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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article is not suspect to be able to induce genetic toxicity.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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
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.
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.
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.
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November from the 07th to the 23rd, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 ± C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.
- Culturing: the CHO K1 cells for the study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
125, 250, 500, 1000 and 2000 µg/ml
Vehicle / solvent:
- Vehicle: the test item was dissolved in Ham's F12 medium and diluted prior to treatment.
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability was confirmed with the available laboratory’s historical database.
- Preparation: test item was prepared in a concentration of 25 mg/ml in Ham's F12 medium (stock solution) at the first step. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of solvent was added and the formulation was stirred until homogeneity is reached. The formulation was diluted by serial dilutions to obtain the dosing formulations for lower doses. The appropriate amount of these dosing formulations were diluted with Ham's F12 medium or Ham's F12 medium + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.
Untreated negative controls:
yes
Remarks:
Ham's F12 medium
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
PROCEDURES
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 x10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Replicates: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: after the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

PLATING SURVIVAL
Following adjustment of the cultures to 10^5 cells/ml, samples from these cultures were diluted to 40 cells/ml.
A total of 5 ml (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies.
Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.

EXPRESSION OF MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 cells were taken
on days 1, 3, 6 and evaluated on day 8.

SELECTION OF THE MUTANT PHENOTYPE: at the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).

PLATING OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.

FIXATION AND STAINING OF COLONIES: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
A GLP Pre-test on Toxicity was performed. During the Pre-test on Toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5x106 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 µl/ml) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration was adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing. Colonies were then fixed with methanol and was stained with Giemsa and the colonies were counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH and osmolality was considered for dose level selection. The results of Pre-test on cell toxicity were used for dose selection of the test item concentrations used in the Main Mutation Assay. Five concentrations were selected for the treatment without and with metabolic activation system, respectively

METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
all results are inside the distribution of the historical negative control data (based 95 % control limit).
Statistics:
Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
On Day 1, there was no cytoxicity observed at the tested concentrations with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. There were no significant differences between treatment and control groups and no dose-response relationships were noted.

SOLVENT CONTROL
All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

POSITIVE and NEGATIVE CONTROLS
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 µl/ml) and 7,12-Dimethyl benz[a]anthracene (20 µg/ml). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.

OSMOLALITY and pH
The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.

SOLUBILITY
A clear solution was obtained up to a concentration of 25 mg/ml. For examined test item concentrations no precipitation in the medium was noted.
Conclusions:
The test item is considered as being non-mutagenic in the system.
Executive summary:

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in Ham's F12 medium and the following concentrations were selected on the basis of cytotoxicity investigations (made in a preliminary study without and with metabolic activation): 125, 250, 500, 1000 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)).

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µl/ml) and 7, 12-Dimethyl benzanthracene (20 µg/ml) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion

The substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

Thus, the test item is considered as being non-mutagenic in the system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January the 09th to the 29th, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro cytogenicity / chromosome aberration
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures).
- Storange: the cell stocks were kept in liquid nitrogen.
- Subculture: Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture.
- Storage conditions: the laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5 % CO2.
- Growing conditions: the V79 cells were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/ml penicillin, 10 mg/ml streptomycin and 25 µg/m amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

MEDIA USED
- Periodically checked for Mycoplasma contamination: the cell stocks were routinely checked for mycoplasma infections.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time 500, 1000 and 2000 µg/ml test item, with and without mix
Experiment B with 20/20 h and with 20/28 h treatment/sampling time 31.3, 62.5, 125 and 250 µg/ml test item without mix
Experiment B with 3/28 h treatment/sampling time 500, 1000 and 2000 µg/ml test item with S9 mix
Vehicle / solvent:
- Vehicle: Dulbecco’s Modified Eagle’s (DME) medium
- Justification for choice of solvent/vehicle: vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Cell density at seeding: 5 x 10^5 cells per culture were seeded for each group.

NUMBER OF REPLICATIONS
Dduplicate cultures were used at each test item concentration.

EXPOSURE
Experiment A: the exposure period was 3 hours. After test item exposure the cells were washed with DME (Dulbecco’s Modified Eagle’s) medium and then growth medium was added.
Experiment B: the exposure period without metabolic activation was 20 hours; the exposure period with metabolic activation was 3 hours.

SAMPLING
Experiment A: sampling was made at 20 hours after start of treatment (approximately 1.5 normal cell cycles from the beginning of treatment).
Experiment B: sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

PH AND OSMOLALITY
The pH value and osmolality of negative (solvent) control and test item treatment solutions (for every treatment concentrations) were measured in the main cytogenetic experiments.

PREPARATION OF CHROMOSOMES
Cell cultures were treated with colchicine (0.2 µg/ml) methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

ANALYSIS OF METHAPHASE CELLS
All slides were independently coded before microscopic analysis and scored blind.
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored.

DETERMINATION OF CYTOTOXICITY
The cells were seeded into 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 10^5 cells each and were incubated for 24 hours in 10 ml of DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/ml) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment) using a Bürker chamber.

Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. At harvest the cells were trypsinised, collected and cell counts were determined. Based on the cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. The volume of culture medium was 5 ml/dish for each per group.

The results obtained were used for dose selection of the test item used in the chromosome aberration assays (experiments A and B). In addition, the pH and osmolality were measured in this pre-test.

MAMMALIN MICROSOMAL FRACTION S9 MIX
The protein concentrations of the S9 batch used in the experiments were 35.7 and 33.8 mg/ml.
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH. Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.
The complete S9 Mix was freshly prepared containing components with the following ratios: S9 fraction 3 ml, HEPES 2 ml, KCl 1 ml, MgCl2 1 ml, NADP 1 ml, Glucose-6-phosphate 1 ml, DME medium 1 ml.

ACCEPTABILITY OF THE ASSAY
The chromosome aberration assay is considered acceptable because it meets the following criteria:
- the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
- concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
- cell proliferation in the solvent control is adequate,
- adequate number of cells and concentrations are analyzable,
- all requested experimental conditions were tested unless one resulted in a positive result
- the criteria for the selection of top concentration are fulfilled.
Rationale for test conditions:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
Clear cytotoxicity of about 50 % was observed after 20-hour test item treatment in experiment B. In the experiment A and in the experiment B after 3-hour treatment low toxicity was observed. In this part of experiment the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration.
No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

CONTROLS
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate and Cyclophosphamide caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

SOLUBILITY AND DOSE SELECTION
A clear solution of test item was obtained in in Dulbecco’s Modified Eagle’s (DME) medium up to a concentration of 25 mg/ml. There was no precipitation in the medium at any concentration tested.
Conclusions:
The test item is considered as being non-clastogenic, under tested conditions.
Executive summary:

The test item, dissolved in Dulbecco’s Modified Eagle’s (DME) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

Experiment A with 3/20 h treatment/sampling time 500, 1000 and 2000 µg/ml test item, with and without mix

Experiment B with 20/20 h and with 20/28 h treatment/sampling time 31.3, 62.5, 125 and 250 µg/ml test item without mix

Experiment B with 3/28 h treatment/sampling time 500, 1000 and 2000 µg/ml test item with S9 mix

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/ml) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50 % was observed after 20-hour test item treatment in experiment B. In the experiment A and in the experiment B after 3-hour treatment low toxicity was observed. In this part of experiment the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for lower-cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 473 (2016)).

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion

The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro AMES tests, in vitro gene mutation on mammalian cells and in vitro chromosomal aberration.

BACTERIA GENE MUTATION

The test item was assessed 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). The test item belongs to azo-dyes therefore the modified protocol proposed by Prival and Mitchell was applied.

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.

MAMMALIAN CELL GENE MUTATION

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in Ham's F12 medium and the following concentrations were selected on the basis of cytotoxicity investigations (made in a preliminary study without and with metabolic activation): 125, 250, 500, 1000 and 2000 µg/ml, with and without S9-mix.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

CHROMOSOMAL ABERRATION

The test item, dissolved in Dulbecco’s Modified Eagle’s (DME) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments.

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50 % was observed after 20-hour test item treatment in experiment. In the experiments, after 3-hour treatment, low toxicity was observed.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggests that test substance did not show any reasons of concern from the genotoxicity point of view.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC) No 1272/2008.