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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

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 Jul 2016
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)
The particle characterization in cell culture medium was not performed under GLP.
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: nanoform
Specific details on test material used for the study:
Batch identification: 0004501436
Content: 99.4 %
physical state: solid; brown
storage at room temperature


Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- 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).
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
mitomycin C
other: Tungsten Carbide-Cobalt
WC was used as a particle control for nanomaterials
Details on test system and experimental conditions:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

- Test substance added in medium

- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

- 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:

- 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).


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).

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
Cytotoxicity / choice of top concentrations:
other: homogeneous dispersion of nanomaterial
Vehicle controls validity:
not applicable
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
- 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.

- 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

Preparation interval
Test groups
nucleated cells**
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
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.
3.0 A
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.
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.
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
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