2-(2-ethoxyethoxy)ethyl acrylate

Genetic toxicity: in vitro

Administrative data

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:

31 March 2017 - 24 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethyl acrylate

EC Number:

230-811-7

EC Name:

2-(2-ethoxyethoxy)ethyl acrylate

Cas Number:

7328-17-8

Molecular formula:

C9H16O4

IUPAC Name:

2-(2-ethoxyethoxy)ethyl acrylate

Test material form:

other: clear, colorless liquid

Details on test material:

- Name of test material (as cited in study report): EOEOEA
- Physical state: clear colourless liquid
liquid
- Analytical purity: 2- (2-ethoxyethoxy)ethyl acrylate, CAS RN 7328-17-8: 92.1% (by GLC)
- Purity test date:
- Lot/batch No.: EA633
- Expiration date of the lot/batch: 01 June 1999
- Stability under test conditions: Stable
- Storage condition of test material: In refrigerator in the dark

Specific details on test material used for the study:

- Name of test material (as cited in study report): MIRAMER M170
- Physical state: Clear colorless liquid
- Analytical purity: 96.20% ( CAS No.: 7328-17-8; EC No.: 230-811-7)
- Impurities (identity and concentrations): Ethyl Carbitol 0.91%, 2-Ethoxy ethyl acrylate(EOEA) 0.21%, EOEOA/AA 1.06%, EOEOEA dimer 1.56%.
- Lot/batch No.: 161225177
- Expiration date of the lot/batch: 24 December 2017
- Storage condition of test material: At ambient temperature (10 to 30 degree celcius) and protected from light (although could be used for formulation in light).
Supplier: Miwon

Method

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

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:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

Test concentrations with justification for top dose:

The test item was tested using the following method with and without metabolic activation. The maximum concentration was 5000 µg/plate (the maximum recommended dose level).

Experiment 1: Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2: Eight concentrations of the test item (5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
Supplier: Fisher Scientific
Batch number (purity): Experiment 1: 1684307 (> 99%) and Expiry 03/22 Experiment 2: 1690734 (> 99%) Expiry 03/22

- 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 (3.8%)

Controls

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:

Mutation Test - Experiment 1 (plate incorporation method):
Eight concentrations of 2-phenoxyethyl acrylate (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. 0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

The same procedure as described above was performed with metabolic activation except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

All of the plates were incubated at 37 ± 3oC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
All testing for this experiment was performed in triplicate.

Mutation Test - Experiment 2 (Pre-Incubation Method):
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation. Seven doses (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, with and without metabolic activation. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 oC for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.

The same procedure as described above was performed with metabolic except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 oC for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media.

All of the plates were incubated at 37 ± 3 oC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
All testing for this experiment was performed in triplicate.

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
- there should be a minimum of four non-toxic test item dose levels.
- there should be no evidence of excessive contamination

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:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

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:

The maximum dose level of the test item in the first and second experiment were selected as the maximum recommended dose level of 5000μg/plate. In the first and second mutation test, the test item induced toxicity as a visible reduction in the frequency of revertant colonies of all of the Salmonella tester strains (without a weakening of the bacterial background lawns), at 5000μg/plate in both the presence and absence of metabolic activation (S9-mix).In addition, slight reductions in colony frequency at 1500μg/plate in both the absence and presence of S9-mix. No reductions in colony frequency were noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix.

Any other information on results incl. tables

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 2015 and 2016 was included in the test report. The results of this study was in accordance with the history profile.

Applicant's summary and conclusion

Conclusions:

The mutagenic potential of Ethoxy ethoxy ethyl acrylate was tested in the reverse mutation in accordance with OECD Test Guideline 471.
Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The mutagenic potential of Ethoxy ethoxy ethyl 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 in accordance with OECD Test Guideline 471 using the plate incorporation and pre-incubation methods at seven to eight doses (1.5 - 5000 µg/plate), in triplicate, both with and with out present of S9-mix.

The maximum dose level of the test item in the first and second experiment were selected as the maximum recommended dose level of 5000μg/plate. In the first and second mutation test, the test item induced toxicity as a visible reduction in the frequency of revertant colonies of all of the Salmonella tester strains (without a weakening of the bacterial background lawns), at 5000μg/plate in both the presence and absence of metabolic activation (S9-mix). In addition, slight reductions in colony frequency at 1500μg/plate in both the absence and presence of S9-mix. No reductions in colony frequency were noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix.

Neither experiment one or two showed any increase in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Thus Ethoxy ethoxy ethyl acrylate (CAS No. 7328-17-8) was considered to be non-mutagenic under the conditions of this test.