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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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]dec-4-yl)ethyl methacrylate
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Controlsopen allclose all
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
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
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
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
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
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 resultsopen allclose all
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
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
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
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
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.