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EC number: 226-970-7 | CAS number: 5580-57-4
- 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
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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:
- 3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]
- EC Number:
- 226-970-7
- EC Name:
- 3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]
- Cas Number:
- 5580-57-4
- Molecular formula:
- C43H35Cl5N8O6
- IUPAC Name:
- 3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis[4-chloro-N-(3-chloro-2-methylphenyl)benzamide]
- Test material form:
- solid: nanoform
- Details on test material:
- Physical state/ appearance: yellow powder
Constituent 1
- Specific details on test material used for the study:
- Batch no.: 0004460012
Purity: 99.3 wt%
Date of production: 18 Jan 2011
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: ambient (RT)
Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations
Storage stability: The stability of the test substance was guaranteed until 18 Jan 2031 as indicated by the sponsor, and the sponsor holds this responsibility.
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.
- Cytokinesis block (if used):
- Cytochalasin B (6 µg/mL)
- Metabolic activation:
- without
- 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 were obtained in concentrations of 5.0, 10.0 and 20.0 mg/mL. The highest concentration, which could be homogenously formulated in 0.05% w/v BSA-water was 2.56 mg/mL corresponding to 256 µg/mL in culture medium (1:10 dilution). - 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 (nanomaterial positive control)
- Details on test system and experimental conditions:
- TIME SCHEDULE:
Day 1: Activation of the cells with Phytohemagglutinin
Day 3: Test substance incubation (approx. 48 hours after activation)
Day 4: Removal of test substance by intense washing; treatment with Cyt B
Day 5: Preparation of the slides
Since a pulse treatment (as described in OECD 487) does not allow enough time for the nanoparticles to enter the cell, only continuous treatment protocol was used.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate (two flasks with two slides prepared for each)
- Number of independent experiments: one
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h (stimulating with phytohemagglutinin)
- Exposure duration/duration of treatment: 20 h
- Harvest time after the end of treatment: 20 h
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
cytokinesis blocking: 6 µg/mL Cytochalasin B (Cyt B), 20 h
- Methods of slide preparation and staining technique: The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step was repeated twice. After the last fixation step, the cells were centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted.
- Number of cells spread and analysed per concentration: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (1976).
- 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.
Slides were coded randomly before microscopic analysis with an appropriate computer program. Cultures with few isolated cells were analyzed for micronuclei.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in 500 cells per culture (1000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerization inhibitor Cyt B.
CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / (Total number of cells)
The CBPI was used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) - a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
% Cytostasis = 100 - 100 {(CBPIT - 1) / (CBPIC - 1)}
T = test substance treated culture
C = vehicle control culture
pH value:
At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each. - 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:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- 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: 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 (if applicable):
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
In the Main experiment all evaluated values of the test substance (0.3 – 0.7% micronucleated cells) were within the range of the 95% control limit of the historical negative control data (0.2 – 0.9% micronucleated cells). A statistical significance compared to the concurrent vehicle control value (0.7% micronucleated cells) or dose dependency was not observed.
The positive control substances MMC (0.04 μg/mL) and Colchicine (0.05 μg/mL) induced statistically significantly increased micronucleus frequencies. In this study, the frequencies of micronucleated cells (7.9% and 3.7% micronucleated cells (MMC and Col, respectively) were compatible to the historical positive control data range.
The particular positive control substance Tungsten Carbide-Cobalt (WC-Co) induced an increased micronucleus frequency at 60 μg/mL (1.2%) and exceeded the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells). This increase was, however, not statistically significant.
Results from cytotoxicity measurements: 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)
See: Any other information (Tables 5 and 6)
Any other information on results incl. tables
Table 1: Summary Table:
Exp. | Exposure/ Recovery/ Preparation interval [h] |
Test groups [µg/mL] |
Micro- nucleated cells [%] |
Cytotoxicity Proliferation index cytostasis [%] |
1 | 20/0/20 | Vehicle control1 | 0.7 | 0.0 |
|
| 1.0 | n.d. | 1.6 |
|
| 3.0 | n.d. | -1.3 |
|
| 10.0 | 0.7 | -11.7 |
|
| 30.0 | 0.3 | -1.6 |
|
| 60.0 | n.d. | -1.1 |
|
| 100.0 | 0.3 | -7.1 |
|
| 256.0 | 0.3 | -4.2 |
|
| WC-Co 30 | 0.7 | 51.8 |
|
| WC-Co 60 | 1.2 | 58.5 |
|
| WC-Co 100 | n.d. | 65.2 |
|
| Positive control2 | 7.9s | 6.5 |
|
| Positive control3 | 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
n.s. Not scorable due to strong cytotoxicity
1 0.05% BSA-water (w/v) 2 MMC 0.04 µg/mL 3 Col 0.05 µg/mL
Table 2: Proliferation index (CBPI) - Main experiment
Test group [µg/mL] | Culture | Mononucleated cells | Binucleated cells | Multinucleated cells | CBPI absolute | CBPI cyto- stasis [%] |
Vehicle control* | A | 90 | 399 | 11 | 1.79 | 0.0 |
B | 153 | 327 | 20 | |||
1.0 | A | 110 | 385 | 5 | 1.78 | 1.6 |
B | 141 | 338 | 21 | |||
3.0 | A | 141 | 341 | 18 | 1.80 | -1.3 |
B | 114 | 351 | 35 | |||
10.0 | A | 134 | 354 | 12 | 1.88 | -11.7 |
B | 87 | 324 | 89 | |||
30.0 | A | 135 | 357 | 8 | 1.80 | -1.6 |
B | 101 | 370 | 29 | |||
60.0 | A | 115 | 380 | 5 | 1.80 | -1.1 |
B | 115 | 363 | 22 | |||
100.0 | A | 112 | 373 | 15 | 1.84 | -7.1 |
B | 97 | 365 | 38 | |||
256.0 | A | 117 | 368 | 15 | 1.82 | -4.2 |
B | 102 | 373 | 25 | |||
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 |
* 0.05% w/v BSA-water
Table 3: Analysis of micronuclei - Main experiment
Test group [µg/mL] | Culture | No. of evaluated cells | Cells containing Micronuclei | ||
[n] | [n] | [%] | |||
Vehicle control* | A | 1000 | 7 | 14 | 0.7 |
B | 1000 | 7 | |||
1.0 | A |
n.d. | |||
B | |||||
3.0 | A | ||||
B | |||||
10.0 | A | 1000 | 6 | 13 | 0.7 |
B | 1000 | 7 | |||
30.0 | A | 1000 | 4 | 6 | 0.3 |
B | 1000 | 2 | |||
60.0 | A | n.d. | |||
B | |||||
100.0 | A | 1000 | 5 | 6 | 0.3 |
B | 1000 | 1 | |||
256.0 | A | 1000 | 2 | 6 | 0.3 |
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 |
* 0.05% w/v BSA-water
S Frequency statistically significantly higher than corresponding control values
Table 4: Linear trend test
Linear trend-test | Slope | One-sided p-value* |
Main experiment | -0.00124 | 0.94854 |
* The linear trend-test testing for an increased number of micronucleated cells is significant (significance level of 5%), if the one-sided p-value is lower than 0.05.
Table 5: Hitorcial negative control data (2018 - 2020)
Micronucleated cells [%] | |
Exposure period | 20 hrs |
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 |
Table 6: Historical positice control data (2018-2020)
Micronucleated cells [%] | ||
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 |
Applicant's summary and conclusion
- Conclusions:
- Thus, under the experimental conditions chosen here, the conclusion is drawn that the test item has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence of metabolic activation.
- Executive summary:
The test substance was tested for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity) in this study according OECD 487 and in complince with GLP. One experiment was carried out, incubating the cells for 20 h (20 h harvest time) with the test substance at concentrations in the range of 1.0 to 256 µg/mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, 0.05% w/v BSA-water was selected as vehicle. The characterization of the nanomaterial in cell culture medium showed, that the particles were successfully dispersed into a stable suspension with partial agglomeration, that did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine (Col) and the nanomaterial positive control Tungsten Carbide-Cobalt (WC-Co), led to the expected increase in the number of cells containing micronuclei.
The test substance was formulated in the given vehicle according to the NANOGENOTOXProject (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018.
In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant in the number of cells containing micronuclei.
Thus, under the experimental conditions described, test material is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.
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