Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From JUN 16 2011 to 26 JUL 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD guideline 473, and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital / beta-naphtoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0

Without metabolic activation:
Experiment I: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0
Experiment II: 0.2, 0.7, 2.1, 6.2, 18.5, 55.6, 166.7, 500.0
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


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


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: at least 100 per culture, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:
Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) and relative cell numbers were determined.

In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases)
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The highest treatment concentration in this study, 500.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 15.6 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In Experiment II precipitation was observed at 6.2 µg/mL and above in the absence of S9 mix and at 12.5 µg/mL and above in the presence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH value was observed. The osmolarity and pH-value were determined in the solvent control and the maximum concentration of Experiment I and II without metabolic activation:

Exp. Solvent control Test item 500.0 µg/mL
I Osmolarity (mOsm) 380 390
pH-value 7.3 7.3
II Osmolarity (mOsm) 395 392
pH-value 7.5 7.4

No cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after treatment with the test item .
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.3 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.3 - 3.9 %) as compared to the rates of the solvent controls (1.3 - 3.6 %).
In both experiments, either EMS (600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating concentrations.
Remarks on result:
other: strain/cell type: V79 cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results of the chromosome aberration study with the test substance

Exp.

Preparation

Test item

Endomitotic

Polyploid

Cell numbers

Mitotic indices

Aberrant cells

 

interval

concentration

cells

cells

in %

in %

in %

 

 

in µg/mL

in %

in %

of control

of control

incl. gaps*

excl. gaps*

with exchanges

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

0.0

3.6

100.0

100.0

1.0

1.0

0.0

 

 

Positive control2#

n.d.

n.d.

n.d.

91.0

33.0

33.0S

20.0

 

 

3.9

0.0

3.4

82.0

95.3

0.5

0.5

0.5

 

 

7.8

0.0

3.0

115.6

100.0

0.5

0.5

0.5

 

 

15.6P

0.0

2.7

92.7

111.4

1.5

1.0

0.0

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

0.0

2.4

100.0

100.0

3.0

2.0

0.0

 

 

Positive control3

n.d.

n.d.

n.d.

71.2

18.0

17.5S

10.0

 

 

0.7

0.0

2.7

106.6

101.8

1.0

1.0

0.0

 

 

2.1

0.0

1.9

76.4

119.6

1.0

1.0

0.0

 

 

6.2P

0.0

1.9

75.7

83.2

2.5

2.0

0.0

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

0.1

3.6

100.0

100.0

1.5

1.0

0.5

 

 

Positive control4

n.d.

n.d.

n.d.

87.0

14.0

13.0S

4.5

 

 

3.9

0.1

2.4

79.3

115.1

1.5

1.5

0.0

 

 

7.8

0.1

3.6

78.9

92.5

3.0

2.5

0.5

 

 

15.6P

0.3

3.9

113.1

102.5

2.5

2.0

0.5

II

18 hrs

Solvent control1

0.0

2.3

100

100

2.5

2.5

0.0

 

 

Positive control4

n.d.

n.d.

n.d.

71.4

16.0

14.5S

7.0

 

 

3.1##

0.0

1.9

111.9

78.3

4.0

3.3

1.3

 

 

6.3##

0.0

2.0

90.5

99.2

3.0

3.0

0.8

 

 

12.5P

0.0

1.3

76.8

112.0

2.5

2.5

2.5

*     Inclusive cells carrying exchanges

#     Evaluation of 50 metaphases per culture

##    Evaluation of 200 metaphases per culture

n.d. Not determined

P     Precipitation occurred at the end of treatment

S     Aberration frequency statistically significant higher than corresponding control values

1     DMSO    0.5 % (v/v)

2           EMS  1000.0 µg/mL

3           EMS   600.0 µg/mL

4           CPA       1.4 µg/mL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I

Exp. II

Exposure period

4 hrs

18 hrs

4 hrs

4 hrs

Recovery

14 hrs

-

14 hrs

14 hrs

Preparation interval

18 hrs

18 hrs

18 hrs

18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (500.0 µg/mL, approx. 1.4 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The evaluated experimental points and the results are summarised in Table1.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred.

No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

No relevant increase in endomitotic cells was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.