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EC number: 258-605-2 | CAS number: 53523-90-3
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
- EC Number:
- 258-605-2
- EC Name:
- Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
- Cas Number:
- 53523-90-3
- Molecular formula:
- C30H20Li4N4O12S2
- IUPAC Name:
- tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
- Reference substance name:
- Unknown impurities, none of which are present at ≥1%
- Molecular formula:
- Unknown impurities, none of which are present at ≥1%
- IUPAC Name:
- Unknown impurities, none of which are present at ≥1%
- Test material form:
- liquid
Constituent 1
impurity 1
- Specific details on test material used for the study:
- Identification: Bayscript Gelbkomponente
CAS-No.: 53523-90-3
Chemical Name: Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]
Empirical Formula: C30H20N4O12S2Li4
Molecular Mass: 720.4 g/mol
Appearance: Red-brown powder
Storage Conditions: At room temperature, moisture protected
Stability in Solvent: Stable for 4 and 24 hours at room temperature in the light in deion. Water (based on analytical study number PV29HC)
Expiry Date: 27 February 2017
Method
- Target gene:
- histidine locus
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system
- Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
with and without metabolic activation.
Since no toxic effects were observed, 5000 μg/plate were chosen as maximal concentration. - Vehicle / solvent:
- Deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/plate, TA 1535, TA 100
- Positive control substance:
- sodium azide
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionized water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 10 µg/plate in strain TA 98. 50 µg/plater in strain TA 1537
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 2.0 µL/plate TA 102
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 2.5 µg/plate for all strains except TA 102 (10.0 µg/plate)
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- Precultures
The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. This nutrient medium contains per litre:
8 g Nutrient Broth 5 g NaCl
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
Experimental Performance
For each strain and dose level, including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
Experiment I (Plate Incorporation)
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
Experiment II (Pre-Incubation)
In the pre-incubation assay 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.
Data Recording
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager. - Evaluation criteria:
- Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The test item Bayscript Gelbkomponente was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.
Any other information on results incl. tables
Summary of Experiment I
Study Name: 1747203 |
Study Code: Envigo 1747203 |
Experiment: 1747203 VV Plate |
Date Plated: 11/03/2016 |
Assay Conditions: |
Date Counted: 14/03/2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|
|
|
|
|
|
|
|
|
Without Activation |
Deionised water |
|
|
13 ± 3 |
7 ± 2 |
25 ± 5 |
160 ± 24 |
520 ± 8 |
Untreated |
|
|
12 ± 1 |
10 ± 5 |
23 ± 1 |
160 ± 7 |
506 ± 6 |
|
Bayscript |
3 µg |
|
14 ± 2 |
9 ± 1 |
26 ± 6 |
170 ± 27 |
516 ± 12 |
|
Gelbkomponente |
10 µg |
|
15 ± 5 |
7 ± 2 |
20 ± 7 |
144 ± 19 |
539 ± 23 |
|
|
33 µg |
|
13 ± 5 |
8 ± 1 |
21 ± 1 |
151 ± 16 |
511 ± 26 |
|
|
100 µg |
|
14 ± 2 |
7 ± 2 |
24 ± 10 |
149 ± 17 |
495 ± 78 |
|
|
333 µg |
|
13 ± 3D |
11 ± 2D |
24 ± 10D |
168 ± 8D |
519 ± 37D |
|
|
1000 µg |
|
12 ± 3D |
8 ± 1D |
25 ± 3D |
142 ± 10D |
539 ± 29D |
|
|
2500 µg |
|
13 ± 3D |
7 ± 1D |
20 ± 5D |
152 ± 12D |
530 ± 26D |
|
|
5000 µg |
|
10 ± 5D |
7 ± 2D |
21 ± 6D |
143 ± 16D |
503 ± 39D |
|
NaN3 |
10 µg |
|
1223 ± 29 |
|
|
2143 ± 132 |
|
|
4-NOPD |
10 µg |
|
|
|
390 ± 31 |
|
|
|
4-NOPD |
50 µg |
|
|
66 ± 12 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
|
5562 ± 1178 |
|
|
|
|
|
|
|
|
|
|
With Activation |
Deionised water |
|
|
10 ± 1 |
11 ± 4 |
34 ± 2 |
153 ± 35 |
624 ± 42 |
Untreated |
|
|
11 ± 4 |
16 ± 7 |
36 ± 7 |
119 ± 8 |
657 ± 12 |
|
Bayscript |
3 µg |
|
10 ± 3 |
10 ± 2 |
27 ± 2 |
131 ± 8 |
762 ± 28 |
|
Gelbkomponente |
10 µg |
|
9 ± 3 |
11 ± 4 |
27 ± 10 |
134 ± 26 |
754 ± 55 |
|
|
33 µg |
|
8 ± 1 |
7 ± 1 |
26 ± 6 |
142 ± 5 |
713 ± 63 |
|
|
100 µg |
|
13 ± 6 |
10 ± 4 |
32 ± 3 |
128 ± 5 |
733 ± 130 |
|
|
333 µg |
|
14 ± 2D |
10 ± 2D |
34 ± 4D |
132 ± 13D |
734 ± 15D |
|
|
1000 µg |
|
11 ± 4D |
6 ± 1D |
26 ± 7D |
122 ± 15D |
692 ± 48D |
|
|
2500 µg |
|
11 ± 5D |
10 ± 3D |
26 ± 4D |
129 ± 8D |
675 ± 82D |
|
|
5000 µg |
|
14 ± 2D |
10 ± 5D |
37 ± 1D |
143 ± 7D |
666 ± 103D |
|
2-AA |
2.5 µg |
|
426 ± 17 |
112 ± 8 |
4516 ± 613 |
2495 ± 184 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
1715 ± 215 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA MMS 4-NOPD |
sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine |
D |
Densely coloured plate |
Summary of Experiment II
Study Name: 1747203 |
Study Code: Envigo 1747203 |
Experiment: 1747203 HV2 Pre |
Date Plated: 24/03/2016 |
Assay Conditions: |
Date Counted: 30/03/2016 |
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
|
|
|
|
|
|
|
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|
|
|
|
|
|
|
|
|
Without Activation |
Deionised water |
|
|
9 ± 2 |
8 ± 1 |
21 ± 2 |
192 ± 7 |
451 ± 31 |
Untreated |
|
|
12 ± 2 |
8 ± 2 |
24 ± 3 |
196 ± 14 |
471 ± 30 |
|
Bayscript |
33 µg |
|
9 ± 3 |
8 ± 2 |
22 ± 6 |
215 ± 22 |
453 ± 25 |
|
Gelbkomponente |
100 µg |
|
8 ± 3 |
8 ± 1 |
23 ± 4 |
177 ± 17 |
443 ± 11 |
|
|
333 µg |
|
10 ± 3D |
8 ± 2D |
24 ± 3D |
194 ± 9D |
451 ± 70D |
|
|
1000 µg |
|
8 ± 2D |
7 ± 2D |
23 ± 3D |
201 ± 4D |
453 ± 33D |
|
|
2500 µg |
|
9 ± 2D |
7 ± 1D |
19 ± 4D |
197 ± 12D |
395 ± 36D |
|
|
5000 µg |
|
10 ± 2D |
9 ± 4D |
25 ± 1D |
207 ± 21D |
351 ± 62D |
|
NaN3 |
10 µg |
|
1057 ± 47 |
|
|
1977 ± 31 |
|
|
4-NOPD |
10 µg |
|
|
|
408 ± 30 |
|
|
|
4-NOPD |
50 µg |
|
|
109 ± 15 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
|
5449 ± 739 |
|
|
|
|
|
|
|
|
|
|
With Activation |
Deionised water |
|
|
11 ± 2 |
10 ± 5 |
29 ± 2 |
199 ± 27 |
603 ± 18 |
Untreated |
|
|
7 ± 1 |
11 ± 4 |
38 ± 2 |
180 ± 18 |
643 ± 47 |
|
Bayscript |
33 µg |
|
9 ± 3 |
10 ± 5 |
30 ± 1 |
185 ± 23 |
648 ± 49 |
|
Gelbkomponente |
100 µg |
|
10 ± 3 |
9 ± 2 |
35 ± 8 |
198 ± 10 |
585 ± 5 |
|
|
333 µg |
|
12 ± 4D |
11 ± 1D |
34 ± 6D |
202 ± 15D |
563 ± 118D |
|
|
1000 µg |
|
11 ± 4D |
11 ± 2D |
25 ± 7D |
202 ± 15D |
583 ± 27D |
|
|
2500 µg |
|
14 ± 2D |
10 ± 2D |
26 ± 3D |
211 ± 11D |
551 ± 31D |
|
|
5000 µg |
|
15 ± 2D |
9 ± 3D |
28 ± 3D |
181 ± 8D |
449 ± 7D |
|
2-AA |
2.5 µg |
|
417 ± 45 |
106 ± 13 |
4377 ± 111 |
2370 ± 210 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
1872 ± 278 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA MMS 4-NOPD |
sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine |
D |
Densely coloured plate |
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Executive summary:
This study was performed to investigate the potential of Bayscript Gelbkomponente to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item, dissolved in deionized water, was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed an increasing dense color from 333 to 5000 μg/plate, which had no impact on evaluation of the plates.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Bayscript Gelbkomponente at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Conclusion
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Bayscript Gelbkomponente is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
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