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Diss Factsheets

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: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012.07.02 - 2012.09.17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed in accordance with recognized testing guidelines with no deviations.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-phenoxyethyl acrylate
EC Number:
256-360-6
EC Name:
2-phenoxyethyl acrylate
Cas Number:
48145-04-6
Molecular formula:
C11H12O3
IUPAC Name:
2-phenoxyethyl prop-2-enoate
Test material form:
liquid: viscous
Details on test material:
SR 339 C, Lot number HZI 1511, clear liquid.

Method

Target gene:
Thymidine kinase gene (TK+/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media).RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of male Sprague-Dawley rats. These had each received, orally, three consecutive daily doses of phenobarbital/b-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 7.51, 15.02, 30.03, 60.06, 120.13, 240.25, 480.5, 961, 1922 μg/ml.
Mutation tests-experiment I: 0, 1.25,2.5, 5, 10, 20, 25, 30 μg/ml.
Mutation tests-experiment II: 0, 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 μg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Following solubility checks, DMSO was chosen as vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with 2-phenoxyethyl acrylate at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, due to the equivocal response observed
in the presence of metabolic activation in Experiment 1, the cells were treated with 2-phenoxyethyl acrylate at eight dose levels using a repeat of the 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated at 37°C with 5% CO 2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 105 cells/ml.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37°C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/ml in phosphate buffered saline (PBS), was added to each well of the mutation
plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Based on the scoring, calculation of percentage Relative Suspension Growth (%RSG), Day 2 Viability (%V), Relative Total Growth (RTG) and the Mutation Frequency (MF) was performed.
Evaluation criteria:
A mutagenic is concluded when a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value is observed. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method.

Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely,
when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Small significant increases designated by the UKEMS statistical package was reviewed using the above criteria, by the Study Director and could be disregarded at the Study Director's discretion.
Statistics:
UKEMS statistical package

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:There was no marked change in pH when 2-phenoxyethyl acrylate was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm (Scott et al 1991).
- Evaporation from medium: No information but considered to not relevant.
- Water solubility: Formulations prepared in DMSO, not soluble in water.
- Precipitation: The maximum proposed dose level in the solubility test was set at 1922 μg/ml, the approximate 10 mM limit dose. The purity of 2-phenoxyethyl acrylate was 85.5% and was therefore accounted for when formulating the dosing solutions.

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/ml, using a 4-hour exposure period both with and without metabolic activation (20% S9-mix), and at 1.5 x 105 cells/ml using a 24-hour exposure period without metabolic activation. The dose range used in the preliminary toxicity test was 7.51 to 1922 μg/ml for all three of the exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle and positive control data were in accordance with the historical Vehicle and Positive Control Mutation Frequencies obtained by the testing laboratorie (Harlan Laboratories).

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. 2-phenoxyethyl acrylate did not induce any reproducible toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Applicant's summary and conclusion

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

2-phenoxyethyl acrylate was non-mutagenic and did not induce any clastogenic effects to L5178Y mouse lymphoma cells treated in vitro.
Executive summary:

2-phenoxyethyl acrylate was tested in vitro in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells (OECD 476). Two independent experiments were performed. In Experiment 1, mouse lymphoma cells were treated with 2-phenoxyethyl acrylate at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, due to the equivocal response observed in the presence of metabolic activation in Experiment 1, the cells were treated with 2-phenoxyethyl acrylate at eight dose levels using a repeat of the 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range was selected following the results of a preliminary toxicity test and was 1.25 to 30 μg/ml in the absence of metabolic activation, and 7.5 to 240 μg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 0.63 to 40 μg/ml in the absence of metabolic activation, and 60 to 240 μg/ml in the presence of metabolic activation.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

2-phenoxyethyl acrylate did not induce any reproducible toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. Hence, 2-phenoxyethyl acrylate was considered to be non-mutagenic to L5178Y cells under the conditions of the test.