[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES, OECD 471:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
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 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

 

In vitro Micronucleus test, OECD 487:

The test substance was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system according to the OECD guideline 487. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.
In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 55 ± 5% reduction in cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 60 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 200 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 200 μg/mL in the non-activated and S9-actvated 4-hour exposure groups, and at doses ≥ 600 μg/mL in the non-activated 24-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 2.79 to 76.8 μg/mL for the non-activated 4-hour exposure group, from 10.5 to 150 μg/mL for the S9-activated 4-hour exposure group, and from 2.79 to 61.4 μg/mL for the non-activated 24-hour exposure group.
In the micronucleus assay, cytotoxicity (≥ 55 ± 5% reduction in CBPI relative to the vehicle control), was observed at doses ≥ 49.1 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 96 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 31.4 μg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the exposure groups.
The doses selected for evaluation of micronuclei were 25.1, 39.3 and 49.1 μg/mL for the non-activated 4-hour exposure group; 31.4, 61.4, and 96 μg/mL for the S9-activated 4-hour exposure group; and 8.37, 25.1, and 31.4 μg/mL for the non-activated 24-hour exposure group.
Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.
The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.
These results indicate the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

 

HPRT, OECD 476:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of the test substance to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.
DMSO (Dimethyl sulfoxide) was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1
5-hour treatment in the presence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
5-hour treatment in the absence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.

Assay 2
5-hour treatment in the presence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
24-hour treatment in the absence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.
In Assays 1 and 2, insolubility (precipitate or minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment at 2000-666.67 and/or 222.22 μg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In the Assay 1, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.006)). This experiment is considered to be negative.
In the Assay 1, in the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.052)). This experiment is considered to be negative.

In the Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. Statistically significant increase in the mutation frequencies (at p < 0.05 level) were observed in this experiment at the concentrations of 74.07 and 2.74 μg/mL, although the observed values were within the general historical control range. Furthermore, the observed mutant frequencies (12.1 and 8.5 x 10-6) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment (R2 = 0.141)). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment is concluded as negative. Moreover, it confirmed the result of the Assay 1.
In the Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.137)). This experiment is considered to be negative.
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least four evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

In conclusion, no mutagenic effect of the test substance was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2021 to 13 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

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

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cell line is selected to be used in the study as it is one of the preferred cell lines for in vitro Mammalian Cell Gene Mutation Test based on the OECD No. 476 guideline, and it was also used in the method validation study of the Test Facility.

Metabolic activation:

with and without

Metabolic activation system:

The S9-mix was prepared as follows:
Concentration of the stock solution
Concentration in the mix
HEPES* - Concentration of the stock solution: 20 mM - Concentration in the mix: 0.2 mL/mL
KCl - Concentration of the stock solution: 330 mM - Concentration in the mix: 0.1 mL/mL
MgCl2 - Concentration of the stock solution: 50 mM - Concentration in the mix: 0.1 mL/mL
NADP** - Concentration of the stock solution: 40 mM - Concentration in the mix: 0.1 mL/mL
D-Glucose-6-phosphate - Concentration of the stock solution: 50 mM - Concentration in the mix: 0.1 mL/mL
F12-10 - Concentration of the stock solution: - - Concentration in the mix: 0.1 mL/mL
S9 fraction - Concentration of the stock solution: - - Concentration in the mix: 0.3 mL/mL

*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate

Prior to addition to the culture medium the S9-mix was kept in an ice bath.
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 μg/mL (the recommended maximum concentration).
Insolubility (precipitate, minimal amount of precipitate or oily film) was detected in the preliminary experiment in the final treatment medium at the end of the treatment at 2000-125 μg/mL concentration range in case of 5-hour treatment in the presence and absence of S9-mix and at 2000-500 μg/mL concentration range in case of 24-hour treatment in the absence of S9-mix. The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Test according to the OECD No. 476 guideline instructions (up to the recommended maximum concentration and the solubility limit). Seven test item concentrations were selected for the main experiments.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

In Assay 1, 5-hour treatment was performed with and without metabolic activation (in the presence and absence of S9-mix). In Assay 2, 5-hour treatment was performed with metabolic activation (in the presence of S9-mix) and 24-hour treatment without metabolic activation (in the absence of S9-mix).

Treatment of the cells:
For the 5-hour treatments, at least 2x106 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x107 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 μL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium and incubated for approximately 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x105 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
For the 24-hour treatment, at least 2x106 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x107 cells were placed in flasks and incubated for approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% (v/v) serum (F12-5). A suitable volume (100 μL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with F12-10 medium and once with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x105 cells/mL (if possible). Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.

Plating for survival:
Following adjustment of the cultures to 2x105 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium. Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.

Expression period:
Cultures were maintained in dishes for 7 days, during which time the HPRT-mutation was expressed. During this expression period, the cultures were sub-cultured and maintained at 2x105 cells/dish twice (whenever possible) (on Days 1, 3, 6 and 8) to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell density was determined by haemocytometer and cells were plated for viability and for selection of the mutant phenotype.

Plating for viability:
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x105 cells/mL, then further diluted to 40 cells/mL using F12-10 medium.
Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.

Plating for selection of the mutant phenotype (6-TG resistance):
At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x105 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 μg/mL 6-thioguanine (abbreviation: 6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 μg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.

Fixation and staining of colonies:
After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

ANALYSIS OF THE RESULTS
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x106 cells: 5 plates at 4x105 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 106 clonable cells.
The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).
In the statistical analysis, negative trends were not considered significant.

Rationale for test conditions:

According to OECD guideline 476

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The general historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary).
According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

The following computerized systems were used in the study.
Provantis v9.3 for test item receipt
SPSS PC+4.0 for statistical evaluation
Moreover, data were recorded on the appropriate forms from the relevant SOPs of Charles River Laboratories Hungary

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the mutation assays, cells were exposed to the test item for 5 hours with and without metabolic activation system (±S9-mix) or for 24 hours without metabolic activation system (-S9-mix) then the cells were plated for determination of survival and in parallel sub-cultured without test item for 7 days to allow the expression of the genetic changes (if any occurred). At the end of the expression period, cells were allowed to grow and form colonies in culture dishes with and without selective agent (6-TG) for determination of mutations and viability.

Assay 1
In Assay 1, a 5-hour treatment with metabolic activation (in the presence of S9-mix) and a 5-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence and absence of S9-mix, the following concentrations were examined: 2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
In Assay 1, insolubility (precipitate or minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment at 2000-666.67 and/or 222.22 μg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases (see Appendix 13 of the study report attached).
In the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data (see Table 5 of Appendix 6) and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.006)). This experiment is considered to be negative.
In the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data (see Table 5 of Appendix 6) and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.052)). This experiment is considered to be negative.
Data of Assay 1 are presented for survival (Table 1 of Appendix 4 and Appendix 7), viability (Table 3 of Appendix 5 and Appendix 9) and mutagenicity (Table 5 of Appendix 6 and Appendix 11). Observations (including pH and osmolality) after treatment are summarized in Appendix 13 of the study report attached.

Assay 2
In Assay 2, 5-hour treatment with metabolic activation (in the presence of S9-mix) and 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed.
For the 5-hour treatment in the presence of S9-mix and the 24-hour treatment in the absence of S9-mix, the following concentrations were examined: 2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
In Assay 2, insolubility (precipitate or minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment at 2000-666.67 and/or 222.22 μg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases (see Appendix 13 of the study report attached).
In the presence of S9-mix (5-hour treatment), similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. Statistically significant increase in the mutation frequencies (at p < 0.05 level) were observed in this experiment at the concentrations of 74.07 and 2.74 μg/mL, although the observed values were within the general historical control range (see Table 6 of Appendix 6). Furthermore, the observed mutant frequencies (12.1 and 8.5 x 10-6) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment (R2 = 0.141)). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment is concluded as negative. Moreover, it confirmed the result of the Assay 1.
In the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data (see Table 6 of Appendix 6) and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.137)). This experiment is considered to be negative.
Data of Assay 2 are presented for survival (Table 2 of Appendix 4 and Appendix 8), viability (Table 4 of Appendix 5 and Appendix 10*) and mutagenicity (Table 6 of Appendix 6 and Appendix 12*). Observations (including pH and osmolality) after treatment are summarized in Appendix 13 of the study report attached.

Validity of the study:
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays (Tables 5 and 6 of Appendix 6*), and the observed values were in the expected range (5-20 x 10-6) as shown in the OECD No. 476 guideline.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls (Tables 5 and 6 of Appendix 6*) and were in good harmony with the historical data in all assays (for historical control data see Appendix 15*).
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays (Tables 1-4 of Appendices 4-5*).
The tested concentration range in the study was considered to be adequate as the highest evaluated concentration was the recommended maximum concentration.
Seven test item concentrations were evaluated in duplicate in each experiment.
The overall study was considered valid.

*Refer to the study report attached

Conclusions:

The HPRT Assay with the test substance performed on CHO K1 Chinese hamster ovarian cells was considered to be valid and reflect the real potential of the test item to cause mutations in the cultured mammalian cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9) in this study.
Treatment with the test item resulted in a statistically significant increase in mutation frequencies in the presence of a rat metabolic activation system (S9) in Assay 2; the observed values were within the general historical control range and was not part of a dose response. Furthermore, the observed mutant frequencies were within the expected range of the negative control samples according to the relevant OECD guideline.

In conclusion, no mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of the test substance to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.
DMSO (Dimethyl sulfoxide) was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1
5-hour treatment in the presence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
5-hour treatment in the absence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.

Assay 2
5-hour treatment in the presence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.
24-hour treatment in the absence of S9-mix:
2000, 666.67, 222.22, 74.07, 24.69, 8.23 and 2.74 μg/mL.

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.
In Assays 1 and 2, insolubility (precipitate or minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment at 2000-666.67 and/or 222.22 μg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In the Assay 1, in the presence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.006)). This experiment is considered to be negative.
In the Assay 1, in the absence of S9-mix (5-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.052)). This experiment is considered to be negative.

In the Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. Statistically significant increase in the mutation frequencies (at p < 0.05 level) were observed in this experiment at the concentrations of 74.07 and 2.74 μg/mL, although the observed values were within the general historical control range. Furthermore, the observed mutant frequencies (12.1 and 8.5 x 10-6) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment (R2 = 0.141)). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment is concluded as negative. Moreover, it confirmed the result of the Assay 1.
In the Assay 2, in the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment (R2 = 0.137)). This experiment is considered to be negative.
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least four evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

In conclusion, no mutagenic effect of the test substance was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January to 18 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Due to calculation error in the dilution scheme, the definitive micronucleus assay was dosed at the concentration 0.836 mg/mL instead of 0.837 mg/mL. It has been concluded that this minor difference in concentration had no impact on study integrity.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers, females and aged 25 years.
The donors had no recent history of radiotherapy, viral infection, or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals (Clare et al., 2006).

Cytokinesis block (if used):

Cytochalasine B (6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 4375, Exp. Date: 10 Dec 2022) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use as indicated below:
Component and Final Concentration in Culture Medium (RPMI 1640 Serum-free medium supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin)
- NADP (sodium salt) 1 mM
- Glucose-6-phosphate 1 mM
- Potassium chloride 6 mM
- Magnesium chloride 2 mM
- S9 homogenate 20 μL/mL

Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.

Test concentrations with justification for top dose:

Based on the following preliminary toxicity test:
- Visible precipitate was observed in treatment medium at the following doses:
1) Non activated - 4h treatment time - visible precipitate at the beginning of treatment period >= 200 microg/mL - visible precipitate at the conclusion of treatment period >= 200 microg/mL
2) Non activated - 24h treatment time - visible precipitate at the beginning of treatment period >= 200 microg/mL - visible precipitate at the conclusion of treatment period >= 600 microg/mL
3) S9 activated - 4h treatment time - visible precipitate at the beginning of treatment period >= 200 microg/mL - visible precipitate at the conclusion of treatment period >= 200 microg/mL

The osmolality of the test substance doses in treatment medium was considered acceptable (< 120% of vehicle). The pH of the highest dose of test substance in treatment medium was 7.5.
Cytotoxicity [≥ 55 ± 5% reduction in cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 60 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 200 μg/mL in the S9-activated 4-hour exposure group.
Based upon the results of the preliminary toxicity assay, the doses selected for the micronucleus assay were as follows:
1) Non activated - 4h treatment time - 20h recovery time - doses in microg/mL : 2.79, 8.37, 25.1, 31.4, 39.3, 49.1, 61.4, 76.8
2) Non activated - 24h treatment time - 0h recovery time - doses in microg/mL : 2.79, 8.37, 25.1, 31.4, 39.3, 49.1, 61.4
3) S9 activated - 4h treatment time - 20h recovery time - doses in microg/mL : 10.5, 31.4, 61.4, 76.8, 96, 120, 150

Vehicle / solvent:

The vehicle for the test item was DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

other: water

Details on test system and experimental conditions:

Solubility Determination
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Preparation of Target Cells
Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Identification of Test System
Prior to treatment, the culture tubes were identified by the BioReliance study number, dose level, test phase, treatment condition, activation system and/or replicate design.

Exogenous metabolic activation system
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 4375, Exp. Date: 10 Dec 2022) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use as indicated below:
Component and Final Concentration in Culture Medium (RPMI 1640 Serum-free medium supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin)
- NADP (sodium salt) 1 mM
- Glucose-6-phosphate 1 mM
- Potassium chloride 6 mM
- Magnesium chloride 2 mM
- S9 homogenate 20 μL/mL

Frequency and route of administration
Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.

Preliminary Toxicity Test for Selection of Dose Levels
HPBL were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

Micronucleus assay
The doses selected for testing in the micronucleus assay were based on the results of the preliminary toxicity test.

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the following:
4h non S9 activated - 55 +/- 5% cytotoxicity (CBPI relative to the vehicle control)
4h S9 activated - 55 +/- 5% cytotoxicity (CBPI relative to the vehicle control)
24h non S9 activated - 55 +/- 5% cytotoxicity (CBPI relative to the vehicle control)

Two additional doses were included in the evaluation of micronuclei.

Treatment of Target Cells (Preliminary Toxicity Test and Micronucleus Assay)
The pH of the highest dose of dosing solution in the treatment medium was measured using test tape. Treatment was carried out by refeeding the cultures as mentioned in the table in 'Any other information on materials and methods incl. tables' section.

After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 μg/mL and returned to the incubator under standard conditions. For the 24-hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.

Collection of Cells (Preliminary Toxicity Test and Micronucleus Assay)
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4-hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and were stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Micronucleus Assay)
For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:
CBPI = (1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells)/Total number of cells scored

% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

Micronucleus Scoring (Micronucleus Assay)
The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

Statistical Analysis
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Rationale for test conditions:

Test conditions were based on OECD guideline.

Evaluation criteria:

Vehicle Controls:
The frequency of cells with micronuclei should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).

Positive Controls:
The percentage of micronucleated cells must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation:
The CBPI of the vehicle control at harvest must be ≥ 1.4.

Test Conditions:
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 24-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations
At least 2000 binucleated cells from at least three appropriate test substance concentrations.

Evaluation of Test Results:
The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Electronic systems used for the collection or analysis of data included, but not limited to the following (version numbers are maintained in the system documentation):
LIMS Labware System for Test Substance Tracking
Excel (Microsoft Corporation) for Calculations
Kaye Lab Watch Monitoring system (Kaye GE) for Environmental Monitoring
BRIQS for Deviation and audit reporting

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

Visible precipitate was observed in treatment medium at the following doses:
1) Non activated - 4h treatment time - visible precipitate at the beginning of treatment period : None - visible precipitate at the conclusion of treatment period : None
2) Non activated - 24h treatment time - visible precipitate at the beginning of treatment period : None - visible precipitate at the conclusion of treatment period : None
3) S9 activated - 4h treatment time - visible precipitate at the beginning of treatment period >= 96 microg/mL - visible precipitate at the conclusion of treatment period : None

The pH of the highest dose of test substance in treatment medium was 7.5. Cytotoxicity (≥ 55 ± 5% reduction in CBPI relative to the vehicle control), was observed at doses ≥ 49.1 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 96 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 31.4 μg/mL in the non-activated 24-hour exposure group. Cytotoxicity at the highest dose evaluated was observed as follows:
1) Non activated - 4h treatment time - highest evaluated dose 49.1 microg/mL - Cytotoxicity 52%
2) Non activated - 24h treatment time - highest evaluated dose 31.4 microg/mL - Cytotoxicity 51%
3) S9 activated - 4h treatment time - highest evaluated dose 96 microg/mL - Cytotoxicity 58%

Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.
The results of cytotoxicity and micronucleus data for the untreated controls were comparable to that of the vehicle control. Therefore, use of organic solvent (DMSO) had no adverse effect.
The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Conclusions:

Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test substance was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system according to the OECD guideline 487. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO) was used as the vehicle.
In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 55 ± 5% reduction in cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 60 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 200 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 200 μg/mL in the non-activated and S9-actvated 4-hour exposure groups, and at doses ≥ 600 μg/mL in the non-activated 24-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 2.79 to 76.8 μg/mL for the non-activated 4-hour exposure group, from 10.5 to 150 μg/mL for the S9-activated 4-hour exposure group, and from 2.79 to 61.4 μg/mL for the non-activated 24-hour exposure group.
In the micronucleus assay, cytotoxicity (≥ 55 ± 5% reduction in CBPI relative to the vehicle control), was observed at doses ≥ 49.1 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 96 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 31.4 μg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was not observed at any dose in any of the exposure groups.
The doses selected for evaluation of micronuclei were 25.1, 39.3 and 49.1 μg/mL for the non-activated 4-hour exposure group; 31.4, 61.4, and 96 μg/mL for the S9-activated 4-hour exposure group; and 8.37, 25.1, and 31.4 μg/mL for the non-activated 24-hour exposure group.
Neither statistically significant nor dose-dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data.
The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.
These results indicate the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June 2021 to 24 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and Tryptophan

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System:
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 S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly. The composition of solution refers to 1000 mL.

- Induction of Liver Enzymes
Male Wistar rats (502-719 g, animals were 26-29 weeks old and 410-708 g, animals were 9-25 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 28 June 2021 (Charles River Laboratories Hungary code: E13612) and 11 January 2021 (Charles River Laboratories Hungary code: E13455).

- Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized, and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The dates of preparation of S9 fractions for this study was 01 July 2021 (E13612, Expiry date: 01 July 2023) and 14 January 2021 (E13455, Expiry date: 14 January 2023).
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the S9 fraction used was determined to be 25.1 g/L (E13612) and 21.1 g/L (E13455).
Note: E13455 was used in Assay 1-2 and E13612 was used in Assay 3.
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

- The S9 Mix (containing 10 % (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. 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:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4: 500 mL
Rat liver homogenate (S9): 100 mL
Salt solution for S9 Mix (see above): 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

- 0.2 M Sodium Phosphate Buffer, pH 7.4

Solution A:
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

Solution B:
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

0.2M Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL

Test concentrations with justification for top dose:

Based on on the Sponsor's information, a 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in Assay 1 were 5000, 3750, 1581, 500, 158.1, 50 and 15.81 μg/plate at all examined bacterial strains with and without metabolic activation. Examined concentrations in Assay 2 were 5000, 3750, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate at all Salmonella typhimurium TA98 and TA1537 bacterial strains with metabolic activation and at Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation. Examined concentrations in Assay 3 were 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation; 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium TA100 and TA1535 strains without metabolic activation.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as vehicle for the study (based on the Sponsor's information). The obtained stock formulation (50 μL) with the solution of top agar (section 10.2.4.) and phosphate buffer (section 10.3.4.) was examined in a test tube without test bacterium suspension.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:

Remarks:

with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other:

Remarks:

without S9

Details on test system and experimental conditions:

TEST SYSTEM
BACTERIAL STRAINS
1) Origin
Date of arrival and origin:
Salmonella typhimurium TA98 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 1)
17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 2 and Assay 3)
Salmonella typhimurium TA100 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 1)
17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 2 and Assay 3)
Salmonella typhimurium TA1535 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 1)
17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 2 and Assay 3)
Salmonella typhimurium TA1537 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 1)
17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 2 and Assay 3)
Escherichia coli WP2 uvrA 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 1)
17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA (in Assay 2 and Assay 3)
The true copies of original certificates and other documents of the strains are collected and stored in the Microbiological Laboratory of Charles River Laboratories Hungary Kft.

2) Genotypes
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains used for mutagenicity testing are summarized in Table 4 of the final report attached.

3) Storage
The strains are stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of Charles River Laboratories Hungary Kft. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

4) Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of Charles River Laboratories Hungary Kft.

5) Spontaneous Reversion of Tester Strains
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without and with metabolic activation were in the period of 2015-2020 were (as guide) as follows: Salmonella typhimurium TA98: 11-50, 13-54, TA100: 67-152, 67-152, TA1535: 1-33, 3-39, TA1537: 2-26, 1-29, Escherichia coli WP2 uvrA: 14-77, 16-89. More detailed historical control data are shown in Appendix 6.

6) Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 (Section 10.2.2. of the final report) for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C in a Gyrotory water bath shaker.

7) Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 105, 106, 107 and 108 dilutions prepared by sterile physiological saline on Nutrient Agar (Section 10.2.3. of the final report) plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.

MEDIA
The supplier, batch number and expiry date of the used chemicals described in sections 10.2.2.-10.2.5. are summarized in Table 2 (Chemicals used in the experiments table in Section 8. of the final report). The composition of media refers to 1000 mL.

1) The 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 13.0 g
Distilled water q.s. ad1 1000 mL
Minimal glucose agar plates (Batch number: 558662, Expiry date: 01 September 2021 was used in Assay 1 and Batch number: 603137, Expiry date: 14 December 2021 was used in Assay 2-3) were provided by Merck. Certificate of Analysis were obtained from the Supplier.

2) Nutrient Broth No.2
Nutrient Broth No.2 25.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

3) Nutrient Agar
Nutrient Agar 20.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

4) Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.31) 122.2 mg
L-Histidine x·HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL
Agar solution 900 mL

5) Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.
Complete Top Agar for Escherichia coli strain:
Nutrient Broth 2 (see section 10.2.2.) 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution (see section 10.2.4.) 947.5 mL


TEST PROCEDURE
1) Concentrations
Concentrations for the main tests were selected on the basis of the available information. In the main tests different concentrations were used.

2) Preliminary Compatibility Test
Dimethyl sulfoxide (DMSO) was selected as vehicle for the study (based on the Sponsor's information). The obtained stock formulation (50 μL) with the solution of top agar (section 10.2.4.) and phosphate buffer (section 10.3.4. of the final report) was examined in a test tube without test bacterium suspension. The results of the Preliminary Compatibility Test are summarized in Table 5 of the final report.

3) Test Item Concentrations in the Mutagenicity Tests (Assay 1, Assay 2 and Assay 3)
Based on on the Sponsor's information, a 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in Assay 1 were 5000, 3750, 1581, 500, 158.1, 50 and 15.81 μg/plate at all examined bacterial strains with and without metabolic activation. Examined concentrations in Assay 2 were 5000, 3750, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate at all Salmonella typhimurium TA98 and TA1537 bacterial strains with metabolic activation and at Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation. Examined concentrations in Assay 3 were 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation; 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium TA100 and TA1535 strains without metabolic activation.

4) Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and/or without metabolic activation were included in Assay 1, Assay 2 and Assay 3. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects. The control groups used in the tests are listed in Table 6 of the final report.

5) Experimental Method
The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al. , Venitt and Parry , OECD Guideline No. 471, 2020 , Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of Charles River Laboratories Hungary Kft.

6) Procedure for Exposure in the Assay 1
Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2 as described in Section 10.2.2.) 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 labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 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 minimal agar plates. 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, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 66 hours.

7) Procedure for Exposure in the Assay 2 and Assay 3
Assay 2 and Assay 3 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1 (see details in Section 16. of the final report).
Bacteria (cultured in Nutrient Broth No.2. as described in section 10.2.2. of the final report) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 10.1.6. of the final report) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.

Rationale for test conditions:

Test conditions follow the OECD 471 guideline.

Evaluation criteria:

- Criteria for Validity
The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.

- Criteria for a Positive Response
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:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines [5][6][7][8], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

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

Statistics:

Not used.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Assay 1, the plate incorporation method was used. In Assay 2 and Assay 3, the pre-incubation method was used. The main tests were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Assay 1, Assay 2 and Assay 3 were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in Salmonella typhimurium TA98 and TA1537 bacterial strains without metabolic activation; Salmonella typhimurium TA100 and TA1535 bacterial strains with and without metabolic activation at several concentrations. In these cases, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains with and/or without metabolic activation were repeated during the Experimental Period III (Assay 3) using the pre-incubation method and a modified concentration range (Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation at 158.1, 50, 15.81, 5, 1.581 and 0.5 μg test item/plate; Salmonella typhimurium TA100 and TA1535 strains without metabolic activation at 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg test item/plate). Results of the invalid experiment was not reported; however, all data are kept and archived in the raw data binder.
The examined test concentrations:
- in Assay 1 were 5000, 3750, 1581, 500, 158.1, 50 and 15.81 μg/plate at examined bacterial strains with and without metabolic activation,
- in Assay 2 were 5000, 3750, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate at Salmonella typhimurium TA98 and TA1537 bacterial strains with metabolic activation and at Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation,
- in Assay 3 were 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation; 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium TA100 and TA1535 strains without metabolic activation.
Precipitate was detected on the plates (precipitate / microdrops (oily drops)) in Assay 1 all Salmonella typhimurium bacterial strains with and without metabolic activation and in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000, 3750 and 1581 μg/plate concentrations.

Inhibitory, cytotoxic effect of the test item (slight reduced / reduced background lawn development) was observed:
- in the Assay 2 in all Salmonella typhimurium TA 98 and TA1537 bacterial strains with metabolic activation at 1581 μg/plate concentration,
- in the Assay 3 in Salmonella typhimurium TA98 and TA1537 bacterial strain without metabolic activation and in Salmonella typhimurium TA100 and TA1535 bacterial strain with metabolic activation at 158.1 μg/plate concentration; in Salmonella typhimurium TA100 and TA1535 bacterial strain without metabolic activation at 15.81 μg/plate concentration.
Note: Due to the observed precipitate the background lawn development could not be properly evaluated at the 5000 and 3750 μg/plate concentrations with and without metabolic activation in the Assay 1 and Assay 2 during microscopic evaluation. Colony counting was unaffected.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5000 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.22). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
In Assay 2 and 3 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 0.5 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.30). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Validity of the tests:

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
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 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-incubation Method) and an Assay 3 (Pre-Incubation Method).
Based on the available information, the test item was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. The examined test concentrations:
- in Assay 1 were 5000, 3750, 1581, 500, 158.1, 50 and 15.81 μg/plate at all examined bacterial strains with and without metabolic activation,
- in Assay 2 were 5000, 3750, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate at Salmonella typhimurium TA98 and TA1537 bacterial strains with metabolic activation and at Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation,
- in Assay 3 were 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate at Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation; 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg/plate at Salmonella typhimurium TA100 and TA1535 strains without metabolic activation.
In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in Salmonella typhimurium TA98 and TA1537 bacterial strains without metabolic activation; Salmonella typhimurium TA100 and TA1535 bacterial strains with and without metabolic activation at several concentrations. In these cases, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains with and/or without metabolic activation were repeated during the Experimental Period III (Assay 3) using the pre-incubation method and a modified concentration range (Salmonella typhimurium TA98 and TA1537 strains without metabolic activation and Salmonella typhimurium TA100 and TA1535 strains with metabolic activation at 158.1, 50, 15.81, 5, 1.581 and 0.5 μg test item/plate; Salmonella typhimurium TA100 and TA1535 strains without metabolic activation at 15.81, 5, 1.581, 0.5, 0.1581 and 0.05 μg test item/plate). Results of the invalid experiment was not reported; however, all data are kept and archived in the raw data binder.
Precipitate was detected on the plates in the Assay 1 and Assay 2 in all examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item was observed in the Assay 2 and Assay 3 in all Salmonella typhimurium bacterial strains with and/or without metabolic activation at higher concentrations.
In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
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 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the study results which are negatives, the test substance should not be classified as mutagenic nor clastogenic according to the EU CLP regulation (No 1272/2008 and its adaption 286/2011) and GHS.