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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 02, 2005 - June 09, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The test item C 010 was assessed for the potential to induce genmutation in the salmonella typhimurium reverse mutation test.
First test: plate incorporation method
Second test: pre-incubation method
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Basic yellow 57
- Molecular formula (if other than submission substance): C19-H22-N5-O.Cl
- Molecular weight (if other than submission substance): 371.87 g/mol
- Smiles notation (if other than submission substance): N1(C([C@@H](\N=N\c2cc([N+](C)(C)C)ccc2)C(=N1)C)=O)c1ccccc1.[ClH-]
- InChl (if other than submission substance): 1S/C19H22N5O.ClH/c1-14-18(19(25)23(22-14)16-10-6-5-7-11-16)21-20-15-9-8-12-17(13-15)24(2,3)4;/h5-13,18H,1-4H3;1H/q+1;/p-1/b21-20+;
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid orange powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride)
- Lot/batch No.of test material: 00656555101
- Expiration date of the lot/batch: 20.08.2024
- Purity: 100 area%

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, avoid direct light.
- Stability under test conditions: No data
- Solubility: Ethanol 10 g/l
DMSO 125mg/ml (as determined in a pre-test)
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Six stock solutions of C 010 were prepared freshly in DMSO on the day of the experiment.
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

OTHER SPECIFICS: No data

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver: 2 mg/mI
Test concentrations with justification for top dose:
The test item C 010 was applied at concentrations of 1, 0.316, 0.1, 0.0316, 0.01 and 0.00316 mg/plate (both experiments) with and without metabolic aetivation (S9 rat liver: 2 mg/mI).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
According to information obtained in pre-tests, the test item DMSO was found to be suitable for dissolving the test item.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For strain : S. typh. T A98 without (-S9) metabolic activation
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For strain : S. typh. T A100 and TA1535 without (-S9) metabolic activation
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
For strain : S. typh. T A102 without (-S9) metabolic activation
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
Remarks:
For strain : S. typh. T A1537 without (-S9) metabolic activation
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene
Remarks:
For strain : S. typh. TA98 and TA100 with (+S9) metabolic activation.
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthrachinon
Remarks:
For strain : S. typh. TA102 with (+S9) metabolic activation.
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
For strain : S. typh. TA1535 with (+S9) metabolic activation.
Untreated negative controls:
yes
Remarks:
10 µl of H20
Negative solvent / vehicle controls:
yes
Remarks:
10 µl of solvent (DMSO) have been used.
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene
Remarks:
For strain : S. typh. TA1537 with (+S9) metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
First test (plate incorporation method):
a)Plating out: After mixing the test item mix with 2 ml of HBT topagar (50 ± 5°C), the whole volume was poured onto a minimal agar plate immediately.
b)Curing: minimum 10min. at room temperature
c)Incubation of plates: 69.5 h at 37 ± 2°C
d)Storage until counting: 2-8°C up to 72 hours.

Second test (pre-incubation method):
a) Preincubation period: 60 - 70 minutes in an agitated water bath at 37 ± 0.5 degC;
b)Plating out: After mixing the test item mix with 2 ml of HBT topagar (50 ± 5°C), the complete pre-incubation volume was poured onto a minimal agar plate
c)Curing: minimum 10min. at room temperature
d)Incubation of plates: 71 h at 37±2°C
e)Storage until counting: 2-8°C up to 72 hours.

REAGENTS:
a)Metabolic activating enzyme system:
First test: Rat S9
Second test: Rat S9
In accordance with the OECD Guideline TG 471 cofactor-supplemented post mitochondrial fraction (S9) derived from liver of phenobarbitone/ßnaphtoflavone treated rats was used as the metabolie activating enzyme system.The final protein concentration in the incubation mixtures has been 2 mg/mI.

b)Sterile minimal agar plates: (1 st and 2nd test)

c)Cofactor solution :
Final concentrations in the incubation mixture:
5.5 mM +/- 10 % Glucose-6-phosphat
3.9 mM +/- 10 % NADP-Na

d) HBT –topagar
Concentrations before addition to the test item mix:
6.55 % w/v Bacto-Agar (Difco)
100 mM NaCI
23 mM Sodium phosphate, pH 7.4
41.0 µM L-Histidine
46.5 µM D-Biotin
22.3 µM L-Tryptophane

e) KCP solution:
10 mM Sodium phosphate, pH 7.4
150 mM KCl

NUMBER OF CELLS EVALUATED:
Between 1 x 10E8 and 1 x 10E9 cells of the respective test systems have been applied per plate.
The number of cells per plate was checked during concurrent plating experiments performed specifically for the determination of the number of viable cells.

For each test system the number of viable cells was determined with and without ampicillin in parallel.

NUMBER OF PLATINGS PER DOSE LEVEL
For each dose level of test item, positive control and negative control triplicates have been tested.

- OTHER:
Signs of precipitation: A possible formation of precipitates was determined by visual control (unaided eye) of plates tested with the highest test item concentration. In case of precipitation further plates with lower test item concentrations were checked until there were no more signs of precipitation.

DETERMINATION OF CYTOTOXICITY: A possible cytotoxic effect (decrease in number of so called ,microcolonies') was determined by microscopic analysis (l 00 - 200 fold magnification) of the plates tested with the highest test item concentration. In case of cytotoxic effects further plates with lower test item concentrations were checked until there were no more signs of cytotoxicity.

Signs of contamination: The absence of contaminations with fungi or other bacteria was checked by individual visual control (unaided eye) of all plates.

Determination of number of revertant colonies: The number of revertant colonies has been counted for each plate either by
• using the colony counter or,
• manually, if the semiautomatic counting method was not applicable.
Evaluation criteria:
Acceptance criteria
The number of revertants of negative, solvent and positive control should be in the range of historical data. The number of viable cells per plate should be in the range of 1 x 10E8 and 1 x 10E9 cells.

Evaluation criteria
Cytotoxicity
Abbreviation "T": A strong reduction of the normal bacterial background colony number (,microcolonies '), determined by microscopic analysis at 100 - 200 fold magnification, resulting in an increased diameter ofthe remaining colonies. The respective plates were not evaluated due to the risk of false positive or negative results.
Abbreviation "(T)": A moderate reduction of the normal bacterial background colony number (,microcolonies'), determined by microscopic analysis at 100 - 200 fold magnification, not resulting in an increased diameter of the remaining colonies. An evaluation of the respective plates was perfonned.

Mutagenicity
A mutagenic potential of the test item was assumed if one quotient of the mean value of the respective revertant colonies over the mean value of the respective solvent control was 2.0 for strains TA98, TA100, and TA102 and 3.0 for strains TA1535 and TA1537 or higher. A dose effect relationship could underline this conclusion.
Otherwise no mutagenic potential of a test item was assumed.
Statistics:
Data analysis was performed using MSEXCEL spreadsheets.

Data analysis included the calculation of the mean of the revertant colonies per plates per dose level, the respective standard deviation and the quotient of the mean value of the respective revertant colonies over the mean value of the respective solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
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:
not specified
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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:First and second experiment: The microscopical control of the plates after the incubation phase showed signs of precipitation at concentrations of 0.316 and 1 mg/plate for incubations with and without metabolic activation in both tests.

RANGE-FINDING/SCREENING STUDIES:
As result of a solubility test, DMSO was found to be suitable for dissolving the test item.
In a range finding pre-test, test item showed signs of precipitation at concentrations of 0.5 mg/plate and above (with and without S9). Cytotoxic effects were observed at 2 mg/plate for strain TA98 and 0.5 mg/plate for strain TA100 in incubations without metabolic activation.
Due to these facts the maximum test item concentration set in this test was 1 mg/plate with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See table below
- Negative (solvent/vehicle) historical control data: See table below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Signs of cytotoxicity
First experiment:
Cytotoxicity was observed on plates from incubations without S9 treatment. Depending on the bacteria strain, the concentration at which cytotoxicity occurred varied from 0.1 mg/plate to 1 mg/plate. On plates from incubations with S9 treatment no cytotoxie effects were visible.

Second experiment:
Plates from incubations without S9 treatment showed cytotoxicity at concentrations of 1 mg/plate, 0.316 mg/plate and, depending on the bacteria strain, at 0.1 mg/plate. On plates from incubations with S9 treatment for all strains cytotoxic effects were visible at a concentration of 1 mg/plate and in case of TA1535 also at 0.316 mg/plate.

Determination of the number (titer) of viable cells
First experiment: The number of viable cells (titer) obtained from concurrent incubations (1.7 to 3.1 x 10E8 cells per plate) was in accordance with the OECD test guidelines TG 471.
Second experiment: The number ofviab1e cells (titer) obtained from concurrent incubations (2.2 to 3.1 x 10E8 cells per plate) was in accordance with the OECD test guidelines TG 471.

Number of revertant colonies
The respective negative and solvent controls produced a low number of revertants and the spontaneous revertant colony counts were generally inside the historical range. Slight deviations were considered negligible.

In the first experiment the strain-specific positive controls for TA98, TAlOO, TA102 and TA1535 produced the expected increase in revertant colonies, with and without metabolic activation, respectively. The positive control for TA1537 (without S9) 9-Aminoacridine failed to induce an increase of revertant colonies. This failure did not query the functionality of the test system to indicate genmutations, as 2-Aminofluorene induced an increase of revertants of TA1537 after S9 treatment. Therefore, the failure of the 9-Aminoaeridine can be considered as negligible.

In the second experiment all strain-specific positive controls were shown to produce the expected increase in revertant colonies, with and without metabolic activation, respectively.In both experiments of the main test no significant increase in the number of revertant colonies was observed for the test item C 010, neither with nor without metabolic activation.

Dose-effect relationship
Comparing the number of revertant colonies of increasing test item concentrations with those of the corresponding solvent controls revealed no evidences far a dose dependent effect / increase in the number of revertant colonies.
Also the respective quotients (mean number of revertant colonies per test item concentration over the mean number of revertant colonies seen for the solvent control) show ed no evidence for a dose dependent inerease in the number of revertant colonies.
This allows the conclusion that no dose-effect relationship exists.

Any other information on results incl. tables

Historical data for the test systems

The historical data are based on results which were obtained by using the pre-incubation method.

The pre-incubation method is more sensitive than the plate incorporation method. Therefore slightly lower mean number of revertants could be expecte d for the plate incorporation method.  

 

Historical data of S. typh.TA98: Revertant colonies

 

 

mean number

SD*

range

Solvent control (- S9)

19

7

11 - 40

Solvent control (+ S9)

29

6

16 - 45

Pos. control (- S9),

2-Nitrot1uorene, 0.12 µg/plate

279

83

155-432

Pos. control (+ S9),

2-Aminofluorene, 10 µg/plate

2968

1429

906-8233

*SD standard deviation

 

Historical data of S. typh. TA100: Revertant colonies

 

 

mean number

SD*

range

Solvent control (- S9)

92

13

71- 115

Solvent control (+ S9)

105

9

88 - 123

Pos. control (- S9),

Sodium azide, 1.0 µg/plate

347

79

265 583

Pos. control (+ S9),

2-Aminofluorene, 10 µg/plate

1056

690

434 - 2633

*SD standard deviation

 

Historical data of S. typh. TA102: Revertant colonies

 

 

mean number

SD*

range

Solvent control (- S9)

185

23

147-220

Solvent control (+ S9)

233

34

179 - 283

Pos. control (- S9),

Mitomycin C, 0.1 µg/plate

791

244

347 - 1133

Pos. control (+ S9),

18-Dihydroxyanthraquinone

697

172

402 - 967

*SD = standard deviation

 

Historical data of S. typh. TA1535: Revertant colonies

 

 

mean number

SD*

range

Solvent control (- S9)

13

10

7 - 56

Solvent control (+ S9)

15

10

9 - 56

Pos. control (- S9),

Sodium azide, 1 µg/plate

278

51

195-417

Pos. control (+ S9),

2-Aminoanthracene, 10 µg/plate

297

116

52 - 477

*SD = standard deviation

 

Historical data of S. typh. TA1537: Revertant colonies

 

 

mean number

SD*

range

Solvent control (- S9)

9

3

6 - 13

Solvent control (+ S9)

15

3

9 - 24

Pos. control (- S9),

9-Arninoacridine, 2.5 µg/plate

16806

6541

7200-29000

Pos. control (+ S9),

2-Aminofluorene, 50 µg/plate

70

12

51 - 93

*SD = standard deviation

 

Applicant's summary and conclusion

Conclusions:
According to the OECD Guideline 471 no biological relevant mutagenic potential of the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl) azo]- N,N,N-trimethylanilinium chloride) was determined under the conditions of this test.
Executive summary:

The test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was assessed for the potential to induce genmutation in the salmonella typhimurium reverse mutation test.

The test reported here was performed according to the OECD Guideline TG 471 for the bacterial reverse mutation test. Following bacterial strains have been used in this study: S. typhimurium TA98, TA100, TA102, TA1535 and TA1537. In the first experiment of the main test exposure of the test system has been performed using the plate incorporation method. In the second experiment the pre-incubation method was adopted.

The test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) was applied at concentrations of 1, 0.316, 0.1, 0.0316, 0.01 and 0.00316 mg/plate (both experiments) with and without metabolic activation (S9 rat liver: 2 mg/mI).

Depending on the bacterial strain, precipitates and/or cytotoxicity was observed in higher concentrations. Therefore, according to the OECD Guideline TG 471 the tested concentration range was justified.

The respective negative controls produced a low number of revertants.

All but one strain-specific positive controls were shown to produce the expected increase in revertant colonies, with or without metabolic activation, respectively. The failure of 9- Aminoacridine (without S9) in the 1st experiment was negligible, as the functionality of the strain TA1537 to indicate genmutations was confirmed by 2-Aminofluorene (with S9) in the same experiment.

In the second experiment all strain-specific positive controls were shown to produce the expected increase in revertant colonies, with or without metabolic activation, respectively.

Both experiments showed no significant inerease in the number of revertant colonies independent of metabolic activation - in any bacteria strain. Furthermore no dose-effect relationship could be determined.

Taken together, no mutagenic potential of the test item C 010 (3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-N,N,N-trimethylanilinium chloride) w as determined under the conditions of this test.