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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
February 1998
according to guideline
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
according to guideline
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl acrylate
EC Number:
EC Name:
tert-butyl acrylate
Cas Number:
Molecular formula:
tert-butyl acrylate
Specific details on test material used for the study:
- Name of test material: tert.-Butylacrylate
- Physical state: liquid, colourless, clear
- Lot/batch No.: 011631eda0


Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

- Type and composition of media, CO2 concentration, humidity level, temperature: MEM (minimal essential medium) containing Hank’s salts
supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cell cultures were incubated at 37°C in a
1.5 % carbon dioxide atmosphere (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-orthophosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration in the culture: 0.75 mg/mL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
pre test: 10 - 1280 µg/mL (with and without S9-mix)
The dose range of the main experiments was set according to the data generated in the preexperiment. The individual concentrations were generally spaced by a factor of 2.0. Narrower spacing was used at higher concentrations.

Experiment 1:
160, 320, 640, 960, 1280 µg/mL (with and without S9 mix)
Experiment 2:
320, 640, 800, 960, 1120 µg/mL (without S9 mix);
640, 800, 960, 1120, 1280 µg/mL (with S9 mix)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The final concentration of DMSO in the culture medium was 0.5% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:

- Preincubation period: 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (incubation with a selection agent): 8 days

SELECTION AGENT: 6-thioguanine (11 μg/mL)
Rationale for test conditions:
- The pre-experiment was performed in the presence and absence (4 hours treatment) of metabolic activation. The highest concentration of the preexperiment was chosen with regard to the purity (99.83%) and the molecular weight (128.169 g/mol) of the test item.
- To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations. The cultures at the two lowest concentrations in experiment I with and without metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines. In experiment II the cultures at the lowest concentration with metabolic activation were not continued for the same reason. The cultures at the highest concentration without metabolic activation were not continued to due exceedingly severe cytotoxic effects.
Evaluation criteria:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
True negative controls validity:
not examined
Positive controls validity:
Additional information on results:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 640 μg/mL and above without metabolic activation and at 960 μg/mL and above with metabolic activation. In experiment II relevant cytotoxic effects were noted at 640 μg/mL and above without metabolic activation. Cytotoxicity was more severe and the maximum concentration without metabolic activation was not analyzable. The relative cloning efficiency I was still acceptable with 16.3% and 17.0% but the cell density at the first subcultivation after treatment was too low to proceed. In the presence of metabolic activation cytotoxicity as described above was noted at 800 μg/mL and above. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation.

- No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.
- The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment at 960 μg/mL without metabolic activation. This effect however, was judged as biologically irrelevant, since it was based upon the rather low solvent control of 4.9 mutant colonies/10^6 cells and was not reproduced in the parallel culture under identical conditions.
- In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.9 up to 19.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 4.5 up to 31.3 mutants per 10^6 cells.
- The viability (cloning efficiency II) of the solvent control of culture I of the first experiment with metabolic activation and of culture I of the second experiment with metabolic activation reached but did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit.
- EMS (150 μg/mL) and DMBA (1.1 μg/mL in experiment I, 2.2 μg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.
- Concurrent vehicle negative and positive control data: Please refer to the attached background material.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Applicant's summary and conclusion