[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February from the 04th to the 13rd, 2014

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:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

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):

- Source: Molecular Toxicology Inc.,Boone, USA.
- Storage: the strains are stored at -80 ± 10 ºC. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO is added as a cryoprotective agent.
- Confirmation of Phenotypes of Tester Strains: the phenotypes of the tester strains, as well as spontaneous mutation frequencies, are checked regularly according to Ames et al.
- Spontaneous Reversion of Tester Strains: measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- Procedure for Bacterial Cultures: the frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37 °C Benchtop Incubator Shaker.
- Viability and the Cell Count of the Testing Bacterial Cultures: the viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by plating experiments.

Metabolic activation:

with and without

Metabolic activation system:

hamster and rat liver S9

Test concentrations with justification for top dose:

5000, 1581, 500, 158, 50 and 15.8 μg/plate (both first and second experiment)

Vehicle / solvent:

- Vehicle used: DMSO.
- Justification for choice of vehicle: vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.

Controls

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine TA98, without MA. 2-aminoanthracene TA98; TA100; TA1535; TA1537 and WP2 uvrA, with MA rat and hamster liver S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate incorporation procedure for the first experiment; pre-incubation procedure in the second experiment.

MEDIA
- Typical Composition (g/1000 ml) of Minimal Glucose Agar: Glucose 20.0 g, Magnesium sulfate 0.2 g, Citric acid 2.0 g, di-Potassium hydrogenphosphate 10.0 g, Sodium ammonium hydrogenphosphate 3.5 g, Agar agar 15.0 g, Distilled water ad 1000 ml.
- Nutrient Broth No. 2: Nutrient broth No. 2. 25.0 g, Ultrapure water ad 1000.0 ml. Sterilization for 20 minutes was performed at 121˚C in an autoclave.
- Nutrient Agar: Nutrient Agar 20.0 g, Ultrapure water ad 1000.0 ml. Sterilization for 20 minutes was performed at 121˚C in an autoclave.
- Top Agar for Salmonella typhimurium Strains: Agar Bacteriological 4.0 g, NaCl 5.0 g, Ultrapure water ad 1000.0 ml. Sterilization for 20 minutes was performed at 121˚C in an autoclave.
- Histidine – Biotin solution (0.5 mM): D-Biotin 122.2 mg, L-Histidine•HCl H2O 104.8 mg, Ultrapure water ad 1000.0 ml. Sterilization was performed by filtration through a 0.22 μm membrane filter.
- Complete Top Agar for Salmonella typhimurium strains: Histidine – Biotin solution (0.5 mM) 100.0 ml, Agar solution 900.0 ml.
- Top Agar for Escherichia coli Strain: L-Tryptophan 2000.0 mg, Ultrapure water ad 1000.0 ml. Sterilization was performed by filtration through a 0.22 μm membrane filter.
- Complete Top Agar for Escherichia coli strain: Nutrient Broth by 5.4.2 50.0 ml, Tryptophan solution (2 mg/mL) 2.5 ml, Agar solution by 5.4.4 947.5 ml.

NUMBER OF REPLICATIONS: two experiments were performed. 3 plates per control and per concentration level were used.

FIRST EXPERIMENT
Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 ml of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labeled. The test item and other components were prepared fresh and added to the overlay (45°C).
The typical content of the tubes: top agar 2000 μl, vehicle or solution of test item or reference controls 100 μl, overnight culture of test strain 100 μl, phosphate buffer (pH: 7.4) or S9 mix 500 μl.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates (3 plates per control or concentration level). For activation studies, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37°C for 48 hours.

SECOND EXPERIMENT
A second experiment was performed, as a confirmatory mutationtest because of the unequivocal negative results of the first experiment. Before the overlaying of the test item, the bacterial culture and the S9 mix (containing hamster liver S9) or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 30 min at 37 ºC in a shaking incubator. After the incubation period, 2 ml of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for about 48 hours.

TEST CONCENTRATIONS
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test.
In the solubility test the test item behaviour was investigated in the applied test system. Based on the results of the preliminary tests, a stock solution with a concentration of 50 mg/ml was prepared from the test item with dimethyl sulfoxide (DMSO), that was diluted by serial dilutions each separated by approximately √10 factor to obtain six dosing solutions for lower doses. At the concentration choice the guideline proposal for soluble, non-toxic test compounds was taken into consideration, where the recommended maximum test concentration 5 mg/plate or 5 μl/plate.
At this test the maximum test concentration was 5000 μg test item/plate (±S9 Mix).

RANGE-FINDING TEST
Based on the solubility test, the stock solution with a concentration of 50 mg/ml was prepared in dimethyl sulfoxide (DMSO) and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate of the test item.
The preliminary test was performed under activation and non-activation conditions (in presence and absence of metabolic activation (S9 Mix)) with appropriate positive and negative controls. In the test the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

METABOLIC ACTIVATION
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

RAT LIVER S9 FRACTION
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH. The sensitivity, reliability and promutagen activation potential (on ethidium bromide, cyclophosphamide, benzo(a)pyrene and 2-aminoanthracene) of each batch of used S9 was verified by the supplier.
- The S9 Mix (with Rat Liver S9): NADP Na 7.66 g 4 mM, D-glucose-6 phosphate Na 3.53 g 5 mM, MgCl2 1.90 g 8 mM, KCl 6.15 g 33 mM, Ultrapure water ad 1000 ml. Sterilized by filtration through a 0.22 μm membrane filter.
- The complete S9 Mix was freshly prepared containing components as follows (per 1000 ml): Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml, Rat liver homogenate (S9) 100 ml, Salt solution for S9 Mix 400 ml. The S9 Mix was kept in an ice bath before it was added to the culture medium.

HAMSTER LIVER S9 FRACTION
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH. The sensitivity, reliability and promutagen activation potential (on ethidium bromide, cyclophosphamide, benzo(a)pyrene and 2-aminoanthracene) of each batch of used S9 was verified by the supplier.
- The S9 Mix (with Hamster Liver S9): NADP Na 15.33 g 4 mM, NADH Na2 x H2O 7.27 g 2 mM, FMN (Riboflavine-5’-phosphate-sodium salt) x H2O 4.96 g 2 mM, D-glucose-6 phosphate Na 28.21 g 20 mM, MgCl2 3.81 g 8 mM, KCl 12.30 g 33 mM, Ultrapure water ad 1000 ml. Sterilization was performed by filtration through a 0.22 μm membrane filter.
- The complete S9 Mix was freshly prepared containing components as follows (per 1000 ml): Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml, Hamster liver homogenate (S9) 300 ml, Salt solution for S9 Mix 200 ml, D-glucose-6-phosphate-dehydrogenase 2800 U, Before adding to the culture medium the S9 Mix was kept in an ice bath.

VALIDITY OF THE TEST
The tests are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers is in the 109 cells/ml order.
- The types and batches of S9 used in this study show the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
[A dose level is considered toxic if the reduced revertant colony numbers are observed compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
the reduced revertant colony numbers are below the historical control data range and / or pinpoint colonies appear and / or reduced background lawn development occurs.]

Evaluation criteria:

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control

Criteria for a Negative Response
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

both first and second experiments

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

sporadic increases in revertant colony numbers compared to the vehicle control without any biological significance

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the performed experiments the observed revertant colony number increases were of minor intensity, far below the biologically relevant thresholds for being positive. Most of the obtained increases remained in the historical control data ranges and were considered as to reflect the biological variability of the test system.
The revertant colony numbers remained in the vehicle control data range; however were above the historical control data range without any biological significance in the second experiment in S. typhimurium TA1537 at 15.8 μg/plate (+S9 Mix).
The obtained revertant colony numbers were in the vehicle control data range; however were below the historical control data range without any biological significance in the second experiment in S. typhimurium TA98 at 50 μg/plate (-S9 Mix).
The obtained lower revertant colony numbers than the revertant colony numbers of the vehicle control plates remained in the corresponding historical control data ranges and no signs of inhibition were obtained in any case.

CONTROLS
In the first and second experiments the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
The revertant colony numbers of the untreated and ultrapure water control plates in the different experimental phases were slightly higher or lower than the DMSO control plates. These higher or lower revertant counts of these controls remained mostly in the corresponding historical control data ranges. In the second experimentt in the case of Salmonella typhimurium TA1537 the revertant colony numbers of the untreated control, with addition of metabolic activation (+S9 Mix) were above the historical control data range; however this change was considered as acceptable, without any influence on the final conclusion of the study.

RANGE-FINDING TEST
In the Informatory Toxicity Test unequivocal cytotoxicity (assessed as a reduction in the number of revertant colonies, a clearing or diminution of the background lawn) was not noted up to and including the highest concentration tested, thus 5000 μg/plate.
The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control and were below the corresponding historical control data range in S. typhimurium TA98 at 5000 μg/plate, without metabolic activation (-S9 Mix). The lower counts were unique neither additional sign of cytotoxicity nor dose-related tendencies accompanied them.
In comparison with the revertant colony numbers of the vehicle control plates, slightly higher revertant colony counts (within the corresponding historical control data ranges) were observed in S. typhimurium TA98 at 5000 μg/plate, with addition of metabolic activation (+S9 Mix) and in TA100 at 158 and 15.8 μg/plate, without metabolic activation (-S9 Mix).
These obtained changes were considered as reflecting the biological variability of the applied test system.

VALIDITY OF THE PERFORMED EXPERIMENTS
Valid tests were performed since the tester strains (used in this study) demonstrated the specific phenotype characteristics, agreed with the corresponding historical control data ranges, and showed the adequate strain culture titer. Each batch of the S9 fraction (induced rat liver S9 as well as uninduced hamster liver S9) used in this test had the appropriate biological activity (according to the provided Certificates) and was active in the applied system (2AA treatments).
Each of the investigated reference mutagens showed the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all experimental phases.
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay, following the procedures outlined in the OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, and the tryptophan-requiring auxotroph strain of Escherichia coli, stain WP2 uvrA, in the presence and absence of a post mitochondrial supernatant (S9). In the plate incorporation test (first experiment) S9, prepared from livers of phenobarbital/β-naphthoflavone-induced rats, was used; in the pre-incubation test S9, prepared from uninduced hamster liver, was used. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test, a first experiment (Plate Incorporation Test), and a second experiment (Pre-Incubation Test). In the Range Finding Test as well as in the first experiment the plate incorporation method was used. The test item belongs to azo-dyes therefore the modified protocol proposed by Prival and Mitchell was applied. The modified protocol differed from the standard plate incorporation assay in 5 ways: (1) uninduced hamster liver S9 rather with phenobarbital/β-naphthoflavone induced rat liver S9 was used; (2) the hamster liver S9 Mix contained 30 % hamster liver extract instead of the usual 10 % liver extract content of the standard procedure; (3) flavin mononucleotide (riboflavin 5’-phosphate: FMN) was added to the cofactor mix; (4) the cofactor mix was modified to include exogenous glucose 6-phosphate dehydrogenase, NADH, and 4 times the standard amount of glucose 6-phosphate; (5) a 30 min. pre-incubation step was used before addition of top agar. Based on the results of the Solubility Test and the Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO) in a concentration of 50 mg/ml. Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the first and second experiments: 5000, 1581, 500, 158, 50 and 15.8 μg/plate. In the first and second experiments the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. No substantial increases or decreases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in the second experiment (Pre-Incubation Test) in S. typhimurium TA98 at 5000 μg/plate, without metabolic activation (-S9 Mix). The mutation rate was 1.78. This value remained far below the genotoxicological threshold for being positive.

The revertant colony numbers of vehicle control (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

Conclusion

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.