Benzyl cinnamate

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:

29.05.2015 - 19.06.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl cinnamate

EC Number:

203-109-3

EC Name:

Benzyl cinnamate

Cas Number:

103-41-3

Molecular formula:

C16H14O2

IUPAC Name:

benzyl 3-phenylacrylate

Method

Target gene:

Salmonella typhimurium: histidine (his)
Escherichia coli: tryptophan (trp)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

- Genotype: his C 3076; rfa-; uvrB-
- Type of mutations indicated: frame shift mutations

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Genotype: his G 46; rfa-; uvrB-
- Type of mutations indicated: base-pair substitutions

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Genotype: his D 3052; rfa-; uvrB-; R-factor
- Type of mutations indicated: frame shift mutations

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Genotype: his G 46; rfa-; uvrB-; R-factor
- Type of mutations indicated: base-pair substitutions

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Genotype: trp-; uvrA-
- Type of mutations indicated: base-pair substitutions

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Two independent reverse mutation assays were performed. Experiment I was a plate incorporation assay. Since a negative result was obtained in this experiment, Experiment II was performed as a pre-incubation assay.

The test substance was mixed in a test tube and poured onto selective agar plates.

DURATION:
- Preincubation period: 37 °C for 60 minutes
- Expression time (cells in growth medium): 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3

DATA RECORDING: colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager (v.1.21).

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data of the test facility
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:

A test item is considered mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

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:

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity

Remarks:

With S9 mix: only in experiment II, minor toxic effects were observed at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

Additional information on results:

- The undissolved particles of precipitated test item had no influence on the data recording.
- Plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains.
- Only minor toxic effects were observed in experiment II in strain TA 100 with S9 mix at 5000 μg/plate.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Remarks on result:

other: precipitation at 2500 µg/plate

Any other information on results incl. tables

Summary of Results

Experiment I

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535	TA1537	TA98		TA100		WP2uvrA
DMSO	n.a.	9±1	12±4	22±3		167±7		42±9
Untreated	n.a.	11±2	10±5	21±7		196±5		43±7
Test item	3	8±4	13±4	24±2		153±16		44±13
	10	10±4	14±2	26±6		160±8		44±10
	33	12±5	12±2	28±9		151±14		36±9
	100	11±3	16±3	23±7		164±17		42±5
	333	9±6	15±2	24±5		156±6		35±12
	1000	11±3	16±1	27±5		154±16		37±6
	2500	11±3P	12±2PM	28±4		153±4P		44±7P
	5000	7±2PM	11±3PM	22±2		169 ± 27P		40±4P
NaN3	10	1017 ± 64				1646 ± 226
4-NOPD	10			229±36
4-NOPD	50		74±4
MMS	2.0							830±44
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537		TA98		TA100		WP2uvrA
DMSO	n.a.	9±1	18±1		33±4		161±13		47±6
Untreated	n.a.	12±2	18±5		39±9		170±16		56±2
Test item	3	9±3	20±4		36±5		154±12		55±8
	10	9±1	22±2		32±6		166±31		38±8
	33	11±2	16±4		28±3		138±19		48±5
	100	11±3	17±9		32±1		161±20		50±16
	333	12±0	20±3		33±4		141±8		47±7
	1000	14±2	19±5		34±7		132±16		32±10
	2500	12±6	17±5		34±4		126±4		26 ± 10
	5000	12±1P	19±5P		30±7P		146±26P		28 ± 9P
2-AA	2.5	362±20	200 ± 11		4321 ± 308		4120 ± 59
2-AA	10								310 ± 27

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

M Manual count

Experiment II

 

Without Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean±SD)
		TA1535	TA1537	TA98		TA100		WP2uvrA
DMSO	n.a.	10±3	8±0	22±3		127±12		40±7
Untreated	n.a.	9±1	9±1	24±6		169±23		44±11
Test item	33	10±3	10±1	20±5		94±7		38±11
	100	8±1	9±3	20±1		97±8		35±5
	333	7±3	7±3	23±2		96±4		37±5
	1000	7±2	9±3	18±3		73±9		30±3
	2500	9±1P	9±1P	29±6P		88±10P		34 ± 4P
	5000	8±1PM	4±2PM	18±2PM		81 ± 6P M		35 ± 11P
NaN3	10	1107 ± 17				1869 ± 141
4-NOPD	10			353±20
4-NOPD	50		82±6
MMS	2.0							533±64
With Metabolic Activation
Test Group	Dose level (µg/plate)	Revertant Colony Counts (mean +/- SD)
		TA1535	TA1537		TA98		TA100		WP2uvrA
DMSO	n.a.	11±1	9±2		25±5		108±11		48±7
Untreated	n.a.	11±2	9±4		31±6		161±23		61±16
Test item	33	14±2	11±3		33±4		107±9		45±3
	100	9±3	8±1		27±2		115±11		50±9
	333	7±3	12±2		29±3		114±7		43±8
	1000	8±2	1±2		23±2		84±19		33±6
	2500	8±4P	14±3P		20±7P		61±6P		24±3P
	5000	7±1P	12±4P		28±1P		40±3PM		26±5P
2-AA	2.5	378±31	98 ± 14		4148 ± 434		3196 ± 225
2-AA	10								375 ± 60

 

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

M Manual count

Applicant's summary and conclusion

Conclusions:

The test item was considered not genotoxic under the conditions of the current test.

Executive summary:

In the current study the genotoxic potential of the test item was assessed according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study was conducted according to OECD 471 and GLP.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: (a) Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate, (b) Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate.

The test item precipitated under some conditions, however, the undissolved particles had no influence on the data recording. Additionally, the plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix. No toxic effects occurred in any strain with and without metabolic activation. Only in experiment II in strain TA 100 with S9 mix minor toxic effects were observed at 5000 μg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Therefore, under the experimental conditions of the current test the test item did not induce gene mutations and thus is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.