Registration Dossier
Registration Dossier
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EC number: 290-904-3 | CAS number: 90268-98-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item was not mutagenic in bacterial cells in the Ames test.
The test substance was not clastogenic in the chromosome aberration assay in Chinese hamster lung cells.
The test item was not mutagenic in vitro in mammalian cells (Chinese hamster ovary cells).
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:
- October 26 - December 12, 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Justification of concentrations:
Selection of the concentration range was done on the basis of a Solubility Test and a Concentration Range Finding Test (Informatory Toxicity Test). - Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- July, 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction: S9
- Test concentrations with justification for top dose:
- The concentrations investigated in the Initial and Confirmatory Mutation Tests:
5000, 1600, 500, 160, 50, 16 and 5 µg/plate.
(reported as corrected concentrations considering the dye content of the test item) - Vehicle / solvent:
- Name: Dimethyl sulfoxide (DMSO)
Supplier: SIGMA-ALDRICH
Batch Numbers: SZBF3070V and SZBG0830V
Retest date: 18 October 2018 and 08 March 2019
Storage: Room temperature - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-1,2-phenylenediamine, NPD;2-aminoanthracene, 2AA
- Details on test system and experimental conditions:
- Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH ;Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.
Storage of Tester Strains
The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.
Confirmation of Phenotypes of Tester Strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.
Spontaneous Reversion of Tester Strains
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Procedure for Bacterial Cultures
The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-13 hours in a 37oC Benchtop Incubator Shaker.
Viability and the Cell Count of the Testing Bacterial Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates The viable cell number of the cultures was determined by manual colony counting.
Media
The Minimal Glucose Agar (MGA) Plates
Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis* were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
* Batch No.: 140149; Expiry date: 26 December 2016; (used in the Informatory Toxicity Test)
139475; Expiry date: 13 November 2016; (used in the Initial and Confirmatory Mutation Tests)
Nutrient Broth No. 2
Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.
Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.
Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.
Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.
Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL
Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.
Complete Top Agar for Escherichia coli strain:
Nutrient Broth by 5.4.2 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by 5.4.4 947.5 mL
Metabolic Activation System
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).
Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier. The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots are given in Appendix VIII. The following lots of the S9 were applied:
Lot Number: 3647; Expiry date: June 09, 2018; Protein content: 40.2 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in all experimental phases of the study).
The S9 Mix (with Rat Liver S9)
Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.
The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.
Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL
Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter. - Rationale for test conditions:
- Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test).
Based on the solubility test, the stock solution with a concentration of 50 mg/mL was prepared in the vehicle and diluted in at least 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains. - Evaluation criteria:
- The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- 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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA98 strain at 160 μg/plate, in the absence of metabolic activation ( S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 2.14, which was far below the genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain only, in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains, mostly in the absence of exogenous metabolic activation. The cytotoxicity was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. For the cytotoxicity tendency (it did not show a clear dose-dependent tendency), the effect of precipitate formation was supposed. The cytotoxicity results are summarized in Table 7. The table contains the unequivocal results where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or affected background lawn development occurred. All of the further observed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system.
In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity.
When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration of 5000 µg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures.
For confirmation of manual evaluations (made by naked eye) all of the plates were checked for colony and background lawn development by microscope at 40X magnification. At this magnification test item particles were noticed down to and including the concentration of 50 µg/plate (±S9 Mix), following the plate incorporation test and down to and including the concentration of 16 µg/plate (±S9 Mix) following the pre-incubation procedure. - Conclusions:
- The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
- Executive summary:
The test item dissolved in DMSO was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test. The following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests (reported as corrected concentrations considering the dye content of the test item)
: 5000;1600; 500; 160; 50; 16 and 5 µg/plate. The selection of the concentration range was based on the recommendations in OECD 471 guideline. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimuriumTA1537 strain in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains, mostly in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 July- 22 November, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 29th July, 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 14 February 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Chromosomal aberration in vitro
- Specific details on test material used for the study:
- Date of production: 22.05.2015
Expiration date: 22.05.2020 - Target gene:
- Chromatid and chromosome type aberrations in metaphase cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%. - Cytokinesis block (if used):
- Cell cultures were treated with colchicine (0.2 µg/mL) 2.5 hours prior to harvesting
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.
- Test concentrations with justification for top dose:
- Experiment A (3/20h): 15.6,1 31.3, 62.5, 125 and 1802 μg/mL test item with and without S9 mix
Experiment B (20/20h and 20/28h): 3.9, 1 7.8, 15.6, 31.3 and 452 μg/mL test item without S9 mix
Experiment B (3/28h): 15.6, 1 31.3, 62.5, 125 and 180 μg/mL test item with S9 mix
(reported as corrected concentrations considering the dye content of the test item) - Vehicle / solvent:
- Dimethyl sulfoxide DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- other: DME (Dulbecco’s Modified Eagle’s) medium
- Details on test system and experimental conditions:
- The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
Mammalian Microsomal Fraction S9 Mix
An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 40.3 and 33.8 mg/mL.
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.
The S9 Mix (with Rat Liver S9)
The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= β-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath. - Rationale for test conditions:
- Acceptability of the Assay
The chromosome aberration assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration are fulfilled. - Evaluation criteria:
- Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.
Interpretation of Results
Providing that all acceptability criteria are fulfilled, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance should be considered together.
There is no requirement for verification of a clearly positive or negative response. - Statistics:
- For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Solubility and Dose Selection
A homogeneous suspension of the test item was obtained in DMSO up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix). Based on cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with the test item are presented in Table 2A - 2C.
Based on the results of the cytotoxicity assay the following concentrations were selected for the chromosome aberration assay:
Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802 *g/mL test item
with S9 mix: 15.6,1 31.3, 62.5, 125 and 180 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9, 1 7.8, 15.6, 31.3 and 452 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6, 1 31.3, 62.5, 125 and 180 *g/mL test item
1These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next higher concentration and sufficient number of concentrations.
2These concentrations were tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.
All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.
Chromosome Aberration Assay
No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item (Tables 13 and 14).
In both experiments, clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.
No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation
In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values (5-6 aberrant cells excluding
gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells). However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data .Thus, the study is considered valid. - Remarks on result:
- other: In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
- Remarks:
- test item is considered as being non-clastogenic in this system
- Conclusions:
- The test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
- Executive summary:
The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations (reported as corrected concentrations considering the dye content of the test item) were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.
Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802g/mL test item
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-10-17 to 2017-11-13
- 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)
- Version / remarks:
- 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian cell gene mutation test: HPRT Assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: 106134974
- Expiration date of the lot/batch: 22.05.2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared in DMSO and diluted prior to treatment. - Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- Sub-line (K1)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: suitable cell line for this type of assay
MEDIA USED
- Type and identity of media including CO2 concentration: For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- Test concentrations with justification for top dose:
- The concentration range for the pre-test for cytotoxicity was chosen based on results of a preliminary screening test, where the concentrations were: 62.5, 125, 250, 500, 1000, 2500 and 5000 μg/mL.
Main test with metabolic activation:
test item concentration: 125, 250, 500, 750, 100 µg/mL; positive control 7,12-Dimethyl benzanthracene: 20 µg/mL
Main test without metabolic activation:
test item concentration: 62.5, 125, 250, 350, 450 µg/mL; positive control Ethyl methanesulfonate (EMS): 1 µL/mL
(reported as corrected concentrations considering the dye content of the test item) - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: This solvent was chosen based on the results of the preliminary solubility test and its suitability is confirmed with the available laboratory’s historical database. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x10E6 cells/dish
DURATION
- Exposure duration: h hours
- Expression time: 19 h
- Selection time: 8 days
SELECTION AGENT: selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)
STAIN: Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
NUMBER OF CELLS EVALUATED: 200 /dish
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (based 95% control limit).
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system. - Statistics:
- Statistical Analysis was performed with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups
• The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- Sub-line (K1)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- evidence of toxicity was seen at the highest tested concentration with the test item in presence and absence of metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: not observed
RANGE-FINDING/SCREENING STUDIES:
The concentrations for the main mutation assay were selected on the basis of preliminary cytotoxicity investigations (without and with metabolic activation using S9-mix, see table 1). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. - Conclusions:
- The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.
- Executive summary:
The test item suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations (reported as corrected concentrations considering the dye content of the test item) were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:
Mutation Assay 5-hour treatment period without S9-mix: 62.5, 125, 250, 350 and 450 µg/mL
Mutation Assay 5-hour treatment period with S9-mix: 125, 250, 500, 750 and 1000 µg/mL
In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure. In the absence and presence of metabolic activation clear cytotoxicity (survival approximately 16%) of the test item was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.
Referenceopen allclose all
Summary of the Inhibitory Tendencies in the Initial and Confirmatory Mutation Tests
Initial Mutation Test |
||||||||||
Concentrations (µg/plate) |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
|
5000 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
< B |
< B |
‑ |
‑ |
1600 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
SB |
SB |
‑ |
‑ |
500 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
SB |
SB |
‑ |
‑ |
160 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
50 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
16 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
5 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
Confirmatory Mutation Test |
||||||||||
Concentrations (µg/plate) |
Salmonella typhimurium |
Escherichia coliWP2uvrA |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
|||||||
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
‑S9 |
+S9 |
|
5000 |
SB |
<< |
SB |
‑ |
SB |
‑ |
<< B |
‑ |
‑ |
‑ |
1600 |
< SB |
‑ |
SB |
‑ |
< SB |
‑ |
<< B |
‑ |
‑ |
‑ |
500 |
< SB |
‑ |
<< SB |
‑ |
< SB |
‑ |
B0 |
‑ |
‑ |
‑ |
160 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
50 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
16 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
5 |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
‑ |
<: Revertant colony numbers below the vehicle control data range
<<: Revertant colony numbers below the vehicle and historical control data ranges
B: Reduced background lawn development
SB: Slightly reduced background lawn development
B0: Reduced background lawn development and absent revertant colonies
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
3-hour treatment without and with S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
6700000 |
7000000 |
6862500 |
4943750 |
100,00 |
0,00 |
- |
B |
– |
6850000 |
6900000 |
|||||
test item |
31.3 |
A |
– |
6800000 |
6800000 |
6800000 |
4881250 |
98,74 |
1,26 |
62.5 |
A |
– |
5400000 |
5150000 |
5275000 |
3356250 |
67,89 |
32,11 |
|
125 |
A |
– |
4300000 |
4400000 |
4350000 |
2431250 |
49,18 |
50,82 |
|
250 |
A |
– |
3150000 |
3000000 |
3075000 |
1156250 |
23,39 |
76,61 |
|
500 |
A |
– |
2500000 |
2450000 |
2475000 |
556250 |
11,25 |
88,75 |
|
EMS 1 µL/mL |
A |
– |
4600000 |
4600000 |
4600000 |
2681250 |
54,24 |
45,76 |
|
Solvent control (DMSO) |
- |
A |
+ |
6600000 |
6850000 |
6700000 |
4781250 |
100,00 |
0,00 |
- |
B |
+ |
6650000 |
6700000 |
|||||
test item |
31.3 |
A |
+ |
6600000 |
6600000 |
6600000 |
4681250 |
97,91 |
2,09 |
62.5 |
A |
+ |
5800000 |
6100000 |
5950000 |
4031250 |
84,31 |
15,69 |
|
125 |
A |
+ |
4400000 |
4200000 |
4300000 |
2381250 |
49,80 |
50,20 |
|
250 |
A |
+ |
3600000 |
3700000 |
3650000 |
1731250 |
36,21 |
63,79 |
|
500 |
A |
+ |
3250000 |
3250000 |
3250000 |
1331250 |
27,84 |
72,16 |
|
Cycl. 5µg/mL |
A |
+ |
4350000 |
4500000 |
4425000 |
2506250 |
52,42 |
47,58 |
|
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
Cycl: Cyclophosphamide monohydrate
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix / 20-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
5400000 |
5500000 |
5487500 |
3568750 |
100,00 |
0,00 |
- |
B |
– |
5600000 |
5450000 |
|||||
test item |
7.8 |
A |
– |
5500000 |
5450000 |
5475000 |
3556250 |
99,65 |
0,35 |
15.7 |
|
|
4250000 |
4500000 |
4375000 |
2456250 |
68,83 |
31,17 |
|
31.3 |
A |
– |
3600000 |
3700000 |
3650000 |
1731250 |
48,51 |
51,49 |
|
62.5 |
A |
– |
3000000 |
3000000 |
3000000 |
1081250 |
30,30 |
69,70 |
|
125 |
A |
– |
1800000 |
1700000 |
1750000 |
-168750* |
-4,73** |
104,73*** |
|
250 |
A |
– |
1250000 |
1150000 |
1200000 |
-718750* |
-20,14** |
120,14*** |
|
500 |
A |
– |
900000 |
1000000 |
950000 |
-968750* |
-27,15** |
127,15*** |
|
EMS 1 µL/mL |
A |
– |
3600000 |
3550000 |
3575000 |
1656250* |
46,41** |
53,59*** |
|
RICC=Relative Increase in Cell Counts
Cytotoxicity= 100-RICC
DME: (Dulbecco’s Modified Eagle’s)medium
EMS: Ethyl methanesulfonate (EMS)
*: cell number decrease,
**: zero RICC value,
***:100% cytotoxicity
Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY
20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time
Test group |
Concentration |
Parallels |
S9-mix |
Cell counts |
Mean cell counts |
Increase in cell counts |
RICC (%) |
Cytotoxicity |
|
First count |
Second count |
||||||||
Initial cell count |
- |
A |
– |
1850000 |
1900000 |
1918750 |
- |
- |
- |
- |
B |
– |
2050000 |
1950000 |
|||||
- |
C |
– |
2000000 |
1800000 |
|||||
- |
D |
– |
1850000 |
1950000 |
|||||
Solvent control (DMSO) |
- |
A |
– |
8600000 |
8350000 |
8450000 |
6531250 |
100,00 |
0,00 |
- |
B |
– |
8500000 |
8350000 |
|||||
test item |
7.8 |
A |
– |
8200000 |
8300000 |
8250000 |
6331250 |
96,94 |
3,06 |
15.7 |
A |
– |
6950000 |
6750000 |
6850000 |
4931250 |
75,50 |
24,50 |
|
31.3 |
A |
– |
5150000 |
5000000 |
5075000 |
3156250 |
48,33 |
51,67 |
|
62.5 |
A |
– |
3850000 |
3600000 |
3725000 |
1806250 |
27,66 |
72,34 |
|
125 |
A |
– |
1900000 |
1850000 |
1875000 |
-43750* |
-0,67** |
100,67*** |
|
250 |
A |
– |
1300000 |
1300000 |
1300000 |
-618750* |
-9,47** |
109,47*** |
|
500 |
A |
– |
700000 |
600000 |
650000 |
-1268750* |
-19,43** |
119,43*** |
|
EMS 1 µL/mL |
A |
– |
4900000 |
5050000 |
4975000 |
3056250* |
46,79** |
53,21*** |
|
Solvent control (DMSO) |
- |
A |
+ |
8800000 |
8950000 |
8762500 |
6843750 |
100,00 |
0,00 |
- |
B |
+ |
8600000 |
8700000 |
|||||
test item |
31.3 |
A |
+ |
8800000 |
8550000 |
8675000 |
6756250 |
98,72 |
1,28 |
62.5 |
A |
+ |
7600000 |
7500000 |
7550000 |
5631250 |
82,28 |
17,72 |
|
125 |
A |
+ |
5200000 |
5300000 |
5250000 |
3331250 |
48,68 |
51,32 |
|
250 |
A |
+ |
4400000 |
4600000 |
4500000 |
2581250 |
37,72 |
62,28 |
|
500 |
A |
+ |
3900000 |
4000000 |
3950000 |
2031250 |
29,68 |
70,32 |
|
Cycl. 5µg/mL |
A |
+ |
5150000 |
4950000 |
5050000 |
3131250 |
45,75 |
54,25 |
|
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
incl. gaps |
excl. gaps |
||||
Negative (Solvent) control |
- |
3 h |
20 h |
7 |
3 |
test item |
|||||
31.3 µg/mL |
- |
3 h |
20 h |
12 |
5 |
62.5 µg/mL |
- |
3 h |
20 h |
12 |
5 |
125 µg/mL |
- |
3 h |
20 h |
11 |
6 |
Pos. Control |
- |
3 h |
20 h |
41** |
31** |
Negative (Solvent) control |
+ |
3 h |
20 h |
8 |
4 |
test item |
|||||
31.3 µg/mL |
+ |
3 h |
20 h |
10 |
5 |
62.5 µg/mL |
+ |
3 h |
20 h |
12 |
5 |
125 µg/mL |
+ |
3 h |
20 h |
14 |
6 |
180 µg/mL |
+ |
3 h |
20 h |
10 |
5 |
Pos. Control (Cyclophosphamide) |
+ |
3 h |
20 h |
45** |
41** |
Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)
Positive control (+S9): Cyclophosphamide (5.0g/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|||
incl. gaps |
excl. gaps |
||||||
|
Negative (Solvent) control |
- |
20 h |
20 h |
7 |
3 |
|
|
test item |
|
|||||
|
7.8 µg/mL |
- |
20 h |
20 h |
8 |
3 |
|
|
15.6 µg/mL |
- |
20 h |
20 h |
8 |
4 |
|
|
31.3 µg/mL |
- |
20 h |
20 h |
8 |
4 |
|
|
Pos. Control |
- |
20 h |
20 h |
47** |
38** |
|
|
Negative (Solvent) control |
- |
20 h |
28 h |
7 |
3 |
|
|
test item |
|
|||||
|
7.8 µg/mL |
- |
20 h |
28 h |
8 |
3 |
|
|
15.6 µg/mL |
- |
20 h |
20 h |
7 |
3 |
|
|
31.3 µg/mL |
- |
20 h |
28 h |
7 |
4 |
|
|
Pos. Control |
- |
20 h |
28 h |
48** |
37** |
|
Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)
**= p < 0.01 to the concurrent negative control and to the historical control
TABLE 9 continued
MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B
Concentration |
S9 mix |
Treatment |
Harvesting time |
Mean aberrant cells/150cells |
|
|
incl. gaps |
excl. gaps |
|
||||
Negative (Solvent) control |
+ |
3 h |
28 h |
7 |
4 |
|
test item |
||||||
31.3 µg/mL |
+ |
3 h |
28 h |
8 |
3 |
|
62.5 µg/mL |
+ |
3 h |
28 h |
10 |
5 |
|
125 µg/mL |
+ |
3 h |
28 h |
10 |
3 |
|
180 µg/mL |
+ |
3 h |
28 h |
11 |
5 |
|
Pos. Control (Cyclophosphamide) |
+ |
3 h |
28 h |
48** |
39** |
|
Cyclophosphamide: 5.0g/mL
**= p < 0.01 to the concurrent negative control and to the historical control
APPENDIX IV
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT A
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
3/20 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
62.5 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
- |
3/20 h |
0.0 |
0.0 |
Pos. Control |
- |
3/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
+ |
3/20 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
180 µg/mL |
+ |
3/20 h |
0.0 |
0.0 |
Pos. Control (Cyclophosphamide) |
+ |
3/20 h |
0.0 |
0.0 |
Ethyl methanesulphonate: 1.0mL/mL
Cyclophosphamide: 5.0g/mL
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
- |
20/20 h |
0.0 |
0.0 |
test item |
||||
7.8 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
15.6 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20/20 h |
0.0 |
0.0 |
Pos. Control |
- |
20/20 h |
0.0 |
0.0 |
Negative (Solvent) control |
- |
20/28 h |
0.0 |
0.0 |
test item |
||||
7.8 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
15.6 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
31.3 µg/mL |
- |
20/28 h |
0.0 |
0.0 |
Pos. Control |
- |
20/28 h |
0.0 |
0.0 |
Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
TABLE 11 Continued
NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS
EXPERIMENT B
Concentration |
S9 mix |
Treatment/Harvesting |
Polyploid Cells (mean) |
Endoredup-lication (mean) |
Negative (Solvent) control |
+ |
3/28 h |
0.0 |
0.0 |
test item |
||||
31.3 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
62.5 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
125 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
180 µg/mL |
+ |
3/28 h |
0.0 |
0.0 |
Pos. Control |
+ |
3/28 h |
0.0 |
0.0 |
Cyclophosphamide: 5.0g/mL
The number of polyploid and endoreduplicated cells was determined
in
300 cells of each test group.
In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed.
TABLE 1. SUMMARIZED RESULTS OF THE PRE-TEST ON TOXICITY 8% HOUR TREATMENT WITHOUT S9-MIX
Test group |
Dose |
S9-mix |
Treatment/ |
Number of colonies/200cells/dish |
Mean |
Relativea |
||
dish 1 |
dish 2 |
dish 3 |
||||||
Untreated Control |
– |
– |
- |
203 |
201 |
205 |
203,0 |
99 |
Solvent Control (DMSO) |
– |
– |
5 |
205 |
206 |
206 |
205,7 |
100 |
Test item |
31.3 |
– |
5 |
202 |
198 |
201 |
200,3 |
97 |
62.5 |
– |
5 |
194 |
197 |
198 |
196,3 |
95 |
|
125 |
– |
5 |
170 |
165 |
166 |
167,0 |
81 |
|
250 |
– |
5 |
77 |
72 |
72 |
73,7 |
36 |
|
500 |
– |
5 |
15 |
14 |
12 |
13,7 |
7 |
|
750 |
– |
5 |
6 |
7 |
8 |
7,0 |
3 |
|
1000 |
– |
5 |
5 |
4 |
3 |
4,0 |
2 |
|
Untreated Control |
– |
+ |
- |
201 |
205 |
200 |
202,0 |
100 |
Solvent Control (DMSO) |
– |
+ |
5 |
206 |
201 |
200 |
202,3 |
100 |
Test item |
31.3 |
+ |
5 |
202 |
201 |
203 |
202,0 |
100 |
62.5 |
+ |
5 |
202 |
197 |
201 |
200,0 |
99 |
|
125 |
+ |
5 |
188 |
190 |
190 |
189,3 |
94 |
|
250 |
+ |
5 |
97 |
95 |
95 |
95,7 |
47 |
|
500 |
+ |
5 |
75 |
77 |
74 |
75,3 |
37 |
|
750 |
+ |
5 |
63 |
60 |
61 |
61,3 |
30 |
|
1000 |
+ |
5 |
33 |
31 |
30 |
31,3 |
15 |
|
aRelative to Solvent Control
Table 2:CHO/HPRT MUTAGENESIS ASSAY RESULTS; MAIN MUTATION ASSAY/a, b, c and d
(5-hour Treatment without S9-Mix)
Study code: |
805-476-2615 |
|
|
|||||||||||||||||||||||||||||||||||||||||||||
Test item: |
without S9-mix |
Batch number: |
106134974 |
|||||||||||||||||||||||||||||||||||||||||||||
Test date of Main Mutation Assay: |
October 17, 2017 – November 02, 2017 |
Expression period: |
8 days |
|||||||||||||||||||||||||||||||||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4g/mL 6-thioguanine |
|||||||||||||||||||||||||||||||||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||||||||||||||||||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
||||||||||||||||||||||||||||||||||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
||||||||||||||||||||||||||||||||||||||||||
Solvent control a |
201.0 |
± |
2.65 |
100 |
100 |
1 |
0 |
1 |
2 |
3 |
7 |
101 |
6.93 |
|||||||||||||||||||||||||||||||||||
Pos. control |
57.7 |
± |
2.52 |
29 |
64 |
198 |
203 |
187 |
188 |
191 |
967 |
65 |
1487.69** |
|||||||||||||||||||||||||||||||||||
TEST ITEM |
|
|||||||||||||||||||||||||||||||||||||||||||||||
62.5g/mL a |
190,0 |
± |
3,00 |
95 |
99 |
1 |
2 |
1 |
1 |
0 |
5 |
101 |
4.95 |
|||||||||||||||||||||||||||||||||||
125g/mL a |
159,3 |
± |
2,08 |
79 |
99 |
3 |
0 |
1 |
3 |
0 |
7 |
100 |
7.00 |
|||||||||||||||||||||||||||||||||||
250g/mL a |
79,0 |
± |
1,00 |
39 |
98 |
2 |
0 |
1 |
1 |
1 |
5 |
100 |
5.00 |
|||||||||||||||||||||||||||||||||||
350g/mL a |
69,0 |
± |
1,73 |
34 |
100 |
1 |
2 |
2 |
2 |
2 |
9 |
101 |
8.91 |
|||||||||||||||||||||||||||||||||||
450g/mL a |
30,7 |
± |
1,15 |
15 |
97 |
1 |
3 |
3 |
2 |
0 |
9 |
98 |
9.18 |
|||||||||||||||||||||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||||||||||||||||||||||||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||||||||||||||||||||||||||||||||
Solvent control b |
200.3 |
± |
1.53 |
100 |
100 |
1 |
2 |
1 |
2 |
1 |
7 |
101 |
6.93 |
|
||||||||||||||||||||||||||||||||||
Pos. control |
58.0 |
± |
3.00 |
29 |
65 |
194 |
205 |
186 |
200 |
195 |
980 |
65 |
1507.69** |
|
||||||||||||||||||||||||||||||||||
TEST ITEM |
|
|
||||||||||||||||||||||||||||||||||||||||||||||
62.5g/mL b |
190,7 |
± |
1,15 |
95 |
99 |
1 |
2 |
1 |
0 |
2 |
6 |
100 |
6.00 |
|
||||||||||||||||||||||||||||||||||
125g/mL b |
160,7 |
± |
2,08 |
80 |
99 |
1 |
2 |
3 |
2 |
1 |
9 |
100 |
9.00 |
|
||||||||||||||||||||||||||||||||||
250g/mL b |
77,0 |
± |
1,00 |
38 |
99 |
1 |
1 |
1 |
1 |
1 |
5 |
100 |
5.00 |
|
||||||||||||||||||||||||||||||||||
350g/mL b |
70,3 |
± |
0,58 |
35 |
100 |
2 |
1 |
3 |
0 |
2 |
8 |
101 |
7.92 |
|
||||||||||||||||||||||||||||||||||
450g/mL b |
32,0 |
± |
2,00 |
16 |
99 |
3 |
2 |
2 |
1 |
2 |
10 |
99 |
10.10 |
|
||||||||||||||||||||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||||||||||||||||||||||||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||||||||||||||||||||||||||||||||
Solvent control c |
203.0 |
± |
2.0 |
100 |
100 |
1 |
1 |
2 |
1 |
2 |
7 |
101 |
6.93 |
|
||||||||||||||||||||||||||||||||||
Pos. control |
55.0 |
± |
1.73 |
27 |
66 |
199 |
205 |
202 |
191 |
197 |
994 |
67 |
1483.58** |
|
||||||||||||||||||||||||||||||||||
TEST ITEM |
|
|
||||||||||||||||||||||||||||||||||||||||||||||
62.5g/mL c |
194,3 |
± |
2,08 |
96 |
99 |
0 |
2 |
1 |
1 |
1 |
5 |
100 |
5.00 |
|
||||||||||||||||||||||||||||||||||
125g/mL c |
164,0 |
± |
1,00 |
81 |
98 |
3 |
3 |
2 |
0 |
1 |
9 |
99 |
9.09 |
|
||||||||||||||||||||||||||||||||||
250g/mL c |
82,0 |
± |
2,00 |
40 |
101 |
1 |
2 |
2 |
1 |
1 |
7 |
102 |
6.86 |
|
||||||||||||||||||||||||||||||||||
350g/mL c |
69,3 |
± |
2,89 |
34 |
99 |
2 |
0 |
2 |
1 |
0 |
5 |
100 |
5.00 |
|
||||||||||||||||||||||||||||||||||
450g/mL c |
34,7 |
± |
2,08 |
17 |
98 |
1 |
2 |
3 |
3 |
1 |
10 |
99 |
10.10 |
|
||||||||||||||||||||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||||||||||||||||||||||||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||||||||||||||||||||||||||||||||
Solvent control d |
202.0 |
± |
1.00 |
100 |
100 |
0 |
2 |
2 |
2 |
1 |
7 |
101 |
6.93 |
|
||||||||||||||||||||||||||||||||||
Pos. control |
53.3 |
± |
1.53 |
26 |
66 |
207 |
205 |
196 |
188 |
194 |
990 |
66 |
1500.00** |
|
||||||||||||||||||||||||||||||||||
TEST ITEM |
|
|
||||||||||||||||||||||||||||||||||||||||||||||
62.5g/mL d |
193,0 |
± |
1,00 |
96 |
100 |
2 |
1 |
1 |
1 |
2 |
7 |
100 |
7.00 |
|
||||||||||||||||||||||||||||||||||
125g/mL d |
163,0 |
± |
0,00 |
81 |
99 |
1 |
1 |
3 |
1 |
1 |
7 |
99 |
7.07 |
|
||||||||||||||||||||||||||||||||||
250g/mL d |
76,0 |
± |
2,65 |
38 |
100 |
3 |
0 |
1 |
3 |
1 |
8 |
100 |
8.00 |
|
||||||||||||||||||||||||||||||||||
350g/mL d |
70,7 |
± |
0,58 |
35 |
99 |
2 |
2 |
1 |
2 |
0 |
7 |
100 |
7.00 |
|
||||||||||||||||||||||||||||||||||
450g/mL d |
32,7 |
± |
0,58 |
16 |
99 |
3 |
2 |
1 |
3 |
1 |
10 |
99 |
10.10 |
|
||||||||||||||||||||||||||||||||||
a = parallel for mutation.
b = parallel for mutation.
c = parallel for mutation.
d = parallel for mutation.
abs.C.E. = Absolute Cloning Efficiency
EMS=Ethyl methanesulfonate
** = p < 0.01 to the concurrent negative control and to the historical control
Table 3:CHO/HPRT MUTAGENESIS ASSAY RESULTS, MAIN MUTATION ASSAY/a, b, c and d
(5-hour Treatment with S9-Mix)
Study code: |
805-476-2615 |
|
|
|||||||||||||||
Test item: |
with S9-mix |
Batch number: |
106134974 |
|||||||||||||||
Test date of Main Mutation Assay: |
October 17, 2017 – November 02, 2017 |
Expression period: |
8 days |
|||||||||||||||
Solvent: |
DMSO |
Selective agent: |
3.4g/mL 6-thioguanine |
|||||||||||||||
Cells seeded for analysis: |
2x105cells /dish for mutant selection: 200 cells/dish for C.E. |
|
||||||||||||||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||
Solvent control a |
199.0 |
± |
1.73 |
100 |
100 |
2 |
1 |
1 |
2 |
2 |
8 |
100 |
8.00 |
|
||||
Pos. control |
122.3 |
± |
2.08 |
61 |
76 |
119 |
109 |
108 |
115 |
114 |
565 |
76 |
743.42** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125g/mL a |
192,3 |
± |
2,08 |
97 |
100 |
2 |
0 |
1 |
2 |
1 |
6 |
100 |
6.00 |
|
||||
250g/mL a |
88,0 |
± |
1,00 |
44 |
98 |
3 |
1 |
0 |
1 |
2 |
7 |
98 |
7.14 |
|
||||
500g/mL a |
69,3 |
± |
1,15 |
35 |
100 |
0 |
1 |
2 |
1 |
1 |
5 |
100 |
5.00 |
|
||||
750g/mL a |
57,0 |
± |
1,00 |
29 |
99 |
1 |
2 |
2 |
2 |
1 |
8 |
99 |
8.08 |
|
||||
1000g/mL a |
31,3 |
± |
1,15 |
16 |
97 |
3 |
3 |
2 |
0 |
0 |
8 |
97 |
8.24 |
|
||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||
Solvent control b |
200.7 |
± |
1.15 |
100 |
100 |
1 |
2 |
1 |
1 |
1 |
6 |
100 |
6.00 |
|
||||
Pos. control |
124.0 |
± |
1.00 |
62 |
75 |
117 |
112 |
108 |
105 |
107 |
549 |
76 |
722.37** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125g/mL b |
194,7 |
± |
2,08 |
97 |
99 |
3 |
0 |
1 |
2 |
1 |
7 |
100 |
7.00 |
|
||||
250g/mL b |
90,3 |
± |
0,58 |
45 |
99 |
1 |
1 |
2 |
0 |
2 |
6 |
99 |
6.06 |
|
||||
500g/mL b |
71,7 |
± |
1,53 |
36 |
99 |
2 |
1 |
2 |
1 |
2 |
8 |
100 |
8.00 |
|
||||
750g/mL b |
58,3 |
± |
1,53 |
29 |
99 |
3 |
3 |
0 |
1 |
1 |
8 |
99 |
8.08 |
|
||||
1000g/mL b |
32,0 |
± |
1,00 |
16 |
98 |
1 |
2 |
3 |
2 |
0 |
8 |
98 |
8.16 |
|
||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||
Solvent control c |
199.0 |
± |
1.00 |
100 |
100 |
3 |
2 |
1 |
0 |
1 |
7 |
101 |
6.93 |
|
||||
Pos. control |
127.0 |
± |
2.00 |
64 |
76 |
110 |
112 |
108 |
106 |
117 |
553 |
77 |
718.18** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125g/mL c |
189,0 |
± |
1,00 |
95 |
99 |
2 |
3 |
1 |
1 |
0 |
7 |
100 |
7.00 |
|
||||
250g/mL c |
90,3 |
± |
0,58 |
45 |
99 |
1 |
1 |
3 |
3 |
1 |
9 |
100 |
9.00 |
|
||||
500g/mL c |
69,0 |
± |
1,73 |
35 |
97 |
2 |
2 |
2 |
2 |
1 |
9 |
98 |
9.18 |
|
||||
750g/mL c |
57,0 |
± |
1,73 |
29 |
100 |
1 |
2 |
1 |
3 |
2 |
9 |
101 |
8.91 |
|
||||
1000g/mL c |
33,3 |
± |
0,58 |
17 |
97 |
2 |
2 |
3 |
1 |
0 |
8 |
98 |
8.16 |
|
||||
NON |
SURVIVAL TO TREATMENT |
REL. POPU- |
MUTANT COLONIES |
TOTAL |
ABSOLUTE |
MUTANT |
|
|||||||||||
MEAN COLONY |
PERCENT |
1 |
2 |
3 |
4 |
5 |
|
|||||||||||
Solvent control d |
200.0 |
± |
1.00 |
100 |
100 |
3 |
1 |
1 |
2 |
0 |
7 |
101 |
6.93 |
|
||||
Pos. control |
126.7 |
± |
0.58 |
63 |
76 |
106 |
104 |
118 |
112 |
111 |
551 |
76 |
725.00** |
|
||||
TEST ITEM |
|
|
||||||||||||||||
125g/mL d |
190,7 |
± |
1,53 |
95 |
99 |
0 |
0 |
2 |
3 |
2 |
7 |
100 |
7.00 |
|
||||
250g/mL d |
91,7 |
± |
1,15 |
46 |
99 |
1 |
1 |
1 |
2 |
1 |
6 |
100 |
6.00 |
|
||||
500g/mL d |
69,3 |
± |
1,53 |
35 |
99 |
3 |
2 |
3 |
1 |
0 |
9 |
99 |
9.09 |
|
||||
750g/mL d |
58,3 |
± |
0,58 |
29 |
100 |
2 |
2 |
1 |
2 |
2 |
9 |
100 |
9.00 |
|
||||
1000g/mL d |
32,3 |
± |
1,15 |
16 |
99 |
1 |
0 |
0 |
3 |
1 |
5 |
99 |
5.05 |
|
||||
a = parallel for mutation.
b = parallel for mutation.
c = parallel for mutation.
d = parallel for mutation.
abs.C.E. = Absolute Cloning Efficiency
DMBA=7,12-Dimethyl benzanthracene
** = p < 0.01 to the concurrent negative control and to the historical control
Table 4: Day 1 Cloning efficiencies -Main Mutation Assay (5-hour treatment without And WITH S9-Mix)
Test Group a and b |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
||||||||||||
Negative (Solvent Control) |
- |
– |
5 |
204 |
200 |
199 |
201,0 |
101 |
100 |
||||||
TEST ITEM- Group a |
62.5 |
– |
5 |
193 |
187 |
190 |
190,0 |
95 |
95 |
||||||
125 |
– |
5 |
160 |
161 |
157 |
159,3 |
80 |
79 |
|||||||
250 |
– |
5 |
80 |
78 |
79 |
79,0 |
40 |
39 |
|||||||
350 |
– |
5 |
70 |
67 |
70 |
69,0 |
35 |
34 |
|||||||
450 |
– |
5 |
32 |
30 |
30 |
30,7 |
15 |
15 |
|||||||
EMS (1µL/mL) |
– |
5 |
60 |
55 |
58 |
57,7 |
29 |
29 |
|||||||
Negative (Solvent Control) |
- |
– |
5 |
202 |
200 |
199 |
200,3 |
100 |
100 |
||||||
TEST ITEM - Group b |
62.5 |
– |
5 |
192 |
190 |
190 |
190,7 |
95 |
95 |
||||||
125 |
– |
5 |
159 |
160 |
163 |
160,7 |
80 |
80 |
|||||||
250 |
– |
5 |
76 |
77 |
78 |
77,0 |
39 |
38 |
|||||||
350 |
– |
5 |
71 |
70 |
70 |
70,3 |
35 |
35 |
|||||||
450 |
– |
5 |
32 |
34 |
30 |
32,0 |
16 |
16 |
|||||||
EMS (1µL/mL) |
– |
5 |
58 |
55 |
61 |
58,0 |
29 |
29 |
|||||||
Test Group c and d |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
||||||||||||
Negative (Solvent Control) |
- |
– |
5 |
201 |
203 |
205 |
203,0 |
102 |
100 |
||||||
Test item- Group c |
62.5 |
– |
5 |
196 |
192 |
195 |
194,3 |
97 |
96 |
||||||
125 |
– |
5 |
164 |
165 |
163 |
164,0 |
82 |
81 |
|||||||
250 |
– |
5 |
80 |
82 |
84 |
82,0 |
41 |
40 |
|||||||
350 |
– |
5 |
71 |
71 |
66 |
69,3 |
35 |
34 |
|||||||
450 |
– |
5 |
34 |
37 |
33 |
34,7 |
17 |
17 |
|||||||
EMS (1µL/mL) |
– |
5 |
57 |
54 |
54 |
55,0 |
28 |
27 |
|||||||
Negative (Solvent Control) |
- |
– |
5 |
201 |
202 |
203 |
202,0 |
101 |
100 |
||||||
Test item - Group d |
62.5 |
– |
5 |
193 |
192 |
194 |
193,0 |
97 |
96 |
||||||
125 |
– |
5 |
163 |
163 |
163 |
163,0 |
82 |
81 |
|||||||
250 |
– |
5 |
79 |
75 |
74 |
76,0 |
38 |
38 |
|||||||
350 |
– |
5 |
71 |
71 |
70 |
70,7 |
35 |
35 |
|||||||
450 |
– |
5 |
33 |
32 |
33 |
32,7 |
16 |
16 |
|||||||
EMS (1µL/mL) |
– |
5 |
55 |
52 |
53 |
53,3 |
27 |
26 |
|||||||
Test group a and b |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|
||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
|
|||||||||||
Negative (Solvent Control) |
- |
+ |
5 |
200 |
197 |
200 |
199,0 |
100 |
100 |
|
|||||
Test item- Group a |
125 |
+ |
5 |
193 |
194 |
190 |
192,3 |
96 |
97 |
|
|||||
250 |
+ |
5 |
89 |
88 |
87 |
88,0 |
44 |
44 |
|
||||||
500 |
+ |
5 |
70 |
68 |
70 |
69,3 |
35 |
35 |
|
||||||
750 |
+ |
5 |
57 |
58 |
56 |
57,0 |
29 |
29 |
|
||||||
1000 |
+ |
5 |
32 |
30 |
32 |
31,3 |
16 |
16 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
124 |
123 |
120 |
122,3 |
61 |
61 |
|
||||||
Negative (Solvent Control) |
- |
+ |
5 |
200 |
202 |
200 |
200,7 |
100 |
100 |
|
|||||
Test item-Groupb |
125 |
+ |
5 |
197 |
194 |
193 |
194,7 |
97 |
97 |
|
|||||
250 |
+ |
5 |
90 |
91 |
90 |
90,3 |
45 |
45 |
|
||||||
500 |
+ |
5 |
72 |
73 |
70 |
71,7 |
36 |
36 |
|
||||||
750 |
+ |
5 |
57 |
58 |
60 |
58,3 |
29 |
29 |
|
||||||
1000 |
+ |
5 |
31 |
33 |
32 |
32,0 |
16 |
16 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
125 |
123 |
124 |
124,0 |
62 |
62 |
|
||||||
Test group c and d |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|
||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
|
|||||||||||
Negative (Solvent Control) |
- |
+ |
5 |
199 |
198 |
200 |
199,0 |
100 |
100 |
|
|||||
Test item- Group c |
125 |
+ |
5 |
190 |
188 |
189 |
189,0 |
95 |
95 |
|
|||||
250 |
+ |
5 |
91 |
90 |
90 |
90,3 |
45 |
45 |
|
||||||
500 |
+ |
5 |
67 |
70 |
70 |
69,0 |
35 |
35 |
|
||||||
750 |
+ |
5 |
58 |
55 |
58 |
57,0 |
29 |
29 |
|
||||||
1000 |
+ |
5 |
33 |
33 |
34 |
33,3 |
17 |
17 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
125 |
129 |
127 |
127,0 |
64 |
64 |
|
||||||
Negative (Solvent Control) |
- |
+ |
5 |
199 |
201 |
200 |
200,0 |
100 |
100 |
|
|||||
Test item-Groupd |
125 |
+ |
5 |
192 |
191 |
189 |
190,7 |
95 |
95 |
|
|||||
250 |
+ |
5 |
91 |
93 |
91 |
91,7 |
46 |
46 |
|
||||||
500 |
+ |
5 |
68 |
69 |
71 |
69,3 |
35 |
35 |
|
||||||
750 |
+ |
5 |
58 |
59 |
58 |
58,3 |
29 |
29 |
|
||||||
1000 |
+ |
5 |
31 |
33 |
33 |
32,3 |
16 |
16 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
126 |
127 |
127 |
126,7 |
63 |
63 |
|
||||||
EMS= Ethyl methanesulfonate; DMBA= 7,12-Dimethylbenzanthracene
Table 5: Day 8 Cloning efficiencies -Main Mutation Assay (5-hour treatment without and With S9-Mix)
Test Group a and b |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
||||||||||||
Negative (Solvent Control) |
- |
– |
5 |
202 |
205 |
201 |
202,7 |
101 |
100 |
||||||
Test item- Group a |
62.5 |
– |
5 |
202 |
199 |
203 |
201,3 |
101 |
99 |
||||||
125 |
– |
5 |
200 |
198 |
204 |
200,7 |
100 |
99 |
|||||||
250 |
– |
5 |
198 |
202 |
198 |
199,3 |
100 |
98 |
|||||||
350 |
– |
5 |
201 |
204 |
202 |
202,3 |
101 |
100 |
|||||||
450 |
– |
5 |
199 |
196 |
195 |
196,7 |
98 |
97 |
|||||||
EMS (1µL/mL) |
– |
5 |
128 |
130 |
131 |
129,7 |
65 |
64 |
|||||||
Negative (Solvent Control) |
- |
– |
5 |
201 |
202 |
201 |
201,3 |
101 |
100 |
||||||
Test item - Group b |
62.5 |
– |
5 |
200 |
199 |
200 |
199,7 |
100 |
99 |
||||||
125 |
– |
5 |
199 |
199 |
202 |
200,0 |
100 |
99 |
|||||||
250 |
– |
5 |
200 |
200 |
200 |
200,0 |
100 |
99 |
|||||||
350 |
– |
5 |
201 |
202 |
201 |
201,3 |
101 |
100 |
|||||||
450 |
– |
5 |
200 |
199 |
197 |
198,7 |
99 |
99 |
|||||||
EMS (1µL/mL) |
– |
5 |
130 |
129 |
133 |
130,7 |
65 |
65 |
|||||||
Test Group c and d |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
||||||||||||
Negative (Solvent Control) |
- |
– |
5 |
202 |
199 |
203 |
201,3 |
101 |
100 |
||||||
Test item- Group c |
62.5 |
– |
5 |
200 |
201 |
196 |
199,0 |
100 |
99 |
||||||
125 |
– |
5 |
196 |
198 |
197 |
197,0 |
99 |
98 |
|||||||
250 |
– |
5 |
205 |
204 |
200 |
203,0 |
102 |
101 |
|||||||
350 |
– |
5 |
198 |
202 |
197 |
199,0 |
100 |
99 |
|||||||
450 |
– |
5 |
198 |
198 |
196 |
197,3 |
99 |
98 |
|||||||
EMS (1µL/mL) |
– |
5 |
133 |
134 |
133 |
133,3 |
67 |
66 |
|||||||
Negative (Solvent Control) |
- |
– |
5 |
201 |
200 |
203 |
201,3 |
101 |
100 |
||||||
Test item - Group d |
62.5 |
– |
5 |
198 |
201 |
202 |
200,3 |
100 |
100 |
||||||
125 |
– |
5 |
199 |
200 |
197 |
198,7 |
99 |
99 |
|||||||
250 |
– |
5 |
202 |
200 |
199 |
200,3 |
100 |
100 |
|||||||
350 |
– |
5 |
199 |
201 |
198 |
199,3 |
100 |
99 |
|||||||
450 |
– |
5 |
200 |
199 |
197 |
198,7 |
99 |
99 |
|||||||
EMS (1µL/mL) |
– |
5 |
132 |
133 |
133 |
132,7 |
66 |
66 |
|||||||
Test group a and b |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|
||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
|
|||||||||||
Negative (Solvent Control) |
- |
+ |
5 |
198 |
201 |
200 |
199,7 |
100 |
100 |
|
|||||
Test item- Group a |
125 |
+ |
5 |
201 |
198 |
200 |
199,7 |
100 |
100 |
|
|||||
250 |
+ |
5 |
197 |
195 |
194 |
195,3 |
98 |
98 |
|
||||||
500 |
+ |
5 |
200 |
200 |
200 |
200,0 |
100 |
100 |
|
||||||
750 |
+ |
5 |
198 |
197 |
196 |
197,0 |
99 |
99 |
|
||||||
1000 |
+ |
5 |
195 |
192 |
194 |
193,7 |
97 |
97 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
155 |
153 |
150 |
152,7 |
76 |
76 |
|
||||||
Negative (Solvent Control) |
- |
+ |
5 |
201 |
201 |
200 |
200,7 |
100 |
100 |
|
|||||
Test item-Groupb |
125 |
+ |
5 |
199 |
198 |
200 |
199,0 |
100 |
99 |
|
|||||
250 |
+ |
5 |
197 |
200 |
199 |
198,7 |
99 |
99 |
|
||||||
500 |
+ |
5 |
198 |
199 |
200 |
199,0 |
100 |
99 |
|
||||||
750 |
+ |
5 |
198 |
200 |
196 |
198,0 |
99 |
99 |
|
||||||
1000 |
+ |
5 |
197 |
198 |
194 |
196,3 |
98 |
98 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
151 |
152 |
150 |
151,0 |
76 |
75 |
|
||||||
Test group c and d |
Concentration |
S9-mix |
Treatment |
Number of colonies/200cells/dish |
Cloning |
% of Control |
|
||||||||
dish 1 |
dish 2 |
dish 3 |
Mean |
|
|||||||||||
Negative (Solvent Control) |
- |
+ |
5 |
203 |
202 |
201 |
202,0 |
101 |
100 |
|
|||||
Test item- Group c |
125 |
+ |
5 |
200 |
198 |
202 |
200,0 |
100 |
99 |
|
|||||
250 |
+ |
5 |
201 |
200 |
196 |
199,0 |
100 |
99 |
|
||||||
500 |
+ |
5 |
194 |
198 |
198 |
196,7 |
98 |
97 |
|
||||||
750 |
+ |
5 |
202 |
202 |
204 |
202,7 |
101 |
100 |
|
||||||
1000 |
+ |
5 |
199 |
196 |
194 |
196,3 |
98 |
97 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
155 |
149 |
155 |
153,0 |
77 |
76 |
|
||||||
Negative (Solvent Control) |
- |
+ |
5 |
202 |
201 |
201 |
201,3 |
101 |
100 |
|
|||||
Test item-Groupd |
125 |
+ |
5 |
199 |
198 |
201 |
199,3 |
100 |
99 |
|
|||||
250 |
+ |
5 |
199 |
200 |
198 |
199,0 |
100 |
99 |
|
||||||
500 |
+ |
5 |
199 |
198 |
198 |
198,3 |
99 |
99 |
|
||||||
750 |
+ |
5 |
200 |
202 |
200 |
200,7 |
100 |
100 |
|
||||||
1000 |
+ |
5 |
199 |
199 |
197 |
198,3 |
99 |
99 |
|
||||||
DMBA (20µg/mL) |
+ |
5 |
154 |
150 |
154 |
152,7 |
76 |
76 |
|
||||||
EMS= Ethyl methanesulfonate; DMBA= 7,12-Dimethylbenzanthracene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames Test:
The test item dissolved in DMSO was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotrophic strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotrophic strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). The following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000;1600; 500; 160; 50; 16 and 5 µg/plate (corrected concentrations on the basis of dye content in the test item). The selection of the concentration range was based on the recommendations in OECD 471 guideline. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimuriumTA1537 strain in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains, mostly in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains.
Chromosome Aberration Assay:
The test item suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations (corrected concentrations on the basis of the dye content of the test item) were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.
Experiment A with 3/20 h treatment/sampling time
without: 15.6,1 31.3, 62.5, 125 and 1802g/mL test item
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item
Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.
HPRT Assay
The test item suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations (reported as corrected concentrations considering the dye content of the test item) were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:
Mutation Assay 5-hour treatment period without S9-mix:
62.5, 125, 250, 350 and 450 µg/mL
Mutation Assay 5-hour treatment period with S9-mix:
125, 250, 500, 750 and 1000 µg/mL
In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure. In the absence and presence of metabolic activation clear cytotoxicity (survival approximately 16 %) of the test item was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95 % confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95 % confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. The test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.
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. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.
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