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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:
The study was conducted between 15 December 2006 and 12 January 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: "Standards for Mutagenicity Tests using Microorganisms" (Notification No. 77, September 1, 1988 & Notification No. 67, June 2, 1997, Ministry of Labour, Japan)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Magenta T-43
IUPAC Name:
Magenta T-43
Test material form:
solid: particulate/powder
Details on test material:
Name: [Magenta T-43]
Appearance at room temperature: Dark red powder
Condition of storage: Room temperature
BML-CODE: BML-9923
Lot No. H186-5
Purity: 90.1 wt%
CAS No. 909094-45-7
Stability: Not reactive to light or to air

Method

Target gene:
Histidine for Salmonella and Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital and 5,6-benzoflavone induced rat liver)
Test concentrations with justification for top dose:
Preliminary test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.
Main Test:
Concentration range in the main test (with and without metabolic activation): 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water for injection was used as the solvent for preparation.
Controls
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of bacterial strains
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3 -(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine (ICR-191); 2-Aminoanthracene (2AA)
Remarks:
AF-2, NaN3, ICR-191 used without S9 mix. B[a]P and 2AA used with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation

DURATION
- Preincubation period for bacterial strains: 20 minutes
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Criteria for judgement:
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. 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results and discussion:
Preliminary Test:
growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. Precipitate of the test substance was not observed either with or without metabolic activation.

Main Test:
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 ± 3 SD) 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:
negative with metabolic activation
negative without metabolic activation

From the results, it is concluded that Magenta T-43 was not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.
Executive summary:

The mutagenicity potential of Magenta T-43 was assessed with Salmonella typhimurium TAl00, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA.

In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

From the above, it is judged that Magenta T-43 has no mutagenicity forward to bacteria under the described study conditions.