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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication, which meets basic scientific principles

Data source

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

Materials and methods

Principles of method if other than guideline:
The experimental materials, methods, and procedures as explained below are based on those described by Ames et al.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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.

Method

Target gene:
his-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2) 10E9 cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al.
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2) 10E9 cells/mL. On the day of use, all tester strain cultures were checked for genetic integrity as recommended by Ames et al.
Metabolic activation:
with and without
Metabolic activation system:
Each chemical was tested without metabolic activation and with liver S9 preparations from Sprague-Dawley rats and Syrian golden hamsters. Unless limited by toxicity, each chemical was tested to the upper limit of 10000 µg/plate.
Test concentrations with justification for top dose:
10, 33, 100, 333, 667, 1000, 3333 µg (if no cytotoxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays):

OTHER:
S9-Preparation:
Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The postmitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. (26). The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added.
Evaluation criteria:
The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No genotoxicty was observed at any of the doses tested (without S9, with rat S9, with Hamster S9)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
No genotoxicty was observed at any of the doses tested (without S9)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
A clear positive result (genotoxicty) was observed at all of the doses tested (with rat S9, with Hamster S9), This test was repeated and this confirmed the obtained results.
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1:

Chemical Name CAS # Dose TA98 TA100 TA102 TA1535 TA1537 TA1538 TA97
no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9
Benxylideneacetone (Methyl styryl ketone) Positive 122-57-6 Negative Negative Negative Negative Positive 2x Positive 2x
DMSO 42 ± 9 42 ± 4 46 ± 3 159 ± 7 161 ± 7 188 ± 14
10ug 39 ± 7 --- --- 170 ± 16 --- ---
33ug 36 ± 2 41 ± 6 35 ± 4 153 ± 19 149 ± 16 164 ± 11
100ug 42 ± 9 41 ± 6 39 ± 6 146 ± 9 208 ± 4 206 ± 27
333ug 41 ± 15 29 ± 5 39 ± 21 145 ± 11 343 ± 35 422 ± 25
667ug --- 25 ± 3 41 ± 6 --- 580 ± 31 800 ± 84
1000ug 31 ± 10 23 ± 3 39 ± 5 152 ± 15 665 ± 42 1043 ± 85
3333ug 10 ± 3 3 ± 1 17 ± 2 6 ± 4 151 ± 49 288 ± 86
Positive 307 ± 21 858 ± 61 1183 ± 81 687 ± 6 903 ± 31 1294 ± 35
DMSO --- --- --- --- 119 ± 12 145 ± 12
33ug --- --- --- --- 175 ± 33 151 ± 20
100ug --- --- --- --- 240 ± 45 200 ± 21
333ug --- --- --- --- 366 ± 20 372 ± 11
667ug --- --- --- --- 587 ± 24 441 ± 40
1000ug --- --- --- --- 708 ± 72 640 ± 13
3333ug --- --- --- --- 70 ± 18 107 ± 91
Positive --- --- --- --- 614 ± 11 910 ± 68

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation A clear positive result (2 times elevated) was obtained in TA100 with metabolic activation

benxylideneacetone was found to induce a clearly positive result in Salmonella typhimurium strain TA 100 in an AMES-test. 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. Benxylideneacetone (CAS 122 -57 -6) is given with a positive result in an AMES test in the Salmonella typhimurium strain TA 100 with metabolic activation (either rat S9 or hamster S9). Moreover, benxylideneacetone 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.