Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 310-132-3 | CAS number: 61902-31-6 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53235
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 OECD guideline study 471 (bacterial reverse mutation assay).
Two further genetic toxicity studies were performed with the read across substance Leuco Sulphur Yellow 22 (CAS 90268-98-7) using mammalian cells: the chromosomal aberration test according to OECD 473 and the HPRT assay according to OECD and 476. No genetic toxicity was observed in both studies.
In conclusion, the test item is considered to have no genotoxic potential.
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:
- 28 March - 12 June, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 30 May, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August, 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: – ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use,
- Version / remarks:
- June, 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Date of production: 29.11.2016
Expiration date: 29.11.2021
Storage: Room temperature (15-25°C) - Target gene:
- his/trp
In addition to histidine (his) or tryptophan (trp) 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:
- S9 mix of phenobarbital and β-naphthoflavone-induced rat liver
- Test concentrations with justification for top dose:
- 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.
Selection of the concentration range was done on the basis of a solubility test and a concentration range finding test (informatory toxicity test). - Vehicle / solvent:
- Solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: Based on the results of a soliubility test, dimethyl sulfoxide (DMSO) was found to be the most appropriate solvent for preparing the test item stock formulations and dilutions. The solvent was chosen due to its non-toxicity to the bacteria and the compatibility to the S9 activity based on the available laboratory’s historical control database. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylenediamine
- Remarks:
- TA98, without S9 mix, 4 µg/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA100 and TA1535, without S9 mix, 2 µg/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537, without S9 mix, 50 µg/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- E.coli WP2, without S9 mix, 2 µL/plate
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- All tested Salmonella strains, with S9 mix, 2 µg/plate; E.coli WP2, with S9 mix, 50 µg/plate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: number of revertat colonies, background lawn growth
- 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:
- cytotoxicity
- Remarks:
- without S9 mix, 500 µg/plate with S9 mix, 5000 µg/plate
- 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:
- cytotoxicity
- Remarks:
- Without S9 mix, 1600 µg/plate
- 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:
- cytotoxicity
- Remarks:
- without S9 mix, 500 µg/plate
- 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:
- cytotoxicity
- Remarks:
- without S9 mix, 500 µg/plate; with S9 mix, 1600 µg/plate
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures. - Conclusions:
- In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Executive summary:
The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (97.4 % dyestuff) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on 97.4 % dyestuff) were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, 16, and 5 µg/plate. 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 concentrations of 5000 and 1600 µg/plate in absence and in the presence of S9 mix following the plate incorporation and pre-incubation procedures. Inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain in the absence and also in the presence of S9 mix, in the confirmatory mutation test in all S. typhimurium strains in the absence of S9; in TA98 and TA1537 also in the presence of S9. The inhibitory effect 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). In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent 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 mostly 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 at any concentration level, either in the presence or absence of S9 mix.The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains, under the test conditions used in this study.
- 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:
- in vitro mammalian chromosome aberration test
- 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 - 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:
- The 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 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 - 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 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
Table 4: Summary Table of the Results of the Initial Mutation Test
Initial Mutation Test (Plate Incorporation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichiacoli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
17.7 |
1.02 |
23.0 |
0.92 |
100.0 |
1.04 |
109.0 |
1.18 |
10.0 |
0.97 |
14.3 |
1.02 |
7.3 |
1.22 |
7.3 |
0.96 |
32.0 |
0.99 |
39.3 |
1.18 |
DMSO Control |
17.3 |
1.00 |
25.0 |
1.00 |
96.3 |
1.00 |
92.3 |
1.00 |
10.3 |
1.00 |
14.0 |
1.00 |
6.0 |
1.00 |
7.7 |
1.00 |
32.3 |
1.00 |
33.3 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
90.0 |
1.00 |
– |
– |
11.3 |
1.00 |
– |
– |
– |
– |
– |
– |
36.3 |
1.00 |
– |
– |
5000 |
12.0 |
0.69 |
16.3 |
0.65 |
71.0 |
0.74 |
82.0 |
0.89 |
12.3 |
1.19 |
12.3 |
0.88 |
0.0 |
0.00 |
1.0 |
0.13 |
18.3 |
0.57 |
25.0 |
0.75 |
1600 |
23.0 |
1.33 |
19.7 |
0.79 |
89.7 |
0.93 |
100.3 |
1.09 |
9.3 |
0.90 |
13.0 |
0.93 |
1.7 |
0.28 |
6.0 |
0.78 |
28.3 |
0.88 |
31.3 |
0.94 |
500 |
21.7 |
1.25 |
35.7 |
1.43 |
96.0 |
1.00 |
113.3 |
1.23 |
14.3 |
1.39 |
12.0 |
0.86 |
5.3 |
0.89 |
6.3 |
0.83 |
29.0 |
0.90 |
29.7 |
0.89 |
160 |
20.3 |
1.17 |
57.3 |
2.29 |
90.0 |
0.93 |
117.3 |
1.27 |
8.7 |
0.84 |
11.3 |
0.81 |
5.3 |
0.89 |
8.3 |
1.09 |
32.7 |
1.01 |
31.3 |
0.94 |
50 |
19.3 |
1.12 |
50.0 |
2.00 |
87.0 |
0.90 |
121.3 |
1.31 |
9.3 |
0.90 |
11.7 |
0.83 |
4.7 |
0.78 |
6.3 |
0.83 |
31.3 |
0.97 |
24.3 |
0.73 |
16 |
21.0 |
1.21 |
42.7 |
1.71 |
88.3 |
0.92 |
110.7 |
1.20 |
15.7 |
1.52 |
12.0 |
0.86 |
6.7 |
1.11 |
6.3 |
0.83 |
32.7 |
1.01 |
29.7 |
0.89 |
5 |
23.7 |
1.37 |
27.7 |
1.11 |
86.0 |
0.89 |
109.0 |
1.18 |
12.7 |
1.23 |
12.7 |
0.90 |
7.0 |
1.17 |
5.3 |
0.70 |
28.0 |
0.87 |
37.7 |
1.13 |
NPD (4mg) |
202.7 |
11.69 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1234.7 |
13.72 |
– |
– |
765.3 |
67.53 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
676.0 |
112.67 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
918.7 |
25.28 |
– |
– |
2AA (2mg) |
– |
– |
2429.3 |
97.17 |
– |
– |
1632.0 |
17.68 |
– |
– |
228.3 |
16.31 |
– |
– |
239.3 |
31.22 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
197.3 |
5.92 |
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.
Table 5 :Summary Table of the Results of the Confirmatory Mutation Test
Confirmatory Mutation Test (Pre-Incubation Test) |
||||||||||||||||||||
Concentrations (mg/plate) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
WP2uvrA |
||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
21.3 |
1.25 |
29.3 |
1.16 |
87.3 |
1.08 |
111.3 |
1.17 |
14.3 |
1.48 |
10.7 |
0.94 |
5.0 |
0.88 |
9.7 |
0.88 |
24.3 |
1.18 |
25.7 |
0.71 |
DMSO Control |
17.0 |
1.00 |
25.3 |
1.00 |
81.0 |
1.00 |
95.3 |
1.00 |
9.7 |
1.00 |
11.3 |
1.00 |
5.7 |
1.00 |
11.0 |
1.00 |
20.7 |
1.00 |
36.0 |
1.00 |
Ultrapure Water Control |
– |
– |
– |
– |
79.0 |
1.00 |
– |
– |
11.7 |
1.00 |
– |
– |
– |
– |
– |
– |
32.7 |
1.00 |
– |
– |
5000 |
8.0 |
0.47 |
14.3 |
0.57 |
45.0 |
0.56 |
89.0 |
0.93 |
5.7 |
0.59 |
7.7 |
0.68 |
0.0 |
0.00 |
2.7 |
0.24 |
16.7 |
0.81 |
24.3 |
0.68 |
1600 |
8.3 |
0.49 |
23.3 |
0.92 |
57.0 |
0.70 |
77.7 |
0.81 |
3.0 |
0.31 |
13.0 |
1.15 |
0.0 |
0.00 |
4.0 |
0.36 |
21.3 |
1.03 |
28.7 |
0.80 |
500 |
11.3 |
0.67 |
28.7 |
1.13 |
91.0 |
1.12 |
91.0 |
0.95 |
2.0 |
0.21 |
9.7 |
0.85 |
2.0 |
0.35 |
7.3 |
0.67 |
23.0 |
1.11 |
39.7 |
1.10 |
160 |
23.0 |
1.35 |
57.7 |
2.28 |
86.7 |
1.07 |
104.0 |
1.09 |
7.0 |
0.72 |
9.3 |
0.82 |
5.0 |
0.88 |
8.0 |
0.73 |
23.3 |
1.13 |
30.3 |
0.84 |
50 |
21.3 |
1.25 |
37.7 |
1.49 |
89.3 |
1.10 |
93.3 |
0.98 |
11.3 |
1.17 |
11.3 |
1.00 |
7.7 |
1.35 |
11.3 |
1.03 |
26.7 |
1.29 |
32.7 |
0.91 |
16 |
15.7 |
0.92 |
27.7 |
1.09 |
79.7 |
0.98 |
109.3 |
1.15 |
12.0 |
1.24 |
11.0 |
0.97 |
3.7 |
0.65 |
11.0 |
1.00 |
27.0 |
1.31 |
33.7 |
0.94 |
5 |
18.0 |
1.06 |
25.3 |
1.00 |
88.3 |
1.09 |
111.7 |
1.17 |
10.0 |
1.03 |
9.3 |
0.82 |
6.7 |
1.18 |
9.3 |
0.85 |
24.7 |
1.19 |
30.3 |
0.84 |
NPD (4mg) |
241.3 |
14.20 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
SAZ (2mg) |
– |
– |
– |
– |
1146.7 |
14.51 |
– |
– |
1314.7 |
112.69 |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
9AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
303.0 |
53.47 |
– |
– |
– |
– |
– |
– |
MMS (2mL) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
1058.7 |
32.41 |
– |
– |
2AA (2mg) |
– |
– |
2122.7 |
83.79 |
– |
– |
2373.3 |
24.90 |
– |
– |
178.0 |
15.71 |
– |
– |
193.7 |
17.61 |
– |
– |
– |
– |
2AA (50mg) |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
– |
168.0 |
4.67 |
MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.
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 was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content was made in the main experiments (97.4 % dyestuff).Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on 97.4 % dyestuff) were prepared and investigated in the initial and confirmatory mutation tests: 5000,1600, 500, 160, 50, 16, and 5 µg/plate. 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 concentrations of 5000 and 1600 µg/plate in absence and in the presence of S9 mix following the plate incorporation and pre-incubation procedures. Inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain in the absence and also in the presence of S9 mix, in the confirmatory mutation test in all S. typhimurium strains in the absence of S9; in TA98 and TA1537 also in the presence of S9. The inhibitory effect 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). In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent 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 coloniesand the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin 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 at any concentration level, either in the presence or absence of S9 mix. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains, under the test conditions used in this study.
No experimental data on the clastogenic or mutgenic potential of the test item in mammalian cells are available. However, experimental data Leuso Sulphur Yellow 22 are available and used in a read across apporach.
Chromosome Aberration Assay (read across from Leuco Sulphur Yellow 22, CAS 90268 -98 -7)
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 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 (read across from Leuco Sulphur Yellow 22, CAS 90268 -98 -7)
The test item suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations 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.
The test item did not show mutagenic properties in a bacteiral reverse mutation assay. In a read-across approach, a suitable source substance did not show clastogenic or mutagenic properties in mammalian cells. It is concluded that the test substance does not contain clastogenic or mutagenic properties.
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 twelfth time in Regulation (EU) No 2019/52.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
