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Toxicological information

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:
weight of evidence
Study period:
From September 08th to 27th, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: ICH S2A and S2B
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

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:
S9 mix
Test concentrations with justification for top dose:
- First experiment (plate incorporation method): 52, 164, 512, 1600, 5000 µg/plate; on the basis of the international guidelines recommendations and relevant findings observed in the preliminary experiment, the test item was evaluated up to the maximum recommended dose level of 5000 µg/plate.
- Second experiment (pre-incubation method): 492, 878, 1568, 2800, 5000 µg/plate; on the basis of the international guidelines recommendations and relevant findings observed in the first experiment, the test item was evaluated up to the maximum recommended dose level of 5000 µg/plate.
Vehicle / solvent:
Water for injection
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: t-Butyl hydroperoxide TA 102, without S9 mix, 100 µg/plate; 2-Aminoanthracene all strains, with S9 mix, 5 µg/plate Except for TA102: 25 µg/plate).
Details on test system and experimental conditions:
TEST SYSTEM
- Origin of strains: B. Ames Laboratory, University of California, Berkeley - USA
- Storage: stock strains were maintained deep frozen in liquid nitrogen
CULTURE MEDIA
- Preparation of the test cultures: Nutrient Broth no. 2
- Molten overlay agar: Agar (0.6 %, w/v), NaCl (0.5 %, w/v), Biotin and L histidine (0.05 mM).
- Vogel-Bonner (VB) medium: Agar (1.5 %, w/v), glucose (2 %, w/v), E medium (2 % v/v).
- E medium: MgSO4 ∙ 7H2O (1 %, w/v), C6H8O7 ∙ H2O (10 %, w/v), K2HPO4 anhydrous (50 %, w/v), NaNH4 HPO4 ∙ 4H2O (17.5 %, w/v).

PRE-TREATMENT PROCEDURES
- Preparation of S9 mix: Commercially available S9 fraction kept below -70 °C. Each batch was checked by the manufacturer for sterility, protein content and enzymatic activities. This induced S9 fraction was obtained from rat liver. The mixture of metabolic activation S9 Mix was prepared immediately prior to the test and kept on ice during the test. This solution was used at the volume of 0.5 mL per plate containing the test or control items. For the plates tested without metabolic activation, phosphate buffer was used instead of S9 Mix, at the same volume.
- Test item preparation: A stock solution was prepared in the vehicle at 50 mg/mL giving a dark blue solution. The other dosing solutions were obtained by serial dilution from this stock solution. While in use, all the preparations were maintained at room temperature and protected from light in brown glass flasks.
- Test culture preparation: for each experiment, the test strain cultures were prepared in nutrient broth from frozen stock(s) and incubated at 37 ± 2 °C in a gyratory incubator to allow the cultures to grow up to the late exponential or early stationary phase of growth (approximately 10E8-10E9 cells per mL). The optical density of each culture was used to check the cell density.
- Identification of the culture: the culture tubes were labelled with the strain, the code of the frozen stock(s) used and the date of the culture initiation.

EXPERIMENTAL DESIGN:
Two independent experiments were conducted to evaluate the mutagenicity of the test item, using the plate incorporation method and the pre-incubation method, both with and without metabolic activation.

TREATMENT:
- Plate incorporation method: After treatment each tube of molten overlay agar contained: 2 mL of molten agar; 100 μL of bacterial culture; 100 μL of the test/control item solution; 500 μL of sterile buffer (without metabolic activation) or 500 μL of S9 Mix (with metabolic activation). This mixture was poured immediately onto VB medium and allowed to solidify at room temperature. The Petri dishes were incubated at 37 ± 2 °C for at least 2 days to allow growth of the colonies.
- Pre-incubation method: Each incubation tube received in the order indicated below: 500 μL of sterile buffer (without metabolic activation) or 500 μL of S9 Mix (with metabolic activation); 100 μL of bacterial culture; 100 μL of the test/control item solution. This mixture was incubated for approximately 20-30 minutes at 37 ± 2 °C under stirring. After the incubation period, 2 mL of molten agar was added to the incubation tubes, then poured onto VB medium and allowed to solidify at room temperature. The Petri dishes were incubated at 37 ± 2 °C for at least 2 days to allow growth of the colonies.

ELECTRONIC SYSTEM FOR DATA ACQUISITION
- PROVANTIS v.8
- SORCERER v.2.2
- AMES STUDY MANAGER v.1.21
Evaluation criteria:
Biological relevance of the results should be considered first. Statistical methods (Dunnett’s test at p < 0.05 and Correlation Coefficient) are used only as an aid in evaluating the test results and are not the only criterion for the determination of a positive/equivocal response. The dose level relationship should be also considered. The following thresholds, generally accepted for significant factor of increase in the number of revertants, may be also used as an aid in the discussion of the results:
- Strains TA98, TA100 and TA102: 2-fold increase over the concurrent negative controls (vehicle and/or untreated).
- Strains TA1535 and TA1537: 3-fold increase over the concurrent negative controls (vehicle and/or untreated).
Statistics:
Statistical methods (Dunnett’s test at p < 0.05 and Correlation Coefficient) are used only as an aid in evaluating the test results and are not the only criterion for the determination of a positive/equivocal response.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
biologically significant increases in the number of revertants
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Plate incorporation method: In the strain TA98 in the presence of metabolic activation, increases were noted at the two highest tested dose levels of 1600 and 5000 μg/plate. The increase observed at the highest dose level of 5000 μg/plate was above the threshold of 2-fold increase accepted as being biologically relevant in this strain. In addition, these increases were dose related. Therefore this finding was considered as being representative of a biological significant increase in the number of revertants.
- Pre-incubation method: In the strain TA98 in the presence of metabolic activation, increases were noted at the three highest tested dose levels of 1568, 2800 and 5000 μg/plate. All the increases were above the threshold of 2-fold increase accepted as being biologically relevant in this strain. In addition, they were dose related. Therefore this finding was considered as being representative of a biological significant increase in the number of revertants.
Remarks on result:
other: Plate incorporation method

Applicant's summary and conclusion

Conclusions:
Based on the results of these experiments, it is concluded that the test substance induced biologically significant increases in the number of revertants in the strain TA98 in the presence of metabolic activation at 5000 µg/plate.
Executive summary:

In the bacterial reverse mutation test (Ames test), the test item Patent Blue V was tested as a dark blue solution in water for injection up to the maximum recommended dose level of 5000 μg/plate.

No cytotoxicity and non observable precipitate were noted in any strain. Under the experimental conditions used, when tested using both the plate incorporation and the pre-incubation methods, the test item Patent Blue V induced biologically significant increases in the number of revertants in the strain TA98 in the presence of metabolic activation.