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EC number: 915-932-1 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
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- Toxicological Summary
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- Acute Toxicity
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In recent GLP guideline studies conducted to examine the genetic toxicity (mutagenicity and chromosome abberations) of the test substance in vitro in bacterial cells and mammalian cells (Chinese hamster V79 cells), both with and without S9 metabolic activation, the substance does exhibit positive genotoxic effects in the mammalian assays. In a mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, with and without S9 metabolic activation, the substance showed no signs of mutagenicity without metabolic activation up to and including doses that produced biologically relevant growth inhibition but was shown to be mutagenic with metabolic activation only at the highest dose tested. In a mammalian cell chromosome abberation test in Chinese Hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay but did induce structural chromosomal aberrations in the long-term assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 July 1990 - 17 September 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Includes most, but not all, currently recommended bacterial strains
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- Includes most, but not all, currently recommended bacterial strains
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Includes most, but not all, currently recommended bacterial strains
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- yes
- Remarks:
- Includes most, but not all, currently recommended bacterial strains
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Test Substance Name: DPS
Product Number: 016513
Appearance: White powder
Storage: Room temp
Chemical name: Diphenylsulfone-3-sulfonic acid, Potassium-salt
Content:
- 86.5% DPS
- 5.4% By-product I
- 1.6% By-product III
- 0.8% Diphenylsulfone
- 4.2% H2O
Note: The substance described above is believed to be the same or similar to the reaction mass being registered but was not described as such in 1991. - Target gene:
- Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Test concentrations used were 8, 40, 200, 1000 and 5000 ug/Plate
- Vehicle / solvent:
- The solvent employed was deionized water and, for the positive controls, DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: See Remarks
- Remarks:
- Positive Controls (without S9): sodium azide (TA1535); nitrofurantoin (TA100); 4-nitro-1,2-phenylene diamine (TA1537, TA98); Postive Controls (with S9) 2-aminoantrachene
- Details on test system and experimental conditions:
- For the mutant count, four plates were used, both with and without 59 mix, for each strain and dose. The same number of plates, filled with thee solvent minus the test substance, comprised the negative control. Each positive control also contained four plate per strain. The doses for the first trial were routinely determined on the basis of a standard protocol: 5000 ug or 5 ul per plate were used as the highest dose, if not limited by solubility. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. In case of a positive response, however, or if at least three doses could be evaluated for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first <0 experiment.
- Evaluation criteria:
- The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which did not comply with at least on of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound. - 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:
- valid
- 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:
- valid
- 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:
- valid
- 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:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item is not matagenic in the bacterial reverse mutation assay.
- Executive summary:
In a GLP guideline bacterial reverse mutation assay using the plate incorporation method conducted according to OPPTS 870.5100, OECD 471, and EC method B.13/14, the substance showed no indications of mutagenic effects at doses up to 5000 uq per plate in any of the Salmonella typhimurium strains used.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 February 2018 - 03 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- Name: KSS-FR
Chemical Name: Potassium 3-(phenylsulphonyl)benzenesulphonate
Batch No.: KSS-1711-02
Composition: 70.8% KSS (CAS No.: 63316-43-8)
20.9% DKSS (CAS No.: 63316-33-6)
8.3% other
pH: 7.11
Physical State: powder
Color: white
Expiry Date: November 2019
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety - Target gene:
- Hprt and xprt genes
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- exogenous metabolic
- Test concentrations with justification for top dose:
- Without Metabolic activation: 2000, 2500, 2750 and 3250 µg/mL
With metabolic activation: 2250, 2750, 3000 and 3500 µg/mL - Vehicle / solvent:
- No
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- other: Not Applicable
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (via PCR), stable spontaneous mutant frequency as well as stability of the modal chromosome number. Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37°C incubation temperature. For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine.
Approx. 2 - 6 million cells per treatment group were seeded in complete culture medium in a 175 cm2 culture flask. Approx. 24 h after seeding, the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the cultures were checked for precipitation and the treatment medium containing the test item (MEM without FBS) was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2 x 106 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment. - Rationale for test conditions:
- V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.
- Evaluation criteria:
- A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Highest dose only.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%. The positive controls, DMBA (1.0 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.
- Conclusions:
- The test item was considered to be mutagenic only at the highest dose tested in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells.
- Executive summary:
In this GLP guideline study conducted according to OECD 476, EU Method B.17 and OPPTS 870.5300 to assess the test item's potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster, with and without S9 metabolic activation, the substance showed no signs of mutagenicity without metabolic activation up to and including doses that produced biologically relevant growth inhibition but was shown to be mutagenic with metabolic activation only at the highest dose tested. Positive controls showed the expected biologically relevant effects in mutation frequency, thus supporting the validity of the study. Given these results, the test item should be considered mutagenic in the in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells and this result should be weighed accordingly with other in vitro and any in vivo data to determine the substance's mutagenicity potential.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 February 2018 - 30 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Name: KSS-FR
Chemical Name: Potassium 3-(phenylsulphonyl)benzenesulphonate
Batch No.: KSS-1711-02
Composition: 70.8% KSS (CAS No.: 63316-43-8)
20.9% DKSS (CAS No.: 63316-33-6)
8.3% other
pH: 7.11
Physical State: powder
Color: white
Expiry Date: November 2019
Storage Conditions: at room temperature - Target gene:
- Entire Chromosome
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Experiment I (without metabolic activation): 1000, 2000 and 2500 µg/mL
Experiment I (with metabolic activation): 2500, 3000 and 3500 µg/mL
Experiment II (without metabolic activation): 500, 1000 and 1500 µg/mL - Vehicle / solvent:
- None
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- other: Not Applicable
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the Eurofins Munich V79 in stock cultures: 12 - 14 h) and a high plating efficiency of untreated cells (normally more than 50%). These facts are necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections was carried out before freezing.
For the experiment thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10E5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made 3-4 days after seeding.
Complete Culture Medium: MEM medium supplemented with:
- 10% (v/v) Fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES
- Also used for the long-term treatment and the post incubation.
Treatment Medium (short-term exposure): Complete culture medium without FBS.
The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.
Three or four days old stock cultures (in exponential growth) more than 50% confluent were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.05% trypsin in Ca-Mg-free PBS at 37 °C for about 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared. About 1 x 104 cells/mL were seeded into cell culture flasks with complete culture medium.
Treatment parameters/conditions were as follows:
Exp. I (without S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours
Exp. II (without S9 mix):
- Treatment Period: 21 hours
- Recovery Time: TBD
- Preparation interval: 21 hours
Exp. I (with S9 mix):
- Treatment Period: 4 hours
- Recovery Time: 17 hours
- Preparation interval: 21 hours - Rationale for test conditions:
- A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix: 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL. The results of this pre-experiment were used to select the concentrations for the main experiment.
- Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system. - Statistics:
- Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control. Aberrant cells without gaps were only used for the calculation. Gaps are recorded separately and reported but generally not included in the total aberration frequency calculation according to the guideline. For the trend test, Statistical significance at the 5% level (p < 0.05) was evaluated by the x² test. The p value was used as a limit in judging for significance levels
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- @1000 ug/mL and higher
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- @1000 ug/mL and higher
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Toxicity: In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 3500 µg/mL and higher (58% at 3500 µg/mL and 6% at 4000 µg/mL. With metabolic activation, no biologically relevant decrease of the RICC was observed. In experiment II without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1000 µg/mL and higher (53% at 1000 µg/mL, 48% at 1500 µg/mL, 25% at 2000 µg/mL, 11% at 2500 µg/mL and 0% at 3000 µg/mL.
Clastogenicity: In experiment I without metabolic activation, the aberration rate of the negative control (1.7%) and two test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). A slight induction of aberrant cells was noted at a concentrations of 2000 µg/mL (4.0%). However, this increase was considered as not biologically relevant due to the lack of a dose response relationship. With metabolic activation, the aberration rates of the negative control (2.7%) and all dose groups treated with the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps. In experiment II without metabolic activation, the aberration rate of the negative control (1.3%) and the lowest evaluated concentration of 500 µg/mL (0.7%) were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). A dose-dependent increase of aberrant cells was noted in the two next higher concentrations of 1000 µg/mL (5.7%) and 1500 µg/mL mM (16.4%). The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the experiment I without and with metabolic activation. In experiment II, the concentrations 1000 µg/mL and 1500 µg/mL which were above the historic control range, were also statistically significantly increased. The X² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation. However, a statistically significant dose-dependent increase was noted in experiment II without metabolic activation. EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
Polyploid Cells: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item. - Conclusions:
- The test substance induced structural chromosomal aberrations in the V79 Chinese hamster cell line in the long-term experiment.
- Executive summary:
In this GLP guideline study, conducted according to OECD 472, EC method B.10, and US EPA Method OPPTS 870.5375, to investigate in vitro the potential for the test substance to induce structural chromosome abberations in chinese hamster V79 cells the substance did not induce structural chromosomal aberrations in the short-term assay (Experiment I) but did induce structural chromosomal aberrations in the long-term assay (Experiment II).
In experiment I without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 3500 µg/mL and higher (58% at 3500 µg/mL and 6% at 4000 µg/mL. With metabolic activation, no biologically relevant decrease of the RICC was observed. The aberration rate of the negative control (1.7%) and two test item concentrations were within the historical control data of the testing facility (-0.28 to 3.70% aberrant cells exclusive gaps). A slight induction of aberrant cells was noted at a concentrations of 2000 µg/mL (4.0%). However, this increase was considered as not biologically relevant due to the lack of a dose response relationship. With metabolic activation, the aberration rates of the negative control (2.7%) and all dose groups treated with the test item were within the historical control data of the testing facility (-0.23 to 3.95% aberrant cells exclusive gaps. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the experiment I without and with metabolic activation.
In experiment II without metabolic activation, a biologically relevant decrease in cell count (decrease below 70% RICC) was noted at 1000 µg/mL and higher (53% at 1000 µg/mL, 48% at 1500 µg/mL, 25% at 2000 µg/mL, 11% at 2500 µg/mL and 0% at 3000 µg/mL. The aberration rate of the negative control (1.3%) and the lowest evaluated concentration of 500 µg/mL (0.7%) were within the historical control data of the testing facility (-0.20 to 2.71% aberrant cells exclusive gaps). A dose-dependent increase of aberrant cells was noted in the two next higher concentrations of 1000 µg/mL (5.7%) and 1500 µg/mL mM (16.4%). These concentrations were statistically significantly increased and were found to be statistically significant dose-dependent increases.
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
Given these results, the test substance did induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the long-term experiment and the need for in vivo testing should be considered.
Referenceopen allclose all
The Salmonella/microsome test, employing doses up to 5000 ug per plate, showed the substance not to produce bacteriotoxic or mutagenic effects. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. No inhibition of growth was noted even at the limit dose. Positive controls, at comparatively low doses, resulted in the expected increases of the mutant counts to well over double those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects could be found at assessable doses up to 5000 ug per plate in any of the Salmonella typhimurium strains used.
Main Experiment – Toxicity, without metabolic activation
Dose Group |
Concen-tration [µg/mL] |
Number of cells at the |
Number of colonies per flask |
CE[%] |
Adjusted CE [%] |
Relative Survival (RS) [%] |
|||
beginning of treatment |
end of treatment |
I |
II |
mean |
|||||
NC1 |
0 |
10000000 |
9588000 |
191 |
173 |
182 |
91 |
87 |
100 |
NC2 |
10000000 |
10268000 |
163 |
185 |
174 |
87 |
89 |
||
1 |
500 |
10000000 |
10132000 |
133 |
141 |
137 |
69 |
69 |
79 |
2 |
1000 |
10000000 |
11628000 |
104 |
109 |
107 |
53 |
62 |
70 |
3 |
1500 |
10000000 |
10336000 |
116 |
129 |
123 |
61 |
63 |
72 |
4 |
2000 |
10000000 |
11560000 |
94 |
130 |
112 |
56 |
65 |
73 |
5 |
2500 |
10000000 |
10676000 |
203 |
152 |
178 |
89 |
95 |
107 |
6 |
2750 |
10000000 |
10812000 |
37 |
45 |
41 |
21 |
22 |
25 |
7 |
3000 |
20000000 |
18564000 |
10 |
11 |
11 |
5 |
5 |
6 |
8 |
3250 |
20000000 |
11050000 |
17 |
34 |
26 |
13 |
7 |
8 |
EMS |
300 |
10000000 |
13005000 |
145 |
120 |
133 |
66 |
86 |
98 |
NC:negative control; CE:cloning efficiency; EMS:Ethylmethanesulfonate
Main Experiment – Mutagenicity, without metabolic activation
|
CE in non-selective medium |
CE in selective medium |
|
|||||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of colonies per flask |
CE[%] |
Number of colonies per flask |
CE[%] |
Mutant Frequency per 106cells |
||||||||
I |
II |
mean |
I |
II |
III |
IV |
V |
mean |
SD |
|||||
NC1 |
0 |
178 |
160 |
169 |
85 |
1 |
4 |
5 |
5 |
2 |
3.4 |
1.6 |
0.0009 |
10.1 |
NC2 |
185 |
157 |
171 |
86 |
6 |
7 |
8 |
6 |
7 |
6.8 |
0.7 |
0.0017 |
19.9 |
|
4 |
2000 |
158 |
168 |
163 |
82 |
7 |
6 |
6 |
7 |
4 |
6.0 |
1.1 |
0.0015 |
18.4 |
5 |
2500 |
161 |
163 |
162 |
81 |
6 |
4 |
5 |
4 |
2 |
4.2 |
1.3 |
0.0011 |
13.0 |
6 |
2750 |
162 |
160 |
161 |
81 |
2 |
8 |
4 |
11 |
8 |
6.6 |
3.2 |
0.0017 |
20.5 |
8 |
3250 |
150 |
157 |
154 |
77 |
10 |
5 |
10 |
7 |
6 |
7.6 |
2.1 |
0.0019 |
24.8 |
EMS |
300 |
161 |
175 |
168 |
84 |
80 |
74 |
91 |
78 |
93 |
83.2 |
7.5 |
0.0208 |
247.6 |
NC:negative control; CE:cloning efficiency; EMS:Ethylmethanesulfonate
Main Experiment – Toxicity, with metabolic activation
Dose Group |
Concen-tration [µg/mL] |
Number of cells at the |
Number of colonies per flask |
CE[%] |
Adjusted CE [%] |
Relative Survival (RS) [%] |
|||
beginning of treatment |
end of treatment |
I |
II |
mean |
|||||
NC1 |
0 |
10000000 |
13464000 |
162 |
156 |
159 |
80 |
107 |
100 |
NC2 |
10000000 |
13396000 |
137 |
134 |
136 |
68 |
91 |
||
1 |
500 |
10000000 |
13617000 |
130 |
122 |
126 |
63 |
86 |
87 |
2 |
750 |
10000000 |
13991000 |
117 |
136 |
127 |
63 |
88 |
89 |
3 |
1000 |
10000000 |
11951000 |
126 |
119 |
123 |
61 |
73 |
74 |
4 |
2250 |
10000000 |
9197000 |
122 |
121 |
122 |
61 |
56 |
56 |
5 |
2750 |
10000000 |
9214000 |
103 |
93 |
98 |
49 |
45 |
46 |
6 |
3000 |
10000000 |
9163000 |
74 |
78 |
76 |
38 |
35 |
35 |
7 |
3500 |
20000000 |
16014000 |
57 |
38 |
48 |
24 |
19 |
19 |
8 |
4000 |
20000000 |
15640000 |
18 |
19 |
19 |
9 |
7 |
7 |
9 |
4250 |
20000000 |
14654000 |
6 |
4 |
5 |
3 |
2 |
2 |
10 |
4500 |
20000000 |
10914000 |
3 |
0 |
2 |
1 |
0 |
0 |
DMBA |
1.0 |
10000000 |
13073000 |
171 |
171 |
171 |
86 |
112 |
113 |
NC:negative control; CE:cloning efficiency; DMBA:7,12-dimethylbenz(a)anthracene
Main Experiment – Mutagenicity, with metabolic activation
|
CE in non-selective medium |
CE in selective medium |
|
|||||||||||
Dose Group |
Concen-tration [µg/mL] |
Number of colonies per flask |
CE[%] |
Number of colonies per flask |
CE[%] |
Mutant Frequency per 106cells |
||||||||
I |
II |
mean |
I |
II |
III |
IV |
V |
mean |
SD |
|||||
NC1 |
0 |
162 |
159 |
161 |
80 |
12 |
13 |
10 |
9 |
16 |
12.0 |
2.4 |
0.0030 |
37.4 |
NC2 |
141 |
147 |
144 |
72 |
8 |
9 |
16 |
7 |
9 |
9.8 |
3.2 |
0.0025 |
34.0 |
|
4 |
2250 |
153 |
146 |
150 |
75 |
8 |
10 |
7 |
12 |
8 |
9.0 |
1.8 |
0.0023 |
30.1 |
5 |
2750 |
161 |
163 |
162 |
81 |
15 |
12 |
12 |
10 |
5 |
10.8 |
3.3 |
0.0027 |
33.3 |
6 |
3000 |
157 |
159 |
158 |
79 |
11 |
11 |
6 |
10 |
8 |
9.2 |
1.9 |
0.0023 |
29.1 |
7 |
3500 |
149 |
139 |
144 |
72 |
12 |
18 |
12 |
14 |
12 |
13.6 |
2.3 |
0.0034 |
47.2 |
DMBA |
1.0 |
144 |
143 |
144 |
72 |
101 |
115 |
100 |
118 |
99 |
106.6 |
8.2 |
0.0267 |
371.4 |
NC:negative control; CE:cloning efficiency; DMBA:7,12-dimethylbenz(a)anthracene
Experiment I (short-term) - Summary of Cytotoxicity Data
Dose Group |
Concentration [µg/mL] |
Cell Count |
Precipitate (+/-) |
|||
Culture |
RICC |
|||||
1 |
2 |
Mean |
[%] |
|||
without metabolic activation |
||||||
C |
0 |
160.20 |
174.84 |
167.52 |
100 |
- |
1 |
1000 |
119.20 |
158.24 |
138.72 |
82 |
- |
2 |
2000 |
146.53 |
134.81 |
140.67 |
83 |
- |
3 |
2500 |
121.15 |
140.67 |
130.91 |
77 |
+ |
4 |
3000 |
127.98 |
130.91 |
129.45 |
76 |
+ |
5 |
3500 |
94.70 |
107.48 |
101.09 |
58 |
+ |
6 |
4000 |
23.44 |
16.61 |
20.02 |
6 |
+ |
EMS |
600 |
143.60 |
162.15 |
152.87 |
91 |
- |
with metabolic activation |
||||||
C |
0 |
142.62 |
204.12 |
173.37 |
100 |
- |
1 |
2000 |
235.36 |
175.81 |
205.59 |
120 |
- |
2 |
2500 |
155.31 |
172.89 |
164.10 |
94 |
- |
3 |
3000 |
155.31 |
170.93 |
163.12 |
94 |
- |
4 |
3500 |
173.86 |
155.31 |
164.59 |
95 |
+ |
5 |
4000 |
186.55 |
87.87 |
137.21 |
78 |
+ |
6 |
4500 |
137.74 |
78.11 |
107.92 |
60 |
+ |
7 |
5000 |
72.25 |
29.30 |
50.77 |
25 |
+ |
CPA |
0.83 |
44.91 |
162.15 |
103.53 |
57 |
- |
RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group.
C: Negative Control (Culture Medium)
EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)
CPA: Positive Control (with metabolic activation: Cyclophosphamide)
Experiment I – Summary of Aberration Rates
Dose Group |
Concentration (µg/mL) |
Treatment Time (hour) |
Fixation Interval |
Mean % Abeerrant Cells |
Precipitation (+/-) |
|
incl. Gaps |
excl. Gaps |
|||||
without metabolic activation |
||||||
C |
0 |
4 |
21 |
3.0 |
1.7 |
- |
1 |
1000 |
4 |
21 |
4.7 |
2.7 |
- |
2 |
2000 |
4 |
21 |
5.3 |
4.0 |
- |
3 |
2500 |
4 |
21 |
3.3 |
2.0 |
+ |
EMS |
600 |
4 |
21 |
9.3 |
8.0 |
- |
with metabolic activation |
||||||
C |
0 |
4 |
21 |
5.3 |
2.7 |
- |
2 |
2500 |
4 |
21 |
5.0 |
3.3 |
- |
3 |
3000 |
4 |
21 |
4.3 |
1.3 |
- |
4 |
3500 |
4 |
21 |
4.3 |
2.7 |
+ |
CPA |
0.83 |
4 |
21 |
14.2 |
12.9 |
- |
300 cells evaluated for each concentration, except for the positive control CPA (225 cells) due to a clearly positive increase in chromosomal aberrations.
C: Negative Control (Culture Medium)
EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)
CPA: Positive Control (with metabolic activation: Cyclophosphamide)
Experiment II - Summary of Cytotoxicity Data
Dose Group |
Concentration [µg/mL] |
Cell Count |
Precipitate (+/-) |
|||
Culture |
RICC |
|||||
1 |
2 |
Mean |
[%] |
|||
without metabolic activation |
||||||
C |
0 |
215.00 |
217.00 |
216.00 |
100 |
- |
S |
0 |
215.00 |
217.00 |
216.00 |
100 |
- |
1 |
500 |
236.00 |
226.00 |
231.00 |
107 |
- |
2 |
1000 |
107.00 |
131.00 |
119.00 |
53 |
- |
3 |
1500 |
98.00 |
118.00 |
108.00 |
48 |
- |
4 |
2000 |
59.00 |
63.00 |
61.00 |
25 |
- |
5 |
2500 |
28.00 |
39.00 |
33.50 |
11 |
- |
6 |
3000 |
1.90 |
4.70 |
3.30 |
0 |
- |
EMS |
400 |
189.00 |
180.00 |
184.50 |
85 |
- |
RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group.
C: Negative Control (Culture Medium)
EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)
Experiment II - Summary of Aberration Rates
Dose Group |
Concentration (µg/mL) |
Treatment Time (hour) |
Fixation Interval |
Mean % Abeerrant Cells |
Precipitation (+/-) |
|
incl. Gaps |
excl. Gaps |
|||||
without metabolic activation |
||||||
C |
0 |
21 |
21 |
3.3 |
1.3 |
- |
1 |
500 |
21 |
21 |
1.0 |
0.7 |
- |
2 |
1000 |
21 |
21 |
8.3 |
5.7 |
- |
3 |
1500 |
21 |
21 |
18.4 |
16.4 |
- |
EMS |
400 |
21 |
21 |
18.0 |
16.0 |
- |
300 cells evaluated for each concentration, except for the positive controls (EMS: 150 cells) and the concentration 1500 µg/mL (250 cells) due to a clearly positive increase in chromosomal aberrations.
C: Negative Control (Culture Medium)
EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
The relevance of these in vitro findings to humans is unknown.
Additional information
Justification for classification or non-classification
Despite the positive findings in vitro, the substance does not meet the GHS criterial for classification as a Germ Cell Mutagen on the basis that there is no positive evidence in vivo in either germ cells or somatic cells and there is no known chemical structure activity relationship to known germ cell mutagens.
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