Phenethyl 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:

weight of evidence

Study period:

30 August 2002 to 19 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Modern, guideline and GLP compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenethyl acetate

EC Number:

203-113-5

EC Name:

Phenethyl acetate

Cas Number:

103-45-7

Molecular formula:

C10H12O2

IUPAC Name:

2-phenylethyl acetate

Method

Target gene:

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains.
The strains TA98 and TA1537 are capable of detecting frameshift mutagens, strains TA100, TA1535 and TA102 are capable of detecting base-pair substitution mutagens

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

In the direct plate assay the doses (µg/plate) used with and without S-9 mix for the TA1535, TA1537, TA98, TA100 and TA102 tester strains were:
3, 10, 33, 100, 333, 1000, 3330 (and 5000 strain TA100 only)
The same doses were used inthe pre-incubation assay.

Vehicle / solvent:

0.1 ml dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: daunomycin and cumene hydroperoxide

Remarks:

positive controls used in plate incorporation and preincubation assays without 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 (2AA) and 1,8-dihydroxanthraquinone (Danthron)

Remarks:

positive controls usd with metabolic activation in plate incorporation and preincubation assays

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
plate incorporation
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide and either 0.5 ml 59-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assay. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant colonies were counted.

pre-incubation
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were preincubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml 59-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (10'cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide. After the preincubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37°C for 48 h. After this period revertant colonies were counted.

OTHER:
The characteristics of the different Salmonella typhimurium strains were as follows:
(Strain, Histidine mutation, Plasmid, Mutation type)
TA1537, hisC3076, -, Frameshift
TA98, hisD30521R-factor, pKM101, Frameshift
TA1535, hisG46, - , Base-pair substitutions
TA100, hisG46/R-factor, pKM101, Base-pair substitutions
TA102, hisG428, pAQ1, Base-pair substitutions

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chi : mutation in nitrate reductase
bio : defective biotin synthesis .
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98, TA100 and TA102), tetracycline resistance (TA102), UV-sensitivity and the number of spontaneous revertants.

Evaluation criteria:

The revertant colonies were counted automatically with a Pratos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

The reverse mutation assay is acceptable if:
the negative control data are within laboratury background data from historical studies (these data are presented in the study report) for each tester strain
the positive controls should produce responses that are within their historical background ranges (also presented in the report) for each strain.
The selected dose range should include a clearly toxic concentration or should exhibit limited solubility or the dose range should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one repeated experiment.

Statistics:

No data: not required

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

The negative and strain-specific positive control values were within the laboratory background historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 in the absence of S9-mix (second experiment; positive control). However, since this value was just outside the limit of the range and a more than three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected.

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related. two-fold, increase in the number of revertants in two separate experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that phenyleythyl acetate is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

Phenylethyl acetate was tested in the Salmonella typhimunum reverse mutation assay with five histidine-requiring strains of Salmonella typhimunum (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). In the direct plate assay, at first phenylethyl acetate was tested in a dose range finding study up to concentrations of 5000 µg/plate in strain TA100. Phenylethyl acetate precipitated on the plates at dose levels of 3330 and 5000 µg/plate. Toxicity was observed at dose levels of 3330 and 5000 µg/plate. Phenylethyl acetate was tested up to concentrations of 3330 µg/plate in the strains TA1535, TA1537, TA98 and TA102. Phenylethyl acetate precipitated on the plates at this dose level. No biologically relevant decrease in the number of revertants was observed.

In the preincubation assay phenylethyl acetate was tested in a dose range finding study in the strain TA100. Phenylethyl acetate was tested up to concentrations of 5000 µg/plate. Toxicity was observed at dose levels of 1000 µg/plate and upwards. After that, phenylethyl acetate was tested up to concentrations of 3330 µg/plate in the strains TA1535, TA1537, TA98 and TA102. Toxicity was observed in all tester strains. Phenylethyl acetate did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that phenylethyl acetate is not mutagenic in the Salmonella typhimurium reverse mutation assay.