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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
1) AMES-Test (S. typhimurium TA 98, TA 100), Seifried, 2006), positive with and without activation in TA 100. 2) Mouse lymphoma mutation assay, Seifried, 2006, positive without metabolic activation at a dose of 50 µg/mL 3) micronuclues assay in bone marrow cells of the mouse, Roth, 2012, 500, 1000 and 2000 mg/kg bw, negative 4) micronuclues assay, NTP, negative
Link to relevant study records
Reference
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
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
Target gene:
his-
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
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.
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

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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Seifried et al. 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 carcinogenicity 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 Carcinogenesis 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. 4 -phenylbutenone (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, 4 -phenylbutenone 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.

The test item 4-phenylbutenone was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Roth, 2012).The test item was dissolved in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. and 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. As estimated by pre-experiments 2000 mg 4-phenylbutenone per kg b.w. was suitable as top dose.The following dose levels of the test item were investigated:24 h preparation interval: 500, 1000, and 2000 mg/kg b.w. and 48 h preparation interval: 2000 mg/kg b.w..

As a result , the mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that 4-phenylbutenone did not have any cytotoxic properties in the bone marrow. However, one male (animal no. 40) of the high dose group (48 h treatment interval) died approximately 3 hours after treatment with the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 4-phenylbutenone were near to the value of the vehicle control group. However, the statistically significant increase above the vehicle control group observed in the low dose group was considered to have no biological relevance, as the value was well within the laboratory’s historical vehicle control data. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages.

Cyclophosphamide administered once orally (40 mg/kg b.w.) was used as positive control which showed a substantial and biologically relevant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, 4-phenylbutenone is considered to be non-mutagenic in this micronucleus assay.

A micronucleus test conducted with B6C3F1 mice was obtained from the National Toxicology Program, which collects data for possible dangerous substances. The test is considerd to be reliable with restrictions (Klimisch2). The test revealed no positive results for methyl styryl ketone (CAS No. 122 -57 -6).

Justification for classification or non-classification

Benzalacetone caused mutations inSalmonella typhimurium TA 100 with and without S9. Benzalacetone was shown to cause a mutagenic response in mouse lymphoma cells treated with doses of 50 µg/mL in the absence of S9. This shows, that in developed cells – like in the human body – where a highly differentiated metabolic system exists, the substance will most likely not show mutagenic potential. In addition, as the test material did not induce chromosome aberrations in an in vivo micronucleus assay, it does not meet the criteria for classification and will not require labelling as a mutagen in accordance with European regulation (EC) No. 1272/2008.