg/plate)	Number of Revertants Per Plate
	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Vehicle	26	4	118	14	10	2	8	4	33	2
Positive control	409	24	2013	189	160	11	108	25	188	5
333	20	7	120	7	11	4	9	4	36	8
667	25	3	121	9	12	5	7	4	34	4
1000	20	5	134	20	11	6	11	4	35	5
3333	20	5	114	9	10	7	7	3	32	5
5000	14	4	107	18	7	2	3	3	31	2
Experiment No: E-1 Plate Aliquot: 100 µL

Applicant's summary and conclusion

Conclusions:

It was concluded that the test substance was negative in this in vitro test.

Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

The dose levels selected for the mutagenicity test were 333, 667, 1000, 3333, and 5000 μg per plate.

No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. Toxicity was observed as a reduction in background lawn starting at 3333 μg per plate with tester strain TA100 in the presence of S9 activation and strain TA1537 both in the presence and absence of S9 activation. A >50% reduction in revertant colonies was also observed for tester strain TA1537 starting at 3333 μg per plate in the absence of S9 activation and at 5000 μg per plate in the presence of S9 activation.

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9.