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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20-05-2015 to 07-07-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In vitro Mammalian Cell Gene Mutation assay was performed for Methyl phenylacetate in the presence of S9 metabolic activation system
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl phenylacetate
EC Number:
202-940-9
EC Name:
Methyl phenylacetate
Cas Number:
101-41-7
Molecular formula:
C9H10O2
IUPAC Name:
methyl phenylacetate
Details on test material:
- Name of test material: Methyl phenylacetate
- Molecular formula: C9H10O2
- Molecular weight: 150.176 g/mol
- Smiles notation:c1(CC(OC)=O)ccccc1
- Substance type: Organic
- Physical state: Colourless liquid
Specific details on test material used for the study:
- Name of test material: Methyl phenylacetate
- Molecular formula: C9H10O2
- Molecular weight: 150.176 g/mol
- Smiles notation:c1(CC(OC)=O)ccccc1
- Substance type: Organic
- Physical state: Colourless liquid

Method

Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays):
6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies
Statistics:
No data available

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Preliminary dose-finding/toxicity test
Completed with S9 metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assayMTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 5.0 mM. Since the test chemical was dissolved in ethanol, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of ethanol. The test chemical could only be dissolved in 99.5% ethanol.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:
Methyl phenylacetate in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the presence of S9 metabolic activation system and hence does not classify as a gene mutant in vitro.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to Methyl phenylacetate in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested Methyl phenylacetate concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that Methyl phenylacetate in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation and hence dose not classify as a gene mutant in vitro.