Methyl cinnamate

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:

August 28, 2012 to October 10, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Methyl cinnamate

EC Number:

203-093-8

EC Name:

Methyl cinnamate

Cas Number:

103-26-4

Molecular formula:

C10H10O2

IUPAC Name:

methyl 3-phenylacrylate

Test material form:

solid: crystalline

Method

Target gene:

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- — his+ and trp- — trp+ reversions, respectively. The Salmonella typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).
Genotype:
TA1537: his C 3076; rfa-; uvrB-; TA98 :his D 3052; rfa-; uvrB-; R-factor ; TA 1535: his G 46; rfa-; uvrB-; TA 100 his G 46; rfa-; uvrB-; R-factor; WP2 uvrA trp-; uvrA-.
Type of mutations indicated:
frame shift mutations : TA 1537 and TA 98;
base-pair substitutions: TA 1535, TA 100 and WP2 uvrA;

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item Methyl Cinnamate was dissolved in DMSO (MERCK, 64293 Darmstadt/Germany; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

with s9 mix

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 µg/plate (WP2 uvrA)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9 mix

Positive control substance:

sodium azide

Remarks:

10 µg/plate for TA 1535, TA 100

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9 mix

Positive control substance:

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

Remarks:

10 µg/plate in TA 98, 50 µg/plate in TA 1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

without s9 mix

Positive control substance:

methylmethanesulfonate

Remarks:

3 µL/plate for WP2 uvrA

Details on test system and experimental conditions:

Storage :
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, 64293 Darmstadt/Germany) in liquid nitrogen.
Precultures:
From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL
Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin
(25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Nutrient Broth (MERCK, 64293 Darmstadt/Germany)
5 g NaCl (MERCK, 64293 Darmstadt/Germany) The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (108-109 cells/mL).
Mammalian Microsomal Fraction S9 Mix:
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damagingmetabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix.
Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2, 33 mM KCl ,5 mM Glucose-6-phosphate, 4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Pre-Experiment for Toxicity:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
Dose Selection:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed eight concentrations were tested in experiment II and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Evaluation criteria:

A test item is considered as a 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

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in revertant colony numbers of any of the five tester strains was observed following treatment with Methyl Cinnamate at any dose level, 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 of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed reduced background growth occurred in the test groups at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500 - 5000	5000	333 - 5000	2500 - 5000
TA 1537	2500 - 5000	5000	333 - 5000	2500 - 5000
TA 98	2500 - 5000	2500 - 5000	1000 - 5000	2500 - 5000
TA 100	2500 - 5000	5000	1000 - 5000	2500 - 5000
WP2 uvrA	5000	5000	1000 - 5000	2500 - 5000

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	2500 - 5000	5000
TA 1537	2500 - 5000	5000	333 - 5000	2500 - 5000
TA 98	5000	2500 - 5000	2500 - 5000	2500 - 5000
TA 100	2500 - 5000	5000	2500 - 5000	2500 - 5000
WP2 uvrA	5000	5000	1000 - 5000	2500 - 5000

Applicant's summary and conclusion

Conclusions:

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

Based on the available data, test item caused negative gene mutagenic response in the Ames system.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed reduced background growth in all strains at higher concentrations. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation at higher concentrations.

No increase in revertant colony numbers of any of the five tester strains was observed following treatment with Methyl Cinnamate at any dose level, 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.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.