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EC number: 252-772-5 | CAS number: 35869-64-8
- 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 cytogenicity / micronucleus study
- Remarks:
- adapted for nanomaterials
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 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 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 29 Jul 2016
- Deviations:
- yes
- Remarks:
- additional characterization of particles in cell culture medium; no 4h exposure (too short for insoluble particles)
- Principles of method if other than guideline:
- Adapations (test material preparation, particle characterization) NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The particle characterization in cell culture medium was not performed under GLP.
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
- EC Number:
- 252-772-5
- EC Name:
- N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
- Cas Number:
- 35869-64-8
- Molecular formula:
- C40H23Cl3N8O8
- IUPAC Name:
- N,N'-(2-chloro-1,4-phenylene)bis{4-[(4-chloro-2-nitrophenyl)diazenyl]-3-hydroxy-2-naphthamide}
- Test material form:
- solid: nanoform
Constituent 1
- Specific details on test material used for the study:
- Batch identification: 0004501436
Content: 99.4 %
physical state: solid; brown
storage at room temperature
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: primary human lymphocytes (buffy coat cells) Fresh Blood was collected from a single donor for each experiment.
Only healthy, non-smoking donors and not receiving medication were used. In this study, in the main experiment a 28 year old male donor was used.
The lymphocytes of each donor have previously been shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.
- Suitability of cells:
- Normal cell cycle time (negative control):
- Cytokinesis block (if used):
- Cytochalasin B (6 µg/mL)
- Metabolic activation:
- not applicable
- Test concentrations with justification for top dose:
- The test material fulfills the criteria of an insoluble nanomaterial and shall be present in the cell culture medium as a homogenous stable nanoparticle dispersion.
In a pretest, inhomogeneous suspensions (aggregation) of the test substance in the vehicle 0.05% w/v BSA in water was tested. 1 mg/L was the highest concentration which remained in a homogenous state as determined by an
Analytical Ultracentrifugation (AUC) method.
1, 3, 10, 30, 60, 100, 1000 mg/L - Vehicle / solvent:
- 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 6 May 2018, 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium was 10% (v/v).
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- colchicine
- mitomycin C
- other: Tungsten Carbide-Cobalt
- Remarks:
- WC was used as a particle control for nanomaterials
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 per culture (2000 per concentration)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (14).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: In case of toxicity, the top concentration should produce 55 ± 5% cytotoxicity: reduction of the
proliferation index (CBPI) to 45 ± 5% of the concurrent vehicle control. The CBPI was determined in 500 cells per culture (1000 cells per test group).
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: - Evaluation criteria:
- Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged
acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
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 number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit). - Statistics:
- The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test).
In addition, a statistical trend test (SAS procedure REG (16)) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (17).
The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: homogeneous dispersion of nanomaterial
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: not determined
- Possibility of evaporation from medium: not applicable
- Water solubility: insoluble
- Precipitation and time of the determination: The test material was tested as a stable dispersion of particles in the nano-size range.
RANGE-FINDING/SCREENING STUDIES: Non-GLP experiments on the stability of the test material dispersion and the particle size distribution were carried out to determine the adequate concentrations.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : yes
Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o No cytotoxicity observed for the test material (CBPI)
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 4
- Negative (solvent/vehicle) historical control data: see table 5
Any other information on results incl. tables
Table 1: Summary of results
Exposure/Recovery/ Preparation interval |
Test groups [μg/mL] |
Micro- nucleated cells** [%] |
Cytotoxicity Proliferation index cytostasis [%] |
20/0/20 | Vehicle control (0.05% BSA-water (w/v)) | 0.7 | 0.0 |
1.0 | n.d. | -2.0 | |
3.0 | n.d. | -8.2 | |
10.0 | 0.8 | -0.9 | |
30.0 | 0.5 | 6.3 | |
60.0 | n.d. | -0.4 | |
100.0 | 0.5 | 2.4 | |
1000.0 | 0.5 | -3.9 | |
WC-Co 30 | 0.7 | 51.8 | |
WC-Co 60 | 1.2S | 58.5 | |
WC-Co 100 | n.d. | 65.2 | |
Positive control (MMC 0.04 μg/mL) | 7.9S | 6.5 | |
Positive control (Col 0.05 μg/mL) | 3.7S | 19.0 |
*Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
S Frequency statistically significantly higher than corresponding control values
n.d. Not determined
Table 2: Proliferation Index (CBPI)
Test group [µg/mL] | Culture | Mononucleated cells | Binucleated cells | Multinucleated cells | CBPI absolute |
CBPI cyto- stasis [%] |
BSA | A | 90 | 399 | 11 | 1.79 | 0.0 |
B | 153 | 327 | 20 | |||
1.0 | A | 113 | 371 | 16 | 1.80 | -2.0 |
B | 124 | 351 | 25 | |||
3.0 | A | 84 | 386 | 30 | 1.85 | -8.2 |
B | 114 | 365 | 21 | |||
10.0 | A | 102 | 375 | 23 | 1.80 | -0.9 |
B | 145 | 336 | 19 | |||
30.0 | A | 153 | 339 | 8 | 1.74 | 6.3 |
B | 126 | 365 | 9 | |||
60.0 | A | 114 | 371 | 15 | 1.79 | -0.4 |
B | 131 | 348 | 21 | |||
100.0 | A | 109 | 368 | 23 | 1.77 | 2.4 |
B | 153 | 339 | 8 | |||
1000.0 | A | 119 | 363 | 18 | 1.82 | -3.9 |
B | 98 | 384 | 18 | |||
WC-Co 30 | A | 348 | 142 | 10 | 1.38 | 51.8 |
B | 284 | 214 | 2 | |||
WC-Co 60 | A | 354 | 139 | 7 | 1.33 | 58.5 |
B | 326 | 174 | 0 | |||
WC-Co 100 | A | 362 | 130 | 8 | 1.27 | 65.2 |
B | 373 | 126 | 1 | |||
MMC 0.04 | A | 159 | 322 | 19 | 1.74 | 6.5 |
B | 141 | 341 | 18 | |||
Col 0.05 | A | 255 | 230 | 15 | 1.64 | 19.0 |
B | 134 | 354 | 12 |
Table 3: Analysis of micronuclei
Test group Culture [µg/mL] | No. of evaluated cells | Cells containing Micronuclei | |||
BSA | A | 1000 | 7 | 14 | 0.7 |
B | 1000 | 7 | |||
1.0 | A | n.d. | |||
B | |||||
3.0 | A | ||||
B | |||||
10.0 | A | 1000 | 9 | 15 | 0.8 |
B | 1000 | 6 | |||
30.0 | A | 1000 | 4 | 9 | 0.5 |
B | 1000 | 5 | |||
60.0 | A | n.d. | |||
B | |||||
100.0 | A | 1000 | 7 | 10 | 0.5 |
B | 1000 | 3 | |||
1000.0 | A | 1000 | 5 | 9 | 0.5 |
B | 1000 | 4 | |||
WC-Co 30 | A | 1000 | 5 | 14 | 0.7 |
B | 1000 | 9 | |||
WC-Co 60 | A | 1000 | 4 | 23 | 1.2 |
B | 1000 | 19 | |||
WC-Co 100 | A | n.d. | |||
B | |||||
MMC 0.04 | A | 1000 | 81 | 157 | 7.9S |
B | 1000 | 76 | |||
Col 0.05 | A | 1000 | 42 | 74 | 3.7S |
B | 1000 | 32 |
S Frequency statistically significantly higher than corresponding control values
Table 4 Historical control data (positive control)
Exposure period | 20 hrs | 20 hrs |
Substance and concentration |
MMC 0.04 µg/mL | Col 0.05 µg/mL |
Mean | 4.1 | 4.0 |
Minimum | 2.1 | 2.4 |
Maximum | 7.1 | 7.2 |
Standard Deviation | 0.93 | 1.05 |
No. of Experiments | 48 | 45 |
Table 5: historical control data (vehicle)
Micronucleated cells [%] | |
Exposure period of 20h | |
Mean | 0.5 |
Minimum | 0.2 |
Maximum | 1.2 |
Standard Deviation | 0.17 |
95% Lower Control Limit | 0.2 |
95% Upper Control Limit | 0.9 |
No. of Experiments | 54 |
Applicant's summary and conclusion
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