2-methoxyethyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Sep - 01 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxyethyl acrylate

EC Number:

221-499-3

EC Name:

2-methoxyethyl acrylate

Cas Number:

3121-61-7

Molecular formula:

C6H10O3

IUPAC Name:

2-methoxyethyl acrylate

Details on test material:

- Name of test material (as cited in study report): 2-methoxyethyl acrylate
- Physical state: clear colourless liquid
- Analytical purity: 99.9%
- Lot/batch No.: C220424SP6
- Expiration date of the lot/batch: 2013-05-31
- Storage condition of test material: room temperature in the dark under nitrogen

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: cleansing medium (THG medium): medium containing thymidine (9 µg/mL), hypoxanthine (15 µg/mL) and glycine (22.5 µg/mL); THGM: TGM medium with methotrexate (0.3 µg/mL); growth medium (R10 medium): 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; R20 medium: growth medium with 20% donor horse serum; R0 medium: growth medium without serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats daily treated with oral doses of a mixture of phenobarbitone (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for 3 consecutive days prior to sacrifice

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
4 h treatment (-S9 mix): 0.64, 1.27, 2.54, 5.08, 10.16, 20.31, 40.63, 81.25 and 162.5 µg/mL
4 h treatment (+S9 mix): 10.16, 20.31, 40.63, 81.25, 162.5, 325, 650, 975 and 1300 µg/mL
24 h treatment (-S9 mix): 0.16, 0.32, 0.64, 1.27, 2.54, 5.08, 10.16, 20.31 and 40.63 µg/mL

Main experiment:
4 h treatment (-S9 mix): 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 µg/mL
4 h treatment (+S9 mix): 20.25, 40.5, 81, 162, 324, 432, 540 and 648 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

cyclophosphamide, 2 µg/mL in R0 medium, +S9; ethylmethanesulphonate, 400 µg/mL in R0 medium, -S9

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (± S9 mix)
- Expression time (cells in growth medium): 2 days after the end of treatment, cells were plated for determination of cell viability and the mutation frequency in 96-well microtitre plates containing TFT-selective medium.
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-16 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures in one experiment in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: relative suspension growth and viability

OTHER EXAMINATIONS: Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal aberrations.

Evaluation criteria:

The test substance was considered mutagenic if:
- a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value was observed.
- any test item concentration has a mutation frequency value that is greater than the corresponding vehicle control by the GEF (global evaluation factor; Moore et al. (2006) Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: Follow-up international workshop on genotoxicity test procedures, New Orleans Louisiana. Environ. Mol. Mutagen. 40: 292-299) of 126E-06 and demonstrates a positive linear trend.

However, if a test item produced a modest increase in mutant frequency, which only marginally exceeded the global evaluation factor (GEF) value and was not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induced modest reproducible increases in the mutation frequencies that do not exceed the GEF value, then scientific judgement was applied. If the reproducible responses were significantly dose-related and included 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 were reviewed using the above criteria, and may be disregarded.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which followed the statistical guidelines recommended by the UKEMSA (Robinson et al., 1989). A test for linear trend was performed to identify a dose-response relationship.

Reference:
Robinson W D et al. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preliminary cytotoxicity test: ≥ 5.08 µg/mL (-S9) and ≥ 162.5 µg/mL (+S9); Main experiment: ≥ 10 µg/mL (-S9) and ≥ 324 µg/mL (+S9)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: based on the toxicity observed in a previous chromosome aberration test, the concentration range used in the preliminary toxicity test was 0.64 to 162.5 µg/mL for the 4-h exposure in the absence of metabolic activation, 10.16 to 1300 μg/mL (corresponding to approx. 10 mM) for the 4-h exposure in the presence of metabolic activation, and 0.16 to 40.63 µg/mL for the 24-h exposure in the absence of metabolic activation. In all three exposure groups, there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. After 4-h treatment, cytotoxicity was observed at concentrations ≥ 5.08 µg/mL without S9 mix and ≥ 162.5 µg/mL with S9 mix. After 24-h exposure in the absence of S9 mix, cytotoxicity was noted at ≥ 2.54 µg/mL (RSG: 72%). Due to the steep nature of the toxicity curve at the higher concentrations (RSG: ≤ 10), it was difficult to achieve the optimum toxicity for all exposure conditions.

COMPARISON WITH HISTORICAL CONTROL DATA: the frequency of chromosomal aberrations in the negative and positive control was within the historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the main experiment, there was evidence of marked concentration-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the reduction in %RSG and RTG (relative total growth) values. Toxicity occurred at concentrations ≥ 10 µg/mL in the absence of S9 mix and at concentrations ≥ 324 µg/mL in the presence of S9 mix. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The toxicity observed at 40 µg/mL in the absence of metabolic activation, and at 648 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, these concentrations were excluded from the statistical analysis.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [mg/mL]	Relative suspension growth [%]	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Mutants per 1E+06 surviving cells		Proportion small colony mutants
					Small colonies	Large colonies
0 (Medium)	100	1	143.78	1	47.8	88.3	0.36
0.63	100	NA	NA	NA	NA	NA	NA
1.25	92	NA	NA	NA	NA	NA	NA
2.5	87	0.76	196.49	1.37	66.9	116.7	0.37
5	85	0.78	193.76	1.35	70.2	110.3	0.40
10	77	0.62	227.23*	1.58	100.7	109.9	0.48
20	49	0.47	240.24*	1.67	90.4	128.3	0.42
30	29	0.13	407.18*	2.83	239.7	132.2	0.63
40#	13	0.04	974.72	6.78	568.0	295.7	0.64
Linear trend			***
EMS, 400	59	0.34	1237.59	8.61	373.3	524.0	0.43

EMS = ethylmethanesulphonate; NA = not analysed; # = excluded from statistical analysis due to overt toxicity

*p < 0.05; ***p < 0.001

Table 2: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [mg/mL]	Relative suspension growth [%]	Relative Total Growth	Mutants per 1E+06 surviving cells	Mutation factor	Mutants per 1E+06 surviving cells		Proportion small colony mutants
					Small colonies	Large colonies
0 (Medium)	100	1	173.24	1	68.0	94.0	0.42
20.25	102	NA	NA	NA	NA	NA	NA
40.5	99	NA	NA	NA	NA	NA	NA
81	93	1.02	167.72	0.97	88.7	67.5	0.56
162	84	0.78	228.559	1.32	123.2	87.2	0.58
324	68	0.76	199.2	1.15	107.1	75.4	0.58
432	43	0.37	297.77**	1.72	141.2	127.2	0.52
540	20	0.11	444.21**	2.56	238.9	163.9	0.59
648#	9	0.03	768.32	4.44	568.0	147.7	0.78
Linear trend			***
CP, 2.0	63	0.35	1328.84	7.67	883.6	193.0	0.78

CP = cyclophosphamide; NA = not analysed; # = excluded from statistical analysis due to overt toxicity

*p < 0.05; ***p < 0.001

DIFFERENTIATION OF LARGE AND SMALL COLONIES

The increases in mutant frequency observed in the absence and presence of metabolic activation were mainly due to small colony formation indicating a clastogenic response (see Table 1 and 2).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive