Registration Dossier
Registration Dossier
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EC number: 433-100-1 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: The test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Chromosome Aberration Test: The substance did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
HPRT Test: The substance did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999-01-13 to 1999-02-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21st July, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated December 29, 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
- Version / remarks:
- June 1996
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-Mix
- Test concentrations with justification for top dose:
- 31.6; 100; 316.2; 1000; 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- S. typhimurium: TA 1535, TA 100 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenyIene-diamine, 4-NOPD
- Remarks:
- S. typhimurium: TA 1537, TA 98 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- S. typhimurium: TA 102 without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene, 2-AA
- Remarks:
- S. typhimurium: TA 1535, TA 1537, TA 98 , TA 100 and TA 102 with metabolic activation
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (> 5000 µg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
- Executive summary:
The test substance was assessed in an Ames test according to EU method B.13/14 and OECD guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 100, TA 98 and TA 102. In two independent experiments several concentrations of test item were used. Each assay was conducted with and without metabolic activation (S9-mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6; 100; 316.2; 1000; 2500 and 5000 µg/plate. No toxic effects of the test effects of the test item were observed in both experiments up to the highest investigated dose in all strains used. No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Therefore, the test substance was considered non-mutagenic in this Ames test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- February 28, 2018 - March 26, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29th July, 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 14 February 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Chinese hamster lung cells (male), ECACC (European Collection of Cell Cultures)
- Suitability of cells: Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture:
The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5 % CO2.
- Cell cycle length, doubling time or proliferation index : high proliferation rates (doubling time 12-14 h)
- Modal number of chromosomes: diploid number, 2n=22
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature
The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5 %. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9
Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA)
- method of preparation of S9 mix
phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver mix
- concentration or volume of S9 mix and S9 in the final culture medium
The protein concentrations of the S9 batch used in the experiments were 33.7 and 37.8 mg/mL.
volume S9 fraction: 3 mL - Test concentrations with justification for top dose:
- The following concentrations were selected on the basis of a pre-test (without and with metabolic activation using rodent S9 mix):
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix 62.5, 125, 250 and 500 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 62.5, 125, 250 and 500 μg/mL test item - Vehicle / solvent:
- - Vehicle used: DMSO
- Justification for choice of vehicle: The vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation, final concentrations of 0.4 and 1.0 μL/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation, final concentration of 5.0 μg/mL
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : two
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10E5 cells per culture were seeded for each group
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h (Experiment A), 20 h (Experiment B)
- Harvest time after the end of treatment: Sampling was made at 20 hours or 28 hours after start of treatment
- Spindle inhibitor: Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting.
- Methods of slide preparation and staining technique used including the stain used:
Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Criteria for scoring chromosome aberrations:
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
In the Pre-test on Toxicity the maximum treatment concentration was 2000 μg/mL instead of 5000 μg/mL (UVCB test item) in the absence and in the presence of metabolic activation. This concentration was chosen on the basis results of the solubility test of the test item.
- Method: relative increase in cell count (RICC) - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance should be considered together. There is no requirement for verification of a clearly positive or negative response. - Statistics:
- For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The data were checked for a linear trend in number of cells with aberrations (without gaps) with treatment dose using the adequate regression analysis by Microsoft Excel software.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no relevant changes in pH after treatment with the test item
- Data on osmolality: There were no relevant changes in osmolality after treatment with the test item
- Precipitation and time of the determination: No
STUDY RESULTS
- Concurrent vehicle negative and positive control data: see tables 1 and 2
- Results from cytotoxicity measurements:
o For cell lines: relative Increase in cell count (RICC)
In both experiments, clear cytotoxicity of about 50 % was observed after test item treatment in the absence and presence of metabolic activation
HISTORICAL CONTROL DATA
see table 3 - Conclusions:
- The substance tested up to the cytotoxic concentrations, without and with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.
- Executive summary:
The substance, suspended in DMSO, was tested in a chromosome aberration assay in V79 cells according OECD TG 473 in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix 62.5, 125, 250 and 500 µg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 µg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 µg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 62.5, 125, 250 and 500 µg/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.
No relevant increase in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.
In the presence of metabolic activation, in experiment A at concentration of 125 µg/mL and in the absence of metabolic activation in experiment B (20 h treatment, 20 h sampling) at concentration of 15.7 µg/mL the values (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.
There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
In conclusion, the substance did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.Thus, the test item is considered as being non-clastogenic in this system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- April 10, 2018 - April 26, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 31/05/2008
- Deviations:
- yes
- Remarks:
- Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 July 2016).
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- Sub-line (KI)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: ECACC (European Collection of Cell Cultures)
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO KI is a sub-line of CHO cell line.
For cell lines:
- Absence of Mycoplasma contamination:
Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary
- Number of passages if applicable:
- Methods for maintenance in cell culture:
Ham's F12 medium (F12-10) supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL Amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %)
MEDIA USED
During the 5-hour treatment with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 µg/mL of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL). - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9
provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen Germany
- method of preparation of S9 mix
from phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- concentration or volume of S9 mix and S9 in the final culture medium
The concentration of S9 was 1.5 % in medium. The protein concentrations of the S9 batch used in the experiments were 35.7, 33.8 and 37.8 mg/mL. - Test concentrations with justification for top dose:
- The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix:
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum suspendable concentration. The 100 mg/mL was the maximum suspendable (homogenous) concentration, which was suitable for the treatment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO, Ham's F12 medium
- Justification for choice of solvent:
The test item was suspended in DMSO at concentration of 250, 200 and 100 mg/mL. The 100 mg/mL was the maximum suspendable (homogenous) concentration, which is suitable for the treatment.
- Justification for percentage of solvent in the final culture medium:
This vehicle is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ham's F12 medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation, 1.0 µL/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- With metabolic activation, 20 µg/mL
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate, triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x10^6 cells/concentration
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment (sampling/recovery times): 48 h
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 19 h
During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 cells were taken on days 1, 3 and 6 and evaluated on day 8
- Selection time:
At the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/mL of thioguanine (6-TG)
- Fixation time (start of exposure up to fixation or harvest of cells): 8 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10^6 cells: 2x5 plates at 2 x 10^5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10^6 clonable cells.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Cloning Efficiency - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria were fulfilled, the test item is considered clearly negative because:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are compatible the distribution of the historical negative control data (based 95 % control limit).
- The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, 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 Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- K1
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay
- Data on osmolality: The osmolality of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay
- Precipitation and time of the determination: not observed
RANGE-FINDING/SCREENING STUDIES: see table 1
HISTORICAL CONTROL DATA: see table 10 - Conclusions:
- The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.
- Executive summary:
The substance, suspended in DMSO, was tested in a Mammalian Gene Mutation Test according OECD TG 476 in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum suspendable concentration. The 100 mg/mL was the maximum suspendable (homogenous) concentration, which was suitable for the treatment.
Phenotypic expression was evaluated up to 8 days following exposure.
In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.
There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values.
The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7,12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
The substance tested without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).
It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.
Referenceopen allclose all
Table 1: Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY (3-hour treatment without and with S9 mix / 20-hour sampling time)
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2150000 |
2150000 |
2081250 |
- |
- |
- |
- |
B |
– |
2100000 |
2200000 |
|||||
- |
C |
– |
2050000 |
2150000 |
|||||
- |
D |
– |
1850000 |
2000000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
7800000 |
7750000 |
7725000 |
5643750 |
100.00 |
0.00 |
- |
B |
– |
7700000 |
7650000 |
|||||
test item |
62.5 |
A |
– |
7600000 |
7700000 |
7650000 |
5568750 |
98.67 |
1.33 |
125 |
A |
– |
6250000 |
6250000 |
6250000 |
4168750 |
73.86 |
26.14 |
|
250 |
A |
– |
5400000 |
5250000 |
5325000 |
3243750 |
57.48 |
42.52 |
|
500 |
A |
– |
4500000 |
4750000 |
4625000 |
2543750 |
45.07 |
54.93 |
|
1000 |
A |
– |
3250000 |
3400000 |
3325000 |
1243750 |
22.04 |
77.96 |
|
2000 |
A |
– |
3000000 |
2750000 |
2875000 |
793750 |
14.06 |
85.94 |
|
EMS |
1 µL/mL |
A |
– |
5000000 |
4800000 |
4900000 |
2818750 |
49.94 |
50.06 |
Solvent control (DMSO) |
- |
A |
+ |
7700000 |
7600000 |
7600000 |
5518750 |
100.00 |
0.00 |
- |
B |
+ |
7600000 |
7500000 |
|||||
test item |
62.5 |
A |
+ |
7350000 |
7400000 |
7375000 |
5293750 |
95.92 |
4.08 |
125 |
A |
+ |
6500000 |
6500000 |
6500000 |
4418750 |
80.07 |
19.93 |
|
250 |
A |
+ |
5950000 |
6000000 |
5975000 |
3893750 |
70.55 |
29.45 |
|
500 |
A |
+ |
4500000 |
4550000 |
4525000 |
2443750 |
44.28 |
55.72 |
|
1000 |
A |
+ |
4400000 |
4400000 |
4400000 |
2318750 |
42.02 |
57.98 |
|
2000 |
A |
+ |
4250000 |
4050000 |
4150000 |
2068750 |
37.49 |
62.51 |
|
Cycl. |
5 µg/mL |
A |
+ |
4800000 |
4900000 |
4850000 |
2768750 |
50.17 |
49.83 |
RICC=Relative Increase in Cell Counts, Cytotoxicity= 100-RICC, EMS: Ethyl methanesulfonate (EMS), Cycl: Cyclophosphamide monohydrate
Table 2: Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY (20-hour treatment without S9 mix / 20-hour sampling time)
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2150000 |
2150000 |
2081250 |
- |
- |
- |
- |
B |
– |
2100000 |
2200000 |
|||||
- |
C |
– |
2050000 |
2150000 |
|||||
- |
D |
– |
1850000 |
2000000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
6850000 |
7200000 |
7012500 |
4931250 |
100.00 |
0.00 |
- |
B |
– |
6900000 |
7100000 |
|||||
test item |
15.7 |
A |
– |
5700000 |
5450000 |
5575000 |
3493750 |
70.85 |
29.15 |
31.3 |
A |
– |
4400000 |
4300000 |
4350000 |
2268750 |
46.01 |
53.99 |
|
62.5 |
A |
– |
2900000 |
3200000 |
3050000 |
968750 |
19.65 |
80.35 |
|
125 |
A |
– |
2200000 |
2500000 |
2350000 |
268750 |
5.45 |
94.55 |
|
250 |
A |
– |
2100000 |
2350000 |
2225000 |
143750 |
2.92 |
97.08 |
|
500 |
A |
– |
1950000 |
1850000 |
1900000 |
-181250* |
-3.68** |
103.68*** |
|
1000 |
A |
– |
1650000 |
1950000 |
1800000 |
-281250* |
-5.70** |
105.70** |
|
2000 |
A |
– |
1900000 |
1600000 |
1750000 |
-331250* |
-6.72** |
106.72** |
|
EMS |
0.4 µL/mL |
A |
– |
4500000 |
4500000 |
4500000 |
2418750 |
49.05 |
50.95 |
RICC=Relative Increase in Cell Counts, Cytotoxicity= 100-RICC, EMS: Ethyl methanesulfonate (EMS)
*: cell number decrease **: zero RICC value, ***:100% cytotoxicity
Table 3:Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY (20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time)
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
2150000 |
2150000 |
2081250 |
- |
- |
- |
- |
B |
– |
2100000 |
2200000 |
|||||
- |
C |
– |
2050000 |
2150000 |
|||||
- |
D |
– |
1850000 |
2000000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
9000000 |
8800000 |
8787500 |
6706250 |
100.00 |
0.00 |
- |
B |
– |
8750000 |
8600000 |
|||||
test item |
15.7 |
A |
– |
7350000 |
7400000 |
7375000 |
5293750 |
78.94 |
21.06 |
31.3 |
A |
– |
5200000 |
5150000 |
5175000 |
3093750 |
46.13 |
53.87 |
|
62.5 |
A |
– |
3800000 |
4050000 |
3925000 |
1843750 |
27.49 |
72.51 |
|
125 |
A |
– |
2900000 |
3050000 |
2975000 |
893750 |
13.33 |
86.67 |
|
250 |
A |
– |
2700000 |
2750000 |
2725000 |
643750 |
9.60 |
90.40 |
|
500 |
A |
– |
2500000 |
2600000 |
2550000 |
468750 |
6.99 |
93.01 |
|
1000 |
A |
– |
2300000 |
2150000 |
2225000 |
143750 |
2.14 |
97.86 |
|
2000 |
A |
– |
2200000 |
2150000 |
2175000 |
93750 |
1.40 |
98.60 |
|
EMS |
0.4 µL/mL |
A |
– |
5400000 |
5500000 |
5450000 |
3368750 |
50.23 |
49.77 |
Solvent control (DMSO) |
- |
A |
+ |
9100000 |
9200000 |
9112500 |
7031250 |
100.00 |
0.00 |
- |
B |
+ |
9000000 |
9150000 |
|||||
test item |
62.5 |
A |
+ |
8900000 |
8850000 |
8875000 |
6793750 |
96.62 |
3.38 |
125 |
A |
+ |
7650000 |
7550000 |
7600000 |
5518750 |
78.49 |
21.51 |
|
250 |
A |
+ |
6950000 |
7100000 |
7025000 |
4943750 |
70.31 |
29.69 |
|
500 |
A |
+ |
5250000 |
5200000 |
5225000 |
3143750 |
44.71 |
55.29 |
|
1000 |
A |
+ |
5000000 |
5000000 |
5000000 |
2918750 |
41.51 |
58.49 |
|
2000 |
A |
+ |
4750000 |
4800000 |
4775000 |
2693750 |
38.31 |
61.69 |
|
Cycl. |
5 µg/mL |
A |
+ |
5500000 |
5750000 |
5625000 |
3543750 |
50.40 |
49.60 |
Table 4: MEAN PERCENTAGE OF CELLS WITH STRUCTURALCHROMOSOME ABERRATION(s) (EXPERIMENT A)
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150 cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control (DMSO) |
- |
3 h |
20 h |
8 |
4 |
test item |
|||||
62.5 µg/mL |
- |
3 h |
20 h |
9 |
3 |
125 µg/mL |
- |
3 h |
20 h |
9 |
4 |
250 µg/mL |
- |
3 h |
20 h |
9 |
4 |
500 µg/mL |
- |
3 h |
20 h |
8 |
4 |
Pos. Control (EMS) |
- |
3 h |
20 h |
43** |
32** |
Solvent control (DMSO) |
+ |
3 h |
20 h |
9 |
4 |
test item |
|||||
62.5 µg/mL |
+ |
3 h |
20 h |
8 |
4 |
125 µg/mL |
+ |
3 h |
20 h |
8 |
5 |
250 µg/mL |
+ |
3 h |
20 h |
8 |
4 |
500 µg/mL |
+ |
3 h |
20 h |
8 |
3 |
Pos. Control (Cycl.) |
+ |
3 h |
20 h |
43** |
37** |
Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL)
Positive control (+S9): Cyclophosphamide (5.0 µg/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
Table 5: MEAN PERCENTAGE OF CELLS WITH STRUCTURALCHROMOSOME ABERRATION(s) (EXPERIMENT B)
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control (DMSO) |
- |
20 h |
20 h |
8 |
4 |
test item |
|||||
7.9 µg/mL |
- |
20 h |
20 h |
8 |
4 |
15.7 µg/mL |
- |
20 h |
20 h |
9 |
5 |
31.3 µg/mL |
- |
20 h |
20 h |
8 |
4 |
Pos. Control (EMS) |
- |
20 h |
20 h |
46** |
38** |
Solvent control (DMSO) |
- |
20 h |
28 h |
9 |
4 |
test item |
|||||
7.8 µg/mL |
- |
20 h |
28 h |
9 |
4 |
15.6 µg/mL |
- |
20 h |
28 h |
7 |
4 |
31.3 µg/mL |
- |
20 h |
28 h |
7 |
3 |
Pos. Control (EMS) |
- |
20 h |
28 h |
45** |
35** |
Positive control (-S9): Ethyl methanesulfonate (0.4µL/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
Table 5: MEAN PERCENTAGE OF CELLS WITH STRUCTURALCHROMOSOME ABERRATION(s) (EXPERIMENT B) continued
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150 cells |
|
incl. gaps |
excl. gaps |
||||
Solvent control (DMSO) |
+ |
3 h |
28 h |
8 |
4 |
test item |
|||||
62.5 µg/mL |
+ |
3 h |
28 h |
10 |
4 |
125 µg/mL |
+ |
3 h |
28 h |
8 |
3 |
250 µg/mL |
+ |
3 h |
28 h |
8 |
4 |
500 µg/mL |
+ |
3 h |
28 h |
8 |
4 |
Pos. Control (Cycl.) |
+ |
3 h |
28 h |
46** |
39** |
Positive control (+S9): Cyclophosphamide (5.0 µg/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
Table 6: NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS (EXPERIMENT A)
Concentration |
S9 mix |
Treatment |
Harvesting time |
Polyploid Cells (mean) |
Endoreduplication (mean) |
Solvent control (DMSO) |
- |
3 h |
20 h |
0.0 |
0.0 |
Test item |
|||||
62.5 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
125 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
250 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
500 µg/mL |
- |
3 h |
20 h |
0.0 |
0.0 |
Pos. Control (EMS)* |
- |
3 h |
20 h |
0.0 |
0.0 |
Solvent control (DMSO) |
+ |
3 h |
20 h |
0.0 |
0.0 |
Test item |
|||||
62.5 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
250 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
500 µg/mL |
+ |
3 h |
20 h |
0.0 |
0.0 |
Pos. Control (Cycl.)** |
+ |
3 h |
20 h |
0.0 |
0.0 |
* :Ethyl methanesulfonate (1.0 µL/mL) ** :Cyclophosphamide (5.0 µg/mL) The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.
Table 7: NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS (EXPERIMENT B)
Concentration |
S9 mix |
Treatment |
Harvesting time |
Polyploid Cells (mean) |
Endoreduplication (mean) |
Solvent control (DMSO) |
- |
20 h |
20 h |
0.0 |
0.0 |
Test item |
|||||
7.9 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
15.7 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20 h |
20 h |
0.0 |
0.0 |
Pos. Control (EMS) |
- |
20 h |
20 h |
0.0 |
0.0 |
Solvent control (DMSO) |
- |
20 h |
28 h |
0.0 |
0.0 |
Test item |
|||||
7.9 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
15.7 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20 h |
28 h |
0.0 |
0.0 |
Pos. Control (EMS) |
- |
20 h |
28 h |
0.0 |
0.0 |
Ethyl methanesulfonate (0.4 µL/mL), The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.
Table 8: NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS (EXPERIMENT B) continued
Concentration |
S9 mix |
Treatment |
Harvesting time |
Polyploid Cells (mean) |
Endoreduplication (mean) |
Solvent control (DMSO) |
+ |
3 h |
28 h |
0.0 |
0.0 |
Reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide |
|||||
62.5 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
250 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
500 µg/mL |
+ |
3 h |
28 h |
0.0 |
0.0 |
Pos. Control (Cycl.)** |
+ |
3 h |
28 h |
0.0 |
0.0 |
** :Cyclophosphamide (5.0 µg/mL), The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.
Table 9: Historical Control Data
3h/20h treatment/sampling time without S9-mix
|
number of aberrant cells/ 150 cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
6.26 |
2.85 |
40.50 |
31.70 |
SD |
0.75 |
0.61 |
3.51 |
3.88 |
Range |
4 - 8 |
2 - 5 |
35-50 |
26-39 |
Lower confidence interval* |
4.70 |
1.59 |
33.22 |
23.64 |
Upper confidence interval* |
7.82 |
4.11 |
47.78 |
39.75 |
n |
23 |
23 |
23 |
23 |
3h/20h treatment/sampling time with S9-mix
|
number of aberrant cells/150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
6.39 |
3.02 |
46.07 |
39.43 |
SD |
0.83 |
0.64 |
2.39 |
2.65 |
Range |
5-9 |
2-5 |
39-51 |
34-46 |
Lower confidence interval* |
4.66 |
1.69 |
41.11 |
33.95 |
Upper confidence interval* |
8.12 |
4.35 |
51.02 |
44.92 |
n |
23 |
23 |
23 |
23 |
20h/20h treatment/sampling time without S9-mix
|
number of aberrant cells/150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
6.17 |
2.93 |
45.26 |
37.85 |
SD |
0.83 |
0.64 |
2.27 |
2.37 |
Range |
4-8 |
2-5 |
38-51 |
33-43 |
Lower confidence interval* |
4.44 |
1.60 |
40.56 |
32.93 |
Upper confidence interval* |
7.90 |
4.27 |
49.96 |
42.77 |
n |
23 |
23 |
23 |
23 |
20h/28h treatment/sampling time without S9-mix
|
number of aberrant cells/ 150cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
6.04 |
2.85 |
45.15 |
37.13 |
SD |
0.83 |
0.61 |
2.25 |
3.36 |
Range |
4-9 |
2-5 |
40-52 |
31-44 |
Lower confidence interval* |
4.31 |
1.59 |
40.48 |
30.16 |
Upper confidence interval* |
7.77 |
4.11 |
49.82 |
44.10 |
n |
23 |
23 |
23 |
23 |
3h/28h treatment/sampling time with S9-mix
|
number of aberrant cells/ 150 cells |
|||
negative control |
positive control |
|||
incl. Gaps |
excl. Gaps |
incl. Gaps |
excl. Gaps |
|
Mean |
6.26 |
3.02 |
45.96 |
38.00 |
SD |
0.63 |
0.53 |
2.41 |
3.68 |
Range |
4-9 |
2-5 |
41-50 |
24-45 |
Lower confidence interval* |
4.96 |
1.92 |
40.95 |
30.36 |
Upper confidence interval* |
7.56 |
4.12 |
50.96 |
45.64 |
n |
23 |
23 |
23 |
23 |
n =number of experiments, SD =standard deviation *:The lower and upper 95% confidence intervals were calculated withC-chart.
Table 1:Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY (5-hour Treatment With and without S9-Mix)
Test group |
Dose |
S9-mix |
Treatment/ |
Number of colonies/200cells/dish |
Mean |
Relativea |
||
dish 1 |
dish 2 |
dish 3 |
||||||
Untreated Control |
– |
– |
5 |
201 |
200 |
198 |
199.7 |
101 |
Solvent Control (DMSO) |
– |
– |
5 |
197 |
199 |
199 |
198.3 |
100 |
Test item |
62.5 |
– |
5 |
201 |
197 |
196 |
198.0 |
100 |
125 |
– |
5 |
195 |
197 |
194 |
195.3 |
98 |
|
250 |
– |
5 |
193 |
189 |
194 |
192.0 |
97 |
|
500 |
– |
5 |
178 |
185 |
184 |
182.3 |
92 |
|
1000 |
– |
5 |
167 |
165 |
165 |
165.7 |
84 |
|
2000 |
– |
5 |
161 |
163 |
166 |
163.3 |
82 |
|
Untreated Control |
– |
+ |
5 |
200 |
198 |
201 |
199.7 |
100 |
Solvent Control (DMSO) |
– |
+ |
5 |
197 |
202 |
198 |
199.0 |
100 |
Test item |
62.5 |
+ |
5 |
197 |
194 |
196 |
195.7 |
98 |
125 |
+ |
5 |
190 |
188 |
185 |
187.7 |
94 |
|
250 |
+ |
5 |
182 |
183 |
185 |
183.3 |
92 |
|
500 |
+ |
5 |
172 |
180 |
178 |
176.7 |
89 |
|
1000 |
+ |
5 |
167 |
173 |
175 |
171.7 |
86 |
|
2000 |
+ |
5 |
155 |
158 |
162 |
158.3 |
80 |
|
aRelative to Solvent Control
Table 2: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-HOUR TREATMENT WITHOUT S9-MIX)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item |
|
|||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control a |
201.7 |
± |
3.06 |
100 |
100 |
2 |
1 |
0 |
2 |
1 |
6 |
100 |
6.00 |
|
||||
Pos. control |
52.7 |
± |
2.52 |
26 |
69 |
200 |
197 |
203 |
199 |
201 |
1000 |
69 |
1449.28** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL a |
194.0 |
± |
2.00 |
96 |
99 |
2 |
0 |
1 |
2 |
2 |
7 |
99 |
7.07 |
|
||||
250 µg/mL a |
189.3 |
± |
2.52 |
94 |
100 |
0 |
2 |
3 |
1 |
0 |
6 |
100 |
6.00 |
|
||||
500 µg/mL a |
184.7 |
± |
3.06 |
92 |
99 |
2 |
0 |
0 |
4 |
0 |
6 |
100 |
6.00 |
|
||||
1000 µg/mL a |
164.3 |
± |
3.06 |
81 |
97 |
1 |
1 |
1 |
0 |
2 |
5 |
97 |
5.15 |
|
||||
2000 µg/mL a |
162.7 |
± |
2.52 |
81 |
97 |
1 |
3 |
0 |
0 |
1 |
5 |
97 |
5.15 |
|
||||
a = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 3: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITHOUT S9-MIX) (continued)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
(without S9-mix) |
|||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control b |
202.3 |
± |
2.08 |
100 |
100 |
0 |
1 |
1 |
1 |
2 |
5 |
101 |
4.95 |
|
||||
Pos. control |
54.3 |
± |
0.58 |
27 |
69 |
188 |
202 |
196 |
186 |
198 |
970 |
70 |
1385.71** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL b |
198.7 |
± |
0.58 |
98 |
99 |
2 |
0 |
1 |
3 |
1 |
7 |
100 |
7.00 |
|
||||
250 µg/mL b |
191.3 |
± |
1.15 |
95 |
99 |
0 |
4 |
0 |
0 |
2 |
6 |
100 |
6.00 |
|
||||
500 µg/mL b |
183.0 |
± |
1.00 |
90 |
99 |
2 |
2 |
0 |
0 |
3 |
7 |
100 |
7.00 |
|
||||
1000 µg/mL b |
168.0 |
± |
1.00 |
83 |
98 |
1 |
1 |
1 |
0 |
2 |
5 |
99 |
5.05 |
|
||||
2000 µg/mL b |
164.7 |
± |
1.53 |
81 |
98 |
2 |
1 |
0 |
0 |
2 |
5 |
99 |
5.05 |
|
||||
b = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 4: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITHOUT S9-MIX) (continued)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
(without S9-mix) |
|||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control c |
202.7 |
± |
1.53 |
100 |
100 |
2 |
1 |
2 |
1 |
0 |
6 |
101 |
5.94 |
|
||||
Pos. control |
52.0 |
± |
2.0 |
26 |
68 |
186 |
190 |
188 |
197 |
190 |
951 |
69 |
1378.26** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL c |
195.0 |
± |
3.61 |
96 |
99 |
0 |
1 |
0 |
1 |
3 |
6 |
100 |
6.00 |
|
||||
250 µg/mL c |
190.0 |
± |
2.00 |
94 |
100 |
2 |
2 |
0 |
0 |
1 |
5 |
100 |
5.00 |
|
||||
500 µg/mL c |
182.3 |
± |
2.08 |
90 |
99 |
3 |
0 |
0 |
1 |
1 |
5 |
100 |
5.00 |
|
||||
1000 µg/mL c |
166.0 |
± |
1.00 |
82 |
97 |
3 |
0 |
0 |
3 |
0 |
6 |
98 |
6.12 |
|
||||
2000 µg/mL c |
159.7 |
± |
1.53 |
79 |
97 |
1 |
2 |
1 |
2 |
0 |
6 |
98 |
6.12 |
|
||||
c = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 5: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITHOUT S9-MIX) (continued)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
|
Batch No.: |
||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control d |
203.0 |
± |
1.00 |
100 |
100 |
2 |
3 |
0 |
0 |
1 |
6 |
102 |
5.88 |
|
||||
Pos. control |
53.7 |
± |
1.15 |
26 |
70 |
202 |
188 |
195 |
199 |
203 |
987 |
72 |
1370.83** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL d |
197.3 |
± |
2.52 |
97 |
99 |
0 |
0 |
0 |
3 |
4 |
7 |
101 |
6.93 |
|
||||
250 µg/mL d |
191.0 |
± |
1.00 |
94 |
99 |
2 |
1 |
1 |
1 |
0 |
5 |
101 |
4.95 |
|
||||
500 µg/mL d |
184.7 |
± |
1.53 |
91 |
98 |
1 |
1 |
1 |
1 |
1 |
5 |
100 |
5.00 |
|
||||
1000 µg/mL d |
167.7 |
± |
0.58 |
83 |
98 |
2 |
2 |
1 |
0 |
2 |
7 |
99 |
7.07 |
|
||||
2000 µg/mL d |
162.0 |
± |
1.00 |
80 |
98 |
1 |
0 |
3 |
2 |
0 |
6 |
100 |
6.00 |
|
||||
d = parallel of second culture
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 6: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITH S9-MIX)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
|
Batch No.: |
||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control a |
202.00 |
± |
3.00 |
100 |
100 |
2 |
0 |
0 |
2 |
1 |
5 |
101 |
5.00 |
|
||||
Pos. control |
118.0 |
± |
2.00 |
58 |
65 |
104 |
96 |
96 |
108 |
102 |
506 |
66 |
766.7** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL a |
192.3 |
± |
2.52 |
95 |
99 |
3 |
0 |
0 |
2 |
0 |
5 |
101 |
4.95 |
|
||||
250 µg/mL a |
182.7 |
± |
2.31 |
90 |
99 |
1 |
1 |
1 |
2 |
1 |
6 |
100 |
6.00 |
|
||||
500 µg/mL a |
173.3 |
± |
1.53 |
86 |
96 |
4 |
0 |
0 |
3 |
0 |
7 |
97 |
7.22 |
|
||||
1000 µg/mL a |
165.0 |
± |
1.00 |
82 |
97 |
2 |
0 |
1 |
1 |
0 |
4 |
98 |
4.08 |
|
||||
2000 µg/mL a |
158.7 |
± |
3.21 |
79 |
91 |
0 |
0 |
2 |
2 |
3 |
7 |
92 |
7.61 |
|
||||
a = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control
Table 7: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITH S9-MIX)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
|
Batch No.: |
||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control b |
202.0 |
± |
1.00 |
100 |
100 |
1 |
1 |
2 |
1 |
2 |
7 |
101 |
6.93 |
|
||||
Pos. control 20 µg/mL) b |
120.3 |
± |
1.15 |
60 |
66 |
95 |
103 |
110 |
104 |
97 |
519 |
67 |
774.63** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL b |
193.3 |
± |
1.53 |
96 |
99 |
2 |
2 |
3 |
0 |
1 |
8 |
100 |
8.00 |
|
||||
250 µg/mL b |
183.7 |
± |
2.08 |
91 |
100 |
1 |
1 |
1 |
0 |
2 |
5 |
101 |
4.95 |
|
||||
500 µg/mL b |
173.7 |
± |
2.52 |
86 |
99 |
2 |
2 |
2 |
1 |
0 |
7 |
100 |
7.00 |
|
||||
1000 µg/mL b |
165.0 |
± |
3.00 |
82 |
98 |
1 |
1 |
2 |
2 |
0 |
6 |
99 |
6.06 |
|
||||
2000 µg/mL b |
161.7 |
± |
2.89 |
80 |
98 |
0 |
2 |
2 |
1 |
1 |
6 |
99 |
6.06 |
|
||||
b = parallel of first culture
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control
Table 8: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITH S9-MIX)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
|
Batch No.: |
||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control c |
202.3 |
± |
3.79 |
100 |
100 |
3 |
0 |
0 |
3 |
0 |
6 |
101 |
5.94 |
|
||||
Pos. control 20 µg/mL) c |
117.7 |
± |
2.52 |
58 |
65 |
106 |
99 |
92 |
106 |
103 |
506 |
65 |
778.46** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL c |
190.0 |
± |
2.65 |
94 |
99 |
1 |
1 |
3 |
1 |
1 |
7 |
100 |
7.00 |
|
||||
250 µg/mL c |
183.7 |
± |
1.53 |
91 |
99 |
2 |
2 |
1 |
0 |
1 |
6 |
100 |
6.00 |
|
||||
500 µg/mL c |
171.0 |
± |
3.61 |
85 |
97 |
2 |
2 |
1 |
3 |
0 |
8 |
98 |
8.16 |
|
||||
1000 µg/mL c |
166.0 |
± |
2.65 |
82 |
97 |
2 |
1 |
1 |
1 |
1 |
6 |
98 |
6.12 |
|
||||
2000 µg/mL c |
159.0 |
± |
3.61 |
79 |
97 |
1 |
1 |
1 |
2 |
1 |
6 |
97 |
6.19 |
|
||||
Table 9: CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d(5-HOUR TREATMENT WITH S9-MIX)
Study code: |
674-476-2609 |
|
|
|||||||||||||||
Test item: |
|
|||||||||||||||||
Test date of Main Mutation Assay: |
April 10, 2018- April 18, 2018 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4 µg/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||
Solvent control d |
202.3 |
± |
1.53 |
100 |
100 |
3 |
0 |
0 |
2 |
2 |
7 |
101 |
6.93 |
|
||||
Pos. control 20 µg/mL) d |
121.3 |
± |
1.53 |
60 |
67 |
108 |
99 |
112 |
98 |
94 |
511 |
67 |
762.69** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125 µg/mL d |
190.3 |
± |
2.52 |
94 |
100 |
0 |
1 |
4 |
0 |
1 |
6 |
100 |
6.00 |
|
||||
250 µg/mL d |
184.7 |
± |
1.53 |
91 |
100 |
1 |
1 |
3 |
0 |
2 |
7 |
100 |
7.00 |
|
||||
500 µg/mL d |
172.0 |
± |
3.61 |
85 |
98 |
0 |
0 |
2 |
2 |
1 |
5 |
99 |
5.05 |
|
||||
1000 µg/mL d |
168.7 |
± |
0.58 |
83 |
98 |
2 |
2 |
0 |
2 |
0 |
6 |
99 |
6.06 |
|
||||
2000 µg/mL d |
162.0 |
± |
1.00 |
80 |
98 |
3 |
3 |
0 |
2 |
0 |
8 |
99 |
8.08 |
|
||||
d = parallel of second culture.
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control
Table 10: Historical Background for Solvent Control Mutant Frequency
|
Without S9 mix |
With S9 mix |
5-hour treatment |
5-hour treatment |
|
Mean |
6.72 |
6.89 |
SD |
0.66 |
0.81 |
Range |
4.90 – 12.12 |
4.12 – 11.76 |
Lower confidence interval |
5.35 |
5.20 |
Upper confidence interval |
8.10 |
8.58 |
n |
21 |
21 |
SD = Standard deviation, n = number of experiments, Solvent: Ham’s F12 medium, DMSO
|
Without S9 mix EMS |
With S9 mix DMBA |
5-hour treatment |
5-hour treatment |
|
Mean |
1517.44 |
745.11 |
SD |
29.86 |
16.86 |
Range |
1357.81 – 1671.46 |
667.11 – 810.29 |
Lower confidence interval |
1455.15 |
709.95 |
Upper confidence interval |
1579.73 |
780.27 |
n |
21 |
21 |
EMS = Ethyl methanesulphonate, DMBA= 7,12-Dimethylbenzanthracene, SD = Standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames-Test:
The test substance was assessed in an Ames test according to EU method B.13/14 and OECD guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 100, TA 98 and TA 102. In two independent experiments several concentrations of test item were used. Each assay was conducted with and without metabolic activation (S9-mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6; 100; 316.2; 1000; 2500 and 5000 µg/plate. No toxic effects of the test item were observed in both experiments up to the highest investigated dose in all strains used. No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Therefore, the test substance was considered non-mutagenic in this Ames test.
Chromosome Aberration Test:
The substance, suspended in DMSO, was tested in a chromosome aberration assay in V79 cells according OECD TG 473 in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix 62.5, 125, 250 and 500 µg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 µg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 7.9, 15.7 and 31.3 µg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 62.5, 125, 250 and 500 µg/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.
Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.
No relevant increase in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.
In the presence of metabolic activation, in experiment A at concentration of 125 µg/mL and in the absence of metabolic activation in experiment B (20 h treatment, 20 h sampling) at concentration of 15.7 µg/mL the values (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.
There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.
The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
In conclusion, the substance did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.Thus, the test item is considered as being non-clastogenic in this system.
In vitro Mammalian Cell Gene Mutation Test:
The substance, suspended in DMSO, was tested in a Mammalian Gene Mutation Test according OECD TG 476 in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 µg/mL
In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum suspendable concentration. The 100 mg/mL was the maximum suspendable (homogenous) concentration, which was suitable for the treatment.
Phenotypic expression was evaluated up to 8 days following exposure.
In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.
There was no precipitation of the test item at any dose level tested. No biologically relevant changes in the osmolality of the test system were noted at the different dose levels tested. The measured pH of treatment solution was similar compared to the control values.
The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7,12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.
The substance tested without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).
It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.
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