[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

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

Test material

Test material information

impurity 1

Reference substance name:

1-(4-methoxyphenyl)-3-phenylpropan-1-one

Cas Number:

684-361-5

Molecular formula:

C16H16O2

IUPAC Name:

1-(4-methoxyphenyl)-3-phenylpropan-1-one

impurity 2

Reference substance name:

(Z)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Cas Number:

183621-24-1

Molecular formula:

C16H16O

IUPAC Name:

(Z)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Constituent 1

Reference substance name:

(E)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Cas Number:

35856-81-6

Molecular formula:

C16H16O

IUPAC Name:

(E)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Test material form:

liquid

Method

Target gene:

hprt locus

Species / strain

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

Controls

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

Results and discussion

Test results

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

Applicant's summary and conclusion

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.