2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

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:

The study was conducted between 1 December 1986 and 12 December 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

EC Number:

224-116-8

EC Name:

2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate

Cas Number:

4203-89-8

Molecular formula:

C14H23NO3

IUPAC Name:

2-{1-oxa-4-azaspiro[4.5]decan-4-yl}ethyl 2-methylprop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Chemical name: Methacrylic acid , 2-(1 oxa-4-aza spiro [4 , 5] dec-4 -yl) ethyl ester
Commercial name/code: Nourycryl MA 128
Batch no . 860930 - DT 4
Storage: At ambient temperature in the dark in the presence of silica gel
Appearance: clear slightly yellow liquid

Method

Target gene:

histidine locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from homogenised liver of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test:
1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
5000 μg/plate is the maximum test substance concentration that should be used, according to the OECD guidelines

Mutagenticity test (experiment 1 and 2)
100, 333, 1000, 3330 and 5000 µg/plate
In the preliminary test, the highest dose tested caused no increase in toxicity

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain TA1538

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/plate for strain TA98

Positive control substance:

other: daunomycine

Remarks:

without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

650 µg/plate for strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Bacterial cultures
Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No.2) and incubated in a shaking water bath (37 °C, 150spm) until the cultures reach an O.D. of 0.4 at 700 nm (10^9 cells /ml) . Freshly grown cultures of each strain are used for a test.

Test procedure
Standard plate test
Top agar in top agar tubes is melted and heated to 45ºC. The following solutions are succesively added to 3 ml of top agar: 0.1ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1ml of a dilution of the test substance in DMSO, and in the case of activation of assays 0.5ml of S9-mix. The ingredients are mixed in a vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidifcation of the top agar, the plates are turned and incubated in the dark at 37ºC for 48h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.
Each concentration was tested in triplicate.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain ay any concentration is not greater than two times the solvent control value with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) is the Ames test if:
a) it induced at least a 2-fold increase in the number of revertants with respect to the number induced by the solvent control in any of the test strains, either with or without metabolic activation. Moreover, the positive response shouls be dose related. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment

The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary test
Nine serial dilutions of the test substance, in approximately half-log steps, were plated with an appropriately diluted TA100 culture (equal numbers of bacterial cells/plate) into non-selective agar (viability determination). The percentage survival of the TA100 culture is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. However, even at the highest test substance concentration used the survival of strain TA100 is not reduced. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate.


Main test
All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values fell within laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considred as not mutagenic in the Ames Salmonella/microsome assay.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related increase in the number of revertants in two independently repeated experiments. Therefore, the test substance can be considered as not mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

Nourycryl MA128 was tested in the Ames Salmonella/microsome test up to 5000 µg/plate. The test substance induced no dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA1538, TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as non mutagenic in this test system.