Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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.
Diss Factsheets
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: 203-116-1 | CAS number: 103-48-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial reverse mutation assay: The test
substance was not mutagenic in the bacterial reverse mutation assay, according
to OECD 471.
HPRT assay: The test substance did not induce gene mutation in the HPRT assay, according to OECD 476.
Micronuclei: The test substance was concluded
to be negative for the induction of micronuclei in both non-acitvated and
S9 -activated test systems in the in vitro micronucleus test, according to
OECD 487.
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:
- 2001-03-16 to 2001-04-04
- 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
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 mix, Aroclor 1254 induced
- Test concentrations with justification for top dose:
- experiment 1: 0, 50, 150, 500, 1500, 5000 µg/plate
experiment 2: 0, 5, 15, 50, 150, 500, 1500 µg/plate - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other: with S9 mix: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- The number of spontaneous revertants observed using each of the five strains was close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.
- Statistics:
- The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each
dosage level was calculated using the X2-test (Mohn and Ellenberger, 1977). - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- 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:
- in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in the absence of S9 mix, bacteriotoxicity was observed at 500 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9-mix the substance was not bacteriotoxic. - Conclusions:
- The test substance was not mutagenic in the bacterial reverse mutation assay.
- Executive summary:
The test substance was tested for its mutagenicity in the bacterial reverse mutation assay. Therefore, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 were used. The test was performed using the standard plate incorporation assay with and without S9 -mix (Aroclor 1254 induced). Two independet experiments were carried out using concentrations of 50 -5000 µg and 5 -1500 µg/plate. In the presence of S9-mix no bacteriotoxicity was observed. In the absence of S9-mix the test substance was bacteriotoxic towards the strain TA102 at 500 µg/plate and towards the strains TA1535, TA1537, TA98, and TA100 at 1500 µg/plate. As mutagenicity controls, sodium azide, 2 -nitrofluorene, 9 -aminoacridine and mitomycin C were used in the absence of S9 -mix and 2 -aminoanthracene was used in the in the presence of S9 mix. As a result of this assay, mutagenicity was not observed.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-06-02 to 2015-07-31
- 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:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: mammalian gene mutation assay (HPRT)
- Target gene:
- Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian microsomal fraction S9 mix
- Test concentrations with justification for top dose:
- Experiment I, exposure period 4 hours:
without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL
with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL
Experiment II, exposure period 24 hous:
without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL
Exposure period 4 hours:
30, 60, 120, 240, 480, 960 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: with metabolic activation: DMBA
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 24 hours or 4 hours
- Expression time (cells in growth medium): 7 days
SELECTION AGENT: 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations of the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) 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. However, both, biological and statistical significance was considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 4 h treatment: at 60.3 µg/mL (with and without S9 mix); after 24 h: at 241.3 µg/mL (without S9 mix)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Phase separation occurred at 241.3 μg/mL and above after 4 and 24 hours treatment without metabolic activation and at 965.0 μg/mL and above with metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).
COMPARISON WITH HISTORICAL CONTROL DATA:
A slight increase of mutant colonies per 106 cells exceeding the range of the historical solvent control data (1.6 - 45.7 mutant colonies/106 cells) was determined in both cultures of the first experiment at 60.3 μg/mL without metabolic activation (46.2 and 48.6 mutant colonies per 106 cells). However, the threshold of three times the corresponding solvent control was not exceeded and the effect was not reproduced in the second experiment without metabolic activation. Consequently, the isolated increases described above were judged as biologically irrelevant. - Conclusions:
- The test item did not induce gene mutations at the HPRT locus in V79 cells.
- Executive summary:
The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay 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 with and 24 hours without metabolic activation. The maximum test item concentration of the pre-experiment (1930 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity (without metabolic activation) and phase separation (with metabolic activation) of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.
The tested concentrations were:
Experiment I, exposure period 4 hours:
without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL
with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL
Experiment II, exposure period 24 hous:
without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL
Exposure period 4 hours:
30, 60, 120, 240, 480, 960 µg/mL
Appropriate reference mutagens, used as positive controls (without metabolic activation: ethylmethane sulfonate, with metabolic activation: DMBA), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Based on the available results the study was considered valid and the tets substance was shown to be not mutagenic in the HPRT assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015 to 2015-04-30
- 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:
- 2010
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes (HPBL)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: human donor
- Sex, age and number of blood donors:
preliminary test and initial assay: healthy non-smoking 27-year old adult female;
repeat assay: healthy non-smoking 24-year old adult female
- Separated lymphocytes were used
MEDIA USED
- Type and identity of media including CO2 concentration: complete medium (RPMI-1640; 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin) with 2% phytohemagglutinin; 37 °C; 5 +/- 1 % CO2
- Properly maintained: yes - Cytokinesis block (if used):
- Cytochalasin B (cytoB) (6 µL/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S9 mix
- Test concentrations with justification for top dose:
- Test concentrations were chosen based on preliminary cytotoxicity test.
Initial test:
without S9, 4 h: 50, 100, 250, 300, 325, 350, 400 µg/mL
without S9, 24 h: 50, 100, 200, 250, 285, 300, 325, 350 µg/mL
with S9, 4 h: 100, 250, 500, 750, 1000 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: recommeded by guideline and the test item was well soluble in the vehicle - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: vinblastine without S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h and 24 h
SPINDLE INHIBITOR: cytochalasine B
STAIN: acridine orange
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were collecetd by centrifugation and resuspended in fixative; fixed cells were applied to glass slides and air dried
NUMBER OF CELLS EVALUATED: min 2000
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 2
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei of a binucleated cell were recoreded it the meet the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: % Cytostasis (cytotoxicity) = 100 - 100 ((CBPI t-1)/(CBPDI c-1))
where CBPI = 1x mononucleated cells + 2 x binucleated cells + 3 x multinucleated cells / total number of cells scored - Rationale for test conditions:
- preliminary cytotoxicity test
- Evaluation criteria:
- The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
- Statistics:
- Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, Cochran-Armitage test was not required to measure dose-responsiveness.
- Key result
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentration > 250 µg/mL without S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH in highest concentration was measured to be 7.5
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: yes, at concentrations > 350 µg/mL without S9 mix and > 500 µg/mL with S9 mix
- Hemolysis was observed at concentrations of > 400 µg/mL without S9 mix
RANGE-FINDING/SCREENING STUDIES: yes
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI - Conclusions:
- Under the conditions of the assay the test item was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
- Executive summary:
The test substance was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system.A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.
Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.In a solubility test the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
In the preliminary toxicity assay, the doses tested ranged from 0.192 to 1920 μg/mL (10 mM).Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 576μg/mL in the non-activated 4 and 24-hour exposure groups.Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 576 μg/mL in the non-activated 4 and 24-hour exposure groups and at 1920 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 50 to 500 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 100 to 1950 μg/mL for the S9-activated 4-hour exposure group.
In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 300 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 250 μg/mL in the non-activated 24-hour exposure group. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group. However, due to technical reasons, the slides from the initial assay were not scorable. Therefore, the micronucleus assay was repeated at dose levels ranging from 50 to 400 μg/mL for the non-activated 4-hour exposure group, from 100 to 1000 μg/mL for the S9-activated 4-hour exposure group, and from 50 to 350 μg/mL for the non-activated 24-hour exposure group.
In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 250 μg/mL in the non-activated 4 and 24-hour exposure groups. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group.
The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.
The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.
Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.
Referenceopen allclose all
Table 1: Number of revertants per plate (mean of three plates), Experiment 1 and 2, without metabolic activation
strain TA 98 |
strain TA 100 |
strain TA 102 |
strain TA 1535 |
strain TA 1537 |
||||||
conc. [µg] |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
control |
35 |
31 |
83 |
99 |
316 |
278 |
22 |
10 |
13 |
11 |
solvent control |
32 |
31 |
88 |
90 |
265 |
257 |
24 |
10 |
11 |
11 |
5 |
33 |
|
87 |
|
183 |
26 |
|
8 |
|
|
15 |
34 |
20 |
79 |
79 |
191 |
165 |
27 |
14 |
9 |
13 |
50 |
39 |
30 |
88 |
78 |
249 |
214 |
29 |
14 |
11 |
13 |
150 |
35 |
28 |
82 |
85 |
239 |
236 |
12 |
11 |
9 |
9 |
500 |
28 |
26 |
86 |
84 |
118T |
143T |
20 |
11 |
7 |
9 |
1500 |
15T |
47T |
85T |
|
7T |
4T |
||||
Sodium azide 0.7 µg |
|
|
528 |
499 |
|
|
841 |
471 |
|
|
2-nitrofluorene 2.5 µg |
549 |
598 |
|
|
|
|
|
|
|
|
9-aminoacridine 50µg |
|
|
|
|
|
|
|
|
115 |
180 |
MitomycinC 0.15 µg |
|
|
|
|
807 |
881 |
|
|
|
|
T: Cytotoxicity
Table 2: Number of revertants per plate (mean of three plates), Experiment 1 and 2, with metabolic activation
strain TA 98 |
strain TA 100 |
strain TA 102 |
strain TA 1535 |
strain TA 1537 |
||||||
conc. [µg] |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
Exp1 |
Exp2 |
control |
27 |
26 |
89 |
90 |
289 |
320 |
12 |
11 |
17 |
10 |
solvent control |
21 |
23 |
80 |
89 |
275 |
299 |
13 |
13 |
14 |
14 |
50 |
23 |
28 |
82 |
89 |
272 |
256 |
7 |
12 |
11 |
15 |
150 |
23 |
23 |
73 |
89 |
265 |
278 |
8 |
13 |
12 |
13 |
500 |
22 |
22 |
75 |
84 |
256 |
259 |
11 |
15 |
10 |
13 |
1500 |
20 |
23 |
74 |
82 |
235 |
273 |
15 |
15 |
9 |
15 |
5000 |
17 |
23 |
72 |
82 |
223 |
228 |
13 |
14 |
14 |
14 |
2-aminoanthracene 0.8 µg/plate |
799 |
1018 |
917 |
753 |
|
|
|
|
|
|
2-aminoanthracene 0.9 µg/plate |
|
|
|
|
538 |
|
308 |
91 |
|
|
2-aminoanthracene 1.0 µg/plate |
|
|
|
|
|
592 |
|
|
|
|
2-aminoanthracene 1.7 µg/plate |
|
|
|
|
|
|
|
|
114 |
203 |
T: Cytotoxicity
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation assay
The test substance was tested for its mutagenicity in the bacterial reverse mutation assay (Symrise 2000485). Therefore, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 were used. The test was performed using the standard plate incorporation assay with and without S9 -mix (Aroclor 1254 induced). Two independet experiments were carried out using concentrations of 50 -5000 µg and 5 -1500 µg/plate. In the presence of S9-mix no bacteriotoxicity was observed. In the absence of S9-mix the test substance was bacteriotoxic towards the strain TA102 at 500 µg/plate and towards the strains TA1535, TA1537, TA98, and TA100 at 1500 µg/plate. As mutagenicity controls, sodium azide, 2 -nitrofluorene, 9 -aminoacridine and mitomycin C were used in the absence of S9 -mix and 2 -aminoanthracene was used in the in the presence of S9 mix. As a result of this assay, mutagenicity was not observed.
HPRT assay
The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay 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 with and 24 hours without metabolic activation. The maximum test item concentration of the pre-experiment (1930μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity (without metabolic activation) and phase separation (with metabolic activation) of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.
The tested concentrations were:
Experiment I, exposure period 4 hours:
without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL
with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL
Experiment II, exposure period 24 hous:
without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL
Exposure period 4 hours:
30, 60, 120, 240, 480, 960 µg/mL
Appropriate reference mutagens, used as positive controls (without metabolic activation: ethylmethane sulfonate, with metabolic activation: DMBA), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Based on the available results the study was considered valid and the tets substance was shown to be not mutagenic in the HPRT assay.
In vitro micronuclei assay
The test substance was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system.A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.
Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.In a solubility test the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.
In the preliminary toxicity assay, the doses tested ranged from 0.192 to 1920 μg/mL (10 mM).Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 576μg/mL in the non-activated 4 and 24-hour exposure groups.Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 576 μg/mL in the non-activated 4 and 24-hour exposure groups and at 1920 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 50 to 500 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 100 to 1950 μg/mL for the S9-activated 4-hour exposure group.
In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 300 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 250 μg/mL in the non-activated 24-hour exposure group. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group. However, due to technical reasons, the slides from the initial assay were not scorable. Therefore, the micronucleus assay was repeated at dose levels ranging from 50 to 400 μg/mL for the non-activated 4-hour exposure group, from 100 to 1000 μg/mL for the S9-activated 4-hour exposure group, and from 50 to 350 μg/mL for the non-activated 24-hour exposure group.
In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 250 μg/mL in the non-activated 4 and 24-hour exposure groups. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group.
The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.
The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.
Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.
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. Based on
available data on genetic toxicity, the
test item is not classified according
to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in
Regulation (EU) No 2016/918.
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.