2-phenoxyethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available on 2-phenoxyethyl methacrylate.

Mutagenicity, in vitro, Salmonella typhimurium and Escherichia Coli:

OECD 471 testing performed with 2-phenoxy acrylate was negative for mutagenic effects both with and without metabolic activation (OECD 471).

Due to the very close structural similarity to 2-phenoxyethyl acrylate, the target substance 2-phenoxyethyl methacrylate should be considered without mutagenic potential in bacteria as well.

Mutagenicity, in vitro, mutations mammalian cells:

OECD 476 testing performed with 2-phenoxyethyl acrylate  was negative for mutagenic effects both in with and without metabolic activation. Due to the very close structural similarity to 2-phenoxyethyl acrylate, the target substance 2-phenoxyethyl methacrylate should be considered without mutagenic potential in mammalian cells in vitro as well.  

Mutagenicity, in vitro, chromosome aberration mammalian cells:

OECD 473 testing performed with 2-phenoxyethyl acrylate was negative both with and without metabolic activation. Due to the very close structural similarity to 2-phenoxyethyl acrylate, the target substance 2-phenoxyethyl methacrylate should be considered without clastogenic potential in mammalian cells in vitro as well.  

Overall, the conducted in vitro mutagenicity testing on 2-phenoxy acrylate do not indicate any mutagenic or clastogenic potential in vitro. Due to the very close structural similarity to 2-phenoxyethyl acrylate, the target substance 2-phenoxyethyl methacrylate can be considered without mutagenic and clastogenic effects in vitro as well.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from chromosome aberation study on one closely structural and physicochemical related substance: 2-Phenoxyethyl acrylate (M140).
For further read-across justification se document attached in section 13.

Reason / purpose for cross-reference:

read-across source

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

Remarks on result:

other: data on read-across subsance

Executive summary:

No data is available for 2-Phenoxyethyl methacrylate (T).

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. 22 -phenoxyethyl acrylate did not induce dose-related or significant increases in the frequency of cells with aberrations, and therefore considered to be non-clastogenic under the conditions of the test. 

Due to structural and pysico-chemical similarity to 2-Phenoxyethyl methacrylate, a non-clastogenic potential can also be concluded for 2-Phenoxyethyl methacrylate as well.

See justification for read-across attached in section 13

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from mammalian cell mutation study on one closely structural and physicochemical related substance: 2-Phenoxyethyl acrylate.
For further read-across justification se document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

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

Remarks on result:

other: data on read-across substance

Executive summary:

No data is available for 2-Phenoxyethyl methacrylate (T).

2-phenoxyethyl acrylate was tested in vitro in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells in accordance with OECD test guideline 476. 2-phenoxyethyl acrylate did not induce dose-related increases in the mutant frequency at any condition. Hence, 2-phenoxyethyl acrylate was considered to be non-mutagenic under the conditions of the test. Due to structural and pysico-chemical similarity to 2-Phenoxyethyl methacrylate, a non-mutagenic potential can also be concluded for 2-Phenoxyethyl methacrylate as well.

See justification for read-across attached in section 13.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from bacterial mutation (AMES) study on one closely structural and physicochemical related substance: 2-Phenoxyethyl acrylate.
For further read-across justification se document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

data on read-across substance

Executive summary:

No data is available for 2-Phenoxyethyl methacrylate (T).

2-phenoxyethyl acrylate was tested in the reverse mutation assay (Ames Test) using Salmonella Typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain (WP2uvrA). The test was performed in accordance with OECD test guideline 471 and EU B13/14. No toxicological significant increases in the frequecy of revertant colonies were recorded for any of the bacterial strains. Hence, 2-phenoxyethyl acrylate was found to be non-mutagenic. 

Due to structural and pysico-chemical similarity to 2-Phenoxyethyl methacrylate this substance can be considered as non-mutagenic in Ames test as well.

See justification for read-across attached in section 13.

Reference 4

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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)

Target gene:

Structural chromosomal aberrations in cultured mammalian cells

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.

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.

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.

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.

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.

Reference 5

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.

Reason / purpose for cross-reference:

reference to same study

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

Target gene:

Thymidine kinase gene (TK+/-)

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

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

Key result

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).

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.

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.

Reference 6

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:

2012.06.19 - 2012.08.13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance with recognized testing guidelines with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Strain Target mutation Mutation type
TA 1535 hisG46; rfa-; uvrB- Base-pair substitution
TA 100 hisG46; rfa-; uvrB-; R-factor Base-pair substitution
TA 98 hisD3052; rfa-; uvrB-, R-factor Frame shift
TA 1537 hisC3076; rfa-; uvrB-; Frame shift
WP2uvrA trp-, urvA- Base-pair substition

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortlemans and Zeiger (2000)). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel (1976)).

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):

NA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction was prepared at Harlan (15 April 2012 (preliminary toxicity test) and 01 July 2012 (Experiments 1 and 2)) from rats induced with Phenobarbitone/Naphthoflavone (80/100 mg/kg/day), orally, for 3 days prior to preparation on day 4.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Mutation tests-experiment I: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Mutation tests-experiment II: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: A solubility check showed that 2-phenoxyethyl acrylate was immiscible in sterile distilled water at 50 mg/ml but fully miscible in DMSO at the same concentration.

- Other: 2-phenoxyethyl acrylate was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (14.5%).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

Without S9-mix: ENNG, 9AA, 4NQO; With S9-mix: 2AA, BP.

Details on test system and experimental conditions:

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of 2-phenoxyethyl acrylate. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of 2-phenoxyethyl acrylate formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the formulation and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of 2-phenoxyethyl acrylate and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of 2-phenoxyethyl acrylate. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1:
Seven concentrations of 2-phenoxyethyl acrylate (5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of 2-phenoxyethyl acrylate formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of 2-phenoxyethyl acrylate both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2:
The second experiment was performed using fresh bacterial cultures, 2-phenoxyethyl acrylate and control solutions, using the pre-incubation method. As it is sound scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation for Experiment 2. The dose range was the same as Experiment 1 (5 to 5000 µg/plate). 2-phenoxyethyl acrylate formulations and vehicle control were dosed using the pre-incubation method as follows: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or 2-phenoxyethyl acrylate formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of 2-phenoxyethyl acrylate both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Evaluation criteria:

Validity criteria:
- negative/positive control data were consistent with historical control data
- positive control showed marked increase over the concurrent negative control
- evaluation was not restricted by loss of plates (e.g. through contamination)
- all bacteria strain must have demonstrated the required charasterics
- the bacterial cell count for each stratin culture should be in the range of 0.9 to 9x109

Evaluation criteria:
- dose-related increases in number of revertant colonies at one or more test points
- increases reproducible between replicate plates
- increases more than twice the corresponding negative control
- biological relevance against in-house historical control ranges
- statistical analysis of data

Statistics:

The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values using the Analysis of
Variance test. When this test showed statistical significanct differences in the data, Dunnett´s test was used to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicate plates.

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment (plate incorporation method), 2-phenoxyethyl acrylate caused a visible reduction in the growth of the bacterial background lawns of all the tester strains dosed in the absence of S9-mix, initially from 1500 μg/plate and to TA100 at 5000 μg/plate in the presence of S9-mix. In the second experiment (pre-incubation method) 2-phenoxyethyl acrylate induced a stronger toxic response with weakened bacterial background lawns initially noted from 500 and 1500 μg/plate in the absence and presence of S9-mix respectively.

The sensitivity of the bacterial tester strains to the toxicity of 2-phenoxyethyl acrylate varied slightly between strain type, exposures with or without S9-mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent 2-phenoxyethyl acrylate being tested up to the maximum recommended dose level of 5000 μg/plate. 2-phenoxyethyl acrylate precipitate (globular in appearance) was noted at 5000 μg/plate under an inverted microscope only; this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of 2-phenoxyethyl acrylate, either with or without metabolic activation or exposure method. Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 at 15 μg/plate (TA100 in the presence of S9-mix) and 1500 μg/plate (WP2uvrA in the absence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.56 times the concurrent vehicle controls.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. 

A history profile of vehicle, untreated and positive control values for 2010 and 2011 was included in the test report. The results of this study was in accordance with the history profile.

Conclusions:

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

2-phenoxyethyl acrylate was found to be non-mutagenic in the reverse mutation assay (Ames Test) using Salmonella Typhimurium and Escherichia Coli with and without metabolic activation.

Executive summary:

2-phenoxyethyl acrylate was tested in the reverse mutation assay (Ames Test) using Salmonella Typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain (WP2uvrA). The test was performed in accordance with OECD test guideline 471 and EU B13/14 using the plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of rat liver homogenate metabolosing system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment.The experiment was repeated on a seperate day (pre-incubatrion method) using the same dose range, fresh cultures of the bacterial strains and fresh 2 -phenoxyethyl acrylate formulations.

No toxicological significant increases in the frequecy of revertant colonies were recorded for any of the bacterial strains, with any dose of 2-phenoxyethyl acrylate, either with or without metabolic activation per exposure method, hence 2-phenoxyethyl acrylate was found to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The three experimental in vitro studies available are of high reliability, GLP studies with no or insignificant deviations.

All studies concluded that 2 -phenoxyethyl acrylate is non-genotoxic:

-         2-phenoxyethyl acrylate was found to be non-mutagenic in the reverse mutation assay (Ames test) using Salmonella Typhimurium and Escherichia Coli. The study was performed in accordance to OECD 471 using the plate incorporation and pre-incubation methods at five dose levels (50 to 5000 ug/plate), in triplicate, both with and without metabolic activation (10% liver S9 in standard co-factors).

-         2-phenoxyethyl acrylate was non-mutagenic and did not induce any clastogenic effects to L5178Y mouse lymphoma cells treated in vitro. The study was performed in accordance to OECD 476. Two experiments were performed using dose levels of 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 (4-hour exposure). In Experiment 2, the dose range was 0.63 to 40 μg/ml in the absence of metabolic activation (24-hour exposure), and 60 to 240 μg/ml in the presence of metabolic activation (4-hour exposure).

 

-         2-phenoxyethyl acrylate was considered to be non-clastogenic and did not induce any toxicological significant increases in the frequency of cells with aberrations, when tested for the potential of inducing chromosome aberrations in cultured mammalian cells (human lymphocytes) in vitro. The study was performed in accordance to OECD 473. The study was performed in accordance to OECD 473. 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.

Justification for selection of genetic toxicity endpoint
The study has high reliability score, GLP study without deviations, and performed recently.

Justification for classification or non-classification

The available in vitro data inidcate that 2-phenoxyethyl acrylate should not be classified for mutagenicity.

Due to the very close structural similarity to 2-phenoxyethyl acrylate, the target substance 2-phenoxyethyl methacrylate is not considered as mutagenic and thus, no classification for mutagenicity shoul be applied.