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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication, which meets basic scientific principles

Data source

Reference Type:
A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays
Seifried, H.E., Seifried, R.M., Clarke, J.J., Junghans, T.B. and San, R.H.C.
Bibliographic source:
Chem. Res. Toxicol. 2006, 19, 627-644

Materials and methods

Principles of method if other than guideline:
The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. S9 mix was prepared according to the procedure of Clive et al.. The mutagenicity assay was performed according to the procedure described by Clive and Spector.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): benxylideneacetone (Methyl styryl ketone), CAS: 122-57-6
- Substance type: organic
- Analytical purity: The tested agents were acquired by NCI contractors [Illinois Institute of Technology Research Institute, Chicago, IL, and Midwest Research Institute (MRI), Kansas City, MO], analyzed for purity by MRI, and supplied as coded samples to the contract laboratory (BioReliance, Inc.,
Rockville, MD).
- Storage condition of test material: The bulk materials were stored at -20 °C, 4 °C, or room temperature as required to maintain compound stability.
Stock solutions of each chemical were prepared in the appropriate solvent immediately prior to use.


Target gene:
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- 3.7.C mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC). The cells were grown in Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). Cells were screened for the presence of mycoplasma after cryopreservation. New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.
Metabolic activation:
with and without
Metabolic activation system:
The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
10, 20 ,30, 40 and 50 µg/ mL (tests without metabolic activation).
20, 30, 40, 50 and 60 µg (tests with metabolic acitvation).
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not given
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
ethyl methylsulfonate at 4.7 * 10E-6 M (or methyl methanesulfonate at 10-20 µg/mL) (test without metabolic activation) & 3-methylcholanthrene at 1.86 * 10-5 M (or dimethylbenz[aanthracene at 0.5 - 4 µg/mL (tests with metabolic activation)
Details on test system and experimental conditions:

- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): trifluorothymidine (TFT, final concentration 3 µg/mL)
- Fixation time (start of exposure up to fixation or harvest of cells): Plates were incubated at 37 (1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

- Method: relative total growth

S9 mix was prepared according to the procedure of Clive et al..
Evaluation criteria:
Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase.
Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from 0.2 to 1.1 mm.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
Cytotoxicity / choice of top concentrations:
at the highest and second highest concentration tested the average viable cell number was reduced ( 82 and 84 or 122 and 128, compared to 143 in the controls, respectively.
Vehicle controls validity:
Positive controls validity:
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
Cytotoxicity / choice of top concentrations:
at the highest concentration tested the average viable cell number was reduced ( 96 and 100, compared to 118 in the controls, respectively)
Vehicle controls validity:
Positive controls validity:

Any other information on results incl. tables

Table 1 - Mouse Lymphoma Test Data
Non-Activated Cultures S9-Activated Cultures
Chemical Name Solvent Dose  (µg/mL) Average TFT Average VC Mut Freq RTG Dose  (µg/mL) Average TFT Average VC  Mut Freq RTG
or µL/mL or µL/mL
Benxylideneacetone (Methyl styryl ketone) Ethanol 10 to 60
  10 20 148 0,27 92 20 23 119 0,39 73
  17 166 0,2 120 18 109 0,33 66
  20 31 130 0,48 66 30 35 129 0,54 35
  33 153 0,43 89 29 125 0,46 37
  30 35 137 0,51 51 40 26 141 0,37 21
  30 139 0,43 63 29 126 0,46 23
  40 46 122 0,75 38 50 32 123 0,52 14
  46 128 0,72 32 32 116 0,55 13
  50 63 82 1,54 11 60 28 100 0,56 8
  61 84 1,45 10 33 96 0,69 8
  Solvent 18 143 0,25 Solvent 21 118 0,34
  Positive 338 71 9,52 37 Positive 150 69 4,35 31

Applicant's summary and conclusion

Positive without metabolic activation: a clear positive result was obtained in the mouse lymphoma assay without metabolic activation at the dose level of 50 µg/mL.
Negative with metabolic activation: a negative results was obtained in the mouse lymphoma assay with metabolic acitvation at dose levels up to 60 µg/mL.

Benzylideneacetone was found to induce a clearly positive result in L5178 TK +/- 3.7C mouse lmyphoma cells at dose levels of 50 µg/mL when the test was conduced without metabolic activation. However, negative results were obtained in the tests with metabolic activation. As the data comes from a well-documented publication and the solvent controls and positive controls were valid, the source is considered to be of high quality and reliability (Klimisch 2).
Executive summary:

Seifried and collagues compiled in their publication the results of genetic mutations assays (AMES-tests) and Mouse-lymphoma Test data of more than 450 chemicals (Seifried et al., 2006). The test results were obtained from the data banc of the National Cancer Institute (NCI), which is responsible for the selection of the most significant chemicals for carinogenicity testing by the National Toxicology Program (NTP). As only some of the testing data available at the NCI has been made available in summary form in the Chemical Carcinogenisis Research Information System

(CCRIS), which is searchable on the NLM TOXNET system, they decided to compile the data of these chemicals in a summary table that presents the results for each compound. Benzylideneacetone (CAS 122 -57 -6) is given with a positive result in the Salmonella typhimurium strain TA 100 with metabolic activation (either rat S9 or hamster S9). Moreover, benzylideneacetone was found to be positive in the non activated mouse lymphoma test (doses 10 - 50 µg/mL, resulted in a lowest effective dose of 20 and 50). In case of test with metabolic activation (20 - 60 µg/mL), the results were either weakly positive or negative.