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EC number: 301-037-8 | CAS number: 93980-59-7
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- Aquatic toxicity
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- Short-term toxicity to fish
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA pKM101, with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in Chinese Hamster Lung (CHL/IU) cells, with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, HPRT test): negative in Chinese Hamster Ovary (CHO) cells, with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 Dec 2020 - 09 Mar 2021
- 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:
- adopted in 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted in 2008
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Ogyéi National Institute of Pharmacy and Nutrition, Budapest, Hungary
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HGPRT
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- Subline K1
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: American Type Culture Collection (ATCC, Manassas, Virginia, United States)
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Cells were cultivated in F12-10 culture medium (see below) and incubated at 37 ± 0.5 °C in a humidified atmosphere at 5 ± 0.3° CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes. Prior to use in the test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium.
MEDIA USED
- F12-1 medium: Treatment medium for 5 h treatment -S9 mix
Ham's F12 medium supplemented with 1% heat inactivated foetal bovine serum (FBS), 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)
- F12-5 medium: Treatment medium for 5 h treatment +S9 mix
Ham's F12 medium supplemented with 5% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)
- F12-10 medium: General culture medium
Ham's F12 medium supplemented with 10% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)
- F12-SEL medium: Culture medium for selection
Hypoxanthine-free Ham's F12 medium supplemented with 10% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Cofactor supplemented post mitochondrial fraction (S9 mix).
- source of S9: in house of the testing facility
- method of preparation of S9 mix: S9 mix was prepared from the livers of male Wistar rats (502 - 672 g) that were 5 months old. The animals were induced with phenobarbital and β-naphthoflavone at 80 mg/kg bw/day by oral gavage for three consecutive days. Liver homogenates were prepared 4 days after induction.
S9 mix was composed of 20 mM HEPES, 330 mM KCl, 50 mM MgCl2, 40 mM NADP, 50 mM D-glucose-6-phosphate and S9 fraction (30% in S9 mix).
- concentration or volume of S9 mix in the final culture medium: 10%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The biological activity of the S9 mix was demonstrated in an Ames test using the reference mutagens 2-aminoanthracene and benzo[a]pyrene. - Test concentrations with justification for top dose:
- Preliminary cytotoxicity test:
With and without metabolic activation: 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (5 h)
Experiment 1:
With and without metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (5 h)
Experiment 2:
With metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (5 h)
Without metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (24 h)
Justification for top concentration: Concentrations were selected based on the results of a preliminary cytotoxicity test. There was no cytotoxicity observed up to and including the maximum dose of 2000 µg/mL. - Vehicle / solvent:
- - Vehicle/solvent used: Dimethyl sulfoxide (DMSO), 1% in the final culture medium
- Justification for choice of solvent/vehicle: In a trial experiment, the test item turned out to be insoluble in water. Formulations in DMSO and in 1% aqueous methyl cellulose solution gave visibly homogeneous suspensions. Therefore, DMSO was selected as vehicle of the study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1% DMSO
- 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: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E6 (in dishes of 100 mm diameter)
- Test substance added in medium (F12-1 for 5 h exposure ± S9 mix, F12-5 for 24 h exposure –S9 mix).
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h with and without S9 mix (Experiments 1 and 2) and 24 h without S9 mix (Experiment 2)
- Expression time: At the end of the treatment period, the cultures were re-plated and incubated for a 7 days expression period. The cells were sub-cultured and maintained at 2 x 10E5 cells/dish twice.
- Selection time: After the expression period, 4 x 10E5 cells were plated in 100 mm dishes (5 per sample) for determination of mutant frequency and incubated for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
SELECTION:
- selection agent: 20 µg/mL 6-thioguanine (6-TG) was added to the medium for the 7-days incubation period of mutant selection.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS) and cloning efficiency
METHODS FOR MEASUREMENTS OF GENOTOXICIY:
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells. - Evaluation criteria:
- The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response. - Statistics:
- The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using MannWhitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency). In the statistical analysis, negative trends were not considered significant.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- In Exp. 2 (5 h exposure +S9 mix) a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The effect was not dose dependent and within historical control data and therefore considered not relevant.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no significant changes in pH after treatment.
- Data on osmolality: There were no significant changes in osmolality after treatment.
- Precipitation and time of the determination: Precipitation was observed in both experiments at 24.69 – 2000 µg/mL at the end of treatment in the presence and absence of metabolic activation. The precipitate did not interfere with the reading of the results.
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed using test item concentrations in the range of 3.906 – 2000 µg/mL in the presence and absence of metabolic activation. The cells were exposed for 5 h ±S9 mix and for 24 h –S9 mix. There was no cytotoxicity observed at any concentration, neither with nor without S9 mix. Precipitation was observed at 15.625 – 2000 µg/mL after 5 h exposure with S9 mix and at 31.25 – 2000 µg/mL after 5 or 24 h without S9 mix.
STUDY RESULTS
Cytotoxicity:
There was no cytotoxicity observed in any experiment up to the highest dose tested, neither in presence nor absence of metabolic activation.
Mutagenicity:
In the first experiment, no statistically significant increase in the mutant frequency was observed at any dose level, neither with nor without S9 mix. In the second experiment after 5 h exposure with S9 mix, a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The finding was not dose-related and fell within the range of the laboratory’s historical control data and was therefore not considered relevant. The test item was concluded to be negative for mutagenicity in mammalian cells in vitro. For details on experimental results, please refer to Tables 1 and 2 under “Any other information on results incl. tables”.
- Concurrent vehicle, negative and positive control data were in accordance with the general historical control data generated in the testing facility for all assays. The positive controls DMBA in the presence of S9 mix and EMS in the absence of S9 mix induced statistically significant increases in the mutant frequency over those of controls, thus confirming the functionality of the S9 mix and demonstrating the sensitivity of the test.
HISTORICAL CONTROL DATA: Please refer to Table 3 under “Any other information on results incl. tables”. - Conclusions:
- Interpretation of results: negative in CHO cells with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 - 29 Dec 2017
- 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 in 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon, trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- Following concentrations were used in the two main experiments (preincubation), based on the results of the range finding study (preincubation):
All strains: 313, 625, 1250, 2500 and 5000 μg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- Justification for choice of solvent/vehicle: A preliminary solubility test showed that it was not possible to dissolve or uniformly suspend the test substance in water, DMSO or acetone. Thus, THF was selected as solvent. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- THF
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- sodium azide
- other: 2-nitrofluorene (2NF), 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation (first and second experiment)
DURATION
- Preincubation period: until solidification takes place
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates in two experiments
DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn and number of revertant colonies - Evaluation criteria:
- Acceptance criteria
The study was considered valid if:
- mean number of revertant colonies of the negative (solvent) and positive controls are in the historical control range
- no contamination
- mean number of revertant colonies in the positive control group is increased at least twice compared to the negative control group
Evaluation criteria
The number of revertant colonies in any strains at one or more doses is increased at least two times compared to the negative control group. There should be dose dependency or reproducibility as dose increases. - Statistics:
- Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated. Statistical analysis was not performed.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A dose range finding study (preincubation) was conducted to determine the highest dose for the main study. The six tested concentrations were ranging from 4.88 until 5000 μg/plate. No signs of growth inhibition by the test substance was evident at any tested concentration in the absence or presence of metabolic activation. The test substance precipitated at a concentration of 19.5 μg/plate or more in all strains in the presence and absence of metabolic activation. However, it did not interfere with the colony counting.
HISTORICAL CONTROL DATA
see Table 3 in "Any other information on results incl. tables" - Conclusions:
- Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Jun - 15 Sep 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- yes
- Remarks:
- no information on pH and osmolality measurements given; only concentrations tested where precipitates were observed
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (CHL/IU cells) were obtained from the Health Science Research Resources Bank, Japan
- Suitability of cells: CHL/IU cells have been recommended in the methods described in OECD guideline 473.
- Doubling time: 15 h
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: Passage 17 was used
- Methods for maintenance in cell culture: The cells were grown in 90-mm diameter petri dishes. Approx. 1.5 x 10E4 cells/mL were seeded and pre-cultured for 2 days prior to each experiment.
- Modal number of chromosomes: 25 per cell
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Not specified
MEDIA USED
- Type and composition of media: Eagle’s MEM medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 0.292 g/L L-glutamine, 1.95 g/L sodium hydrogen carbonate and 10% v/v heat-inactivated newborn calf serum (NBCS, SAFC Biosciences).
- CO2 concentration, humidity level and temperature: The cells were cultured at 37 °C and 5% CO2 in a humidified atmosphere. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix).
- source of S9 mix: IEDA Trading Corporation (Lot no. CAM202003A)
- method of preparation of S9 mix: S9 mix was prepared from the livers of 7 week old male Sprague Dawley rats. The animals had a body weight in the range of 189 - 239 g and were treated with phenobarbital (30 mg/kg bw for one time, 60 mg/kg bw for three times) and 5,6-benzoflavone (80 mg/kg bw one time).
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentent of the S9 fraction was 24.95 mg/mL. S9 mix contained MgCl2 (5 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in HEPES buffer (4 mM, pH 7.2). The amount of S9 mix in the final culture medium was 6.7%. - Test concentrations with justification for top dose:
- Preliminary cell growth inhibition test:
6 h exposure with and without S9 mix: 31.3, 62.5, 125, 500, 1000 and 2000 µg/mL
24 h exposure with and without S9 mix: 31.3, 62.5, 125, 500, 1000 and 2000 µg/mL
Main cytogenicity test:
6 h exposure with and without S9 mix: 500*, 1000* and 2000* µg/mL
24 h exposure without S9 mix: 31.3, 62.5, 125, 500*, 1000* and 2000* µg/mL
*: concentrations selected for chromosome aberration analysis
Justification for top dose: Concentrations of the main cytogenicity test were selected based on the results of the preliminary growth inhibition test. - Vehicle / solvent:
- - Vehicle/solvent used: 0.5% w/v methyl cellulose (MC)
- Justification for choice of solvent/vehicle: The test substance was not suspended in distilled water at 20.0 mg/mL and in DMSO and acetone at 200 mg/mL. The test substance was suspended in 0.5 w/v% carboxymethyl cellulose (CMC) solution and 0.5 w/v% MC solution at 20.0 mg/mL. However, the test substance formulation of 20.0 mg/mL prepared with 0.5 w/v% CMC solution was changed to a heterogeneous formulation at room temperature for 2 h after preparation. The test substance formulation of 20.0 mg/mL prepared with 0.5 w/v% MC solution was considered to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 h after preparation. Therefore, 0.5 w/v% MC solution was selected as a vehicle. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% MC
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: A single experiment was performed.
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 mL cell suspension with 1.5 x 10E4 cells/mL were seeded in 60-mm diameter plastic dishes
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6 and 24 h
- Harvest time after the end of treatment: 24 h after beginning of treatment
SPINDLE INHIBITOR:
50 µL of a 10 µg/mL demecolcine solution was added to each dish 2 h prior to harvest.
SLIDE PREPRATION AND METAPHASE ANALYSIS:
- Methods of slide preparation and staining technique used including the stain used: The cells were trypsinised, centrifuged, counted, centrifuged again and re-suspended in hypotonic solution (0.075 M potassium chloride) at 37 °C for 15 min. Afterwards, the cells were fixed in methanol : acetic acid (3:1) for three times. The suspension was dropped onto a glass slide, dried and stained with 2 vol% Giemsa solution for about 15 min.
- Number of cells spread and analysed per concentration: 300
- Criteria for scoring chromosome aberrations: Metaphase cells containing 25 ± 2 chromosomes were included in the analysis. The total number of cells with structural aberrations and the number of aberrant cells in each aberration category were recorded. The following categories were investigated: chromatid breaks, chromatid exchanges, chromosome breaks, chromosome exchanges and other structural aberrations.
- Determination of polyploidy: Yes, the number of polyploidy cells with triploid or more (38 chromosomes) was recorded among 300 metaphase cells.
- Determination of endoreplication: Yes, the number of endoreduplicated cells was recorded among 300 metaphase cells.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative doubling population (RDP) and relative increase in cell counts (RICC).
METHODS FOR MEASUREMENTS OF GENOTOXICIY: Frequency of cells with structural chromosome aberrations (excluding gaps) when compared to solvent controls. - Evaluation criteria:
- Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be negative if:
- All results were inside the distribution of the historical data of the negative control group.
- All results were outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibited a statistically significant increase compared with the concurrent negative control.
Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be positive if:
- All results were outside the distribution of the historical data of the negative control group.
- There was a statistically significant increase compared with the concurrent negative control.
- The increase of the frequencies of cells with chromosomal aberrations was dose-related. - Statistics:
- Statistical methods were performed in order to evaluate the frequencies of cells with
structural aberrations and numerically aberrant cells. Significance level of these tests was both sides of 1% and 5%. Fisher’s exact test was performed in order to compare in the negative control group with positive control group. Fisher’s exact test was performed in order to compare in the negative control group with test substance group when the frequencies of cells with chromosomal aberrations in each test substance group were outside the distribution of the historical data of the negative control group. When at least one of the test substance doses exhibited a statistically significant increase compared with the negative control group in the Fisher’s exact test, the dose-dependency was evaluated by using Cochran-Armitage trend test. - Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period.
- 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
- Precipitation and time of the determination: Precipitation was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period.
RANGE-FINDING/SCREENING STUDY:
A preliminary growth inhibition test was performed to identify suitable concentrations for the chromosome aberration test. Single cultures of CHL/IU cells were exposed to test item concentrations in the range of 31.3 – 2000 µg/mL for 6 h with and without S9 mix and for 24 h continuous exposure without S9 mix. The relative doubling population (RDP) and the relative increase in cell count (RICC) were used to assess cytotoxicity.
For all treatment conditions and all concentrations, precipitation of the test substance in the culture medium was noted at the start and the end of treatment and at the time of harvest. There was no cytotoxicity noted for any condition at any concentration, neither with nor without S9 mix. There were abundant metaphases observed at 500, 1000 and 2000 µg/mL. Based on these findings, the concentrations for the chromosome aberration test were selected to be in the range of 500 – 2000 µg/mL.
STUDY RESULTS:
- Results from cytotoxicity measurements: There was no cytotoxicity noted for any test item concentration at any treatment period, neither with nor without S9 mix. For details on relative population doubling (RPD) and relative increase in cell count (RICC), please refer to Table 2 under “Any other information on results incl. tables”.
- Genotoxicity results: When compared to the solvent control, there was no statistically significant increase in the frequency of structural chromosome aberrations for any treatment condition or any concentration. A slight increase in structural chromosome aberrations was noted after 24 h continuous exposure at 1000 µg/mL, which was outside the historical control data of the solvent control. As the increase was not statistically significant when compared to the solvent control, the structural aberration was judged to be negative. The frequencies of numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. For details on results, please refer to Table 2 under “Any information on results incl. tables”.
HISTORICAL CONTROL DATA: For details on the historical control data please refer to Table 1 under “Any other information on materials and methods incl. tables”.
- Positive historical control data: The frequency of cells with structural chromosomal aberrations in the positive control group were within the range of the historical data and showed a statistically significant increase compared with the negative control group.
- Negative (solvent/vehicle) historical control data: The frequency of cells with chromosomal aberrations in the negative control group was within the range of the historical data. - Conclusions:
- Interpretation of results: negative in CHL/IU cells with and without metabolic activation.
Referenceopen allclose all
Table 1: Results of Experiment 1
S9 | Treatment | Concentration | Survival | Viability | Mutagenicity | ||||
Total no. of colonies | Cloning efficiency (CE) | Relative survival (%) | Total no. of colonies | Cloning efficiency (CE) | Total no. of colonies | Mutant frequency | |||
+ | 5 | 2000 µg/mL (P) | 1179 | 0.983 | 89 | 1123 | 0.936 | 24 | 6.4 |
666.67 µg/mL (P) | 1177 | 0.981 | 89 | 978 | 0.815 | 19 | 5.8 | ||
222.22 µg/mL (P) | 1202 | 1.002 | 91 | 1142 | 0.952 | 18 | 4.7 | ||
74.07 µg/mL (P) | 1089 | 0.908 | 83 | 1117 | 0.931 | 25 | 6.7 | ||
24.69 µg/mL (P) | 1374 | 1.145 | 104 | 1162 | 0.968 | 26 | 6.7 | ||
8.23 µg/mL | 1260 | 1.050 | 96 | 1157 | 0.964 | 22 | 5.7 | ||
2.74 µg/mL | 1219 | 1.016 | 92 | 1121 | 0.934 | 21 | 5.6 | ||
Negative control | 1319 | 1.099 | 100 | 1141 | 0.951 | 22 | 5.8 | ||
Untreated control | 1196 | 0.997 | 91 | 1092 | 0.910 | 23 | 6.3 | ||
Positive control (DMBA) | 59 | 0.049 | 4 | 1188 | 0.990 | 2244 | 566.3** | ||
- | 5 | 2000 µg/mL (P) | 1152 | 0.960 | 107 | 1126 | 0.938 | 21 | 5.7 |
666.67 µg/mL (P) | 1172 | 0.977 | 109 | 1181 | 0.984 | 32 | 8 | ||
222.22 µg/mL (P) | 1321 | 1.101 | 123 | 1077 | 0.898 | 21 | 5.8 | ||
74.07 µg/mL (P) | 1284 | 1.070 | 120 | 1047 | 0.873 | 19 | 5.4 | ||
24.69 µg/mL (P) | 1306 | 1.088 | 122 | 1248 | 1.040 | 22 | 5.4 | ||
8.23 µg/mL | 1203 | 1.003 | 112 | 1122 | 0.935 | 26 | 7 | ||
2.74 µg/mL | 1189 | 0.991 | 111 | 1020 | 0.850 | 25 | 7.3 | ||
Negative control | 1074 | 0.895 | 100 | 1185 | 0.988 | 26 | 6.6 | ||
Untreated control | 1234 | 1.028 | 115 | 1180 | 0.983 | 20 | 5.1 | ||
Positive control (EMS) | 899 | 0.749 | 84 | 970 | 0.808 | 1089 | 337.0** | ||
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL; EMS = Ethyl methanesulfonate, 0.4 µL/mL; (P): precipitation observed at the end of treatment |
Table 2: Results of Experiment 2
S9 | Treatment | Concentration | Survival | Viability | Mutagenicity | ||||
Total no. of colonies | Cloning efficiency (CE) | Relative survival (%) | Total no. of colonies | Cloning efficiency (CE) | Total no. of colonies | Mutant frequency | |||
+ | 5 | 2000 µg/mL (P) | 1475 | 1.229 | 122 | 1127 | 0.939 | 22 | 5.9 |
666.67 µg/mL (P) | 1235 | 1.029 | 102 | 1051 | 0.876 | 23 | 6.6 | ||
222.22 µg/mL (P) | 1290 | 1.075 | 106 | 1060 | 0.883 | 32 | 9.1 | ||
74.07 µg/mL (P) | 1232 | 1.027 | 102 | 1113 | 0.928 | 21 | 5.7 | ||
24.69 µg/mL (P) | 1264 | 1.053 | 104 | 1048 | 0.873 | 35 | 10.1* | ||
8.23 µg/mL | 1152 | 0.960 | 95 | 1003 | 0.836 | 37 | 11 | ||
2.74 µg/mL | 1194 | 0.995 | 98 | 1090 | 0.908 | 42 | 11.4 | ||
Negative control | 1213 | 1.011 | 100 | 1125 | 0.938 | 29 | 7.7 | ||
Untreated control | 1179 | 0.983 | 97 | 1077 | 0.898 | 34 | 9.5 | ||
Positive control (DMBA) | 95 | 0.079 | 8 | 1115 | 0.929 | 1687 | 453.3** | ||
- | 24 | 2000 µg/mL (P) | 1267 | 1.056 | 97 | 1179 | 0.983 | 24 | 6.1 |
666.67 µg/mL (P) | 1361 | 1.134 | 104 | 1042 | 0.868 | 20 | 5.7 | ||
222.22 µg/mL (P) | 1406 | 1.172 | 108 | 1144 | 0.953 | 23 | 6 | ||
74.07 µg/mL (P) | 1332 | 1.110 | 102 | 1104 | 0.920 | 22 | 6 | ||
24.69 µg/mL (P) | 1256 | 1.047 | 96 | 1129 | 0.941 | 38 | 10 | ||
8.23 µg/mL | 1344 | 1.120 | 103 | 1130 | 0.942 | 21 | 5.6 | ||
2.74 µg/mL | 1258 | 1.048 | 96 | 1016 | 0.847 | 28 | 8.3 | ||
Negative control | 1304 | 1.087 | 100 | 1137 | 0.948 | 30 | 8 | ||
Untreated control | 1223 | 1.019 | 94 | 1213 | 1.011 | 37 | 9.2 | ||
Positive control (EMS) | 768 | 0.640 | 59 | 472 | 0.393 | 1120 | 710.4** | ||
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL; EMS = Ethyl methanesulfonate, 0.4 µL/mL; (P): precipitation observed at the end of treatment |
Table 3: Historical control data generated in the testing facility (updated in Oct 2017 using data of GLP studies)
Mutation frequency (Number of 6-TG resistant mutants per 10E6 clonable cells) |
|||
Untreated control | |||
5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
mean | 18.3 | 20.7 | 19 |
standard deviation | 15.1 | 16.4 | 17.2 |
minimum | 5.1 | 5.5 | 3.3 |
maximum | 64.1 | 55.5 | 58 |
n | 27 | 13 | 14 |
DMSO control | |||
5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
mean | 21.8 | 18.9 | 18.4 |
standard deviation | 15.9 | 11.6 | 14.4 |
minimum | 5.4 | 6.5 | 6.8 |
maximum | 57.3 | 47.4 | 48.5 |
n | 29 | 13 | 14 |
Distilled water / Water based vehicle control | |||
mean | 11.5 | 9.1 | 15.5 |
standard deviation | 3.8 | 3.4 | 5.6 |
minimum | 6.1 | 5.2 | 9.2 |
maximum | 15.8 | 11.6 | 20.1 |
n | 6 | 3 | 3 |
Positive controls | |||
DMBA | EMS | EMS | |
5-hour, S9+ | 5-hour, S9- | 24-hour, S9- | |
mean | 905.2 | 445.6 | 1176.6 |
standard deviation | 562.7 | 118.6 | 610.9 |
minimum | 141.2 | 239.6 | 363.1 |
maximum | 2119.4 | 636.6 | 2449.8 |
n | 27 | 13 | 14 |
Table 1: Test results (experiment 1, preincubation)
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA 98 |
TA 1537 |
TA 100 |
TA 1535 |
WP2 uvrA |
||
– |
Solvent control (MEK) |
20 ± 1 |
9 ± 1 |
109 ± 3 |
14 ± 1 |
121 ± 4 |
313 |
22 ± 2 |
9 ± 1 |
118 ± 9 |
13 ± 1 |
113 ± 4 |
|
625 |
22 ± 2 |
I I ± 2 |
124 ± 2 |
17 ± 2 |
119 ± 5 |
|
1250 |
20 ± 1 |
9 ± 1 |
124 ± 2 |
18 ± 3 |
114 ± 1 |
|
2500 |
21 ± 2 |
8 ± 3 |
125 ± 5 |
17 ± 2 |
112 ± 2 |
|
5000 |
19 ± 2 |
8 ± 1 |
110 ± 2 |
13 ± 2 |
112 ± 2 |
|
Positive controls (µg/plate) |
2NF (5) |
9AA (80) |
SA (1.5) |
SA (1.5) |
4NQO (0.1) |
|
Mean (No. of colonies/plate) |
799 ± 13 |
704 ± 20 |
695 ± 24 |
591 ± 11 |
615 ± 11 |
|
+ |
Solvent control (MEK) |
35 ± 1 |
21 ± 1 |
122 ± 2 |
14 ± 1 |
162 ± 3 |
313 |
39 ± 1 |
22 ± 2 |
133 ± 5 |
14 ± 2 |
161 ± 4 |
|
625 |
38 ± 1 |
19 ± 2 |
129 ± 9 |
15 ± 2 |
163 ± 4 |
|
1250 |
34 ± 3 |
16 ± 3 |
120 ± 5 |
13 ± 1 |
157 ± 2 |
|
2500 |
34 ± 4 |
16 ± 1 |
124 ± 9 |
12 ± 2 |
147 ± 2 |
|
5000 |
31 ± 2 |
18 ± 1 |
118 ± 4 |
12 ± 1 |
152 ± 5 |
|
Positive controls (µg/plate) |
2AA (1) |
2AA (3) |
2AA (2) |
2AA (3) |
2AA (2) |
|
Mean (No. of colonies/plate) |
249 ± 37 |
152 ± 9 |
574 ± 30 |
111 ± 20 |
404 ± 17 |
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
4NQO = 4-nitroquinoline N-oxide
9AA = 9-aminoacridine
SAZ = sodium azide
The test substance precipitated at a concentration of 313 μg/plate or more in all strains in the presence and absence of metabolic activation.
Table 2: Test results (experiment 2, preincubation)
With or without S9 Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA 98 |
TA 1537 |
TA 100 |
TA 1535 |
WP2 uvrA |
||
– |
Solvent control (MEK) |
20 ± 2 |
9 ± 1 |
108 ± 2 |
14 ± 1 |
116 ± 2 |
313 |
19 ± 1 |
8 ± 1 |
114 ± 6 |
14 ± 2 |
117 ± 2 |
|
625 |
20 ± 2 |
7 ± 1 |
110 ± 3 |
13 ± 1 |
116 ± 1 |
|
1250 |
19 ± 2 |
8 ± 2 |
103 ± 4 |
12 ± 1 |
120 ± 2 |
|
2500 |
16 ± 1 |
8 ± 1 |
108 ± 2 |
13 ± 2 |
116 ± 2 |
|
5000 |
31 ± 2 |
7 ± 1 |
103 ± 3 |
11 ± 1 |
111 ± 1 |
|
Positive controls (µg/plate) |
2NF (5) |
9AA (80) |
SA (1.5) |
SA (1.5) |
4NQO (0.1) |
|
Mean (No. of colonies/plate) |
761 ± 8 |
730 ± 23 |
683 ± 24 |
625 ± 13 |
633 ± 9 |
|
+ |
Solvent control (MEK) |
36 ± 2 |
22 ± 1 |
121 ± 2 |
13 ± 2 |
160 ± 1 |
313 |
38 ± 2 |
20 ± 1 |
122 ± 2 |
10 ± 1 |
151 ± 2 |
|
625 |
40 ± 3 |
19 ± 1 |
118 ± 1 |
10 ± 1 |
152 ± 3 |
|
1250 |
38 ± 4 |
15 ± 2 |
118 ± 2 |
10 ± 1 |
149 ± 3 |
|
2500 |
30 ± 2 |
16 ± 1 |
118 ± 6 |
10 ± 1 |
144 ± 2 |
|
5000 |
29 ± 2 |
16 ± 1 |
116 ± 4 |
10 ± 2 |
143 ± 1 |
|
Positive controls (µg/plate) |
2AA (1) |
2AA (3) |
2AA (2) |
2AA (3) |
2AA (2) |
|
Mean (No. of colonies/plate) |
252 ± 13 |
153 ± 7 |
569 ± 43 |
118 ± 12 |
418 ± 14 |
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
4NQO = 4-nitroquinoline N-oxide
9AA = 9-aminoacridine
SAZ = sodium azide
The test substance precipitated at a concentration of 313 μg/plate or more in all strains in the presence and absence of metabolic activation.
Table 3: Historical data (negative and positive controls)
Strain |
TA 98 |
TA100 |
TA 1535 |
TA 1537 |
WP2 uvr A (pKM 101) |
||||||
+/- S9 mix |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+ |
|
Negative controls * |
Range |
10.3 - 26.6 |
16.9 - 37.9 |
59.0 - 113.5 |
69.2 - 124.5 |
5.6 - 16.2 |
5.8 - 14.6 |
4.7 - 10.9 |
7.2 - 22.1 |
81.5 - 165.5 |
16.9 - 199.0 |
Mean ± SD |
18.5 ± 2.9 |
27.4 ± 3.6 |
86.2 ± 10.5 |
96.8 ± 11.6 |
10.9 ± 1.8 |
10.2 ± 1.6 |
7.8 ± 1.1 |
14.7 ± 2.4 |
123.5 ± 16.0 |
156.3 ± 17.3 |
|
Positive controls |
Name |
2NF |
2AA |
SAZ |
2AA |
SAZ |
2AA |
9AA |
2AA |
4NQO |
2AA |
Range |
252.0 - 831.5 |
217.8 - 750.8 |
405.6 - 764.3 |
515.3 - 1818.0 |
348.3 - 617.7 |
84.7 - 223.3 |
144.9 - 767.4 |
135.0 - 373.1 |
55.3 - 1152.0 |
250.1 - 740.9 |
|
Mean ± SD |
541.7 ± 111.8 |
484.3 ± 106.9 |
584.9 ± 63.0 |
1166.5 ± 318.4 |
483.0 ± 48.5 |
154.0 ± 27.1 |
456.2 ± 124.1 |
254.0 ± 49.5 |
603.6 ± 170.8 |
495 ± 93.5 |
* = water for injection, DMSO, acetone, tetrahydrofuran, normal saline injection, sodium phosphate buffer
2AA = 2-aminoanthracene
2NF = 2-nitrofluorene
4NQO = 4-nitroquinoline N-oxide
9AA = 9-aminoacridine
SAZ = sodium azide
Table 2: Results of the chromosome aberration test
Exposure (h) | S9 mix | Substance | Dose (µg/mL) | Cytotoxicity | Structural chromosome aberrations# | Numerical chromosome aberrations# | |||||||
RDP | RICC | Excluding gaps | Gaps | Polyploid cells | Total no. of numerical aberrations | ||||||||
Total no. | Frequency (%) | No. of gaps | Frequency (%) | Total no. | Frequency (%) | Total no. | Frequency (%) | ||||||
6 | - | MC | 0.50% | 100.0 | 100.0 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | - | Test item | 500P | 89.8 | 85.9 | 5 | 1.7 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | - | 1000P | 92.1 | 88.4 | 5 | 1.7 | 0 | 0 | 1 | 0.3 | 1 | 0.3 | |
6 | - | 2000P | 63.8 | 53.8 | 6 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | |
6 | - | MMC | 0.05 | 78.4 | 70.4 | 70** | 23.3** | 0 | 0 | 0 | 0 | 0 | 0 |
6 | + | MC | 0.50% | 100.0 | 100.0 | 2 | 0.7 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | + | Test item | 500P | 85.2 | 77.7 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | + | 1000P | 82.1 | 74.0 | 2 | 0.7 | 0 | 0 | 1 | 0.3 | 1 | 0.3 | |
6 | + | 2000P | 81.5 | 72.6 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
6 | + | CPA | 4 | 59.9 | 47.1 | 73** | 24.3** | 0 | 0 | 0 | 0 | 0 | 0 |
24 | - | MC | 0.50% | 100.0 | 100.0 | 5 | 1.7 | 0 | 0 | 0 | 0 | 0 | 0 |
24 | - | Test item | 500P | 86.7 | 81.2 | 9 | 3 | 0 | 0 | 0 | 0 | 0 | 0 |
24 | - | 1000P | 79.4 | 71.3 | 10 | 3.3 | 2 | 0.7 | 0 | 0 | 0 | 0 | |
24 | - | 2000P | 63.7 | 52.5 | 9 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | |
24 | - | MMC | 0.05 | 67.5 | 56.7 | 174** | 58** | 2 | 0.7 | 0 | 0 | 0 | 0 |
#: 300 metaphase cells scored; **: statistically significant, p < 0.01; CPA: cyclophosphamide, positive control +S9 mix; MC: methyl cellulose, solvent; MMC: mitomycin C, positive control -S9 mix; P: precipitation observed at the start and end of treatment and at the time of harvest |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria:
Sorbitan tridocosanoate was tested for gene mutation in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Riken Vitamin, 2018). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA(pKM101) were exposed to the test item, untreated, solvent (THF) and appropriate positive controls in the presence and absence of metabolic activation (S9 mix).
A preliminary test was performed in order to identify suitable concentrations for the main study. As no cytotoxicity was noted up to the highest tested concentration of 5000 µg/plate, the main bacterial mutagenicity study was conducted at concentrations in the range of 313 – 5000 µg/plate. In the main study, two independent pre-incubation tests using triplicate plates per condition were performed. After 48 h of incubation at 37 °C with and without S9 mix, the bacterial background lawn was inspected and the mean number of revertant colonies was counted for each plate.
Precipitation of the test substance in the agar was observed at all concentrations for all strains, with and without S9 mix. There was no evidence for cytotoxicity at any concentration, neither in the presence nor absence of metabolic activation.
In addition, there was no ≥ 2-fold increase in revertant colony numbers of any of the five tester strains observed following treatment with sorbitan tridocosanoate at any dose level, neither in the presence nor absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations.
The number of revertant colonies induced by the solvent control was within the range of the historical control data for each strain, thus demonstrating an acceptable experimental performance. Appropriate positive control compounds showed a strong increase in the number of revertant colonies, confirming the activity of the S9 mix and the validity of the test system.
Thus, based on the results of the present study and under the experimental conditions chosen, the test item is not mutagenic in bacteria with and without metabolic activation.
In vitro cytogenicity in mammalian cells:
In vitro cytogenicity in mammalian cells was investigated in a chromosome aberration test performed in Chinese hamster lung (CHL/IU) cells in accordance with OECD guideline 473 and in compliance with GLP (Riken Vitamin, 2020). Based on the results of a preliminary growth inhibition test, duplicate cultures were exposed to test item concentrations in the range of 500 – 2000 µg/mL for 6 h with and without metabolic activation (S9 mix) and for test item concentrations in the range of 31.3 – 2000 µg/mL for 24 h continuous exposure in the absence of metabolic activation. For all conditions, solvent (0.5% methyl cellulose, MC) and positive controls (0.05 µg/mL mitomycin C (MMC) and 4 µg/mL cyclophosphamide (CPA)) were included. All cell cultures were harvested 24 h after start of exposure. A total of 300 metaphase cells per condition were scored for the presence of structural and numerical chromosome aberrations. In addition, the relative doubling population (RDP) and relative increase in cell count (RICC) of each culture was assessed to determine cytotoxicity.
Precipitation of the test substance in the culture medium was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period. There was no cytotoxicity, evident as a reduction of cell growth of > 50% noted for any condition at any concentration, neither in the presence nor absence of S9 mix.
All control substances showed the expected results and were within the range of the laboratory's historical control data. MMC and CPA induced statistically significant increases in the number of aberrant cells, thus proving the functionality of the S9 mix and demonstrating the validity of the test system.
After 24 h of exposure at 1000 µg/mL without S9 mix, the test item showed a slight increase in the number of structural chromosome aberrations, which was outside the historical control range for the negative control. The increase was statistically not significant when compared to solvent control cultures and therefore considered not relevant. Also for the other conditions, the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix. In addition, the frequency of polyploid cells and cells with endoreduplicated chromosomes were not affected, neither with nor without metabolic activation.
Under the conditions of the test, sorbitan tridocosanoate is considered negative for clastogenic activity in CHL/IU cells in vitro with and without S9 mix.
In vitro gene mutation in mammalian cells
The potential of sorbitan tridocosanoate to induce gene mutations in mammalian cells was investigated in an HPRT test, in line with OECD guideline 476 and according the GLP (Riken Vitamin, 2021). Two independent experiments were performed in Chinese Hamster Ovary (CHO) cells. Based on the results of a preliminary cytotoxicity test, duplicate cultures were exposed to test item concentrations of 2.74 – 2000 µg/mL for 5 h in the presence and absence of S9 mix (Experiment 1), for 5 h in the presence of S9 mix (Experiment 2) and for 24 h in the absence of S9 mix (Experiment 2).
Solvent (DMSO) and positive controls (ethylmethane sulphonate (EMS, 0.4 µL/mL for 5 and 24 h of exposure without S9 mix) and 7,12-dimethylbenzanthracene (DMBA, 15 µg/mL for 5 h of exposure with S9 mix) were included in each experiment. After exposure, the cells were incubated for 7 days to allow expression of the mutant phenotype. The expression period was followed by a 7-day selection period, in which the cells were incubated in selection medium containing 20 µg/mL 6-thioguanine (6-TG) prior to determination of the mutant frequency.
In both experiments, there was no cytotoxicity up to and including the highest concentration of 2000 µg/mL, neither with nor without metabolic activation. However, precipitation of the test substance in the culture medium was noted in both experiments at 24.69 – 2000 µg/mL at the end of treatment in the presence and absence of metabolic activation.
The mutation frequency of the solvent control was within the range of historical control data. The positive control substances EMS and DMBA markedly increased the mutation frequency in CHO cells, thus demonstrating the sensitivity and validity of the test system and confirming the functionality of the S9 mix.
In the first experiment, no statistically significant increase in the mutant frequency was observed at any dose level, neither with nor without S9 mix. In the second experiment after 5 h exposure with S9 mix, a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The finding was not dose-related, fell within the range of the laboratory’s historical control data and was therefore not considered relevant.
Under the conditions of the test, sorbitan tridocosanoate was considered negative for gene mutation in CHO cells with and without metabolic activation in vitro.
Conclusion:
In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that sorbitan tridocosanoate is neither mutagenic nor clastogenic in vitro.
Justification for classification or non-classification
The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.
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