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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 203-184-2 | CAS number: 104-20-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item did not show any mutagenic effect and was considered not genotoxic.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- May 30, 2008
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II - strains TA100 and WP2 uvr A: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II - other strains: 33, 100, 333, 1000, 2500 and 5000 µg/plate - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylenediamine (4-NOPD) (without metabolic activation, TA1537 and TA98); 2-aminoanthracene (2-AA) (with metabolic activation, all strains)
- Details on test system and experimental conditions:
- CHARACTERISATION OF THE STRAINS:
- TA1537: his C 3076, rfa-, uvrB-; frame shift mutations
- TA98: his D 3052, rfa-, uvrB-, R-factor; frame shift mutations
- TA1535: his G 46, rfa-, uvrB-; base-pair substitutions
- TA100: his G 46, rfa-, uvrB-, R-factor; base-pair substitutions
- WP2 uvrA: trp-; uvrA-; base-pair substitutions and others
Strains were obtained from Trinova Biochem GmbH, 35394 Giessen, Germany.
Strains were regularly checked regarding membrane permeability, ampicillin resistance, UV sensitivity, amino acid requirements, and spontaneous mutation rates.
METHOD OF APPLICATION:
- Experiment I: plate incorporation
- Experiment II: pre-incubation
PRECULTURES:
Thawed bacterial suspensions were added to 50 mL nutrient medium (8 g nutrient broth and 5 g NaCl per litre). A solution of 50 µL of ampicillin (25 µg/mL) was added to strains TA98 and TA100. Incubation at 37°C for 4 hours. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E8-10E9 cells/mL).
METABOLIC ACTIVATION SYSTEM:
Phenobarbital/beta-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Each S9 batch was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 35.0 mg/mL in experiment I and 30.7 mg/mL in experiment II.
S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approx. 10% v/v in the S9 mix. Co-factors are added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium orthophosphate buffer, pH 7.4.
During the experiment, the S9 mix is stored in an ice bath.
The S9 mix substitution buffer contains 700 mL 100 mM sodium orthophosphate buffer, pH 7.4 and 300 mL 0.15 M KCl solution.
During the experiment the S9 mix substitution buffer is stored in an ice bath.
PRE-EXPERIMENT FOR TOXICITY:
- pre-experiment is reported as main experiment I
DOSE SELECTION
- Doses used in experiment I were in the range of 3-5000 µg/plate.
- Since toxic effects were observed in experiment I, at least 6 concentrations were tested in experiment II, with a maximum concentration of 5000 µg/plate.
EXPERIMENTAL PERFORMANCE
Experiment I (plate incorporation):
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 100 µL bacteria suspension
- 2000 µL overlay agar
- Incubated for at least 48 hours at 37°C in the dark.
Experiment II (pre-incubation):
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 100 µL bacteria suspension
- Mixed in a test tube and incubated at 37°C for 60 minutes
- 2000 µL overlay agar (45°C) added to each tube, and poured on minimal agar plates.
- Incubated for at least 48 hours at 37°C in the dark.
Sterile control:
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 2000 µL overlay agar
- Incubated for at least 48 hours at 37°C in the dark. - Evaluation criteria:
- ACCEPTABILITY OF THE ASSAY:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
EVALUATION OF THE RESULTS:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- Not mandatory according to OECD 471.
- 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:
- S. typhimurium TA 1537
- Metabolic activation:
- 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
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate and above
- 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:
- at 5000 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:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 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
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 2500 µg/plate and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item was found to be not mutagenic under the conditions of the Ames test.
- Executive summary:
The potential of the test item to induce gene mutation was assessed according to an Ames test in accordance with OECD 471 and GLP, using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments, both with and without metabolic activation.
The following concentrations were tested, each in triplicate:
- Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment II - strains TA100 and WP2 uvr A: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment II - other strains: 33, 100, 333, 1000, 2500 and 5000 µg/plate
No precipitation of the test item occurred up to the highest investigated dose.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains, except of strain TA 1535.
The negative and positive controls were valid.
Under the experimental conditions reported, the test idem did not induce an increase in revertant colony numbers in any of the strains tested at any dose level, with or without metabolic activation. Hence, it can be stated that the substance is not mutagenic under the conditions of the test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19/11/2015-29/01/2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- Kanpoan n. 287
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT locus in V79 cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix fraction
- Test concentrations with justification for top dose:
- 55.6 µg/mL; 111.3 µg/mL; 222.5 µg/mL; 445 µg/mL; 667.5 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: its solubility properties and its relative non-toxicity to the cells cultures - Untreated negative controls:
- yes
- Remarks:
- DMSO, purity 99.99%
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- DMSO, purity 99.99%
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- CELL CULTURES:
- Origin: Laboratory for Mutagenicity Testing, Technical University, 64287 Darmstadt, Germany.
- Storage: in liquid nitrogen in the cell bank of Envigo CRS GmbH.
- Master cell stock checks: mycoplasm contamination, karyotype stability, spontaneous mutant frequency.
- Preparation: Thawed stock cultures were propagated at 37°C. About 2-3E6 cells were seeded into each flask with 15 ML of minimal essential medium (MEM) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
CULTURE MEDIUM:
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% foetal bovine serum (FBS), and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
MAMMALIAN MICROSOMAL FRACTION S9 MIX:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium orthophosphate buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation was 32.3 mg/mL in the pre-experiment and the main experiment.
TOXICITY PRE-TEST:
- Experimental conditions are the same as for the main experiment
- Colony forming ability of approx. 500 single cells after treatment was compared with the controls
- Duplicate cultures per concentration level
- Test item concentrations ranged from 13.9 to 1782 µg/mL
DOSE SELECTION:
- Cytotoxicity of 50% or below was observed at 891 µg/mL and above, with and without metabolic activation
- Phase separation occurred at 445.5 µh/mL and above, with and without metabolic activation
- Doses selected for main experiment: 27.8, 55.6, 111.3, 222.5, 445.0, 667.5 and 890.0 µg/mL
EXPERIMENTAL PERFORMANCE:
1) Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2E7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
2) Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G". Immediately after the end of treatment the cells were trypsinised as described above and sub- cultivated. At least 2.0E6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm2 flasks containing 30 mL medium. Two additional 25 cm2 flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three or four days after treatment approximately 2.0E6 cells per experimental point were sub-cultivated in 175 cm2 flasks containing 30 mL medium.Following the expression time of 7 days five 75 cm2 cell culture flasks were seeded with about 4 to 5E5 cells each in medium containing 6-TG. Two additional 25 cm2 flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days.
The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
ACCEPTABILITY CRITERIA:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria - Evaluation criteria:
- - A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
- A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
- A mutagenic response is described as follows:
- The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
- The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 445 µg/mL and 890 µg/mL without metabolic activation (experiment II)
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 667.5 µg/mL without and without metabolic activation (experiment I)
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH (without metabolic activation in pre-experiment wih maximum concentration): 7.37
- Effects of osmolality (without metabolic activation in pre-experiment wih maximum concentration): 371
RANGE-FINDING/SCREENING STUDIES: yes
COMPARISON WITH HISTORICAL CONTROL DATA: yes - Conclusions:
- In conclusion, the test substance can be considered to be non-mutagenic in this HPRT assay.
- Executive summary:
The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus V79 cells of the Chinese Hamster.
The assay was performed according to OECD 476 guideline and GLP without deviations.
The test was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours without metabolic activation.
The maximum concentration of the test substance for the pre-experiment was 1782 µg/mL. The concentration range of the main experiment was limited by phase separation of the test substance.
No substantial and reproductible dose dependant increase of the mutation frequency was observed in the main experiment.
The positive controls induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce gene mutation at HPRT locus in V79 cells.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20.04.2016-11.07.2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Strasse 80, 65189 Wiesbaden, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: from healthy non-smoking donors not receiving medication
- Details on mammalian cell type (if applicable):
- - female donor (27 years old) for Experiment I
- male donor (32 years old) for Experiment II
The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B (4 μg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- - Experiment I: 4 hours without S9 mix; 109, 190 and 333 μg/mL
- Experiment I: 4 hrs with S9 mix; 333, 582 and 1018 μg/mL
- Experiment II: 20 hrs without S9 mix; 333, 582 and 1018 μg/mL
- Experiment I: 4 hrs without S9 mix; 109, 190 and 333 μg/mL - Vehicle / solvent:
- DMSO 0.5 % in final concentration
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium with 0.5 % DMSO
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: demecolcin 50.0 ng/mL
- Details on test system and experimental conditions:
- CELL CULTURE CONDITIONS
- Culture medium: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1)
supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen
PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U
.S.P.-U/mL).
- Temperature: 37 °C
- Humidity: 5.5 % CO2 in humidified air
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hour with phytohemeagglutinine (PHA) to stimulate proliferation
- Exposure duration: 4 or 20 hours
- Washing: 2x
- Recovery period: 16 hours
- Harvesting: 40 hours after beginning of treatment
- Post-incubation: at 37 °C for 20 minutes
- Fixation time: 2 x 20 minutes
- Fixative: ico-cold mixture of methanol and glacial acetic acid (19 : 1 parts)
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa
REPLICATES: 2
METHODS OF SLIDE PREPARATION: dropping the cell suspension in fresh fixative onto a clean microscope slide
NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- Frequency of micronucleated cells: reported as % micronucleated cells. To describe a cytotoxic
effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.
A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: publication of Countryman and Heddle (1976).
The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus
should not extend the third part of the area of the main nucleus.
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with
the controls (% cytostasis) by counting 500 cells per culture. The pre-test was performed with 10
concentrations of the test item separated by no more than a factor of √10 and a solvent and positive
control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix).
Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
- OTHER:
- Test Item Preparation: Stock formulations of the test item and serial dilutions were made in DMSO.
All formulations were prepared freshly before treatment and used within two hours of preparation.
The osmolarity and pH were determined by using an osmometer or a pH meter.
ACCEPTANCE CRITERIA:
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range.
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in section ‘Experimental performance’ were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− The criteria for the selection of top concentration are consistent with those described in section ‘Dose selection’. - Rationale for test conditions:
- The highest treatment concentration in this study, 1782 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
- In Experiment I, phase separation of the test item in the culture medium was observed at 333 μg/mL and above in the absence of S9 mix and at 1018 μg/mL and above in the presence of S9 mix at the end of treatment.
- In Experiment II, phase separation occurred in the absence of S9 mix at 1018 μg/mL and above at the end of treatment.
- In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration,
which showed phase separation.
- In Experiment II, clear cytotoxicity was observed at the highest evaluated concentration, which showed phase separation. - Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the 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 item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system. - Statistics:
- Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei
compared to the concurrent solvent control. - Key result
- Species / strain:
- lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- exposure period 4 hours
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to the highest evaluated concentration, which showed phase separation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- exposure period 4 hours
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- up to the highest evaluated concentration, which showed phase separation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- Exposure period 20 hours
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- In both experiments, either Demecolcin (50.0 ng/mL), MMC (1.5 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
- Conclusions:
- Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
The test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating and/or cytotoxic concentrations. - Executive summary:
The test item dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. The assay was performed in accordance to OECD TG 487 and in compliance to GLP.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 1782 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I, phase separation of the test item in the culture medium was observed at 333 μg/mL and above in the absence of S9 mix and at 1018 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 1018 μg/mL and above at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II, clear cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.
In the absence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item.
In the presence of S9 mix, one statistical significant increase in micronucleate cells (0.90 %) was observed in the lowest evaluated concentration (333 μg/mL). This increase can be declared as biologically irrelevant, because the value is well within the range of the historical laboratory control data (95% control limit: 0.08 – 1.20 % micronucleate cells).
In both experiments, either Demecolcin (50.0 ng/mL), MMC (1.5 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
In conclusion and under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
The test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating and/or cytotoxic concentrations.
Referenceopen allclose all
Summary of Experiment I:
| Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ±SD) | ||||
| TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||
| Without | DMSO | 15 ± 2 | 13 ± 1 | 28 ± 7 | 180 ± 20 | 40 ± 9 | |
| Untreated | 14 ± 6 | 18 ± 5 | 26 ± 8 | 190 ± 11 | 41 ± 7 | ||
| Test item | 3 μg | 15 ± 6 | 12 ± 4 | 28 ± 5 | 173 ± 29 | 48 ± 2 | |
| 10 μg | 15 ± 3 | 13 ± 2 | 24 ± 2 | 162 ± 10 | 40 ± 11 | ||
| 33 μg | 15 ± 2 | 13 ± 3 | 30 ± 4 | 172 ± 13 | 46 ± 2 | ||
| 100 μg | 17 ± 3 | 15 ± 2 | 27 ± 8 | 165 ± 13 | 43 ± 3 | ||
| 333 μg | 13 ± 5 | 12 ± 3 | 32 ± 4 | 195 ± 16 | 40 ± 1 | ||
| 1000 μg | 16 ± 4 | 9 ± 3 | 21 ± 6 | 182 ± 14 | 36 ± 8 | ||
| 2500 μg | 14 ± 1 | 7 ± 2 | 24 ± 2 | 150 ± 7 | 22 ± 6 | ||
| 5000 μg | 18 ± 3 | 8 ± 1 | 24 ± 4 | 75 ± 42R | 12 ± 1 | ||
| NaN3 | 10 μg | 1319 ± 68 | 2292 ± 81 | ||||
| 4-NOPD | 10 μg | 384 ± 26 | |||||
| 4-NOPD | 50 μg | 103 ± 13 | |||||
| MMS | 2.0 μL | 603 ± 51 | |||||
| With | DMSO | 13 ± 2 | 14 ± 1 | 29 ± 10 | 163 ± 4 | 53 ± 5 | |
| Untreated | 12 ± 4 | 21 ± 2 | 37 ± 10 | 179 ± 16 | 52 ± 8 | ||
| Test item | 3 μg | 15 ± 2 | 15 ± 1 | 33 ± 10 | 167 ± 89 | 53 ± 7 | |
| 10 μg | 17 ± 4 | 14 ± 1 | 33 ± 9 | 163 ± 3 | 60 ± 8 | ||
| 33 μg | 12 ± 3 | 13 ± 3 | 33 ± 7 | 151 ± 15 | 56 ± 7 | ||
| 100 μg | 13 ± 2 | 15 ± 5 | 33 ± 8 | 154 ± 5 | 44 ± 4 | ||
| 333 μg | 11 ± 2 | 11 ± 4 | 37 ± 11 | 183 ± 15 | 53 ± 6 | ||
| 1000 μg | 12 ± 2 | 10 ± 3 | 36 ± 1 | 174 ± 4 | 41 ± 8 | ||
| 2500 μg | 12 ± 4 | 9 ± 1 | 32 ± 2 | 169 ± 14 | 25 ± 4 | ||
| 5000 μg | 9 ± 4 | 8 ± 1 | 28 ± 4 | 81 ± 9R | 17 ± 1 | ||
| 2-AA | 2.5 μg | 462 ± 22 | 149 ± 6 | 5767 ± 351 | 5421 ± 242 | ||
| 2-AA | 10.0 μg | 940 ± 39 | |||||
Summary of Experiment II:
| Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ±SD) | ||||
| TA 1535 | TA 1537 | TA 98 | TA 100 | WP2 uvrA | |||
| Without | DMSO | 10 ± 0 | 9 ± 3 | 28 ± 3 | 188 ± 29 | 39 ± 3 | |
| Untreated | 8 ± 4 | 9 ± 3 | 27 ± 1 | 206 ± 4 | 45 ± 16 | ||
| Test item | 3 μg | 186 ± 15 | 45 ± 12 | ||||
| 10 μg | 204 ± 6 | 41 ± 8 | |||||
| 33 μg | 12 ± 5 | 11 ± 0 | 24 ± 2 | 221 ± 18 | 51 ± 11 | ||
| 100 μg | 10 ± 3 | 10 ± 3 | 21 ± 7 | 213 ± 2 | 51 ± 4 | ||
| 333 μg | 12 ± 2 | 11 ± 2 | 30 ± 3 | 182 ± 7 | 39 ± 6 | ||
| 1000 μg | 16 ± 3 | 11 ± 5 | 23 ± 7 | 179 ± 21 | 26 ± 4 | ||
| 2500 μg | 17 ± 3 | 8 ± 3 | 26 ± 6 | 90 ± 17 | 18 ± 3 | ||
| 5000 μg | 15 ± 1 | 10 ± 4R | 12 ± 3 | 95 ± 15 | 11 ± 1R | ||
| NaN3 | 10 μg | 1160 ± 53 | 1720 ± 142 | ||||
| 4-NOPD | 10 μg | 424 ± 58 | |||||
| 4-NOPD | 50 μg | 110 ± 25 | |||||
| MMS | 2.0 μL | 722 ± 57 | |||||
| With | DMSO | 12 ± 3 | 13 ± 2 | 44 ± 7 | 201 ± 19 | 58 ± 11 | |
| Untreated | 13 ± 7 | 15 ± 3 | 44 ± 13 | 212 ± 11 | 53 ± 6 | ||
| Test item | 3 μg | 175 ± 35 | 49 ± 2 | ||||
| 10 μg | 175 ± 7 | 56 ± 9 | |||||
| 33 μg | 12 ± 3 | 16 ± 4 | 41 ± 6 | 196 ± 14 | 61 ± 6 | ||
| 100 μg | 16 ± 1 | 13 ± 6 | 44 ± 10 | 178 ± 22 | 54 ± 5 | ||
| 333 μg | 13 ± 1 | 20 ± 1 | 42 ± 5 | 184 ± 14 | 47 ± 2 | ||
| 1000 μg | 11 ± 1 | 18 ± 6 | 36 ± 8 | 148 ± 3 | 44 ± 5 | ||
| 2500 μg | 9 ± 2 | 13 ± 3R | 11 ± 3R | 97 ± 33 | 15 ± 5R | ||
| 5000 μg | 6 ± 3 | 5 ± 1R | 2 ± 2MR | 6 ± 2 | 6 ± 1MR | ||
| 2-AA | 2.5 μg | 407 ± 46 | 147 ± 21 | 4381 ± 574 | 4773 ± 268 | ||
| 2-AA | 10.0 μg | 338 ± 27 | |||||
With:
- NaN3: sodium azide
- 2-AA: 2-aminoanthracene
- 4-NOPD: 4-nitro-o-phenylenediamine
- MMS: methyl methane sulfonate
- R: reduced background growth
- M: manual count
Summary of results:
| Conc. | PS | S9 mix | Relative cloning efficiency I | Relative cell density | Relative adjusted cloning efficiency I | Mutant colonies / 1E6 cells | 95% confidence interval | Relative cloning efficiency I | Relative cell density | Relative adjusted cloning efficiency I | Mutant colonies / 1E6 cells | 95% confidence interval | |
| (µg/mL) | (%) | (%) | (%) | (%) | (%) | (%) | |||||||
| Experiment I; 4h treatement | Culture I | Culture II | |||||||||||
| Solvent control with DMSO | - | 100,0 | 100,0 | 100 | 15.2 | 0.0-29.7 | 100 | 100 | 100 | 24.7 | 0.0-29.7 | ||
| Positive control (EMS) | 225 | - | 90.2 | 90.5 | 81.7 | 208.3 | 0.0-29.7 | 91.3 | 101.5 | 92.7 | 289.1 | 0.0-29.7 | |
| Test item | 27.8 | - | 68.8 | 110.8 | 76.2 | culture not continued # | 96.2 | 100.6 | 96.8 | culture not continued # | |||
| 55.6 | - | 77,0 | 81.9 | 63 | 22.4 | 0.0-29.7 | 94.8 | 103.7 | 98.4 | 40.6 | 0.0-29.7 | ||
| 111.3 | - | 52.4 | 114.7 | 60.1 | 23.2 | 0.0-29.7 | 90.2 | 118.5 | 106.8 | 26.5 | 0.0-29.7 | ||
| 222.5 | - | 23.4 | 68.5 | 16 | 18.4 | 0.0-29.7 | 99.6 | 95.4 | 95.1 | 25.1 | 0.0-29.7 | ||
| 445,0 | - | 65,0 | 81.5 | 53,0 | 49.3 | 0.0-29.7 | 99.3 | 96.7 | 96,0 | 36.5 | 0.0-29.7 | ||
| 667.5 | - | 31.9 | 38.2 | 12.2 | 62.1 | 0.0-29.7 | 93,0 | 98.8 | 91.8 | 29.3 | 0.0-29.7 | ||
| 890,0 | PS | - | 12.3 | 42.2 | 5.2 | culture not continued ## | ## | 4.1 | culture not continued ## | ||||
| Solvent control with DMSO | + | 100,0 | 100,0 | 100,0 | 17,0 | 0.0-27.7 | 100,0 | 100,0 | 100,0 | 13.8 | 0.0-27.7 | ||
| Positive control (DMBA) | 2.2 | + | 98.9 | 97.2 | 96.1 | 172,0 | 0.0-27.7 | 98.1 | 90.8 | 89.1 | 253.9 | 0.0-27.7 | |
| Test item | 27.8 | + | 93.7 | 100,0 | 93.7 | culture not continued # | 97.7 | 104.6 | 102.2 | culture not continued ## | |||
| 55.6 | + | 92.2 | 123.7 | 114,0 | 20.8 | 0.0-27.7 | 101,0 | 90.2 | 91.2 | 13.2 | 0.0-27.7 | ||
| 111.3 | + | 97.4 | 119.6 | 116.5 | 15.1 | 0.0-27.7 | 93.3 | 85.6 | 79.9 | 11.3 | 0.0-27.7 | ||
| 222.5 | + | 91.2 | 108.2 | 98.6 | 28.7 | 0.0-27.7 | 93.9 | 83.9 | 78.8 | 15,0 | 0.0-27.7 | ||
| 445,0 | + | 97.6 | 116.6 | 113.8 | 30.7 | 0.0-27.7 | 97.5 | 63.45 | 61.9 | 26.4 | 0.0-27.7 | ||
| 667.5 | PS | + | 99.1 | 62.5 | 61.9 | 24.3 | 0.0-27.7 | 98.1 | 42.7 | 41.9 | 27.8 | 0.0-27.7 | |
| 890,0 | PS | + | 94.2 | 29.9 | 28.1 | culture not continued ### | 102.3 | 26.1 | 26.7 | culture not continued ### | |||
| Experiment II; 4h treatment | Culture I | Culture II | |||||||||||
| Solvent control with DMSO | - | 100,0 | 100,0 | 100,0 | 19.3 | 0.0-29.7 | 100,0 | 100,0 | 100,0 | 38.7 | 0.0-29.7 | ||
| Positive control (EMS) | 450,0 | - | 84.3 | 97.6 | 82.2 | 392.7 | 0.0-29.7 | 87.4 | 78.5 | 68.6 | 546.7 | 0.0-29.7 | |
| Test item | 27.8 | - | 80.8 | 100.2 | 81,0 | culture not continued # | 85.3 | 79.1 | 67.5 | culture not continued # | |||
| 55.6 | - | 83.9 | 95.8 | 80.4 | culture not continued # | 95.7 | 70,0 | 66.9 | culture not continued # | ||||
| 111.3 | - | 91.4 | 88.3 | 80.7 | 24.9 | 0.0-29.7 | 92.6 | 83.5 | 77.4 | 20.9 | 0.0-29.7 | ||
| 222.5 | - | 90.8 | 90.7 | 82.4 | 26.6 | 0.0-29.7 | 84.2 | 88.1 | 74.1 | 30.8 | 0.0-29.7 | ||
| 445,0 | - | 93.4 | 77.8 | 72.6 | 28.4 | 0.0-29.7 | 85.3 | 55.1 | 47,0 | 21.5 | 0.0-29.7 | ||
| 667.5 | - | 75,0 | 87.7 | 65.8 | 23.3 | 0.0-29.7 | 75,0 | 92.2 | 69.1 | 21,0 | 0.0-29.7 | ||
| 890,0 | PS | - | 102.8 | 65.4 | 67.3 | 34.9 | 0.0-29.7 | 78.4 | 22.8 | 17.8 | 13.7 | 0.0-29.7 | |
With:
PS: phase separation was visible at the end of treatment
#: culture was not continued since a minimum of only 4 concentrations required
##: culture was not continued due to exceedingly severe cytotoxic effects
###: culture was not continued to avoid analysis of too many insoluble concentrations
Overview of results
| Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Binucleated cells with n micronuclei culture 1 |
Sum culture | Binucleated cells with n micronuclei culture 2 |
Sum culture | Sum in 2000 binucelate cells |
% | |||||
| 1 | 2 | >2 | 1 | 1 | 2 | >2 | 2 | |||||||
| Solv. Contr.# | 0.5% | - | 4 / 40 hrs | 2 | 0 | 0 | 2 | 6 | 1 | 0 | 7 | 9 | 0.45 | |
| Pos. Contr.## | 1.5 µg | - | 4 / 40 hrs | 88 | 9 | 1 | 98 | 81 | 4 | 1 | 86 | 184 | 9.20 | |
| Test item | 109 µg | - | 4 / 40 hrs | 7 | 0 | 0 | 7 | 6 | 0 | 0 | 6 | 13 | 0.65 | |
| Test item | 190 µg | - | 4 / 40 hrs | 8 | 0 | 0 | 8 | 5 | 0 | 0 | 5 | 13 | 0.65 | |
| Test item | 333 µg | - | 4 / 40 hrs | 5 | 0 | 0 | 5 | 4 | 0 | 0 | 4 | 9 | 0.45 | |
| Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Binucleated cells with n micronuclei culture 1 |
Sum culture | Binucleated cells with n micronuclei culture 2 |
Sum culture | Sum in 2000 binucelate cells |
% | ||||
| 1 | 2 | >2 | 1 | 1 | 2 | >2 | 2 | ||||||
| Solv. Contr.# | 0.5% | + | 4 / 40 hrs | 1 | 1 | 0 | 2 | 4 | 0 | 0 | 4 | 6 | 0.30 |
| Pos. Contr.### | 17.5 µg | + | 4 / 40 hrs | 38 | 6 | 1 | 45 | 28 | 2 | 0 | 30 | 75 | 3.75 |
| Test item | 333 µg | + | 4 / 40 hrs | 10 | 0 | 0 | 10 | 7 | 1 | 0 | 8 | 18 | 0.90 |
| Test item | 582 µg | + | 4 / 40 hrs | 2 | 0 | 0 | 2 | 8 | 0 | 0 | 8 | 10 | 0.50 |
| Test item | 1018 µg | + | 4 / 40 hrs | 11 | 1 | 0 | 12 | 2 | 0 | 0 | 2 | 14 | 0.70 |
| Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Binucleated cells with n micronuclei culture 1 |
Sum culture | Binucleated cells with n micronuclei culture 2 |
Sum culture | Sum in 2000 binucelate cells |
% | ||||
| 1 | 2 | >2 | 1 | 1 | 2 | >2 | 2 | ||||||
| Solv. Contr.# | 0.5% | - | 20 / 40 hrs | 6 | 1 | 0 | 7 | 10 | 0 | 0 | 10 | 17 | 0.85 |
| Pos. Contr.#### | 50.0 ng | - | 20 / 40 hrs | 22 | 5 | 0 | 27 | 23 | 4 | 4 | 31 | 58 | 2.90 |
| Test item | 333 µg | - | 20 / 40 hrs | 4 | 0 | 0 | 4 | 7 | 1 | 0 | 8 | 12 | 0.60 |
| Test item | 582 µg | - | 20 / 40 hrs | 5 | 2 | 0 | 7 | 6 | 1 | 0 | 7 | 14 | 0.70 |
| Test item | 1018 µg | - | 20 / 40 hrs | 3 | 0 | 0 | 3 | 3 | 0 | 0 | 3 | 6 | 0.30 |
# DMSO
## MMC
### CPA
#### Demecolcin
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
The test item did not show any mutagenic effect and was considered not genotoxic.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Ames test
Two studies are available assessing the genotoxic effects of the test item.
The key study is a recent Ames study according to GLP and OECD 471 using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments, both with and without metabolic activation. Concentrations in the range of 3 -5000 µg/plate were tested.
No precipitation was observed at any of the concentrations. Toxic effects were observed in all strains, except for TA 1535.
The negative and positive controls were valid.
Under the experimental conditions reported, the test idem did not induce an increase in revertant colony numbers in any of the strains tested at any dose level, with or without metabolic activation. Hence, it can be stated that the substance is not mutagenic under the conditions of the test.
In the supporting study (1983), the genotoxicity of the test item was assessed via an Ames test. The test was not GLP or according to OECD as it preceeded the test guidelines. Nevertheless, the test was well performed and documented. 5 tester strains were tested (Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98) with and without S-9 mix. The highest concentration tested was 3.6 mg/plate, and sodium azide and benzo(a)pyrene were used as positive controls. The test item did not show any mutagenic effect and was considered not genotoxic.
In vitro mammalian cell mutation test
One key study is available assessing the potential of the test substance to induce gene mutations at the HPRT locus V79 cells of the Chinese Hamster.
The assay was performed according to OECD 476 guideline and GLP without deviations. The test was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours without metabolic activation. The maximum concentration of the test substance for the pre-experiment was 1782 µg/mL. The concentration range of the main experiment was limited by phase separation of the test substance.
No substantial and reproductible dose dependant increase of the mutation frequency was observed in the main experiment. It can be stated that under the experimental conditions reported, the test substance did not induce gene mutation at HPRT locus in V79 cells.
In vitro mammalian cell micronucleus test
One key study is available assessing the potential of the test substance to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. The assay was performed in accordance to OECD TG 487 and in compliance to GLP.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In Experiment I, phase separation of the test item in the culture medium was observed at 333 μg/mL and above in the absence of S9 mix and at 1018 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 1018 μg/mL and above at the end of treatment.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II, clear cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.
In the absence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item.
In the presence of S9 mix, one statistical significant increase in micronucleate cells (0.90 %) was observed in the lowest evaluated concentration (333 μg/mL). This increase can be declared as biologically irrelevant, because the value is well within the range of the historical laboratory control data (95% control limit: 0.08 – 1.20 % micronucleate cells).
In both experiments, either Demecolcin (50.0 ng/mL), MMC (1.5 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.
In conclusion and under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
The test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating and/or cytotoxic concentrations.
In vivo chormosome aberration test
For chromosome aberration, additional to the key in vitro study, a supporting in vivo micronucleus study is available. The study was not according to an OECD guideline or GLP.
Groups of male and female mice, 4 per dose, were treated once 1426, 713, 356 or 178 mg/kg test item. Control animals were dosed with the vehicle (olive oil) alone. After 30 hours the mice were sacrificed, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. The results are expressed as mean number of micronucleated polychromatic erythrocytes (PE) per 1000 polychromatic erythrocytes assessed.
The test item did not show genetic effects under the conditions of the test.
Overall conclusion
As no mutagenic or genotoxic effects were observed in all available studies, the test item is considered to be not genotoxic.
Justification for classification or non-classification
As no mutagenic or genotoxic effects were observed in all available studies, the test item should not be classified according to the CLP regulation (Table 3.5.1, page 145) and is considered not to be genotoxic.
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.
