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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 242-177-9 | CAS number: 18297-63-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in human lymphocytes with and without metabolic activation
Gene mutation in mammalian cells (OECD 490, Mouse lymphoma assay): negative in mouse lymphoma L5178Y cells with and without metabolic activation
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:
- 19 Jun - 17 Jul 2019
- 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:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 2020
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His operon (S. typhimurium strains) and trp operon (E. coli strain)
- 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:
- Type and composition of metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix).
- method of preparation of S9 mix : S9 mix was prepared from the livers of rats that were induced with phenobarbital / beta-naphthoflavone.
- quality controls of S9: The enzymatic activity of each S9 batch was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- Pre-experiment / Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Justification for top dose: In the pre-experiment the test item concentration range was 3 - 5000 µg/plate. As no cytotoxicity was observed, 5000 µg/plate was selected as highest dose and the pre-experiment were designated as experiment 1. - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD), -S9, 10 µg/plate in DMSO for TA98 and 50 µg/plate for TA1537; 2-aminoanthracene (2-AA), +S9, 2.5 µg/plate in DMSO for TA98, TA100, TA1535 and TA1537 and 10.0 µg/plate for WP2uvrA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2
METHOD OF APPLICATION: in agar (plate incorporation, Experiment 1) and pre-incubation (Experiment 2)
TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period: 60 min
- Exposure duration: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of two-fold or above (strains TA98, TA100, and WP2uvrA) or three-fold or above (strains TA1535 and TA1537) the spontaneous mutation rate of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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 nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: There was no precipitation of the test item observed in the overlay agar at any concentration, neither with nor without S9 mix.
CYTOTOXICITY:
There was no cytotoxicity observed up to and including the highest concentration of 5000 µg/plate in all strains, with and without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: A pre-experiment was performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA according to the plate-incorporation method. Test item concentrations ranging from 3 - 5000 µg/plate were tested in all strains in the presence and absence of metabolic acitivation. There was no cytotoxicity observed for any strain, neither in the presence nor absence of S9 mix. As all acceptability criteria were met, the pre-experiment was reported as experiment 1 of the main mutation assay.
STUDY RESULTS : Please refer to Tables No. 1 and 2 under "Any other information on results incl. tables".
HISTORICAL CONTROL DATA: Please refer to Table No. 3 under "Any other information on results incl. tables". - Conclusions:
- Under the conditions of the test, the test item was not mutagenic in S. typhimuirum strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2uvrA with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 Jun - 15 Jul 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: Human lymphocytes have been used successfully for a long time in in vitro experiments. It has been shown that they are an extremely sensitive indicator of in vitro induced chromosome structural changes. These changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material. A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture; they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations. Furthermore, human lymphocytes have a low spontaneous chromosome aberration frequency.
- Sex, age and number of blood donors: Blood was collected from a female donor (29 years old) for Experiment I and from a male donor (21 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used: Whole blood was used.
- Whether blood from different donors were pooled or not: For each experiment, lymphocytes from a single donor were used.
- Mitogen used for lymphocytes: phytohemagglutinin (PHA, 3 µg/mL)
MEDIA USED
- Type and composition of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) including 200 mM GlutaMAX™, supplemented with penicillin / streptomycin (100 U/mL / 100 µg/mL), PHA (3 µg/mL), 10% fetal bovine serum (FBS), 10 mM HEPES and heparin (125 U.S.P.-U/mL).
- CO2 concentration, humidity level, temperature: The cells were incubated at 37 °C with 5.5% CO2 in humidified air. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix).
- source of S9: Envigo CRS GmbH, Rossdorf, Germany (Lot No. 210618)
- method of preparation of S9 mix: S9 mix was prepared from the livers of rats induced with phenobarbital/beta-naphthoflavone. 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 mix used for this study was 30.4 mg/mL.
- quality controls of S9: Each batch of S9 mix was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- Preliminary cytotoxicity test / Experiment 1:
4 h exposure with and without metabolic activation: 5.2, 9.1, 15.9, 27.9, 48.7, 85.3, 149*, 261*, 457* and 800 µg/mL
Experiment 2:
4 h exposure with metabolic activation: 15.9, 27.9, 48.7, 85.3, 149*, 261*, 457* and 800 µg/mL
22 h without metabolic activation: 15.9, 27.9, 48.7, 85.3, 149, 261*, 457* and 800* µg/mL
*: concentrations used for evaluation of chromosome aberrations
Justification for top dose: The highest concentration used in the preliminary toxicity test / Experiment 1 was chosen with regard to the solubility. Dose selection of Experiment 2 was based on the occurrence of precipitation observed in the preliminary toxicity test / Experiment 1. - Vehicle / solvent:
- - Vehicle / solvent used: anhydrous dimethylsulfoxide (DMSO), 1% in the final culture medium.
- Justification for choice of vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 22 h
- Harvest time: 22 h after beginning of treatment.
SPINDLE INHIBITOR:
0.2 µg/mL colcemid was added approx. 3 h prior to harvest.
SLIDE PREPRATION AND METAPHASE ANALYSIS:
- Methods of slide preparation and staining technique used including the stain used: The cells were harvested by centrifugation, re-suspended in hypotonic solution (0.0375 M potassium chloride) at 37 °C for 20 min and fixed in absolute methanol : glacial acetic acid, 3 : 1). A small amount of cell suspension was then dropped onto wet microscope slides and allowed to dry. All slides were stained with Giemsa, dried and covered with a cover slip.
- Number of cells spread and analysed per concentration: 300 (150 cells per culture)
- Criteria for scoring chromosome aberrations: Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosomal aberrations. Gaps were defined as achromatic lesions of unknown biological relevance and recorded, but not included in the calculation of the aberration rates.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (% of control) - Evaluation criteria:
- A test substance is classified as non-clastogenic if:
- 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 (e.g. Poisson-based 95% control limits).
A test substance is classified as clastogenic if all of the following criteria are met:
- 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 historical negative control data (e.g. Poisson-based 95% control limits). - Statistics:
- The statistical significance was confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics. A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to assess a possible dose dependent increase of mutation frequencies. The number of chromosomal aberrations, obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05.
- Key result
- Species / strain:
- lymphocytes: cultured human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was observed after 4 h exposure with and without S9 mix at 457 and 800 µg/mL and after 22 h exposure without S9 mix at 800 µg/mL.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other: In valid cytotoxicity data of the positive control in the preliminary cytotoxicity test / Experiment 1 in the presence of S9 mix. The experimental part was repeated in Experiment 2, where all acceptability criteria were met.
- Remarks:
- (Data of the preliminary cytotoxicity test / Experiment 1 for 4 h exposure +S9 mix were not shown in the study report)
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant influence on pH was observed.
- Data on osmolality: No relevant influence on osmolality was observed. –the osmolality in general was high (472 mOsm for the solvent control, 470 mOsm for 800 µg/mL test item) when compared to the physiological level of approx. 300 mOsm, but this effect was explained by the final concentration of 1% DMSO in the culture medium.
- Precipitation and time of the determination: Precipitation of the test substance in medium was noted in the first experiment (4 h exposure – S9 mix) at ≥ 457 µg/mL without S9 mix and in the second experiment (4 h exposure + S9 mix and 22 h exposure – S9 mix) at ≥ 457 µg/mL with S9 mix and at 800 µg/mL without S9 mix.
- Definition of acceptable cells for analysis: Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis.
RANGE-FINDING/SCREENING STUDY:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. With regard to solubility properties of the test item, duplicate cultures were exposed to test item concentrations in the range of 5.2 – 800 µg/mL. Precipitation of the test item was observed at the end of treatment at 457 µg/mL and above in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation in the absence of S9 mix, this preliminary test was designated Experiment I. The experimental part with S9 mix was repeated in Experiment II due to invalid cytotoxicity data of the positive control.
STUDY RESULTS:
In the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. In addition, no evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, the positive controls EMS (825 or 770 µg/mL) or CPA (7.5 µg/mL) showed distinct increases in cells with structural chromosome aberrations.
CYTOTOXICITY:
There was no cytotoxicity observed in any experiment up the highest tested concetration of 800 µg/mL, neither with nor without S9 mix. However, precipitation occurred at the highest concentration investigated for chromosome aberrations.
HISTORICAL CONTROL DATA:
The solvent and the positive controls showed the expected results and the obtained values remained within the historical control data. For details on the historical control data please refer to Tables no. 1 and 2 under “Any other information on materials and methods incl. tables”.
OTHER:
In the preliminary cytotoxicity test / Experiment 1 of the main study, the positive control cyclophosphamide produced invalid cytotoxicity data after 4 h of exposure in the presence of S9 mix. The experimental part in the presence of S9 mix was therefore repeated in the second experiment, in which all acceptability criteria were met. The data of the invalid test are not included in the study report. - Conclusions:
- Under the conditions of the test, the test item did not induce chromosomal aberrations in human peripheral lymphocytes.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 May - 15 Jul 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted in 2016
- Deviations:
- yes
- Remarks:
- amount of S9 in final culture medium not specified, selection time 10 - 15 days instead of 10 - 12 days
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Dr. J. Cole, MRC Cell Mutation Unit, Brighton, UK
- Suitability of cells: The L5178Y cell line has been used successfully in in vitro experiments for many years. The cells are characterised by a high proliferation rate and cloning efficiency of untreated cells of 50%, both necessary for the appropriate performance of the study.
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: The cells were maintained in plastic flasks in RPMI 1640 complete culture medium and sub-cultured two times prior to treatment.
- Cell cycle length, doubling time or proliferation index: 10 – 12 h
- Modal number of chromosomes: 40 ± 2
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media:
Complete culture medium: RPMI 1640 medium supplemented with 15% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 220 µg/mL sodium pyruvate and 0.5 – 0.75% amphotericin.
Treatment medium, 4 h exposure: RPMI 1640 medium supplemented with 3% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 220 µg/mL sodium pyruvate and 0.5 – 0.75% amphotericin.
Treatment medium, 24 h exposure: RPMI 1640 medium supplemented with 15% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 220 µg/mL sodium pyruvate and 0.5 – 0.75% amphotericin.
Selection medium: complete culture medium, supplemented with 5 µg/mL trifluorothymidine (TFT).
- Incubation (CO2 concentration, humidity level, temperature): The cells were cultured at 37.0 ± 1.5°C in a humidified atmosphere with 4.5% CO2 and 95.5% ambient air. - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- source of S9: Envigo CRS GmbH, Rossdorf, Germany (Lot no. 210618)
- method of preparation of S9 mix: S9 mix was prepared from the livers of rats induced with phenobarbital/beta-naphthoflavone. 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 mix used for this study was 30.4 mg/mL.
- quality controls of S9: Each batch of S9 mix was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. - Test concentrations with justification for top dose:
- Preliminary cytotoxicity test:
With and without S9 mix: 6.3, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL (4 h exposure)
Without S9 mix: 6.3, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL (24 h exposure)
Experiment 1:
With and without S9 mix: 12.5, 25, 50*, 100*, 200*, 400* and 800* µg/mL (4 h exposure)
Experiment 2:
Without S9 mix: 12.5, 25, 50*, 100*, 200*, 400* and 800* µg/mL (24 h exposure)
*: concentrations selected for evaluation of mutagenicity
Justification for top concentration: The highest concentration used in the preliminary toxicity test was chosen with regard to the solubility of the test item in dry DMSO. The dose range of the main experiments was set according to the data generated in the preliminary cytotoxicity test. - Vehicle / solvent:
- - Vehicle used: dry dimethylsulfoxide (DMSO), 0.5% in the final culture medium.
- Justification for choice of vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2
METHOD OF TREATMENT:
- Cell density at seeding: 1 x 10E7 cells/flask for 4 h exposure and 3 x 10E6 cells/flask for 24 h exposure in 80 cm2 flasks.
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time: 10 - 15 days
- Method used: microtiter plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period, 4 x 10E3 cells/well from each experimental group were seeded into 2 microtiter plates with selective medium including trifluorothymidine (TFT). The viability (cloning efficiency) was determined by seeding 2 cells per well into 2 microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 25% of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).
SELECTION AGENT: 5 μg/mL trifluorothymidine (TFT)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG), calculated by multiplying the relative suspension growth (RSG) by the viability
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF) - Evaluation criteria:
- A test item is considered to be clearly mutagenic if, in any of the experimental conditions examined:
a) the induced mutation frequency (IMF) reproducibly (in both parallel cultures) exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control and
b) a relevant increase of the mutation frequency (MF) is dose-dependent.
A test item is considered to be clearly non-mutagenic if, in all experimental conditions examined:
a) there is no concentration-related response or,
b) if there is an increase in MF, the IMF does not reproducibly exceed a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration. - Statistics:
- A linear regression will be performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be 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 relevance and statistical significance will be considered together.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- Precipitation was noted in both experiments at 800 µg/mL with and without S9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and on osmolality: Both, pH value and osmolarity were determined in the pre-experiment at the maximum concentration of the test item and in the solvent control without metabolic activation. No relevant increase in the osmolarity or pH value was observed.
- Precipitation and time of the determination: Precipitation of test substance in the culture medium after the treatment period was noted in both experiments at 800 µg/mL in the presence and absence of S9 mix.
RANGE-FINDING/SCREENING STUDY:
A preliminary cytotoxicity test was performed in order to determine the concentration range of the mutagenicity experiments. Test item concentrations between 6.3 and 800.0 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 hours of treatment. Precipitation occurred at the highest concentration of 800.0 µg/mL after 4 and 24 hours of treatment without metabolic activation and 4 hours treatment with metabolic activation.
STUDY RESULTS
Please refer to Table No. 2 under “Any other information on results incl. tables”.
HISTORICAL CONTROL DATA: Please refer to Table 1 under "Any other information on materials and methods incl. tables".
- Positive historical control data: The mutation frequencies of both positive controls were 449 mutant colonies per 10E6 cells for 4 h exposure –S9 mix, 408 and 581 mutant colonies per 10E6 cells for 4 h exposure +S9 mix and 353 mutant colonies per 10E6 cells for 24 h exposure –S9 mix and fell within the range of the laboratory's historical control data.
- Solvent historical control data: The mutation frequency of the solvent control DMSO ranged from 58 – 71 mutant colonies per 10E6 cells and were within the recommended range of 50 – 170 mutant colonies per 10E6 cells. - Conclusions:
- Under the conditions of the test the test item did not induce mutations in the Tk locus in the presence or absence of metabolic activation.
Referenceopen allclose all
Table 1: Results of the pre-experiment / Experiment 1 (plate incorporation test)
| Metabolic activation | Compound | Concentration (µg/plate) | TA1535 | TA1537 | TA98 | TA100 | WP2uvrA |
| Without S9 mix | DMSO | / | 13 ± 4 | 14 ± 5 | 31 ± 3 | 111 ± 16 | 54 ± 10 |
| Untreated | / | 12 ± 1 | 13 ± 2 | 24 ± 8 | 114 ± 6 | 57 ± 3 | |
| Test item | 3 | 13 ± 2 | 15 ± 6 | 25 ± 3 | 104 ± 18 | 54 ± 6 | |
| 10 | 15 ± 6 | 18 ± 3 | 26 ± 5 | 123 ± 9 | 62 ± 8 | ||
| 33 | 15 ± 6 | 16 ± 6 | 30 ± 3 | 105 ± 12 | 51 ± 6 | ||
| 100 | 12 ± 5 | 16 ± 5 | 27 ± 5 | 119 ± 6 | 54 ± 9 | ||
| 333 | 14 ± 5 | 14 ± 3 | 24 ± 7 | 107 ± 11 | 59 ± 12 | ||
| 1000 | 14 ± 4 | 15 ± 4 | 23 ± 3 | 106 ± 10 | 55 ± 10 | ||
| 2500 | 11 ± 2 | 16 ± 3 | 27 ± 5 | 105 ± 23 | 56 ± 5 | ||
| 5000 | 11 ± 3 | 11 ± 1 | 30 ± 5 | 99 ± 17 | 49 ± 2 | ||
| NaN3 | 10 | 1112 ± 103 | / | / | 1574 ± 127 | / | |
| 4-NOPD | 10 | / | / | 341 ± 37 | / | / | |
| 4-NOPD | 50 | / | 76 ± 6 | / | / | / | |
| MMS | 2.0 µL/plate | / | / | / | / | 930 ± 66 | |
| With S9 mix | DMSO | / | 16 ± 2 | 18 ± 6 | 45 ± 8 | 120 ± 2 | 66 ± 12 |
| Untreated | / | 16 ± 4 | 19 ± 3 | 39 ± 3 | 113 ± 27 | 60 ± 7 | |
| Test item | 3 | 12 ± 4 | 13 ± 3 | 36 ± 8 | 99 ± 18 | 63 ± 1 | |
| 10 | 14 ± 3 | 17 ± 6 | 40 ± 5 | 118 ± 8 | 61 ± 8 | ||
| 33 | 14 ± 4 | 19 ± 2 | 40 ± 9 | 117 ± 4 | 68 ± 15 | ||
| 100 | 11 ± 4 | 19 ± 6 | 39 ± 3 | 111 ± 12 | 63 ± 8 | ||
| 333 | 11 ± 5 | 18 ± 2 | 34 ± 5 | 104 ± 6 | 58 ± 5 | ||
| 1000 | 14 ± 2 | 17 ± 5 | 35 ± 7 | 108 ± 8 | 67 ± 8 | ||
| 2500 | 13 ± 1 | 13 ± 4 | 41 ± 9 | 91 ± 5 | 57 ± 14 | ||
| 5000 | 11 ± 1 | 14 ± 4 | 36 ± 4 | 92 ± 16 | 60 ± 2 | ||
| 2-AA | 2.5 | 315 ± 25 | 509 ± 16 | 3858 ± 202 | 3870 ± 271 | / | |
| 2-AA | 10 | / | / | / | / | 391 ± 35 | |
| NaN3 sodium azide; 2-AA 2-aminoanthracene; 4-NOPD 4-nitro-o-phenylene-diamine; MMS methyl methane sulfonate |
|||||||
Table 2: Results of Experiment 2 (pre-incubation test)
| Metabolic activation | Compound | Concentration (µg/plate) | TA1535 | TA1537 | TA98 | TA100 | WP2uvrA |
| Without S9 mix | DMSO | / | 13 ± 2 | 13 ± 2 | 30 ± 11 | 95 ± 7 | 43 ± 6 |
| Untreated | / | 15 ± 5 | 13 ± 8 | 29 ± 3 | 115 ± 10 | 52 ± 6 | |
| Test item | 33 | 9 ± 1 | 14 ± 2 | 35 ± 2 | 105 ± 1 | 45 ± 9 | |
| 100 | 12 ± 3 | 14 ± 2 | 33 ± 8 | 93 ± 12 | 40 ± 2 | ||
| 333 | 12 ± 6 | 9 ± 1 | 27 ± 5 | 103 ± 10 | 49 ± 4 | ||
| 1000 | 13 ± 4 | 10 ± 3 | 26 ± 4 | 90 ± 9 | 42 ± 9 | ||
| 2500 | 11 ± 4 | 10 ± 5 | 25 ± 6 | 92 ± 16 | 43 ± 7 | ||
| 5000 | 13 ± 1 | 11 ± 3 | 22 ± 6 | 88 ± 19 | 43 ± 5 | ||
| NaN3 | 10 | 1212 ± 36 | / | / | 1830 ± 144 | / | |
| 4-NOPD | 10 | / | / | 485 ± 26 | / | / | |
| 4-NOPD | 50 | / | 88 ± 3 | / | / | / | |
| MMS | 2.0 µL/plate | / | / | / | / | 716 ± 25 | |
| With S9 mix | DMSO | / | 15 ± 1 | 11 ± 2 | 41 ± 5 | 97 ± 19 | 58 ± 7 |
| Untreated | / | 13 ± 3 | 10 ± 3 | 36 ± 4 | 111 ± 8 | 59 ± 7 | |
| Test item | 33 | 15 ± 3 | 11 ± 6 | 38 ± 11 | 96 ± 8 | 49 ± 3 | |
| 100 | 15 ± 6 | 12 ± 1 | 38 ± 11 | 109 ± 9 | 57 ± 5 | ||
| 333 | 12 ± 3 | 13 ± 2 | 40 ± 7 | 101 ± 3 | 59 ± 11 | ||
| 1000 | 16 ± 5 | 13 ± 3 | 39 ± 6 | 102 ± 8 | 63 ± 6 | ||
| 2500 | 9 ± 6 | 9 ± 4 | 38 ± 13 | 96 ± 3 | 47 ± 6 | ||
| 5000 | 13 ± 3 | 8 ± 3 | 35 ± 6 | 80 ± 9 | 48 ± 12 | ||
| 2-AA | 2.5 | 416 ± 18 | 281 ± 20 | 3358 ± 561 | 3662 ± 645 | / | |
| 2-AA | 10 | / | / | / | / | 404 ± 26 | |
| NaN3 sodium azide, 2-AA 2-aminoanthracene, 4-NOPD 4-nitro-o-phenylene-diamine, MMS methyl methane sulfonate | |||||||
Table 3: Historical control data generated in the testing facility from Nov 2016 - Aug 2018 representing approx. 600 experiments (WP2uvrA historical control data are based on approx. 400 experiments)
| Strain | Compound | without S9 mix | with S9 mix | ||
| Mean ± SD | Range | Mean ± SD | Range | ||
| TA 1535 | Solvent control | 11 ± 2.3 | 6 - 22 | 12 ± 2.3 | 7 - 22 |
| Untreated control | 11 ± 2.9 | 6 - 28 | 12 ± 2.8 | 7 - 23 | |
| Positive control | 1245 ± 161.4 | 367 - 1791 | 398 ± 61 | 183 - 613 | |
| TA 1537 | Solvent control | 10 ± 2.2 | 6 - 19 | 13 ± 3.2 | 7 - 30 |
| Untreated control | 10 ± 2.7 | 5 - 21 | 14 ± 3.6 | 6 - 29 | |
| Positive control | 94 ± 30 | 48 - 231 | 170 ± 64.8 | 81 - 421 | |
| TA 98 | Solvent control | 26 ± 4.2 | 13 - 43 | 36 ± 6.1 | 12 - 56 |
| Untreated control | 27 ± 4.7 | 14 - 40 | 39 ± 6.4 | 12 - 59 | |
| Positive control | 421 ± 176.8 | 196 - 2068 | 3908 ± 815 | 223 - 5918 | |
| TA 100 | Solvent control | 160 ± 29.3 | 79 - 214 | 148 ± 31.3 | 76 - 216 |
| Untreated control | 181 ± 26.1 | 80 - 235 | 171 ± 27.7 | 87 - 218 | |
| Positive control | 2074 ± 262.7 | 511 - 2850 | 3626 ± 981.9 | 553 - 5860 | |
| WP2 uvrA | Solvent control | 39 ± 6.4 | 26 - 60 | 48 ± 7.3 | 28 - 69 |
| Untreated control | 40 ± 5.8 | 22 - 61 | 51 ± 7.4 | 32 - 87 | |
| Positive control | 865 ± 340.9 | 346 - 4732 | 426 ± 111.2 | 184 - 1265 | |
SD = standard deviation
Table 3: Results of the chromosome abberation test
| Substance | Concentration (µg/mL) | Mitotic index (% of control) | Aberrant cells (%) | ||
| incl. gaps# | excl. gaps* | exchanges | |||
| Exposure period 4 hrs without S9 mix, 22 h harvest | |||||
| DMSO | 1% | 100.0 | 1.3 | 1.0 | 0.0 |
| EMS | 825 | 58.9 | 16.3 | 16.3* | 4.3 |
| Test item | 149 | 100.3 | 1.3 | 1.0 | 0.0 |
| 261 | 98.0 | 2.3 | 1.7 | 0.0 | |
| 457P | 97.7 | 1.0 | 1.0 | 0.0 | |
| Exposure period 22 hrs without S9 mix | |||||
| DMSO | 1% | 100.0 | 0.7 | 0.7 | 0.0 |
| EMS | 770 | 54.9 | 25.3 | 24.7* | 10.7 |
| Test item | 261 | 112.7 | 0.7 | 0.7 | 0.0 |
| 457 | 121.0 | 1.7 | 1.3 | 0.0 | |
| 800P | 108.0 | 1.3 | 0.7 | 0.0 | |
| Exposure period 4 hrs with S9 mix, 22 h harvest | |||||
| DMSO | 1% | 100.0 | 1.7 | 1.3 | 0.0 |
| CPA | 7.5 | 53.1 | 10.3 | 10.3* | 3.0 |
| Test item | 149 | 116.2 | 0.7 | 0.3 | 0.0 |
| 261 | 122.1 | 0.7 | 0.7 | 0.0 | |
| 457P | 111.8 | 0.7 | 0.7 | 0.0 | |
| # including cells carrying exchanges P: precipitation at the end of treatment * aberration frequency statistically significantly increased when compared to solvent control EMS: ethylmethanesulphonate, positive control -S9 mix CPA: cyclophosphamide, positive control +S9 mix |
|||||
Table 2: Results of the mouse lymphoma assay
| Concentration (µg/mL) | S9 mix | RTG | Mutant colonies/ 106cells | Threshold | |
| Experiment I / 4 h treatment | |||||
| DMSO | - | 100 | 58 | 184 | |
| MMS | 19.5 | 38.4 | 449 | 184 | |
| Test item | 12.5 | culture was not continued# | |||
| Test item | 25 | culture was not continued# | |||
| Test item | 50 | 114.1 | 60 | 184 | |
| Test item | 100 | 107.5 | 69 | 184 | |
| Test item | 200 | 83.1 | 100 | 184 | |
| Test item | 400 | 79.5 | 88 | 184 | |
| Test item | 800 (P) | 88.4 | 74 | 184 | |
| DMSO | + | 100 | 65 | 191 | |
| CPA | 3 | 37.8 | 408 | 191 | |
| CPA | 4.5 | 9.4 | 581 | 191 | |
| Test item | 12.5 | culture was not continued# | |||
| Test item | 25 | culture was not continued# | |||
| Test item | 50 | 108.4 | 56 | 191 | |
| Test item | 100 | 90.9 | 70 | 191 | |
| Test item | 200 | 108.3 | 49 | 191 | |
| Test item | 400 | 96.7 | 97 | 191 | |
| Test item | 800 (P) | 108 | 56 | 191 | |
| Experiment II / 24 h treatment | |||||
| DMSO | - | 100 | 71 | 197 | |
| MMS | 13 | 26 | 353 | 197 | |
| Test item | 12.5 | culture was not continued# | |||
| Test item | 25 | culture was not continued# | |||
| Test item | 50 | 107.8 | 86 | 197 | |
| Test item | 100 | 97.8 | 102 | 197 | |
| Test item | 200 | 86.5 | 92 | 197 | |
| Test item | 400 | 144.6 | 79 | 197 | |
| Test item | 800 (P) | 90.1 | 68 | 197 | |
| # culture was not continued as a minimum of only four analysable concentrations is required; CPA: cyclophosphamide; RTG: Relative total growth as measure of cytotoxicity; MMS: methylmethanesulphonate; (P): precipitation was noted at the end of treatment | |||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria:
N,N-Bis-(trimethylsilyl)urea was investigated for gene mutation in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Envigo CRS GmbH, 2019c). Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were exposed to the test item, untreated, solvent (DMSO) and appropriate positive controls in the presence and absence of metabolic activation (S9 mix) for at least 48 h at 37 °C.
A pre-experiment was conducted to identify cytotoxic concentrations of the test item. In a plate incorporation test, test item concentrations in the range of 3 to 5000 µg/plate did not show any cytotoxic effects in the presence and absence of metabolic activation. As all acceptability criteria were fulfilled, the pre-experiment was designated Experiment I of the main mutation assay and analysed for the number of revertant colonies. A second main mutation assay was performed using the pre-incubation method and concentrations in the range 33 – 5000 µg/plate. In both experiments, all conditions were tested in triplicates. After at least 48 h of incubation, the bacterial background lawn was inspected and the mean number of revertant colonies was counted for each plate.
Precipitation of the test substance was not reported and no cytotoxicity occurred up to and including the highest tested concentration. In addition, there was no substantial increase in revertant colony numbers in any of the five tester strains observed following treatment with N,N'-Bis(trimethylsilyl)urea at any dose level, neither in the presence nor absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The number of revertant colonies induced by the solvent control was within the range of the historical control data for each strain, thus demonstrating an acceptable experimental performance. Appropriate positive control compounds showed a strong increase in the number of revertant colonies, confirming the activity of the S9 mix and the validity of the test system.
Thus, based on the results of the present study and under the experimental conditions chosen, the test item is not mutagenic in bacteria with and without metabolic activation.
In vitro cytogenicity in mammalian cells
A chromosome aberration test in human peripheral lymphocytes was performed according to OECD guideline 473 and in compliance with GLP (Envigo CRS GmbH, 2019d). A preliminary cytotoxicity test was conducted to identify suitable concentrations for the main cytogenicity test. Duplicate cultures were exposed to the test item, vehicle (DMSO) and positive controls (ethylmethanesulphonate (EMS), 825 µg/mL without S9 mix and cyclophosphamide (CPA), 7.5 µg/mL with S9 mix) for 4 h in the presence and absence of metabolic activation (S9 mix). Test item concentrations ranged from 5.2 – 800 µg/mL, based on solubility of the test item in the vehicle. As no cytotoxicity was observed, and because the acceptability criteria were fulfilled for the experimental condition in the absence of S9 mix, the cultures of the 4 h exposure without S9 mix were examined for chromosome aberrations and the preliminary experiment was designated Experiment 1 of the main cytogenicity test. For the experimental condition in the presence of S9 mix, the cytoxicity criteria were not met for the positive control (data not reported). Thus, in a second experiment, the 4 h exposure in the presence of S9 mix was repeated and an additional 22 h exposure period in the absence of S9 mix was included. Concentrations in the second experiment ranged from 15.9 – 800 µg/mL. As a positive control for continuous exposure in the absence of S9 mix, EMS at a concentration of 770 µg/mL was applied.
In both experiments, the cells were harvested 22 h after start of exposure. A total of 300 metaphase cells per condition were scored for the presence of structural and numerical chromosome aberrations. In parallel, cytotoxicity was assessed as mitotic index in comparison to solvent controls.
There was no cytotoxicity noted in any experiment at any concentration, neither with nor without S9 mix. Precipitation of the test substance in the culture medium was observed after 4 h of treatment with ≥ 457 µg/mL with and without S9 mix and after 22 h of exposure at 800 µg/mL without S9 mix.
All control substances showed the expected results and were within the range of the laboratory's historical control data. EMS and CPA induced statistically significant increases in the number of aberrant cells, thus proving the functionality of the S9 mix and demonstrating the validity of the test system.
N,N-Bis-(trimethylsilyl)urea did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix. In addition, the frequency of polyploid cells and cells with endoreduplicated chromosomes were not affected, neither with nor without metabolic activation.
Under the conditions of the test, N,N-Bis-(trimethylsilyl)urea is considered negative for clastogenic activity in human peripheral lymphocytes in vitro.
In vitro gene mutation in mammalian cells
N,N-Bis-(trimethylsilyl)urea was tested for its potential to induce gene mutations in mammalian cells in a mouse lymphoma assay, in line with OECD guideline 490 and according the GLP (Envigo CRS GmbH, 2019e). Two independent experiments were performed in L5178Y mouse lymphoma cells (thymidine kinase locus). Based on the results of a preliminary cytotoxicity test, duplicate cultures were exposed to test item concentrations of 12.5 – 800 µg/mL for 4 h in the presence and absence of S9 mix (Experiment 1) and for 24 h in the absence of S9 mix (Experiment 2). Solvent (DMSO) and positive controls (methylmethanesulfonate (MMS), 19.5 µg/mL for 4 h of exposure and 13.0 µg/mL for 24 h of exposure without S9 mix and cyclophosphamide (CPA), 3.0 and 4.5 µg/mL with S9 mix) were included in each experiment. After exposure, the cells were incubated for 48 h at 37.0 ± 1.5 °C to allow expression of the mutant phenotype. The expression period was followed by a selection period, in which the cells were incubated in selection medium containing trifluorothymidine (TFT) for 10 – 15 days.
In both experiments, there was no cytotoxicity up to and including the highest concentration of 800 µg/mL, neither with nor without metabolic activation. However, precipitation of the test substance in the culture medium was noted following 4 and 24 h of incubation at 800 µg/mL.
The mutation frequency of the solvent control was within the range of historical control data. The positive control substances CPA and MMS markedly increased the mutation frequency in L5178Y cells, thus demonstrating the sensitivity and validity of the test system. N,N-Bis-(trimethylsilyl)urea did not induce any statistically significant or biologically relevant increase in the mean number of mutant colonies in any of the tested concentrations, neither with nor without S9 mix. No significant dose-dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in both experiments.
Under the conditions of the test, N,N-Bis-(trimethylsilyl)urea does not induce forward mutations in the mouse lymphoma assay with L5178Y cells in the absence or presence of metabolic activation and is therefore considered to be not mutagenic in mammalian cells in vitro.
Conclusion:
In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that N,N-Bis-(trimethylsilyl)urea is neither mutagenic nor clastogenic in vitro.
Justification for classification or non-classification
The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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.
