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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Three experimental studies are available. All studies concluded that 2-phenoxyethyl acrylate is non-genotoxic
Link to relevant study records
Reference
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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. certificate)
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 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 suggest that 2-phenoxyethyl acrylate should not be classified as mutagenic or genotoxic.