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Toxicological information

Genetic toxicity: in vitro

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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:
2006-12-06 to 2007-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 471 and GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
481-170-7
EC Name:
-
Cas Number:
502453-61-4
Molecular formula:
Hill formula: C23H30BrN3O2 CAS formula: C23H30N3O2.Br
IUPAC Name:
dimethyl(3-{[4-(methylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl]amino}propyl)propylazanium bromide
Constituent 2
Reference substance name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
IUPAC Name:
Dimethyl-(3-(4-methylamino-9,10-dioxo-9,10-dihydro-anthracen -1-ylamino)propyl)propylammonium bromide
Details on test material:
- Name of test material (as cited in study report): B119 HC Blue 16
- Molecular formula (if other than submission substance): C232H30N3O2.Br
- Molecular weight (if other than submission substance): 460.42
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: Active
- Physical state: Dark blue powder

Method

Target gene:
Histidine locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see below
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: see below
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment/experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 ug/plate
Experiment 2 and 2A: 10, 33, 100, 333, 1000, 2500, and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: On the day of the experiment, the test substance was suspended in deionised water. The solvent was chosen because of its solubility properties.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
untreated
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: Used for strains TA1535 and TA100 (10 ug/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Remarks:
untreated
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine- used for strains TA1537 (10 ug/plate) and TA98 (50 ug/plate)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
untreated
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation Migrated to IUCLID6: Used for strain TA102 (3 uL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Remarks:
untreated
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene- used for strains TA1535, TA1537, TA98, TA100 (2.5 ug/plate) and TA102 (10 ug/plate
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1-in agar (plate incorporation); experiment 2 and 2A-preincubation

DURATION
- Preincubation period: 60 minutes (experiment 2 and 2A only)
- Exposure duration: 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): L-histidine
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: N/A


DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test substance was measured by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.


OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other: N/A


OTHER: N/A
Evaluation criteria:
The test substance was considered a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged at biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to OECD guideline 471, a statistical analysis of the data was not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA100, TA98, TA1535 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: N/A
- Effects of osmolality: N/A
- Evaporation from medium: N/A
- Water solubility: >20 weight % in water (pH 6.1)
- Precipitation: Precipitation of the test substance was observed in the test tubes from 1000 up to 5000 ug/plate in experiment 1 , and at 2500 and 5000 ug/plate in experiment 2 and 2A. No precipitation was observed on the agar plates.
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test substance a pre-experiment was performed with strains TA1535, TA1537, TA98, TA100 and TA102. Eight concentrations (3-5000 ug/plate) were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the experiment were the same as described for experiment 1 (plate incorporation test) listed above. Toxicity of the test substance was measured by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. This pre-experiment was reported as main experiment 1 (see below for results), since the following criteria was met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used. Based on the toxic effects observed in this experiment, the concentrations used for the second experiment were chosen.


COMPARISON WITH HISTORICAL CONTROL DATA: All positive, vehicle and untreated controls were within historical data ranges.


ADDITIONAL INFORMATION ON CYTOTOXICITY: See below
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 5.0), were observed at the following concentrations (ug/plate):

Strain Experiment 1 Experiment 2
without activation with activation without activation with activation
TA1535 / / 5000 5000
TA1537 / / / /
TA98 5000 2500, 5000 5000 /
TA100 2500, 5000 5000 1000-5000 2500, 5000
TA102 2500, 5000 1000-5000 5000 1000-5000

/no toxic effects observed

In experiment 2, substantial and dose dependent increase in revertant colony numbers were observed following treatment with the test substance in TA1537 in the absence and presence of metabolic activation. The threshold of three times the number of the corresponding solvent control was exceeded at all concentrations equal to and greater than 33 ug/plate without activation and at at all concentrations equal to and greater than 333 ug/plate with activation. To verify these results this part of the second experiment was repeated under identical conditions using strain TA1537. Again, as in the second experiment substantial and dose dependent increases in revertant colony numbers were observed. The required threshold was already reached at 10 ug/plate and well exceeded at 33 ug/plate and above without activation. In the presence of metabolic activation the increase in revertant colony numbers started at100 ug/plate and exceeded the threshold of three times the number of the corresponding solvent control at 333 ug/plate and above. A reduction of the revertant colonies was observed at higher concentrations due to overlapping toxic effects. The remaining strains did not show any increase of the number of the revertant colonies. The repeat experiment was reported as experiment 2A. The positive controls showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

This study was performed to investigate the potential of B119 HC Blue 16 to induce gene mutations according to the plate incorporation (experiment 1) test and to the pre-incubation test (experiment 2 and 2A) using the Salmonella typhimurium strains TA1535, TA1537,TA98, TA100 and TA102. During the described mutagenicity test and under the experimental conditions reported, the test substance induced gene mutations by frameshifts in the genome of the strain TA1537 in the absence and presence of metabolic activation. Therefore, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of B119 HC Blue 16 to induce gene mutations according to the plate incorporation (experiment 1) test and to the pre-incubation test (experiment 2 and 2A) using the Salmonella typhimurium strains TA1535, TA1537,TA98, TA100 and TA102.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test substacne was tested at the following concentrations:

Pre-experiment/experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 ug/plate

Experiment 2 and 2A: 10, 33, 100, 333, 1000, 2500, and 5000 ug/plate

 The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in nearly all strains used.

In experiment 2, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance in strain TA1537 in the absence and presence of metabolic activation. The threshold of three times the number of the corresponding solvent control was exceeded at 33 ug/plate and above without activation and at 333 ug/plate and above with activation. To verify these results this part of the second experiment was repeated under identical conditions using strain TA1537. Again, as in the second experiment substantial and dose dependent increases in revertant colony numbers were observed. The required threshold was already reached at 10 ug/plate and well exceeded at 33 ug/plate and above without activation. In the presence of metabolic activation the increase in revertant colony numbers started at 100 ug/plate and exceeded the threshold of three times the number of the corresponding solvent control at 333 ug/plate. The repeat experiment was reported as experiment 2A. The remaining strains did not show any increase in the number of the revertant colonies.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance induced gene mutations by frameshifts in the genome of the strain TA1537 in the absence and presence of metabolic activation. Therefore, the test substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

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