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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21st of July, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BSL Bioservice Scientific Laboratiories GmbH, Behringstr. 6/8, 82152 Planegg, Germany
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
Expiration Date: October 27, 2019
Physical state, appearance: Solid, yellow
Molecular weight: 916.65 g/mol
Storage conditions: Room temperature
Batch: 4038273

TEM: mostly rods with aspect ratios around 5, diameters from 30 nm to 150 nm
BET (SSA) 57 m2/g

Specific details on test material used for the study:
- Lot/batch No.: 0004038273
- Expiration date of the lot/batch: 27 Oct. 2019
- Storage condition of test material: at room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum essential medium (MEM) supplemented with 10% fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with penobarbital and b-naphthoflavone
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL

Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation: 10, 400, 750 and 900 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
The test item was prepared in cell culture medium (MEM) followed by ultrasound for 5 – 8 minutes (immediately before treatment). After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
other: no solvent/vehicle used
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index in 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

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:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item was suspended in cell culture medium. Consequently, precipitate of the test itern was observed in both experiments (see Tables 1 and 2). Precipitation was assessed after incubation with the test item by unaided eye.

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 250 µg/mL (42%, Table 1). The cell density was decreased at 125 µg/mL and 250 µg/mL down to 64% and 54%, respectively. With metabolic activation a biologically relevant decrease of the relative mitotic index was noted at 500 µg/mL and higher (54% at 500 µg/mL and 58% at 1000 µg/mL, Table 2). The cell density was decreased at 1000 µg/mL down to 54%.
In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index was observed at 500 µg/mL (48%, Table 1). The cell density was not decreased. With metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at 400 µg/mL and higher (49% at 400 µg/mL, 45% at 750 µg/mL and 43% at 900 µg/mL, Table 2). No decrease of the cell density was noted up to the highest concentration evaluated.

Polyploid cells: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Any other information on results incl. tables

Table 1: Experiment I and II, without metabolic activation

 

Dose Group

Concentration [µg/mL]

Relative Mitotic index [%]

Cell Density [%]

Mean % Aberrant Cells

Laboratory Negative Control Range

Precipitation

Incl. Gaps

Excl. Gaps

yes

no

Experiment I 4h treatment, 20h preparation interval

C

0

100

100

1.5

1.0

0.0% – 4.0% aberrant cells

 

X

1

0.975

94

100

2.0

1.0

 

X

4

7.8

81

99

3.5

2.5

X

 

7

62.5

76

83

1.5

1.5

X

 

8

125

76

64

0.5

0.5

X

 

9

250

42

54

0.5

0.5

X

 

EMS

900

97

101

10.5

8.0

 

X

 

Experiment II 20h treatment, 20h preparation interval

C

0

100

100

2.5

1.0

0.0% – 4.0% aberrant cells

 

X

6

62.5

86

99

2.5

0.0

 

X

7

125

95

100

1.0

0.5

X

 

8

250

74

78

1.5

0.5

X

 

9

500

48

71

1.5

0.5

X

 

EMS

400

79

90

12.5

10.5

 

 

 

C: negative control (culture medium)

EMS: Ethylmethanesulfonate

Table 2: Experiment I and II, with metabolic activation

 

Dose Group

Concentration [µg/mL]

Relative Mitotic index [%]

Cell Density [%]

Mean % Aberrant Cells

Laboratory Negative Control Range

Precipitation

Incl. Gaps

Excl. Gaps

yes

no

Experiment I 4h treatment, 20h preparation interval

C

0

100

100

0.5

0.0

0.0% – 4.0% aberrant cells

 

X

4

7.8

109

99

2.5

0.5

 

X

9

250

75

80

2.0

1.5

X

 

10

500

54

72

3.0

2.0

X

 

11

1000

58

54

3.0

1.0

X

 

CPA

0.83

70

99

10.5

9.0

 

X

 

Experiment II 4h treatment, 20h preparation interval

C

0

100

100

4.0

2.5

0.0% – 4.0% aberrant cells

 

X

1

10

93

97

3.0

2.5

 

X

5

400

49

83

2.5

2.0

X

 

7

750

45

89

3.5

1.5

X

 

8

900

43

80

1.0

0.5

X

 

CPA

0.83

99

100

12.0

10.5

 

X

 

C: negative control (culture medium)

CPA: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
negative