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Genetic toxicity: in vitro

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in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
adapted for nanomaterials

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016, specific adaptions for nanomaterials as of - NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018
GLP compliance:
yes (incl. QA statement)
Issued by Landesamt für Umwelt, Rheinland-Pfalz, Germany,
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
EC Number:
EC Name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
Cas Number:
Molecular formula:
tetramethyl 2,2'-{1,4-phenylenebis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}diterephthalate
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation:
- Particle size distribution:

- Shape of particles:
- Surface area of particles: BET = 46.3 m2/g
- Crystal structure: crystalline
- Coating: none
- Batch: 13007010
- solid yellow
Specific details on test material used for the study:
yellow solid
batch 13007010
purity 95.7%
storage at room temperature


Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For lymphocytes:
- 31 year old male donor, non-smoking
- Buffy coat cells were isolated from whole blood
- Whether blood from different donors were pooled or not: not applicable; single donor
- Mitogen used for lymphocytes:Cytochalasin B
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
Based on the solubility properties of the test substance 256 µg/mLwas used as top concentration. Higher concentrations led to inhomogeneous formulations.
1, 3, 10, 30, 60, 100, 256 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 is 10% (v/v).
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
mitomycin C
other: 100, 60 and 30 µg/mL Tungsten Carbide-Cobalt (Nanostructures and Amorphous Material Inc.; Houston, Texas)
Details on test system and experimental conditions:
- Number of cultures per concentration: duplicate
- Number of independent experiments : At least 2 cultures were prepared per test group (referred to as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

- Test substance added in medium;

- Exposure duration/duration of treatment: 20h
- Harvest time after the end of treatment (sampling/recovery times): see treamtent with cyokinesis blocking substance


- Identity of cytokinesis blocking substance: cytoB, 20h exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells are transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells are centrifuged (900 g, 5 min) and suspended in 0.65% KCl (37°C), incubation for 10 minutes at 37°C. After the hypotonic treatment, the cells are fixed by adding of fixative (19 parts methanol and 1 part acetic acid). The cells are centrifuged (900 g, 5 min, 4°C) and fixed suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step is repeated twice. After the last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides are dipped in deionized water, the cells are pipetted on the slide and fixed by passing through a flame. The cells are stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 20 min).

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): criteria of Countryman and Heddle.
- The diameter of the micronucleus is less than 1/3 of the main nucleus
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells will be scored.

- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: 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

The slides were scored microscopically for micronuclei.

The test substance was weighed, pre-wetted with 0.5 vol% 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.
One stock dispersion was prepared (2.56 mg/mL). 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 further concentrations were serially diluted from the stock solution with 0.05% w/v BSA in water to a 10 times higher concentration of the planned doses. Then the test substance formulations were diluted 1:10 in culture medium according to the planned doses. All test substance formulations were prepared immediately before administration.
Rationale for test conditions:
Pre-experiments were performed to characterize the concentration depandent aggllomeration of particles.
Evaluation 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 is obtained.
• A dose-related increase in the number of cells containing micronuclei is observed.
• The number of micronucleated cells exceeds 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 is observed under any experimental condition.
• The number of micronucleated cells 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).

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
not applicable
Cytotoxicity / choice of top concentrations:
other: no cytotoxicity, but tested up the dispersibility limit
poorly soluble nanoparticle
Vehicle controls validity:
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Data on pH: At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each. No influence was observed.
- Data on osmolality: not determined
- Possibility of evaporation from medium: none
- Water solubility: Insoluble; see adaption for pooly soluble nanoparticles

Fractionating techniques with selective detection (AUC, UVVis) were used to characterise the test item preparations in the cell culture medium. Compared to the size of the constituent particles determined independently by TEM, the particles were successfully dispersed into a stable suspension with partial agglomeration that did not change significantly during the genotoxicity testing. The percentiles of the size distribution (D10, D50, D90) did show a trend with dose. The dissolved content at the end of the incubation time of 20h was around 0.05%.

- Concurrent vehicle negative and positive control data are shown in table 1
- Cytotoxicity and number of micronucleated cells are shown in table 1
- Historical control data are provided in the attachment.

Any other information on results incl. tables

Table 1: Summary of results

Test groups
index cytostasis
vehicle (0.05% BSA-water (w/v)) 0.9 0.0
1 n.d. 5.3
3 n.d. 3.6
10 0.5 2.4
30 0.7 9.5
60 n.d. 3.4
100 0.5 6.9
256 0.9 9.4
WC-Co 30 1.1 49.7
WC-Co 60 1.2 59.5
WC-Co 100 n.d. 74.0
Positive control (MMC 0.04 μg/mL) 10.3S 18.1
Positive control (Col 0.05 μg/mL) 3.0S 19.8

* Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S Frequency statistically significantly higher than corresponding control values

The increase in the frequencies of micronuclei induced by the positive control substances MMC and Colchicine demonstrated the sensitivity of the test system. The values were

compatible to the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. The nanomaterial positive control also induced an

increase in the micronucleus frequency, which was in the range of the historical control data, nevertheless the values were not statistically significant. This was due to the

high vehicle control value, which was the same as the upper 95% control limit value. This does not affect the validity of the study.

Applicant's summary and conclusion