Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 July 2016 to 25 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
Reaction mass of trisodium 4-[[ 4-chloro-6-[( 4-sulphonatophenyl)amino ]-1,3 ,5-triazin-2-yl ]amino ]-2-[[ 1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxolH-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino ]-1,3,5-triazin-2-yl]amino ]-2-[[ 1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1 H-pyrazol-4-yl]azo] benzenesulphonate
IUPAC Name:
Reaction mass of trisodium 4-[[ 4-chloro-6-[( 4-sulphonatophenyl)amino ]-1,3 ,5-triazin-2-yl ]amino ]-2-[[ 1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxolH-pyrazol-4-yl]azo]benzenesulphonate and trisodium 4-[[4-chloro-6-[(3-sulphonatophenyl)amino ]-1,3,5-triazin-2-yl]amino ]-2-[[ 1-(2,5-dichloro-4-sulphonatophenyl)-4,5-dihydro-3-methyl-5-oxo-1 H-pyrazol-4-yl]azo] benzenesulphonate
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Orange Powder
- Storage conditions: Room temperature
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test solution of the maximum concentration was prepared, which was fully stirred with mixer and dissolved after the test substance was weighed and the solvent was added. The lower doses were prepared by diluting stepwise from the test solution of the maximum concentration. The test was carried out with the weight converted by multiplying measured weight by 0.820 because the purity of the test substance was 82.0 wt %. Preparation of the test solution was carried out just prior to use under lamps with ultraviolet absorbent filter.

The amount of test substance and the volume of solvent used for the preparation of the test solution of the maximum concentration in each test were as follows.
Preliminary test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount of test substance 113.0 mg x 0.820 = 92.660 mg
- The volume of solvent (Water for injection) 1.853 mL

The first main test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount oftest substance 421.5 mg x 0.820 = 345.630 mg
- The volume of solvent (Water for injection) 6.913 mL

The second main test [Prepared maximum concentration of test solution: 50 mg/mL]
- The amount oftest substance 414.7 mg x 0.820 = 340.054 mg
- The volume of solvent (Water for injection) 6.801 mL

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: S. typhimurium TA100 was supplied by The Division of Mutagenesis, National Institute of Hygienic Sciences, Japan (The Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Japan at present).
Other S. typhimurium TA strains were supplied by Dr. Ames, U.C. Berkeley, CA, U.S.A.
E. coli WP2 uvr A was supplied by Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan.
- Methods for maintenance in cell culture if applicable: The bacterial suspension and DMSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots, and then frozen and stored at -85 to -80 °C.
- Charecterisation: The number of viable cell, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.
- Pre-culture procedure: A bacterial suspension of each strain (20 μL of S. typhimurium TA strains, 5 μL of E. coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until starting incubation, and then incubated while shaking (100 rpm) in a water bath at 37 °C for 8 hours. After incubation, the optical density was measured and the number of viable cell was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until starting the test.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cultures were maintained in nutrient broth no. 2 supplied by Oxoid Limited. Top agar was bacto-agar (Difco Laboratories) and minimal glucose agar plates were Tesmedia AN (Oriental Yeast , Co. Ltd.).
- Properly maintained: Yes
Additional strain / cell type characteristics:
other: Base-pair substitution type: Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA; Frame-shift type: Salmonella typhimurium T A98 and TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 μg/plate.
Definitive test: 313, 625, 1250, 2 500 and 5 000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection.
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was soluble at 100 g/L and more in water. Therefore water for injection was used as solvent for preparation.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Treated only with water that was used to prepare the test solution.
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, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino Jacridine · 2HCl and 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation method
- Without metabolic activation: 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution.

- With metabolic activation: 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer.

DURATION
- Preincubation period: The mixture was pre-incubated in a water bath at 37 °C for 20 minutes while shaking horizontally, and then 2.0 mL 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. And 0.1 mL of the positive control solution was carried out equally.
- Exposure duration: All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

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.

- OTHER: For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2.0 mL of top agar were then added to the tube, 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.
As top agar, the 0.5 mM biotin-0.5 mM L-histidine solution and the 0.5 mM L-tryptophan solution were added to the soft agar solution (0.6 % Agar and 0.5 % NaCl) by volume of 1/10, for the S. typhimurium TA strains and the E. coli strain, respectively.

- Counting procedure: The number of revertant colonies was counted with a colony counter.
Rationale for test conditions:
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 5 000 μg/plate dose was selected for all strains both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.
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. The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
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 historical 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 of the test substance 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:
Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.

Mutagenicity potential of the test material was assessed with Salmonella typhimurium TAl00, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in to main tests. The dose range for the main test was determined from the preliminary test using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 μg/plate. 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 5 000 μg/plate dose was selected for all strains both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

The main test concentrations were 313, 625, 1250, 2500 and 5 000 µg/plate. 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 historical data, indicating that this study was performed correctly.

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.

Under the conditions of this study, the test material was considered to be non-mutagenic in the bacterial reverse mutation assay.