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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:
14/10/2008 to 11/11/2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No further information required.

Method

Target gene:
Not specified
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: Only good strains were used in the test
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: Only good strains were used in the test
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The dose range for the main tests was determined from the preliminary test using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.
Vehicle / solvent:
-Solvent used: Water for injection
-Justification for choice of solvent: Based on the information from the sponsor that the test substance was soluble in water at 10% and more. Therefore water for injection was used as the solvent for the test substance.

For the positive control 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminojacridine.2HCl, 2-Aminoanthracene and Benzo[a]pyrene were dissolved in DMSO (Wako Pure Chemical Industries, Ltd.; Lot No. PEQ4800). Sodium azide was dissolved in water for injection (Fuso Pharmaceutical Industries, Ltd.; Lot No. 71020C). These solutions were subdivided, stored at -35 to 25 degrees C and thawed just prior to use.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, Sodium azide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylaminojacridine.2HCl, 2-Aminoanthracene and Benzo[a]pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation:
For the tests without metabolic activation, 0.5mL of 0.1M Na-phosphate buffer (pH 7.4) and 0.1mL of each fresh bacterial culture were added to each tube containing 0.1mL of the test solution. For the tests with metabolic activation, 0.5mL of the S9 mix was added to each tube instead of the 0.1M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37°C for 20 minutes while shaking horizontally, and then 2.0mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate.
These operations were conducted under lamps with ultraviolet absorbent filter.

DURATION
- Preincubation period: 20 minute
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main tests which were performed at the same doses.

DETERMINATION OF CYTOTOXICITY
In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 5000µg/plate dose was selected for all strains both with and without metabolic activation.
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive.
Statistics:
The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
n/a
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in background data, indicating that this study was performed correctly.

From these results, mutagenicity of the test substance was judged negative.

The growth inhibition of the test strains by the test substance was not observed, and the precipitate on the plates was not observed either with or without metabolic activation.

In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

From the results described above, it is concluded that the test material is not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.
Executive summary:

In an Mutagenicity Study of INK BH11 C with the Bacterial Reverse Mutation Assay (BML study number: 13217) the test material was judged to be negative for mutagenicity.

The study was performed in accordance with the OECD Guideline for the testing of chemicals 471 (Adopted: 21st July, 1997).

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

During the study period, there was no environmental factor that was thought to have affected the reliability of the study, and there was no deviation from the protocol.