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EC number: 272-902-4 | CAS number: 68919-76-6
Genetic toxicity: in vitro
Administrative data
- 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:
- 2012-03-27 to 2012-06-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study has been performed according to OECD guideline 471 and EU method B.13/14 in a GLP certified testing facility.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- 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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Test material form:
- other: liquid
- Details on test material:
- Name 400112
Batch no. 0001273679
Appearance yellow-brown, liquid
Composition UVCB substance
Purity 100% (not purified but used as synthesised)
Production date May 2011
Expiry date May 2013
Storage Room Temperature: 20 ± 5°C
Constituent 1
Method
- Target gene:
- Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535
- Details on mammalian cell type (if applicable):
- mutations: hisG46, uvrB, rfa
- Species / strain / cell type:
- S. typhimurium TA 102
- Details on mammalian cell type (if applicable):
- mutations: hisG428, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 100
- Details on mammalian cell type (if applicable):
- mutations: hisG46, uvrB, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- mutations: hisD3052, uvrB, pKM 101, rfa
- Species / strain / cell type:
- S. typhimurium, other: 97a
- Details on mammalian cell type (if applicable):
- mutations: hisD6610, uvrB, pKM 101, rfa
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 1st experiment: 5031 / 1509 / 503 / 151 / 50 µg/plate
2nd experiment: 61 / 18 / 6 / 1.8 / 0.6 µg/plate
3rd experiment: 51 / 26 / 13 / 6.4 / 3.2 / 1.6 / 0.8 µg/plate - Vehicle / solvent:
- ethanol
Controlsopen allclose all
- Negative solvent / vehicle controls:
- yes
- Remarks:
- H2O, DMSO, ethanol
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- concentration per plate: 20 µg; solvent: DMSO; strains: TA 98; with S9
- Positive controls:
- yes
- Positive control substance:
- other: 2-Amino-anthracene
- Remarks:
- concentration per plate: 1 µg; solvent: DMSO; strains: TA 97a, TA 100, TA 102, TA 1535; with S9
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- concentration per plate: 1 µg; solvent: H2O; strains: TA 100, TA 1535; without S9
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylene diamine
- Remarks:
- concentration per plate: 20 µg; solvent: DMSO; strains: TA 97a, TA 98, TA 102; without S9
- Details on test system and experimental conditions:
- First Experiment
Concentrations tested: 5031 / 1509 / 503 / 151 / 50 µg/plate
Incubation time: 48 hours
Incubation temperature: 37 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method
Second Experiment
Concentrations tested 61 / 18 / 6 / 1.8 / 0.6 µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method plate incorporation method
Third Experiment
Concentrations tested 51 / 26 / 13 / 6.4 / 3.2 / 1.6 / 0.8 µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method pre-incubation method
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium, other: 97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 151 µg/plate and higher
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 151 µg/plate and higher
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 151 µg/plate and higher
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 151 µg/plate and higher
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations of 151 µg/plate and higher
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was dissolved in ethanol. A stock solution containing 50 g/L was prepared.
Signs of toxicity towards the tested strains were observed in the first experiment in the four highest concentrations (5031, 1509, 503 and 151 µg/plate): No growth was found on these plates.
In the second and third experiment no signs of toxicity towards the tested strains were observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.
Mutagenicity
No significant increase in the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase was found over the tested concentration range.
As only one concentration in the first experiment could be evaluated due to the test item’s toxicity, the test was repeated using a lower concentration range.
The test item was considered as not mutagenic under the test conditions.
A third experiment was performed using the pre-incubation method which verifies this result.
Table 1: Mean Revertants in the First Experiment (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
H2O |
Mean |
115 |
117 |
13 |
14 |
112 |
118 |
155 |
134 |
15 |
15 |
sd |
3.6 |
2.2 |
3.3 |
1.9 |
7.8 |
2.2 |
20.1 |
11.0 |
1.7 |
2.5 |
|
DMSO |
Mean |
105 |
112 |
16 |
14 |
106 |
101 |
151 |
139 |
16 |
15 |
sd |
4.8 |
2.2 |
2.5 |
1.0 |
6.8 |
10.2 |
2.6 |
6.4 |
1.0 |
3.3 |
|
Ethanol |
Mean |
105 |
113 |
15 |
15 |
93 |
82 |
141 |
140 |
15 |
16 |
sd |
9.1 |
6.6 |
2.5 |
2.6 |
9.9 |
6.6 |
6.7 |
3.6 |
1.9 |
1.0 |
|
Positive |
Mean |
594 |
590 |
231 |
224 |
467 |
572 |
576 |
574 |
224 |
206 |
sd |
36 |
30 |
4 |
5 |
35 |
23 |
89 |
56 |
7 |
11 |
|
f(I) |
5.66 |
5.27 |
14.44 |
16.00 |
4.17 |
5.66 |
3.81 |
4.13 |
14.93 |
13.73 |
|
5031 µg/pl. |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
1509 µg/pl. |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
503 µg/pl. |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
151 µg/pl. |
Mean |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
sd |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
f(I) |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
0.00 |
|
50 µg/pl. |
Mean |
90 |
103 |
14 |
14 |
77 |
80 |
137 |
137 |
13 |
12 |
sd |
9 |
5 |
3 |
1 |
7 |
8 |
2 |
5 |
3 |
2 |
|
f(I) |
0.86 |
0.91 |
0.93 |
0.93 |
0.83 |
0.98 |
0.97 |
0.98 |
0.87 |
0.75 |
|
f(I) = increase factor
* Different positive controls were used
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
The test item 400112 is considered not mutagenic under the test conditions. - Executive summary:
The mutagenic potential of 400112 has been tested according to OECD guideline 471 and EU method B.13/14 in a GLP certified testing facility.
The test item 400112 is considered not mutagenic under the test conditions.
The test item showed cytotoxicity towards the bacteria in the first experiment in the four highest concentrations (5030.8, 1509.2, 503 and 151 µg/plate). Therefore, a second experiment using the plate incorporation method was performed with lower concentrations (0.6 – 61 µg/plate). No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. Therefore, the test item was considered as not mutagenic under the test conditions.
On the base of the results of this second experiment, a third experiment was performed with the pre-incubation method to verify this result.
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison to the negative controls, as well as showing mutagenic potential of the diagnostic mutagens.
Spontaneous revertants were within the normal range in comparison with the historical data of LAUS GmbH.
For these reasons, the result of the test is considered valid.
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