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EC number: 210-502-3 | CAS number: 617-04-9
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay (reference 7.6.1-1).
The test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells (refrence 7.6.1-2).
The test item was determined to be non-mutagenic in a HPRT assay using V79 cells of Chinese hamster (reference 7.6.1-3).
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-03-08 to 2021-06-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: V79 cell line (from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line.
- Normal cell cycle time (negative control): see below
For cell lines:
- Absence of Mycoplasma contamination: Before freezing each batch is screened for mycoplasm contamination.
- Methods for maintenance in cell culture: Thawed stock cultures are propagated at 37 °C in 175 cm2 plastic flasks. About 5 x 105 cells per flask are seeded in 30 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium is supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells are sub-cultured twice a week. All incubations are done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Cell cycle length, doubling time or proliferation index: doubling time of V79 cells in stock cultures: approximately 13 hours, determined on May 06, 2011
- Modal number of chromosomes: 22 +/- 2
- Periodically checked for karyotype stability: yes, before freezing
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Please refer to “Methods for maintenance in cell culture” above. - Cytokinesis block (if used):
- Cytochalasin B (1.5 μg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9, prepared in study laboratory
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration of S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- With regard to the molecular weight of the test item, 1950 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.7 to 1950 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment I. The experimental part without S9 mix was repeated in Experiment II since the positive control was invalid (too strong cytotoxicity). The experimental part with S9 mix was repeated in Experiment II as confirmatory experiment since a statistically significant increase and dose-dependency were observed in Experiment I. 1950 µg/mL were chosen as top treatment concentration for Experiment II for both experimental parts (4 hours with and without S9 mix). The continuous treatment without S9 mix was performed in Experiment III with the same top dose (1950 µg/mL).
- Vehicle / solvent:
- - Solvent used: deionised water
- Justification for choice of solvent: The test item was sufficiently soluble in the solvent.
- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium did not exceed 10 %. - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Griseofulvin without metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5.0 – 6.0 x 10^5 cells in 25 cm2 plastic flasks
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4 h; Experiment 2: 24 h
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1: 20 h; Experiment 2: none
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis block: Cytochalasin B (1.5 μg/mL), Experiment 1: 20 h; Experiment 2: 24 h
- Methods of slide preparation and staining technique used including the stain used: Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37 °C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) were added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip.
- Number of cells spread and analysed per concentration: duplicate cultures; 500 cells per culture were scored on coded slides for the determination of the CBPI. At least 1000 binucleate cells were scored per culture for micronuclei on coded slides.
- Criteria for scoring micronucleated cells: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): not applicable
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index - Rationale for test conditions:
- The highest treatment concentration in this study, 1950 µg/mL was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
- Evaluation criteria:
- A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)
A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition.
− The results are outside the range of the laboratory historical solvent control data (95 % confidence interval) - Statistics:
- Statistical significance will be confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis will be conducted for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
A linear regression will be performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance will be considered together. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- 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: No relevant influence on pH was observed.
- Data on osmolality: No relevant influence on osmolarity was observed.
- Precipitation and time of the determination: No precipitation of the test item in the culture medium was observed at the end of treatment.
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.
STUDY RESULTS
Either Griseofulvin (7.0 µg/mL), MMC (0.2 µg/mL) or CPA (3.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
- Statistical analysis:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance was considered together.
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
- Genotoxicity results
In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed by using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed.
HISTORICAL CONTROL DATA
The historical control data were generated in accordance with the OECD Guideline 487. For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data). For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min max range of the positive controls was applied for the evaluation of acceptability - Conclusions:
- Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.
- Executive summary:
The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments. Experiment I and II were conducted with and without metabolic activation (S9-mix) with exposure periods of 4 and 24 hours. Experiment III was conducted with out metabolic activation with an exposure period of 24 hours. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1950 µg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021-03-11 to 2021-04-29
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- 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:
- 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 30 May 2008
- 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 (hypoxanthine-guanine phosphoribosyl transferase)
- 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 cell line V79 (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time (negative control): doubling time 12 - 16 h
For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasm contamination.
- Number of passages: not specified
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3Χ10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly.
- Cell cycle length, doubling time or proliferation index : doubling time 12 - 16 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes. Each master cell stock is checked for karyotype stability.
- Periodically ‘cleansed’ of spontaneous mutants: yes. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air. For details on medium see above. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9, prepared in study laboratory
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration of S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- 60.9; 121.9; 243.8; 487.5; 975.0; 1950.0 μg/mL
In the pre-experiment to determine the toxicity of the test item the highest concentration was equal to a molar concentration of approximately 10 mM, with respect to the OECD guideline 476. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2. - Vehicle / solvent:
- - Solvent used: deionised water
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10 % (v/v). This was chosen due to its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- deionised water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment: 14-18 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): approximately 7 days
- Selection time: 7-11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-18 days
- If a selective agent is used indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine (6-TG; 11 μg/mL), approximately 8 days (evaluation for viability) and approximately 9 ± 2 days (mutation analysis)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 0.7 to 1.2×10^7 24 h before treatment; The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
- Any supplementary information relevant to cytotoxicity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. - Rationale for test conditions:
- The test conditions were selected according to OECD TG and results of the pre-experiment.
- Evaluation criteria:
- A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
- 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 historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal. - Statistics:
- The statistical analysis was performed on the mean values of culture I and II for the main experiments.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics. It was performed only for the positive controls, since all mean mutant frequencies of the groups treated with the test item were well within the 95 % confidence interval of our laboratory’s historical negative control data.
However, both, biological and statistical significance will be considered together. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Possibility of evaporation from medium: not specified
- Water solubility: sufficiently soluble
- Precipitation and time of the determination: Neither precipitation nor phase separation was observed up to the highest tested concentration in the absence and in the presence of metabolic activation
- Definition of acceptable cells for analysis: Colonies with more than 50 cells were counted
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES
In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50 % or below was observed up to the highest concentration with and without metabolic activation.
STUDY RESULTS
- Concurrent vehicle negative and positive control data
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Please refer to “Any other information on results”.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 0.7 to 1.2×10^7 were seeded 24 h before treatment. After treatment at least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultivated twice.
o Number of cells plated in selective and non-selective medium: selective: 4 - 5Χ10^5 cells/75 cm^² cell culture flask (5); non-selective: approx. 500 cells/25 cm^² flask (2)
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: : Please refer to “Any other information on results”.
HISTORICAL CONTROL DATA
- Positive historical control data: : Please refer to “Any other information on results”.
- Negative (solvent/vehicle) historical control data: : Please refer to “Any other information on results”. - Conclusions:
- Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
- Executive summary:
A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (1950 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. Based on the results of the pre-experiment the following concentrations were chosen for the main experiment in the presence and absence of metabolic activation: 60.9, 121.9, 243.8, 487.5, 975.0 and 1950.0 μg/mL. Neither precipitation nor phase separation occurred up the highest investigated concentration after four hours treatment. No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50 % (mean value of both parallel cultures) occurred up to the highest concentration. Consequently, the concentrations of 121.9 to 1950 μg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95 % confidence interval of the solvent historical control data. Linear regression analysis showed no statistically significant trend. The outcome of the experiment was clearly negative in the presence and in the absence of metabolic activation. The positive controls induced distinct increases in mutant frequencies, consistent with the laboratory’s historical positive control data, thus demonstrating the sensitivity of the test system and the efficacy of the S9 mix. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2020-10-27 to 2020-12-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium: histidine
Escherichia coli: tryptophan - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: induced rat liver
- volume of S9 mix in the final culture medium: 0.5 mL S9 mix per plate
- quality controls of S9: not specified - Test concentrations with justification for top dose:
- experiment 1 (dose-finding test): 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate
experiment 2 (main test): 5000, 2500, 1250, 625, and 313 µg/plate
Top dose according to guideline. - Vehicle / solvent:
- - Solvent used: water for injection
- Justification for choice of solvent/vehicle:
Water for injection was selected as the solvent because the test substance is water-soluble and dissolves in water at a rate of 100 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2-AA); 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl (ICR-191)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition - Rationale for test conditions:
- Test conditions according to guideline were used.
- Evaluation criteria:
- A positive result was determined when the mean number of revertant colonies in the test substance-treated group increased more than 2-fold compared to the mean number of spontaneous revertant colonies (negative control value) and dose-response and reproducibility were observed, or when the mean number of revertant colonies increased more than 2-fold compared to the negative control value and reproducibility was observed in the dose-finding and main studies even when no clear dose-response was observed.
- Statistics:
- No statistical method was used for the determination.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No effects on pH reported.
- Data on osmolality: Not applicable
- Possibility of evaporation from medium: not specified
- Water solubility: Test item was sufficiently water soluble.
- Precipitation and time of the determination: No precipitation was detected.
- Definition of acceptable cells for analysis: Not applicable
- Other confounding effects: No futher effects observed.
RANGE-FINDING/SCREENING STUDIES: A dose-finding study was conducted and is used as experiment 1.
Ames test:
- Signs of toxicity : No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.
HISTORICAL CONTROL DATA: Please refer to table 3. - Conclusions:
- The test item was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
To investigate whether the test item has the ability to induce gene mutations a bacterial reverse mutation assay according to OECD 471 was conducted. The preincubation method was used withSalmonella typhimurium (hereinafter referred to as S. typhimurium) TA100, TA1535, TA98, TA1537, and Escherichia coli (hereinafter referred to as E. coli) WP2uvrA under the conditions of metabolic activation and non-metabolic activation using S9. Water for injection was used as the solvent for the test substance. In order to establish the test dose, a dose-finding test was conducted with a total of seven doses of 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate diluted in six steps wit the ratio of 4.Results showed that there was no growth inhibition or precipitation of the test substance in any of the strains with or without metabolic activation. Therefore, the main test was conducted at a total of five doses of 5000, 2500, 1250, 625, and 313 µg/plate for all strains with and without metabolic activation. Regardless of the presence or absence of metabolic activation in both the dose-finding study and the main study, there was no increase of more than twice the negative control value in the number of revertant mutant colonies, in either the base-pair-substituted or the frameshifted strains, and no dose-response was observed. Based on the above test results, the test item was judged to have no gene mutagenic activity against bacteria under the test conditions.
Referenceopen allclose all
Summary of results
Exp. | interval | concentration | Proliferation index CBPI | Cytostatsis in %* | Micronucleated cells in %** | 95 % ctrl limit |
Exposure period 4 hrs without S9 mix | ||||||
II | 24 h | Solvent control 1 | 1.93 |
| 0.8 | 0-2.45 |
Positive control 2 | 1.43 | 53.7 | 13.20s |
| ||
637 | 1.92 | 0.9 | 0.65 |
| ||
1114 | 1.92 | 1.2 | 0.65 |
| ||
1950 | 1.90 | 3.2 | 0.85 |
| ||
Trend test: p-value 0.740 | ||||||
Exposure period 24 hrs without S9 mix | ||||||
III | 24 h | Solvent control 1 | 2.21 |
| 1.10 | 0-1.7 |
Positive control 3 | 2.30 | n.c. | 10.90s |
| ||
637 | 2.07 | 11.8 | 0.6 |
| ||
1114 | 2.11 | 8.1 | 0.6 |
| ||
1950 | 2.07 | 11.2 | 0.8 |
| ||
Trend test: p-value 0.740 | ||||||
Exposure period 4 hrs with S9 mix | ||||||
I | 24 h | Solvent control 1# | 1.96 |
| 0.65 | 0.01-1.99 |
Positive control 4 | 1.54 | 43.1 | 6.20s |
| ||
637# | 1.92 | 3.8 | 0.7 |
| ||
1114# | 1.99 | n.c. | 0.83 |
| ||
1950# | 1.96 | n.c. | 1.15s |
| ||
Trend test: p-value 0.040T | ||||||
II | 24 h | Solvent control 1 | 1.87 |
| 1.25 | 0.01-1.99 |
Positive control 4 | 1.34 | 60.5 | 22.20 |
| ||
637 | 1.87 | 0.1 | 0.8 |
| ||
1114 | 1.87 | n.c. | 0.75 |
| ||
1950 | 1.85 | 1.6 | 0.9 |
| ||
Trend test: p-value 0.418 |
* For the positive control groups and the test item treatment groups the values are related to the solvent controls
** The number of micronucleated cells was determined in a sample of 2000 binucleated cells
# The number of micronucleated cells was determined in a sample of 4000 binucleated cells
T Trend analysis via linear regression is significant (p ˂ 0.05)
n.c. Not calculated as the CBPI is equal or higher than the solvent control value
1 Deion. water 10.0 % (v/v)
2 MMC 0.2 µg/mL 3
Griseofulvin 7.0 µg/mL
4 CPA 3.0 µg/m
Table 1 Summary of Results
|
|
|
| relative | relative | rel. adjusted | mutant | 95% | statistical analysis* | ||
| conc. | P/ | S9 | cloning | cell | cloning | colonies/ | confidence | |||
| µg/mL | PS | mix | efficiency I | density | efficiency I | 106 cells | interval | t-test | linear regression | |
|
|
|
| % | % | % |
|
| |||
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
Main Experiment / 4h treatment | Mean values of culture I and II |
|
|
| |||||||
Solvent control with water |
|
| - | 100.0 | 100.0 | 100.0 | 12.4 | 2.9 - 22.4 |
|
| |
Positive control (EMS) | 300.0 |
| - | 101.4 | 84.3 | 85.5 | 236.8 | 2.9 – 22.4 | 0.000S |
| |
Test item | 60.9 | - | - | 104.0 | 94.3 | 98.0 | # |
|
| ||
Test item | 121.9 | - | - | 105.4 | 75.8 | 79.9 | 7.2 | 2.9 – 22.4 | n.c. | 0.769 | |
Test item | 243.8 | - | - | 88.2 | 94.4 | 83.2 | 13.5 | 2.9 – 22.4 | n.c. | ||
Test item | 487.5 | - | - | 102.4 | 81.2 | 83.4 | 7.0 | 2.9 – 22.4 | n.c. | ||
Test item | 975.0 | - | - | 95.4 | 95.7 | 90.9 | 7.4 | 2.9 – 22.4 | n.c. | ||
Test item | 1950.0 | - | - | 89.7 | 102.5 | 91.6 | 10.2 | 2.9 – 22.44 | n.c. | ||
Solvent control with water |
|
| + | 100.0 | 100.0 | 100.0 | 10.3 | 2.9 – 23.7 |
|
| |
Positive control (DMBA) | 2.3 |
| + | 89.6 | 81.5 | 73.3 | 143.7 | 2.9 – 23.7 | 0.000S |
| |
Test item | 60.9 | - | + | 85.3 | 86.4 | 73.5 | # |
|
| ||
Test item | 121.9 | - | + | 91.8 | 92.9 | 85.1 | 6.9 | 2.9 – 23.7 | n.c. | 0.650 | |
Test item | 243.8 | - | + | 91.8 | 83.9 | 77.1 | 5.8 | 2.9 – 23.7 | n.c. | ||
Test item | 487.5 | - | + | 88.6 | 83.4 | 74.2 | 7.7 | 2.9 – 23.7 | n.c. | ||
Test item | 975.0 | - | + | 94.2 | 88.3 | 83.2 | 7.1 | 2.9 – 23.7 | n.c. | ||
Test item | 1950.0 | - | + | 91.4 | 87.3 | 79.8 | 7.2 | 2.9 – 23.7 | n.c. |
* statistical analysis based on the mean values of culture I and II
P/PS = Precipitation/Phase separation at the end of treatment
S = significant trend (p < 0.05)
MF Mutant Frequency
# cultures not continued as a minimum of only four analyzable concentrations are required
Table 2 Cloning Efficiency I (Survival). culture I
| conc. | P/ | S9 | cells | number of colonies per flask | CE I | CE I | cells/mL | cell density | rel. adjusted | ||
Test group | µg/mL | PS | mix | seeded | found | absolute | relative | at 1st | % | CE I | ||
|
|
|
| I/II | I | II | mean |
| % | subcultivation | of control | % |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Solvent control with water |
|
| - | 518 | 398 | 436 | 417.0 | 0.8 | 100.0 | 1254000 | 100.0 | 100.0 |
Positive control with EMS | 300.0 |
| - | 535 | 445 | 438 | 441.5 | 0.8 | 102.5 | 998000 | 79.6 | 81.6 |
Test item | 60.9 | - | - | 509 | 421 | 396 | 408.5 | 0.8 | 99.7 | 1232000 | 98.2 | 97.9 |
Test item | 121.9 | - | - | 519 | 439 | 445 | 442.0 | 0.9 | 105.8 | 884000 | 70.5 | 74.6 |
Test item | 243.8 | - | - | 519 | 357 | 360 | 358.5 | 0.7 | 85.8 | 1256000 | 100.2 | 85.9 |
Test item | 487.5 | - | - | 522 | 432 | 399 | 415.5 | 0.8 | 98.9 | 908000 | 72.4 | 71.6 |
Test item | 975.0 | - | - | 509 | 375 | 316 | 345.5 | 0.7 | 84.3 | 1234000 | 98.4 | 83.0 |
Test item | 1950.0 | - | - | 555 | 366 | 386 | 376.0 | 0.7 | 84.2 | 1344000 | 107.2 | 90.2 |
Solvent control with water |
|
| + | 527 | 305 | 296 | 300.5 | 0.6 | 100.0 | 1422000 | 100.0 | 100.0 |
Positive control with DMBA | 2.3 |
| + | 540 | 330 | 298 | 314.0 | 0.6 | 102.0 | 1188000 | 83.5 | 85.2 |
Test item | 60.9 | - | + | 542 | 302 | 279 | 290.5 | 0.5 | 94.0 | 1192000 | 83.8 | 78.8 |
Test item | 121.9 | - | + | 530 | 268 | 273 | 270.5 | 0.5 | 89.5 | 1432000 | 100.7 | 90.1 |
Test item | 243.8 | - | + | 509 | 286 | 264 | 275.0 | 0.5 | 94.8 | 1234000 | 86.8 | 82.2 |
Test item | 487.5 | - | + | 544 | 257 | 310 | 283.55 | 0.5 | 91.4 | 1318000 | 82.7 | 84.7 |
Test item | 975.0 | - | + | 523 | 275 | 321 | 298.0 | 0.6 | 99.9 | 1268000 | 89.2 | 89.1 |
Test item | 1950.0 | - | + | 535 | 274 | 282 | 278.0 | 0.5 | 91.1 | 1296000 | 91.1 | 83.1 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
Table 3 Cloning Efficiency II (Viability). culture I
| conc. | P/ | S9 | cells | number of colonies per flask | CE II | CE II | cells | cells | ||
Test group | µg/mL | PS | mix | seeded | found | absolute | relative | seeded | survived | ||
|
|
|
| I/II | I | II | mean |
| % |
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
Solvent control with water |
|
| - | 537 | 397 | 412 | 404.5 | 0.8 | 100.0 | 457400 | 344541 |
Positive control with EMS | 300.0 |
| - | 506 | 370 | 361 | 365.5 | 0.7 | 95.9 | 494400 | 357121 |
Test item | 60.9 | - | - | culture was not continued# | |||||||
Test item | 121.9 | - | - | 520 | 453 | 428 | 440.5 | 0.8 | 112.5 | 387000 | 327834 |
Test item | 243.8 | - | - | 524 | 329 | 347 | 338.0 | 0.6 | 85.6 | 388200 | 250404 |
Test item | 487.5 | - | - | 569 | 353 | 336 | 344.5 | 0.6 | 80.4 | 400800 | 242664 |
Test item | 975.0 | - | - | 534 | 413 | 381 | 397.0 | 0.7 | 98.7 | 471600 | 350609 |
Test item | 1950.0 | - | - | 529 | 397 | 415 | 406.0 | 0.8 | 101.9 | 417600 | 320502 |
Solvent control with water |
|
| + | 592 | 411 | 445 | 428.0 | 0.7 | 100.0 | 407400 | 294539 |
Positive control with DMBA | 2.3 |
| + | 562 | 417 | 428 | 422.5 | 0.88 | 104.0 | 437400 | 328828 |
Test item | 60.9 | - | + | culture was not continued# | |||||||
Test item | 121.9 | - | + | 502 | 429 | 448 | 438.5 | 0.9 | 120.8 | 403200 | 352198 |
Test item | 243.8 | - | + | 574 | 420 | 454 | 437.0 | 0.8 | 105.3 | 407400 | 310163 |
Test item | 487.5 | - | + | 548 | 442 | 417 | 429.5 | 0.8 | 108.4 | 404400 | 316952 |
Test item | 975.0 | - | + | 528 | 455 | 423 | 439.0 | 0.8 | 115.0 | 436800 | 363173 |
Test item | 1950.0 | - | + | 553 | 323 | 363 | 343.0 | 0.6 | 85.8 | 408300 | 253249 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
# culture was not continued as a minimum of only four analysable concentrations is required
Table 4 Mutagenicity data (Mutation rates). culture I
| conc. | P/ | S9 | number of colonies per flask | mutant | ||||||
Test group | µg/mL | PS | mix | found after plating in TG medium | standard | colonies | |||||
|
|
|
| I | II | III | IV | V | mean | deviation | per 106 cells |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
Solvent control with water |
|
| - | 5 | 2 | 6 | 3 | 8 | 4.8 | 2.4 | 13.9 |
Positive control with EMS | 300.0 |
| - | 68 | 78 | 66 | 82 | 65 | 71.8 | 7.7 | 201.1 |
Test item | 60.9 | - | - | culture was not continued# | |||||||
Test item | 121.9 | - | - | 2 | 3 | 5 | 3 | 2 | 3.0 | 1.2 | 9.2 |
Test item | 243.8 | - | - | 5 | 1 | 3 | 1 | 2 | 2.4 | 1.7 | 9.6 |
Test item | 487.5 | - | - | 3 | 2 | 1 | 1 | 2 | 1.8 | 0.8 | 7.4 |
Test item | 975.0 | - | - | 4 | 1 | 4 | 4 | 4 | 3.4 | 1.3 | 9.7 |
Test item | 1950.0 | - | - | 2 | 3 | 1 | 4 | 2 | 2.4 | 1.1 | 7.5 |
Solvent control with water |
|
| + | 3 | 5 | 1 | 8 | 1 | 3.6 | 3.0 | 12.2 |
Positive control with DMBA | 2.3 |
| + | 46 | 43 | 51 | 47 | 46 | 46.6 | 2.9 | 141.7 |
Test item | 60.9 | - | + | culture was not continued# | |||||||
Test item | 121.9 | - | + | 4 | 4 | 3 | 2 | 1 | 2.88 | 1.3 | 8.0 |
Test item | 243.8 | - | + | 1 | 2 | 1 | 4 | 2 | 2.0 | 1.2 | 6.4 |
| 487.5 | - | + | 3 | 3 | 1 | 2 | 2 | 2.2 | 0.8 | 6.9 |
Test item | 975.0 | - | + | 2 | 2 | 4 | 3 | 3 | 2.8 | 0.8 | 7.7 |
Test item | 1950.0 | - | + | 2 | 1 | 2 | 1 | 3 | 1.8 | 0.8 | 7.1 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
# culture was not continued as a minimum of only four analysable concentrations is required
Table 5 Cloning Efficiency I (Survival). culture II
| conc. | P/ | S9 | cells | number of colonies per flask | CE I | CE I | cells/mL | cell density | rel. adjusted | ||
Test group | µg/mL | PS | mix | seeded | found | absolute | relative | at 1st | % | CE I | ||
|
|
|
| I/II | I | II | mean |
| % | subcultivation | of control | % |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Solvent control with water |
|
| - | 561 | 440 | 431 | 435.5 | 0.8 | 100.0 | 1982000 | 100.0 | 100.0 |
Positive control with EMS | 300.0 |
| - | 500 | 369 | 410 | 389.5 | 0.8 | 100.3 | 1766000 | 89.1 | 89.4 |
Test item | 60.9 | - | - | 507 | 411 | 442 | 426.5 | 0.8 | 108.4 | 1792000 | 90.4 | 98.0 |
Test item | 121.9 | - | - | 526 | 435 | 423 | 429.0 | 0.8 | 105.1 | 1608000 | 81.1 | 85.2 |
Test item | 243.8 | - | - | 575 | 392 | 417 | 404.5 | 0.7 | 90.6 | 1758000 | 88.7 | 80.4 |
Test item | 487.5 | - | - | 505 | 430 | 400 | 415.0 | 0.8 | 105.9 | 1784000 | 90.0 | 95.3 |
Test item | 975.0 | - | - | 521 | 426 | 435 | 430.5 | 0.8 | 106.4 | 1842000 | 92.9 | 98.9 |
Test item | 1950.0 | - | - | 548 | 419 | 391 | 405.0 | 0.7 | 95.2 | 1938000 | 97.8 | 93.1 |
Solvent control with water |
|
| + | 509 | 337 | 308 | 322.5 | 0.6 | 100.0 | 1232000 | 100.0 | 100.0 |
Positive control with DMBA | 2.3 |
| + | 533 | 267 | 255 | 261.0 | 0.5 | 77.3 | 980000 | 79.5 | 61.5 |
Test item | 60.9 | - | + | 543 | 256 | 271 | 263.5 | 0.5 | 76.6 | 1096000 | 89.0 | 68.1 |
Test item | 121.9 |
| + | 519 | 316 | 303 | 309.5 | 0.6 | 94.1 | 1048000 | 85.1 | 80.1 |
Test item | 243.8 | - | + | 535 | 295 | 307 | 301.0 | 0.6 | 88.8 | 998000 | 81.0 | 71.9 |
Test item | 487.5 | - | + | 522 | 273 | 295 | 284.0 | 0.5 | 85.9 | 914000 | 74.2 | 63.7 |
Test item | 975.0 | - | + | 533 | 292 | 306 | 299.0 | 0.6 | 88.5 | 1076000 | 87.3 | 77.3 |
Test item | 1950.0 | - | + | 509 | 301 | 290 | 295.5 | 0.6 | 91.6 | 1028000 | 83.4 | 76.5 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
Table 6 Cloning Efficiency II (Viability). culture II
| conc. | P/ | S9 | cells | number of colonies per flask | CE II | CE II | cells | cells | ||
Test group | µg/mL | PS | mix | seeded | found | absolute | relative | seeded | survived | ||
|
|
|
| I/II | I | II | mean |
| % |
|
|
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
Solvent control with water |
|
| - | 553 | 368 | 404 | 386.0 | 0.7 | 100.0 | 555600 | 387815 |
Positive control with EMS | 300.0 |
| - | 513 | 358 | 374 | 366.0 | 0.7 | 102.2 | 391800 | 279530 |
Test item | 60.9 | - | - | culture was not continued# | |||||||
Test item | 121.9 | - | - | 523 | 451 | 477 | 464.0 | 0.9 | 127.1 | 523800 | 464710 |
Test item | 243.8 | - | - | 553 | 303 | 325 | 314.0 | 0.6 | 81.3 | 444600 | 252449 |
Test item | 487.5 | - | - | 512 | 280 | 324 | 302.0 | 0.6 | 84.5 | 406200 | 239595 |
Test item | 975.0 | - | - | 564 | 357 | 350 | 353.5 | 0.6 | 89.8 | 444600 | 278663 |
Test item | 1950.0 | - | - | 518 | 362 | 388 | 375.0 | 0.7 | 103.7 | 406200 | 294064 |
Solvent control with water |
|
| + | 541 | 421 | 409 | 415.0 | 0.8 | 100.0 | 401400 | 307913 |
Positive control with DMBA | 2.3 |
| + | 515 | 412 | 415 | 413.5 | 0.8 | 104.7 | 422400 | 339150 |
Test item | 60.9 | - | + | culture was not continued# | |||||||
Test item | 121.9 | - | + | 514 | 402 | 395 | 398.5 | 0.8 | 101.1 | 402300 | 311900 |
Test item | 243.8 | - | + | 556 | 441 | 418 | 429.5 | 0.8 | 100.7 | 394800 | 304976 |
Test item | 487.5 | - | + | 500 | 423 | 405 | 414.0 | 0.8 | 107.9 | 400500 | 331614 |
Test item | 975.0 | - | + | 550 | 411 | 427 | 419.0 | 0.8 | 99.3 | 402000 | 306251 |
Test item | 1950.0 | - | + | 555 | 401 | 416 | 408.5 | 0.7 | 96.0 | 405000 | 298095 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
# culture was not continued as a minimum of only four analysable concentrations is required
Table 7 Mutagenicity data (Mutation rates). culture II
| conc. | P/ | S9 | number of colonies per flask | mutant | ||||||
Test group | µg/mL | PS | mix | found after plating in TG medium | standard | colonies | |||||
|
|
|
| I | II | III | IV | V | mean | deviation | per 106 cells |
Column | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
Solvent control with water |
|
| - | 1 | 3 | 8 | 4 | 5 | 4.2 | 2.6 | 10.8 |
Positive control with EMS | 300.0 |
| - | 88 | 69 | 72 | 72 | 80 | 76.2 | 7.8 | 272.6 |
Test item | 60.9 | - | - | culture was not continued# | |||||||
Test item | 121.9 | - | - | 3 | 3 | 1 | 4 | 1 | 2.4 | 1.3 | 5.2 |
Test item | 243.8 | - | - | 5 | 3 | 6 | 6 | 2 | 4.4 | 1.8 | 17.4 |
Test item | 487.5 | - | - | 1 | 2 | 2 | 1 | 2 | 1.6 | 0.5 | 6.7 |
Test item | 975.0 | - | - | 1 | 1 | 1 | 1 | 3 | 1.4 | 0.9 | 5.0 |
Test item | 1950.0 | - | - | 6 | 3 | 3 | 3 | 4 | 3.8 | 1.3 | 12.9 |
Solvent control with water |
|
| + | 2 | 1 | 3 | 2 | 5 | 2.6 | 1.5 | 8.4 |
Positive control with DMBA | 2.3 |
| + | 40 | 53 | 46 | 57 | 51 | 49.4 | 6.6 | 145.7 |
Test item | 60.9 | - | + | culture was not continued# | |||||||
Test item | 121.9 | - | + | 3 | 2 | 1 | 2 | 1 | 1.8 | 0.8 | 5.8 |
Test item | 243.8 | - | + | 3 | 1 | 1 | 1 | 2 | 1.6 | 0.9 | 5.2 |
Test item | 487.5 | - | + | 2 | 3 | 2 | 5 | 2 | 2.8 | 1.3 | 8.4 |
Test item | 975.0 | - | + | 1 | 1 | 2 | 3 | 3 | 2.0 | 1.0 | 6.5 |
Test item | 1950.0 | - | + | 2 | 4 | 2 | 1 | 2 | 2.2 | 1.1 | 7.4 |
P/PS = Precipitation/Phase separation at the beginning and at the end of treatment
# culture was not continued as a minimum of only four analysable concentrations is required
Table 8 Historical Laboratory Control Data
These values represent the historical control data from 2018-2020
Number of mutant colonies per 106 cells (mean values) | ||
without metabolic activation (4 hours treatment time) | ||
| Positive control EMS 300 µg/mL | Solvent control (medium. acetone. water. DMSO. ethanol. THF) |
Range: | 90 – 406.8 | 5.0 – 30.5 |
Mean value: | 245.3 | 12.6 |
Standard deviation: | 67.5 | 4.9 |
95% confidence interval | - | 2.9 – 22.4 |
Number of studies: | 83 | 83 |
with metabolic activation (4 hours treatment time) | ||
| Positive control DMBA 2.3 µg/mL | Solvent control (medium. acetone. water. DMSO. ethanol. THF) |
Range: | 54.5 – 347.1 | 5.3 – 27.5 |
Mean value: | 145.4 | 13.3 |
Standard deviation: | 66.5 | 5.2 |
95% confidence interval | - | 2.9 – 23.7 |
Number of studies: | 83 | 83 |
The 95 % confidence interval is derived from the mean value plus/minus 2 times the standard deviation.
Table 1: Experiment 1
Dose (µg/plate) | Mean number of revertant colonies/2 replicate plates with different strains of Salmonella typhimurium and E. coli | ||||
TA1535 | TA1537 | TA98 | TA100 | WP2 uvrA | |
Results without S9 | |||||
Water | 12 | 8 | 29 | 124 | 35 |
1.22 | 11 | 6 | 30 | 117 | 30 |
4.88 | 10 | 5 | 27 | 114 | 32 |
19.5 | 13 | 5 | 26 | 116 | 38 |
78.1 | 12 | 6 | 29 | 122 | 36 |
313 | 14 | 6 | 32 | 114 | 30 |
1250 | 13 | 8 | 25 | 115 | 30 |
5000 | 14 | 5 | 21 | 121 | 32 |
NaN3 (0.5) | 521 | ||||
AF-2 (0.01) | 530 | 104 | |||
AF-2 (0.1) | 541 | ||||
ICR-191 | 1487 | ||||
Results with S9 | |||||
Water | 13 | 10 | 45 | 124 | 37 |
1.22 | 13 | 8 | 40 | 121 | 40 |
4.88 | 12 | 10 | 33 | 139 | 31 |
19.5 | 8 | 8 | 42 | 122 | 30 |
78.1 | 12 | 10 | 41 | 107 | 37 |
313 | 7 | 8 | 38 | 104 | 28 |
1250 | 10 | 10 | 39 | 140 | 33 |
5000 | 9 | 8 | 33 | 119 | 34 |
B(a)P (5) | 74 | 335 | 1063 | ||
2-AA (2) | 285 | ||||
2-AA (10) | 542 |
Table 2: Experiment 2
Dose (µg/plate) | Mean number of revertant colonies/2 replicate plates with different strains of Salmonella typhimurium and E. coli | ||||
TA1535 | TA1537 | TA98 | TA100 | WP2 uvrA | |
Results without S9 | |||||
Water | 14 | 10 | 23 | 127 | 21 |
313 | 13 | 5 | 27 | 110 | 20 |
625 | 15 | 2 | 28 | 117 | 24 |
1250 | 16 | 5 | 22 | 134 | 20 |
2500 | 12 | 6 | 26 | 109 | 19 |
5000 | 15 | 3 | 28 | 106 | 18 |
NaN3 (0.5) | 459 | ||||
AF-2 (0.01) | 494 | 98 | |||
AF-2 (0.1) | 459 | ||||
ICR-191 | 1311 | ||||
Results with S9 | |||||
Water | 14 | 8 | 38 | 136 | 27 |
313 | 9 | 9 | 35 | 126 | 22 |
625 | 13 | 7 | 42 | 108 | 22 |
1250 | 11 | 5 | 33 | 125 | 20 |
2500 | 12 | 7 | 42 | 122 | 21 |
5000 | 11 | 4 | 34 | 117 | 17 |
B(a)P (5) | 64 | 397 | |||
2-AA (2) | 295 | 295 | |||
2-AA (10) | 633 |
Table 3: Historical Data (preincubation method)
Period: From June 1, 2020 to September 11, 2020
Strain | S9 mix | Management ranges | Mean | Management ranges | number of plates | |||
-3SD | -2SD | +2SD | +3SD | |||||
TA 100 | - | Solvent control | 74 | 92 | 128 | 165 | 183 | 234 |
Positive control | 413 | 455 | 540 | 624 | 66 | 228 | ||
+ | Solvent control | 78 | 97 | 137 | 176 | 196 | 234 | |
Positive control | 817 | 927 | 1146 | 1364 | 1485 | 228 | ||
TA1535 | - | Solvent control | 2 | 5 | 11 | 18 | 21 | 234 |
Positive control | 277 | 331 | 438 | 546 | 599 | 228 | ||
+ | Solvent control | 1 | 4 | 11 | 18 | 21 | 234 | |
Positive control | 214 | 256 | 340 | 424 | 466 | 228 | ||
WP2uvrA | - | Solvent control | 6 | 12 | 24 | 36 | 42 | 234 |
Positive control | 56 | 72 | 102 | 133 | 148 | 228 | ||
+ | Solvent control | 7 | 14 | 27 | 40 | 47 | 234 | |
Positive control | 320 | 409 | 587 | 766 | 855 | 228 | ||
TA 98 | - | Solvent control | 4 | 11 | 24 | 38 | 45 | 234 |
Positive control | 430 | 464 | 532 | 600 | 634 | 228 | ||
+ | Solvent control | 14 | 22 | 37 | 52 | 59 | 234 | |
Positive control | 248 | 288 | 369 | 449 | 490 | 228 | ||
TA 1537 | - | Solvent control | 1 | 1 | 7 | 13 | 16 | 234 |
Positive control | 291 | 630 | 1310 | 1989 | 2329 | 228 | ||
+ | Solvent control | 1 | 3 | 11 | 19 | 23 | 234 | |
Positive control | 28 | 42 | 70 | 99 | 113 | 228 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test (reference 7.6.1-1)
To investigate whether the test item has the ability to induce gene mutations a bacterial reverse mutation assay according to OECD 471 was conducted. The preincubation method was used with Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA100, TA1535, TA98, TA1537, and Escherichia coli (hereinafter referred to as E. coli) WP2uvrAunder the conditions of metabolic activation and non-metabolic activation using S9. Water for injection was used as the solvent for the test substance. In order to establish the test dose, a dose-finding test was conducted with a total of seven doses of 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate diluted in six steps with the ratio of 4.Results showed that there was no growth inhibition or precipitation of the test substance in any of the strains with or without metabolic activation. Therefore, the main test was conducted at a total of five doses of 5000, 2500, 1250, 625, and 313 µg/plate for all strains with and without metabolic activation. Regardless of the presence or absence of metabolic activation in both the dose-finding study and the main study, there was no increase of more than twice the negative control value in the number of revertant mutant colonies, in either the base-pair-substituted or the frameshifted strains, and no dose-response was observed. Based on the above test results, the test item was judged to have no gene mutagenic activity against bacteria under the test conditions.
Micronucleus test (reference 7.6.1-2)
The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments. Experiment I and II were conducted with and without metabolic activation (S9-mix) with exposure periods of 4 and 24 hours. Experiment III was conducted without metabolic activation with an exposure period of 24 hours. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1950 µg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
HPRT assay (reference 7.6.1-3)
A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (1950 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. Based on the results of the pre-experiment the following concentrations were chosen for the main experiment in the presence and absence of metabolic activation: 60.9, 121.9, 243.8, 487.5, 975.0 and 1950.0 μg/mL. Neither precipitation nor phase separation occurred up the highest investigated concentration after four hours treatment. No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50 % (mean value of both parallel cultures) occurred up to the highest concentration. Consequently, the concentrations of 121.9 to 1950 μg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95 % confidence interval of the solvent historical control data. Linear regression analysis showed no statistically significant trend. The outcome of the experiment was clearly negative in the presence and in the absence of metabolic activation. The positive controls induced distinct increases in mutant frequencies, consistent with the laboratory’s historical positive control data, thus demonstrating the sensitivity of the test system and the efficacy of the S9 mix. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.
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