α-methylenebenzyl acetate

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:

2014-06-10 to 2014-08-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

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)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 2012-11-20/22, Date of signature: 2014-04-14

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α-methylenebenzyl acetate

EC Number:

218-620-7

EC Name:

α-methylenebenzyl acetate

Cas Number:

2206-94-2

Molecular formula:

C10H10O2

IUPAC Name:

1-phenylethenyl acetate

Test material form:

other: liquid

Method

Target gene:

histidine operon (Salmonella strains)
tryptophan operon (E.coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

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:

phenobarbitone/ 5,6-benzoflavone activated rat liver S9

Test concentrations with justification for top dose:

Test 1 : 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2 : 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [TA98]: 15, 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [TA1537]: 50, 150, 500, 1500, 5000 µg/plate
Test 2 repeat [WP2 uvrA]: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Test 3 [TA1535]: 50, 150, 500, 1500, 3000, 4000, 5000 µg/plate
Test 4 [TA1535]: 5, 15, 50, 150, 500, 1500, 3000, 4000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was fully miscible in dimethyl sulphoxide at 50 mg/ml.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 μg/plate for strains TA98 and TA1537, with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

5 μg/plate for strains TA100 and TA1535; 10 μg/plate for strain WP2 uvrA (pKM101), with metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 μg/plate for strain WP2 uvrA (pKM101), without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2 μg/plate for strain TA98, without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 μg/plate for strain TA1537, without metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 μg/plate for strains TA100 and TA1535, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar


NUMBER OF REPLICATIONS: triplicate plates, initial plate incorporation experiment repeated using preincubation


DETERMINATION OF CYTOTOXICITY
- Method other: condition of bacterial lawn, reduction in number of revertants

ACTIVATION

The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water.
0.5 ml of S9 mix was added to 2ml of agar, giving a final concentration of approximately 2%.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

Statistics:

No statistical analysis was performed.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Experiment 1 Plate incorporation without metabolic activation revertants per plate (mean of three plates)

	Ta 98	TA100	TA1535	TA1537	WP2 uvrA
Solv cnt	48.0	182.7	46.7	17.3	108.7
5 µg	47.7	238.3	38.7	14.0	103.7
15 µg	42.3	201.7	44.3	19.3	98.0
50 µg	37.7	221.0	38.3	18.3	105.3
150 µg	36.3	206.7	40.0	12.7	92.3
500 µg	51.7	246.7	42.0	15.3	105.0
1500 µg	46.0	235.7	66.0	16.3	127.3
5000 µg	40.7	230.3	63.0	18.3	79.0
Pos cont	335.0	534.7	939.7	326.0	1397.7

 

 

 

Experiment 1 Plate incorporation with metabolic activation revertants per plate (mean of three plates)

	Ta 98	TA100	TA1535	TA1537	WP2 uvrA
Solv cnt	62.3	203.3	23.0	34.3	120.7
5 µg	50.3	198.0	23.3	38.0	98.0
15 µg	49.7	201.0	17.3	43.3	101.7
50 µg	50.7	208.7	14.3	32.7	117.0
150 µg	47.0	190.7	20.7	32.7	123.3
500 µg	66.7	189.0	17.0	33.7	130.0
1500 µg	58.3	210.7	21.0	38.0	113.3
5000 µg	56.3	102.3	20.0	47.7	84.3
Pos cont	319.7	1258.0	390.3	186.0	421.0

 

 

 

Experiment 2 pre incubation without metabolic activation revertants per plate (mean of three plates)

	Ta 98	TA100	TA1535	TA1537	WP2 uvrA
Solv cnt	63.7	142.0	24.3	49.0	133.7
50 µg	70.7	184.7	26.3	31.7	142.7
150 µg	67.3	191.0	18.0	37.3	151.0
500 µg	60.3	168.7	17.0	36.3	170.0
1500 µg	32.0	166.0	13.7	48.0	76.7
5000 µg	26.3	31.7	78.0	101.0	131.3
Pos cont	532.0	396.0	384.7	1204.0	2254.3

 

 

 

 

Experiment 2 pre incubation with metabolic activation revertants per plate (mean of three plates)

	Ta 98	TA100	TA1535	TA1537	WP2 uvrA
Solv cnt	70.0	205.0	34.0	20.3	73.3
50 µg	55.7	140.3	20.7	31.3	110.7
150 µg	51.3	175.0	25.7	28.7	133.0
500 µg	54.3	199.7	30.7	27.7	37.3
1500 µg	56.7	185.0	31.7	24.7	38.3
5000 µg	58.7	162.3	50.3	18.7	41.7
Pos cont	394.3	702.7	193.0	293.3	510.0

 

Pre incubation, revertants per plate (mean of three plates)- Experiment 2 repeat, experiment 3 and experiment 4 

	Test 2 repeat			Test 3	Test 4
	Without metabolic activation		With metabolic activation	Without metabolic activation	Without metabolic activation
	TA98	TA1537	WP2 uvrA	TA1535	TA1535
Solv cnt	47.0	26.3	202.7	21.0	24.3
5 µg			208.0		19.3
15 µg	47.3		221.3		23.0
50 µg	52.3	20.0	210.7	21.7	24.3
150 µg	41.7	25.3	199.3	19.0	19.3
500 µg	48.0	25.3	193.3	29.7	30.7
1000 µg					15.7
1500 µg	37.0	17.7	199.7	3.3	0.0
3000 µg				0.7	0.0
4000 µg				0.0	0.0
5000 µg	20.0	0.0	118.0	1.3	0.0
Pos cont	447.0	893.0	507.3	1085.3	873.3

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

α-Methylenebenzyl acetate has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, and in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that α-methylenebenzyl acetate is negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

α-Methylenebenzyl acetate has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in the initial or the repeat experiments up to limit concentrations using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.