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EC number: 945-910-7 | CAS number: -
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Endpoint summary
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Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2016
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: September 2015; signature: November 2015
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver microsomal enzymes (S9 homogenate)
- Test concentrations with justification for top dose:
- Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 1: 52, 164, 512, 1600, 5000 μg/plate
In the additional first mutation experiment with tester strain TA98 (absence of S9-mix), the test item concentrations were not corrected for the purity and the following dose range was tested, 1.5, 4.6, 15, 45, 140, 440, 1375 and 4300 μg/plate. TA98 strain did not show any indications of increases including in the arguably more sensitive pre-incubation method (Experiment 2) with/without S9 activation at up to precipitating dose concentrations. The omission of the use of the correction factor in this part of the project had no influence on the study.
Experiment 2: 275, 492, 878, 1568, 2800 μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item could not be dissolved in water. The test item was soluble
in dimethyl sulfoxide. Preparation of test solutions started with solutions of 50 mg/mL applying a mixing step on the vortex, resulting in a clear solution. The lower test concentrations were prepared by subsequent dilutions in dimethyl sulfoxide. Test item concentrations were used within 3 hours of preparation. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191; 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)
DURATION
- Exposure duration:
Dose range finding test and Experiment 1: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted.
Experiment 2: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies were counted. The revertant colonies were counted automatically with a Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants
OTHER:
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item in the subsequent mutation assays was 2500 to 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. An additional direct plate assay was performed with the tester strains TA1537 and TA98 in the absence and presence of S9-mix. Initially the additional experiment with the tester strains TA1537 (with and without S9-mix) and TA98 (without S9-mix) were rejected due to technical reasons (inadequate non-cytotoxic dose levels), this part of the study was repeated. - Evaluation criteria:
- See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
- Statistics:
- No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Test substance was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Test substance was either tested up to cytotoxic or precipitating concentrations or the limit concentration (5000 μg/plate).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 µg/plate or 1600 µg/plate (additional experiment in ) and upwards and at the top dose of 5000 µg/plate at the end of the incubation period.
Experiment 2: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 µg/plate and upwards and at the top dose of 2500 µg/plate and 5000 µg/plate in the absence and presence of S9-mix, respectively at the end of the incubation period.
RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within the laboratory historical control data ranges.The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA98 (absence of S9-mx) in the initial test within Experiment 1. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study. Furthermore, TA98 (absence and presence of S9-mix) repetition for technical reasons prior to Experiment 2 showed all postive controls were within laboratory historic control data ranges this indicates that test conditions were adequate and the metabolic activation system functioned properly.
Tester strain TA98 (Experiment 1), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 52 µg/plate in the presence of S9-mix. However since the mean number of revertant colonies (8 revertants) was equal to the value of 8 revertant colonies observed at the solvent control group, this reduction is caused by incidental fluctuations in the number of revertant colonies. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Under the conditions of this study the test substance is not considered to be mutagenic. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- Executive summary:
The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat; incorporating the plate incorporation and pre-incubation methods). The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional direct plate assay was performed with the tester strains TA1537 and TA98 in the absence and presence of S9-mix. A correction factor was used to correct for the purity of the test item dissolved in dimethyl sulfoxide. In the dose range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at the top dose of 5000 μg/plate. In tester strain WP2uvrA, toxicity was observed at dose levels of 1600 and 5000 μg/plate in the presence of S9-mix. In tester strain TA100, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. Toxicity was observed in the tester strains TA1537 and TA98 in the absence and presence of S9-mix. In the first mutation test, insufficient non-toxic dose levels were present in the tester strains TA1537 and TA98, an additional experiment was performed. In this mutation experiment, the test item was tested at a concentration range of 1.8 to 5000 µg/plate in the absence of S9-mix in tester strain TA1537, at 1.5 to 4300 µg/plate in the absence of S9-mix in tester strain TA98 and at a range of 1.8 to 2500 µg/plate in the presence of S9-mix in both tester strains. The test item precipitated on the plates at dose levels of 2500 μg/plate and above. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second experiment, the test item was tested up to concentrations of 5000 µg/plate in the absence of S9-mix and 2500 µg/plate in the presence of S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at dose levels of 2500 μg/plate and above. In tester strain WP2uvrA, toxicity was observed in the presence of S9-mix. In the other tester strains, no toxicity was observed at any of the dose levels tested. The toxicity observed in the first experiment in the tester strains TA1537 and TA98 was not confirmed in the additional and second mutation experiment. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed in this additional experiment, therefore it is concluded that the toxicity in the first experiment was an incidental finding and that the test item is not toxic for the bacteria up to and including the top dose of 5000 µg/plate in the tester strains TA1537 and TA98. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Under the conditions of this study, it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Reference
Table 1. Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
Direct plate assay
Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain. |
||||||||
Dose (µg/plate) |
TA100 |
|
|
|
WP2uvrA |
|
|
|
Without S9-mix |
|
|
|
|
|
|
|
|
Positive control |
809 |
± |
61 |
|
1605 |
± |
48 |
|
Solvent control |
84 |
± |
14 |
|
13 |
± |
2 |
|
1.7 |
80 |
± |
19 |
|
21 |
± |
3 |
|
5.4 |
74 |
± |
8 |
|
20 |
± |
11 |
|
17 |
73 |
± |
5 |
|
15 |
± |
10 |
|
52 |
93 |
± |
9 |
|
20 |
± |
2 |
|
164 |
68 |
± |
9 |
|
20 |
± |
5 |
|
512 |
85 |
± |
19 |
|
16 |
± |
4 |
|
1600 |
74 |
± |
2 |
NP |
19 |
± |
4 |
NP |
5000 |
81 |
± |
6 |
n SP |
18 |
± |
4 |
n SP |
|
|
|
|
|
|
|
|
|
With S9-mix |
|
|
|
|
|
|
|
|
Positive control |
1447 |
± |
330 |
|
305 |
± |
19 |
|
Solvent control |
88 |
± |
10 |
|
24 |
± |
9 |
|
1.7 |
83 |
± |
8 |
|
27 |
± |
5 |
|
5.4 |
68 |
± |
6 |
|
15 |
± |
4 |
|
17 |
78 |
± |
15 |
|
13 |
± |
3 |
|
52 |
86 |
± |
12 |
|
19 |
± |
5 |
|
164 |
68 |
± |
8 |
|
30 |
± |
7 |
|
512 |
85 |
± |
13 |
|
24 |
± |
4 |
|
1600 |
72 |
± |
7 |
NP |
8 |
± |
6 |
|
5000 |
80 |
± |
5 |
n SP |
1 |
± |
1 |
|
|
|
|
|
|
|
|
|
|
NP: No precipitate
SP: Slight Precipitate
N: Normal bacterial background lawn
Table 2. Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay
Direct plate assay
Dose (µg/plate) |
Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium. |
|||||||||||
Without S9-mix |
TA1535 |
|
|
|
TA1537 |
|
|
|
TA98 |
|
|
|
Positive control |
726 |
± |
506 |
|
53 |
± |
37 |
|
178 |
± |
200 |
|
Solvent control |
19 |
± |
3 |
|
5 |
± |
2 |
|
17 |
± |
13 |
|
52 |
21 |
± |
19 |
|
7 |
± |
4 |
|
21 |
± |
7 |
|
164 |
24 |
± |
10 |
|
3 |
± |
2 |
|
8 |
± |
10 |
|
512 |
28 |
± |
9 |
|
3 |
± |
4 |
|
8 |
± |
7 |
|
1600 |
25 |
± |
4 |
NP |
0 |
± |
0 |
NP |
6 |
± |
9 |
NP |
5000 |
11 |
± |
4 |
n SP |
0 |
± |
0 |
n SP |
5 |
± |
8 |
n SP |
|
|
|
|
|
|
|
|
|
|
|
|
|
With S9-mix |
|
|
|
|
|
|
|
|
|
|
|
|
Positive control |
505 |
± |
14 |
|
412 |
± |
44 |
|
690 |
± |
26 |
|
Solvent control |
25 |
± |
1 |
|
6 |
± |
1 |
|
8 |
± |
8 |
|
52 |
21 |
± |
4 |
|
8 |
± |
6 |
|
8 |
± |
7 |
|
164 |
20 |
± |
3 |
|
8 |
± |
6 |
|
19 |
± |
4 |
|
512 |
23 |
± |
9 |
|
2 |
± |
2 |
|
6 |
± |
2 |
|
1600 |
23 |
± |
6 |
NP |
0 |
± |
1 |
NP |
6 |
± |
5 |
NP |
5000 |
10 |
± |
4 |
n SP |
0 |
± |
1 |
n SP |
1 |
± |
2 |
n SP |
NP: No precipitate
SP: Slight Precipitate
n: Normal bacterial background lawn
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
OECD TG 471, 2016 - The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat; incorporating the plate incorporation and pre-incubation methods. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix (rat liver S9-mix induced Aroclor 1254). An additional direct plate assay was performed with the tester strains TA1537 and TA98 in the absence and presence of S9-mix. A correction factor was used to correct for the purity of the test item dissolved in dimethyl sulfoxide. In the first mutation test, insufficient non-toxic dose levels were present in the tester strains TA1537 and TA98, an additional experiment was performed. In this mutation experiment, the test item was tested at a concentration range of 1.8 to 5000 µg/plate in the absence of S9-mix in tester strain TA1537, at 1.5 to 4300 µg/plate in the absence of S9-mix in tester strain TA98 and at a range of 1.8 to 2500 µg/plate in the presence of S9-mix in both tester strains. The test item precipitated on the plates at dose levels of 2500 μg/plate and above. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second experiment, the test item was tested up to concentrations of 5000 µg/plate in the absence of S9-mix and 2500 µg/plate in the presence of S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at dose levels of 2500 μg/plate and above. In tester strain WP2uvrA, toxicity was observed in the presence of S9-mix. In the other tester strains, no toxicity was observed at any of the dose levels tested. The toxicity observed in the first experiment in the tester strains TA1537 and TA98 was not confirmed in the additional and second mutation experiment. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed in this additional experiment, therefore it is concluded that the toxicity in the first experiment was an incidental finding and that the test item is not toxic for the bacteria up to and including the top dose of 5000 µg/plate in the tester strains TA1537 and TA98. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Under the conditions of this study, it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Justification for selection of genetic toxicity endpoint
Study selected is an in vitro study (Klimisch 1)
Short description of key information:
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2016
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity
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