Vinyl benzoate

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:

21 Jul - 05 Aug 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

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

Version / remarks:

Commission Directive No. 2000/32/EC

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vinyl benzoate

EC Number:

212-214-3

EC Name:

Vinyl benzoate

Cas Number:

769-78-8

Molecular formula:

C9H8O2

IUPAC Name:

vinyl benzoate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80/100 mg/kg bw/day)

Test concentrations with justification for top dose:

Pre-experiment: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without mtabolic activation for TA 100 and WP2 uvr A

Main Experiment 1:
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for WP2 uvr A
5, 15, 50, 150, 500, 1500 and 500 µg/plate with and without metabolic activation for remaining strains and without metabolic activation for WP2 uvr A

Main Experiment 2:
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for WP2 uvr A
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for remaining strains

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information supplied by the sponsor suggested that the test material is insoluble in 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 (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: clearing of the bacterial background lawn

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by UKEMS (Kirkland ( 1989) Sub-committee on Guidelines for mutagenicity Testing, Report-Part III, Cambridge University Press) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Acceptance criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
Spontaneous revertants of tester strain cultures for vehicle and negative control should be in the range of historical control data. The appropriate characteristics for each tester strain have been confirmed (eg rfs cell-wall-mutation, pKM101 plasmid R-factor etc.). All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E09 bacteria per mL. Each mean positive control value should be at least two times the respective vehicle control for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and integrity of the S9-mix. There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

Statistics:

Mean value and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 5000 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Remarks:

at and above 1500 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1500 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the dose levels tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic at and above 1500 μg/plate to TA100 (with and without S9) and WP2uvrA- (without S9) and non-toxic to WP2uvrA- with S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 μg/plate in both the presence and absence of S9. No toxicity was exhibited to Escherichia coli strain WP2 uvr A, in the presence of S9 only, at any test substance dose level. The sensitivity of the tester strains to the toxicity of the test substance varied slightly between strain type, exposure with or without S9 and experiment number. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

Any other information on results incl. tables

Table 1. Results of Experiment 1

	Number of revertant colonies (mean of 3 plates ± SD)
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535			WP2 uvr A	TA 98		TA1537
SC	128 ± 4.6	26 ± 2.6			30 ± 3.8	23 ± 5.5		10 ± 4.9
5	128 ± 7.2	27 ± 2.1			19 ± 7.4	23 ± 8.3		10 ± 6.0
15	130 ± 6.5	25 ± 3.0			22 ± 3.8	20 ± 4.0		6 ± 1.2
50	127 ± 8.6	19 ± 2.6			25 ± 3.2	22 ± 4.4		12 ± 1.5
150	135 ± 12.5	23 ± 6.8			22 ± 7.5	17 ± 5.5		13 ± 2.1
500	126 ± 10.4	28 ± 9.0			25 ± 8.0	13 ± 1.7		13 ± 2.0
1500	75 ± 0.6 s	28 ± 4.2 s			23 ± 4.5	13 ± 3.6 s		7 ± 2.5
5000	0 ± 0.0 v	0 ± 0.0 v			16 ± 2.3 s	0 ± 0.0 v		0 ± 0.0 v
Positive Control	ENNG	ENNG			ENNG	4NQO		9AA
Dose (µg/plate)	3	5			2	0.2		80
Number of revertant colonies/plate	443 ± 67.0	168 ± 32.1			540 ± 27.3	101 ± 4.5		659 ± 69.6
S9-Mix	With
Test substance (µg/plate)	TA 100		TA 1535		WP2 uvr A	TA 98	TA1537
SC	128 ± 4.4		13 ± 2.1		29 ± 6.1	22 ± 1.5	15 ± 2.0
5	107 ± 9.3		13 ± 1.5		N/T	28 ± 7.8	12 ± 2.5
15	102 ± 6.8		13 ± 0.0		N/T	26 ± 4.9	13 ± 3.2
50	108 ± 8.4		11 ± 2.1		28 ± 2.0	21 ± 1.5	10 ± 2.3
150	111 ± 10.2		12 ± 3.1		23 ± 3.0	22 ± 0.6	13 ± 7.8
500	98 ± 14.4		14 ± 1.0		31 ± 9.8	22 ± 7.0	10 ± 3.6
1500	68 ± 14.5 s		9 ± 3.5 s		27 ± 4.0	16 ± 0.6 s	8 ± 1.2
5000	0 ± 0.0 v		5 ± 1.2 s		25 ± 0.6	14 ± 5.1 s	7 ± 0.6 s
Positive Control	2AA		2AA		2AA	BP	2AA
Dose (µg/plate)	1		2		10	5	2
Number of revertant colonies/plate	458 ± 154.0		166 ± 25.1		168 ± 7.0	262 ± 17.0	354 ± 48.8
SC: Solvent Control (dimethyl sulphoxide) ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO: 4-Nitroquinoline-1-oxide 9AA: 9-Aminoacridine 2AA: 2-Aminoanthracene				BP: Benzo(a)pyrene N/T: Not tested at this dose level s: sparse bacterial background lawn v: very weak bacterial background lawn

Table 2: Results of Experiment 2

	Number of revertant colonies (mean of 3 plates ± SD)
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535			WP2 uvr A	TA 98		TA1537
SC	115 ± 18.7	18 ± 2.5			26 ± 3.2	21 ± 1.0		8 ± 0.6
5	123 ± 9.2	19 ± 7.0			N/T	19 ± 3.5		10 ± 2.9
15	118 ± 10.1	20 ± 1.2			N/T	15 ± 3.5		15 ± 6.7
50	122 ± 11.2	19 ± 5.0			27 ± 3.0	17 ± 8.5		10 ± 6.0
150	117 ± 7.0	24 ± 1.7			21 ± 1.5	15 ± 4.6		13 ± 5.7
500	116 ± 14.8	19 ± 3.8			28 ± 4.7	20 ± 7.8		10 ± 1.0
1500	100 ± 4.9 s	25 ± 2.5 s			25 ± 5.7	14 ± 4.2 s		7 ± 4.0
5000	77 ± 13.3 s	17 ± 3.6 v			23 ± 2.5 s	6 ± 1.5 v		8 ± 3.6 s
Positive Control	ENNG	ENNG			ENNG	4NQO		9AA
Dose (µg/plate)	3	5			2	0.2		80
Number of revertant colonies/plate	302 ± 14.2	179 ± 6.0			400 ± 14.7	100 ± 15.0		219 ± 31.7
S9-Mix	With
Test substance (µg/plate)	TA 100		TA 1535		WP2 uvr A	TA 98	TA1537
SC	101 ± 2.5		10 ± 2.1		29 ± 5.8	19 ± 5.9	11 ± 2.0
5	115 ± 20.6		10 ± 2.0		N/T	23 ± 6.1	14 ± 0.6
15	98 ± 12.3		8 ± 1.0		N/T	21 ± 3.1	10 ± 2.5
50	88 ± 8.0		8 ± 3.5		28 ± 8.1	22 ± 7.1	15 ± 3.1
150	98 ± 15.0		9 ± 2.1		31 ± 7.5	17 ± 3.8	16 ± 5.5
500	96 ± 6.4		11 ± 3.1		24 ± 8.0	20 ± 6.0	11 ± 2.1
1500	83 ± 4.4		8 ± 2.5		31 ± 5.9	17 ± 5.1	10 ± 1.5
5000	62 ± 8.0 s		7 ± 3.1		23 ± 2.3	13 ± 3.2 s	13 ± 6.2
Positive Control	2AA		2AA		2AA	BP	2AA
Dose (µg/plate)	1		2		10	5	2
Number of revertant colonies/plate	525 ± 118.2		220 ± 24.5		340 ± 37.0	223 ± 16.4	218 ± 27.4
SC: Solvent Control (dimethyl sulphoxide) ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine 4NQO: 4-Nitroquinoline-1-oxide 9AA: 9-Aminoacridine 2AA: 2-Aminoanthracene				BP: Benzo(a)pyrene N/T: Not tested at this dose level s: sparse bacterial background lawn v: very weak bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to the limit dose of 5000 µg/plate.