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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 701-372-3 | CAS number: -
- 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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Jan 2021 - July 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
- Version / remarks:
- 29 Jul 2016
- Deviations:
- yes
- Remarks:
- Additional investigations for nanomaterials included
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- certified by Landesamt für Umwelt Rheinland-Pfalz, Germany
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Test material form:
- solid: nanoform, no surface treatment
- Details on test material:
- Particle size distribution: D50 = 28.9 nm (TEM)
- Shape of particles: elongated, rod
- Surface area of particles: BET = 100.3
- Crystal structure: crystalline
- Coating: none
- Density: Rel density = 1.442; Bulk density 125 kg/m3
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 14 Feb 2023
- Purity: 98.5%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Due to the use of culture medium (HAM´s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance preparation was performed in accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June, 2018.
The test substance was weighed, pre-wetted with 0.5vol% ethanol (pre-wetting is introduced to enable dispersion of hydrophobic materials in water-based systems) and topped up with the vehicle 0.05% w/v BSA-water to achieve the required concentration of the stock dispersions. Two stock dispersions were prepared (2.56 mg/mL and 2.0 mg/mL).
The stock dispersion of 2.0 mg/mL was handled separately. For further dilutions only the stock dispersion of 2.56 mg/mL were used.
A homogeneous test substance preparation in the vehicle was prepared by using a Branson Sonifier S-550D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a standard 13 mm disruptor horn.
The test substance formulations was diluted according to the planned doses.
All test substance formulations were prepared immediately before administration.
To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before the removal.
FORM AS APPLIED IN THE TEST: Homogeneous dispersion
OTHER SPECIFICS
- physical state, appearance: Solid, blue
- molecular weight: 515 g/mol
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type of cells: CHO (Chinese hamster ovary) cell line
- Source: cell stock of testing facility
- Suitability of cells: yes, as recommended in OECD TG 476
- Normal cell cycle time (negative control): not specified
For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 3
- Methods for maintenance in cell culture: Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS. Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter. Cell suspensions were diluted with complete culture medium to the desired cell count.
- Doubling time: of about 12 - 16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL); 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity
- Metabolic activation:
- with and without
- Metabolic activation system:
- - type and composition of metabolic activation system: exogenous metabolic activation by cofactor-supplemented postmitochondrial fraction (S9 mix)
- source of S9: liver S9 mix from phenobarbital- and β-naphthoflavone induced rats
- method of preparation of S9 mix: the S9 mix was prepared freshly prior to each experiment. 1 part S9 fraction was mixed with 9 parts S9 supplement (consisting of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 8 mL S9 mix and 32 mL test substance preparation (final volume 40 mL) - Test concentrations with justification for top dose:
- 1
st Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL
2
nd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 µg/mL
Dose selection for genotoxicity testing was based on the SOP for Preparing Batch Dispersions
for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement
No 2009 21 01); Version 1.2, dated 12 June 2018. Furthermore, to fulfill the requirements of
the OECD Guidelines for the HPRT assay, the top concentrations in both main Experiments
were defined as followed. 0.05% w/v bovine serum albumin water (BSA-water) was used as
vehicle.
2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid.
1000.0 µg/mL was used as top concentration in the second repeat experiment which was a homogeneous suspension when applied to the test culture. - Vehicle / solvent:
- - Vehicle used: 0.05% w/v bovine serum albumin water (BSA-water)
- Justification for choice of solvent/vehicle: In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018, 0.05% w/v bovine serum albumin water (BSA-water) is used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium is be 10% (v/v).
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- - DMBA with metabolic activation (1.25 μg/mL) - EMS without metabolic activation (400 μg/mL)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 20x10^6 cells in 40 mL medium/flask
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20-24 hours
- Exposure duration/duration of treatment: 4 hours
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time: 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-9 days (cloning efficiency 1); 16 days (cloning efficiency 2)
- Method used: colonies were fixed with methanol and stained with Giemsa
- Selective agent: 6-thioguanine; 10 μg/mL; 6-7 days exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^6 cells from every treatment group were seeded in 20 mL selection medium (175 cm^2 flasks) and the remaining colonies were counted at the end of the selection period
- Criteria for small (slow growing) and large (fast growing) colonies: not applicable
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cloning efficiency
METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Mutant frequency
- OTHER: Check or determination of further parameters:
- pH, osmolality, solubility, cell morphology
-Analytical Ultracentrifugation (AUC) method to determine the agglomeration in genotoxicity test medium.
- Incubation and filtration with analysis by Inductively-Coupled-Plasma Mass Spectrometry (ICPMS) or by UVVis-Spectrometry, to determine the static solubility in genotoxicity test medium. - Rationale for test conditions:
- In the pre-test for toxicity based on the SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018, 2.56 mg/mL 29H,31H-phthalocyanine was used as stock dispersion. The highest tested concentration was 100.0 μg/mL both with and without S9 mix at 4 hour exposure time.
The pre-test was performed following the method described for the main experiment. The Relative Survival (RS) was determined as a toxicity indicator for dose selection.
In the pre-test for dose selection the pH, osmolality and solubility were additionally determined for all or at least some selected doses.
In the pre-test the pH value was not relevantly altered by the test substance. The osmolality was not relevantly influenced by the addition of the test substance preparation to the culture
medium at the concentrations measured. The highest applied concentration of 0.1 mg/mL (Test group: 100 μg/mL) was a homogeneous
dispersion. Test substance dispersions were obtained from 0.025 mg/mL (Test group:25 μg/mL) onward.
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20%.
The test substance is a nanoparticle. Therefore, some considerations need to be taken for the performance of this assay in order to adhere to the current version of the OECD guideline.
The substance is tested at precipitating levels, the highest concentration is based on homogeneity of the formulation. - Evaluation criteria:
- A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit). - Statistics:
- A linear dose-response is evaluated by testing for linear trend. The dependent variable is the corrected mutant frequency and the independent variable is the dose. The calculation is performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on genotoxicity Test procedures Workgroup Report.
A pair-wise comparison of each test group with the control group is carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation is performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Osmolality and pH values were not relevantly influenced by test substance treatment.
In the 1st Experiment in the absence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 25.00 μg/mL and above. In the presence of S9 mix
test substance precipitation was observed from 1.00 μg/mL onward. In the 2nd Experiment in the presence and absence of S9 mix test substance precipitation occurred from 10.00 μg/mL
onward.
After 4 hours treatment neither in the absence nor presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test
group tested for gene mutations.
The results of the characterization confirmed that the study was performed with the compound as nanoparticles. Since the test substance is an insoluble nanoparticle, the visual assessment of concentration of test substance precipitation is misleading. As described in the characterization report the test substance did not dissolve within the tested period. Thus, the documentation of test substance precipitation merely shows that the particulate matter could be observed visually and does not mean that the test substance at lower concentrations were dissolved.
Any other information on results incl. tables
Table 3: Summary of results – experimental parts without S9 mix
Exp. | period Test groups [h] [µg/mL] |
S9 mix |
Prec.* | MFcorr. [per 106 cells] |
RS [%] | CE2 [%] | |
1 | 4 | Vehicle control (BSA-water 0.05% (v/v)) | - | n.d. | 0.00 | 100.0 | 100.0 |
0.125 | - | - | 2.69s | 124.6 | 108.1 | ||
0.25 | - | - | 3.05s | 129.3 | 104.9 | ||
0.50 | - | - | 1.29 | 110.6 | 89.8 | ||
1.00 | - | - | 1.82 | 118.8 | 95.9 | ||
10.00 | - | - | 0.00 | 110.6 | 86.6 | ||
25.00 | - | + | 1.26 | 125.5 | 92.2 | ||
50.00 | - | + | 3.36s | 127.3 | 86.6 | ||
100.00 | - | + | 1.25 | 130.0 | 93.0 | ||
Positive control (EMS 400 μg/mL) | - | n.d | 110.04s | 87.8 | 72.4 | ||
2 | 4 | Vehicle control (BSA-water 0.05% (v/v)) | - | n.d. | 2.27 | 100.0 | 100.0 |
0.125 | - | - | 0.00 | 128.8 | 82.2 | ||
0.250 | - | - | 1.40 | 113.4 | 81.0 | ||
0.500 | - | - | 1.36 | 112.1 | 83.6 | ||
1.000 | - | - | 0.34 | 122.3 | 83.0 | ||
10.000 | - | + | 2.01 | 121.1 | 98.6 | ||
25.000 | - | + | 0.00 | 100.1 | 88.4 | ||
50.000 | - | + | 3.09 | 101.7 | 82.4 | ||
100.000 | - | + | 1.62 | 103.5 | 87.3 | ||
1.000.000 | - | + | 1.99 | 81.8 | 85.3 | ||
Positive control (EMS 400 μg/mL) | - | n.d. | 118.18s | 77.1 | 77.9 |
* Macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)
Table 4: Summary of results – experimental parts with S9 mix
Exp. | Exposure period [h] |
Test groups [µg/mL] | S9 mix |
Prec.* | Genotoxicity** MFcorr. [per 106 cells] |
Cytotoxicity*** | |
RS [%] |
CE2 [%] |
||||||
1 | 4 | Vehicle control (BSA-water 0.05% (v/v)) | + | n.d. | 3.41 | 100.0 | 100.0 |
0.125 | + | - | 0.00 | 129.5 | 94.4 | ||
0.25 | + | - | 1.56 | 122.1 | 99.1 | ||
0.50 | + | - | 0.00 | 128.0 | 91.0 | ||
1.00 | + | + | 0.69 | 118.8 | 89.8 | ||
10.00 | + | + | 0.82 | 125.9 | 75.9 | ||
25.00 | + | + | 1.24 | 100.2 | 74.6 | ||
50.00 | + | + | 2.60 | 97.9 | 71.5 | ||
100.00 | + | + | 0.87 | 95.3 | 71.5 | ||
Positive control (DMBA 1.25 μg/mL) | + | n.d. | 78.87s | 81.5 | 65.9 | ||
2 | 4 | Vehicle control (BSA-water 0.05% (v/v)) | + | n.d. | 5.96 | 100.0 | 100.0 |
0.125 | + | - | 1.45 | 155.9 | 96.8 | ||
0.25 | + | - | 2.45 | 105.4 | 100.4 | ||
0.50 | + | - | 6.41 | 113.5 | 109.5 | ||
1.00 | + | - | 1.61 | 102.3 | 87.4 | ||
10.00 | + | + | 4.15 | 106.6 | 93.0 | ||
25.00 | + | + | 1.79 | 100.4 | 98.2 | ||
50.00 | + | + | 3.08 | 94.0 | 91.2 | ||
100.00 | + | + | 1.15 | 115.9 | 91.2 | ||
1000.00 | + | + | 5.11 | 101.9 | 96.1 | ||
Positive control (DMBA 1.25 μg/mL) | + | n.d. | 114.75s | 80.1 | 76.1 |
* Macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significantly higher than corresponding control values
Particle Characterization in cell culture medium
The size distribution is polydisperse, ranging from below 50 nm to 1000 nm. Due to the selective tracking of pigment particles by the UVVis optics the colloidal components of the cell culture
medium (proteins etc) do not interfere with the analysis.
Across all doses tested (10 - 100 mg/L), the median diameter D50 remained constant. The D90 percentile, representating the largest agglomerates, shows a minimal trend towards larger agglomerates at higher doses (Table 5).
The re-characterisation after 20h finds no change. The size distributions overlap, and consequently also the percentile diameters shows that the particles have not significantly changed their state of agglomeration.
The dissolved content at the end of the incubation time of 20h is below the limit of quantification (below 0.05% of 0.01 mg/mL).
Table 5: Percentile diameters of the measured particle size distributions in cell culture medium
t = 0h | t = 20h | |||
Dose | c=0.01 mg/ml | c=0.03 mg/ml | c=0.10 mg/ml | c=0.10 mg/ml |
D10 [nm] | 31 | 39 | 41 | 41 |
D50 [nm] | 65 | 69 | 70 | 64 |
D90 [nm] | 110 | 118 | 134 | 90 |
(d90-d10)/d50 | 1.21 | 1.15 | 1.33 | 0.76 |
Applicant's summary and conclusion
- Conclusions:
- Pigment Blue 16 was not mutagenic in the HPRT test.
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.
Sebbene gran parte del materiale online fornito dall'ECHA sia accessibile nella tua lingua, alcuni contenuti di questa pagina sono disponibili solo in inglese. Maggiori informazioni sulla prassi dell'ECHA in materia di multilinguismo.
Benvenuti al sito dell'ECHA. Non tutte le funzionalità del presente sito sono fruibili con Internet Explorer 7 (e versioni precedenti). Aggiornare Internet Explorer a una versione più recente.
Questo sito web si avvale di cookie affinché possiate usufruire della migliore esperienza sui nostri siti web.
Per saperne di più su come utilizziamo i cookie.