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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-03-2008 - 08-04-2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
OECD 471, GLP. Study according to relevant guideline.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Isodecyl methacrylate
EC Number:
EC Name:
Isodecyl methacrylate
Cas Number:
Molecular formula:
8-methylnonyl methacrylate
Test material form:
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isodecyl methacrylate
- Supplier: Evonik Röhm GmbH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Aug 26, 2008
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature


Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix fraction of Aroclor 1254-induced, male Sprague-Dawley rats and male Syrian hamster livers
Test concentrations with justification for top dose:
Main experiment:
-S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate
+S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate

According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 µg/plate. The concentration range include two logarithmic decades. Six adequately spaced concentrations
were tested. Two independent experiments were performed.

Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in
the preliminary toxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
Concurrent untreated and solvent control: DMSO were performed
Negative solvent / vehicle controls:
vehicle: THF
True negative controls:
Negative solvent / vehicle controls:
Positive controls:
for TA 1535, TA 100
Positive control substance:
sodium azide
sodium azide (purity: >= 99.0%, supplier: Serva, D-69042 Heidelberg, Germany) dissolved in aqua dest.; concentration: 10 µg/plate
Positive controls:
for TA 1537, TA 98
Positive control substance:
other: 4-nitro-o-phenylenediamine, without metabolic activation
4-nitro-o-phenylenediamine (purity: > 99.9%, supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in DMSO; concentration: 10 µg/plate
Positive controls:
for TA 102
Positive control substance:
methylmethanesulfonate (purity: > 99.0%, supplier: Merck-Schuchardt, D-85662 Hohenbrunn, Germany) dissolved in aqua dest.; concentration: 1.0 µl/plate Migrated to IUCLID6: without metabolic activation
Positive controls:
for TA 1535, TA 1537, TA 98, TA 100, TA 102
Positive control substance:
other: 2-aminoanthracene, with metabolic activation
2-aminoanthracene (purity: > 99%, supplier: MERCK, D-64293 Darmstadt, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate in TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation assay as described by Haworth et al. 1983, with some differences.

- Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley, U.S.A.) and were stored as recommended (Maron and Ames, 1983).
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analyszed prior to their use for mutagenicity assays.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone.
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:

- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.

Range of spontaneous reversion frequencies*
TA 135 TA 1537 TA 98 TA 100 TA 102
10 - 29 5 - 28 15 - 57 77 - 189 121 - 293

* These values refer to the negative control without metabolic activation and represent our historical control range in 1993.

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be
recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible
increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive
response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535,
TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible
increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or not.
According to OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Key result
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Interpretation of results: negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, Isodecyl methacrylate has to be judged as nonmutagenic in the presence and absence of mammalian metabolic activation
according to the Ames test results.
Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimurium were exposed to Isodecyl methacrylate at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

No toxic effects occurred in the test groups with and without metabolic activation.The plates incubated with the test article showed normal background growth up to ug/plate with and without S9-mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with at any concentration level, either in the presence or absence of metabolic activation (S9 -mix). There was also no tendency to higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains.


There was no evidence of induced mutant colonies over background.

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.


This reliable and valid study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.


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