[2-[(2-methyl-1-oxoallyl)oxy]ethyl] hydrogen succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June - July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

no

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-methyl-1-oxoallyl)oxy]ethyl] hydrogen succinate

EC Number:

244-096-4

EC Name:

[2-[(2-methyl-1-oxoallyl)oxy]ethyl] hydrogen succinate

Cas Number:

20882-04-6

Molecular formula:

C10H14O6

IUPAC Name:

4-{2-[(2-methylprop-2-enoyl)oxy]ethoxy}-4-oxobutanoic acid

impurity 1

Reference substance name:

2-hydroxyethyl methacrylate

EC Number:

212-782-2

EC Name:

2-hydroxyethyl methacrylate

Cas Number:

868-77-9

Molecular formula:

C6H10O3

IUPAC Name:

2-hydroxyethyl methacrylate

impurity 2

Reference substance name:

Succinic acid

EC Number:

203-740-4

EC Name:

Succinic acid

Cas Number:

110-15-6

Molecular formula:

C4H6O4

IUPAC Name:

succinic acid

Test material form:

liquid

Details on test material:

- Name as cited in the report: Methacryloyloxyethyl succinate
- EC-name: [2-[(2-methyl-1-oxoallyl)oxy]ethyl] hydrogen succinate
- CAS: 20882-04-6
- EC-No.: 244-096-4
- Batch No.: 3903/64
- Storage: 4-8°C, protected from heat and sunlight
- Description: yellowish liquid
- Density: 1.1884 g/kg
- Molecular weight: approx. 230 g/mol
- Formula: C10H14O6
- Purity: 84% (NMR)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
- Main experiment: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine TA98, TA1537

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

With S9-mix

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL). The nutrient medium consists per litre:
- 8 g Nutrient Broth
- 5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by Eurofins Munich or provided by an appropriate supplier. Quality controls were performed. Sterilisation was performed for 20 min at 121°C in an autoclave.

The overlay agar contains per litre:
- 7.0 g Agar Agar
- 6.0 g NaCI
- 10.5 mg L-histidine x HCI x H2O
- 12.2 mg biotin
Sterilisation was performed for 20 min at 121°C in an autoclave.

Evaluation criteria:

CYTOTOXICITY:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the mean values of the spontaneous reversion frequency of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact values).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as folIows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

No precipitation of the test item was observed in any tester strain used in experiment l and II (with and without metabolic activation).

In experiment I (Plate-incorporation Test) toxic effects of the test item were observed only in tester strain TA 1535 at a concentration of 5.0 µL/plate (with metabolic activation). In tester strain TA 1535 (without metabolic activation) the mutation factor of the negative control was reduced to 0.5. Due to the fact that the mean value as well as all single values of the negative control were within the historical data range and no concomitant reducing of the background lawn was observed this finding was regarded as not biologically relevant and did not influence the results of the experiment.

In experiment II (Pre-incubation Test) toxic effects of the test item were noted only in tester strain TA 98 at a concentration of 5.0µL/plate (with metabolic activation) and in tester strain TA 1537 at concentrations of 2.5 µL/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.4 found in tester strain TA 1535 at a concentration of 0.316 µL/plate and down to a mutation factor of 0.5 at a concentration of 0.100 µL/plate in tester strain TA 1537 (both without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Methacryloyloxyethyl succinate at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. The negative control plates with and without S9 mix were within the historical control data range. The slightly higher spontaneous reversion frequency in tester strain TA 1537 in experiment I (35 revertants instead of 27 revertants) was regarded as not biologically relevant and did not influence the validity of the results.

EXPERIMENT I (Plate-incorporation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	48	40	105	112	9	11	35	24	159	261
solvent control	49	42	90	111	20	9	27	21	179	223
test item 0.0316 µL	48	29	87	121	17	9	22	18	214	266
test item 0.100 µL	40	42	94	112	17	10	27	20	249	270
test item 0.316 µL	37	38	89	112	22	9	26	18	220	251
test item 1.0 µL	28	40	101	101	21	6	37	16	209	219
test item 2.5 µL	33	33	98	112	17	10	32	18	213	248
test item 5.0 µL	37	40	93	103	14	5	27	18	230	304
pos. control	299	2758	844	2045	1143	128	111	258	442	679

EXPERIMENT II (Pre-incubation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	23	34	88	106	14	9	5	10	177	203
solvent control	19	41	66	78	13	9	10	7	201	161
test item 0.0316 µL	28	35	66	82	12	8	7	8	193	199
test item 0.100 µL	19	31	58	72	8	10	5	6	198	166
test item 0.316 µL	16	36	50	67	5	6	6	10	184	140
test item 1.0 µL	18	30	55	77	8	12	7	7	228	163
test item 2.5 µL	20	26	50	79	7	7	5	8	203	173
test item 5.0 µL	23	23	55	72	8	8	5	6	169	181
pos. control	321	1164	327	835	548	55	110	218	663	504

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacryloyloxyethyl succinate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Methacryloyloxyethyl succinate is considered to be non-mutagenic in this bacteriaI reverse mutation assay.

Executive summary:

In order to investigate the potential of Methacryloyloxyethyl succinate for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaIuated (with and without metabolic activation) in experiment I. In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5.0 µL/plate and in tester strain TA 1537 at concentrations of 2.5 µL/plate and higher (both without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Methacryloyloxyethyl succinate at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacryloyloxyethyl succinate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Methacryloyloxyethyl succinate is considered to be non-mutagenic in this bacteriaI reverse mutation assay.