2-phenoxyethyl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

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:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome aberation test in human lymphocytes (In vitro)

Test material

Test material information

Constituent 1

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:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): 2-phenoxyethyl acrylate
- Lot/batch No.: 120517147
- Expiration date of the lot/batch: 16 May 2013
- Stability under test conditions: All formulations (Dimethyl sulphoxide was selected as vehicle) were used within four hours of preparation and were assumed to be stable for this period.
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Structural chromosomal aberrations in cultured mammalian cells

Species / strain

Species / strain / cell type:

other: Human Lymphocytes

Details on mammalian cell type (if applicable):

Human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:
4(20)(four hour exposure and 20 hour expression) without S9; 7.5, 15, 30, 45, 60 and 75 ug/ml
4(20) with S9 (2%); 30, 60, 120, 240, 360, and 480 ug/ml
24 hour without S9; 3.75, 7.5, 15, 30, 60 and 120 ug/ml
4(20) hour with S9 (1%); 30, 60, 120, 240, 360 and 480 ug/ml

For the the analysis of chromosome abberations the follwoing concentrations were used:
In experiment 1, A dose-related inhibition of mitotic index was observed, and 48% mitotic inhibition was achieved at 30 μg/ml in the absence of S9. In the presence of S9, 45% mitotic inhibition was achieved at 240 μg/ml. The maximum dose level selected for metaphase analysis was 30 and 240 μg/ml in the absence and presence of S9, respectively.

In experiment 2, inhibition of mitotic index was observed, and 45% mitotic inhibition was achieved at 15 μg/ml in the absence of S9 and 58% mitotic inhibition was achieved at 240 μg/ml in the presence of S9. The maximum dose level selected for metaphase analysis was therefore, 15 μg/ml and 240 μg/ml in the absence and presence of S9, respectively.

Vehicle / solvent:

The test substance was dissolved in dimethyl sulphoxide.
The positive control used in absence of metabolic activation was mitomycin, dissolved in Minimal Essential Medium
The positive control used in the presence of metabolic activation was cyclophosphamide, dissolved in dimethyl sulphoxide.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Whole blood samples was drawn from the peripheral circulation of a volunteer, screened for suitability. The volunteer had not knowingly been exposed to hazardous chemicals, and have not suffered from viral infections. The cell-cycle time for the lymphocytes was determined using bromodeoxyuridine incorporation to assess the number of first, second and third division metaphase cell, and to calculate the average generation time (AGT). AGT was approximately 16 hours.

Cells were grown in Eagles minimal essential medium with HEPES buffer, supplemented with L-glutamine, penicilin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS) at 37C with 5% CO2 in humified air. The heparinised lymphocytes were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Vehicle and positive control were used in paralel with 2-phenoxyethyl acrylate..

S9 was produced in-house from liver obtained from male rats, these had received three daily doses of a mixture of phenobarbitone (80mg/kg) and B-napthflavone (100mg/kg) prior to S9 preparation on the fourth day. Each batch of S9 was routinely tested for its capability to activate known mutagens in the Ames test.

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30-50% of cells with aberations, slide evaluation weas terminated at 50 cells.

If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Cells with 69 chromosomes or more were scored as polyploid cells, and incidence of polyploid cells (5) were reported.

Statistics:

Frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher´s Exact test.

Results and discussion

Test results

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 2-phenoxyethyl acrylate was accurately weighed, dissolved in dimethyl sulphoxide and serial dilutions prepared. The molecular weight of the test item was given as 192.2, therefore the maximum dose level was 1922 μg/ml, which was calculated to be equivalent to the 10 mM maximum recommended dose level. The purity of the test item was 85.5% and was accounted for in the formulations. There was no significant change in pH when dissolved into dimethyl
sulphoxide.

- Effects of osmolality: The osmolality did not increase by more than 50 mOsm (Scott et al, 1991).

- Evaporation from medium: No information but coinsidered not relevant. 2-phenoxyethyl acrylate was formulated within two hours of it being applied to the test system.

- Water solubility: Low water soulbility, therfore dissolved in DMSO.

- Precipitation:No precipitate or haemolysis observed

RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range used was 7.51, 15.02, 30.03, 60.06, 120.13, 240.25, 480.5, 961 and 1922 μg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. Historical contro data for the vehicle and the positive control is included in the test report.

Any other information on results incl. tables

Preliminary toxicity test:

The dose range for the preliminary toxicity test was 7.51 to 1922 μg/ml. The maximum dose was based on the maximum recommended dose level, 10 mM concentration. Precipitate observations were made from the blood-free cultures. In the exposure groups in the absence of S9, precipitate was noted at the end of exposure at and above 961 μg/ml. However, in the 4(20)-hour exposure group in the presence of S9, precipitate was observed at 1922 μg/ml only. Haemolysis was also observed in the blood cultures at 1922 μg/ml in the 4(20)-hour exposure group (without S9) and at and above 961 μg/ml in the continuous exposure group. No haemolysis was observed in the 4(20)-hour exposure group (with S9). Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 60.06 and 240.25 μg/ml in the 4(20)-hour exposure groups, without and with S9, respectively. In the continuous exposure group metaphase cells were present at and above 30.03 μg/ml. There were no scorable metaphases present at dose levels above these stated in any exposure group. The selection of the maximum dose level was based on toxicity rather than the onset of the precipitate in all exposure groups tested.

Chromosome Aberration Test - Experiment 1:

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the preliminary toxicity test and that there were metaphases suitable for scoring present up to 60 μg/ml in the absence of metabolic activation (S9) and up to 360 μg/ml in the presence of metabolic activation (S9). No precipitate or haemolysis was observed in the blood cultures either in the presence or absence of S9. A dose-related inhibition of mitotic index was observed, and 48% mitotic inhibition was achieved at 30 μg/ml in the absence of S9. In the presence of S9, 45% mitotic inhibition was achieved at 240 μg/ml. The maximum dose level selected for metaphase analysis was 30 and 240 μg/ml in the absence and presence of S9, respectively. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. 2 -phenoxyethyl acrylate did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in the absence of metabolic activation. In the presence of S9 an increase in the frequency of cells with aberrations at 240 μg/ml was observed, but the Fishers Exact Test result was P = 0.052 which is not statistically significant. The response was lopsided between the duplicate cultures being predominantly observed in the more toxic ‘A’ duplicate, was not part of a dose related response and was only marginally outside the historical upper limit. Therefore, with such an equivocal result, a second experiment was performed. 2 -phenoxyethyl acrylate did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2:

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum dose level of 30 μg/ml in the absence of S9 and at 240 μg/ml in the presence of S9. No precipitate or haemolysis was observed at the end of exposure. Inhibition of mitotic index was observed, and 45% mitotic inhibition was achieved at 15 μg/ml in the absence of S9 and 58% mitotic inhibition was achieved at 240 μg/ml in the presence of S9. The maximum dose level selected for metaphase analysis was therefore, 15 μg/ml and 240 μg/ml in the absence and presence of S9, respectively. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. In the presence of S9, a modest but statistically significant increase in the frequency of cells with aberrations only at 240 μg/ml (P = 4.5 X10 -4) was observed. This dose level marginally exceeded the optimal maximum level of 50% (+/- 5) mitotic inhibition (Mitotic Index 42%). However, the aberrations seen were predominantly break-type aberrations but they were consistent between both cultures with only two exchange type aberrations, and no evidence of a response in the lower dose levels, it is possible that the observed aberrations were due to a cytotoxic mechanism rather than a true genotoxic mechanism. Therefore the response may have little biological relevance. However, no statistically significant increases in the frequency of cells with aberrations in the 24-hour continuous exposure group was observed, in the absence of S9, which included a dose level that was within the optimal 50% mitotic inhibition. No statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups was observed.

Applicant's summary and conclusion

Conclusions:

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

2-phenoxyethyl acrylate was tested for the potential of inducing chromosome aberrations in cultured mammalian cells. No toxicological significant increases in the frequency of cells with aberrations was observed, hence 2-phenoxyethyl acrylate was considered to be non-clastogenic.

Executive summary:

2-phenoxyethyl acrylate was tested for the potential of inducing chromosome aberrations in cultured mammalian cells in vitro in accordance with the testing guidelines OECD 473 and EC. B10.

Duplicate cultures of human lymphocytes, treated with 2-phenoxyethyl acrylate were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal lymphocytes, and all positive control items induces significant increases in the frequency of cells with aberrations, indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated.

In the first experiment a small increase in the frequency of cells with aberrations was observed in the presence of S9 only, and associated with the dose level that induced approximately 50% mitotic inhibition. The increases observed were however small and lop-sided with most aberrations occuring in one culture and not both.

In the second experiment, a significant increase in cells with aberrations was only observed in the dose range outside the optimal maximum level of 50% mitotic inhibition. The substance did not induce increases in the frequency of cells with aberrations in the absence of S9, including the dose level that was within the optimal 50% mitotic inhibition.

The responses were not dose-related and not reproducible in a repeat experiment. It was therefore concluded that the clastogenic activity observed at or around the onset of excessive toxicity, could be considered to be of no toxicological relevance.

It was concluded that 2 -phenoxyethyl acrylate did not induce significant increases in the frequency of cells with aberrations, and therefore considered to be non-clastogenic.