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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-09-26 to 2012-12-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study with GLP, RL1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Tellurium dioxide
- Substance type: inorganic

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
V79 cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 °C. The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix (Phenobarbital and ß-naphthoflavone induced).
Test concentrations with justification for top dose:
Assay 1:
3-hr treatment without S9-mix, harvest 20 hours from the beginning of treatment:
Tellurium dioxide: 200, 100, 75, 50, 25, 12.5, 6.25 and 3.125 µg/mL
3-hr treatment with S9-mix, harvest 20 hours from the beginning of treatment:
Tellurium dioxide: 200, 100, 75, 50, 25, 12.5, 6.25 and 3.125 µg/mL
Assay 2:
20-hr treatment without S9-mix, harvest 28 hours from the beginning of treatment:
Tellurium dioxide: 60, 40, 30, 20, 15, 10, 7.5, 5 and 2.5 µg/mL
3-hr treatment with S9-mix, harvest 28 hours from the beginning of treatment:
Tellurium dioxide: 200, 100, 75, 50, 25, 12.5, 6.25 and 3.125 µg/mL


Vehicle:
- Vehicle(s)/solvent(s) used: 1% methyl cellulose
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 and 28 hours


SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 µg/mL)
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF REPLICATIONS: Duplicate cultures for each concentration

NUMBER OF CELLS EVALUATED: 100 metaphases from each culture

DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer.
Results are expressed compared to the negative (solvent) control as % relative survival.

OTHER EXAMINATIONS:
- Determination of polyploidy: metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number
- Determination of endoreplication: metaphases have chromosomes with 4, 8, etc. chromatids




Evaluation criteria:
The assay is considered valid, if the following criteria are met:
- The negative (solvent) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:
For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more
chromosomal aberrations excluding gaps.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no large changes
- Effects of osmolality: no large changes
- Water solubility: substance is water soluble
- Precipitation: Assay 1: minimal amount of insolubility was detected at the end of the treatment period in the final treatment medium at 200 and 100 μg/mL concentrations, with and without S9 mix.
In Assay 2: no insolubility was detected at the end of the treatment period in the final treatment medium in the experiment without metabolic activation, but minimal amount of insolubility was detected at 200 and 100 μg/mL concentrations in the experiment with metabolic activation.


RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Assay 1:
Cytotoxicity was observed at 200, 100 and 75 μg/mL concentrations without metabolic activation (relative survival values were 5, 30 and 45 %, respectively); and at 200 and 100 μg/mL concentrations with metabolic activation (relative survival values were 11 and 30 %, respectively).
Concentrations of 75, 50 and 25 μg/mL were chosen for evaluation in case of the experiment without metabolic activation;
Concentrations of 100, 75 and 50 μg/mL were chosen for evaluation in case of the experiment with metabolic activation.


Assay 2,
Cytotoxicity was observed at 60, 40, 30, 20, 15 and 10 μg/mL concentrations without metabolic activation (relative survival values were 5, 6, 3, 25, 43 and 38 %, respectively); and at 200, 100 and 75 μg/mL concentrations with metabolic activation (relative survival values were 7, 19 and 37 %,
respectively)

Concentrations of 10, 7.5 and 5 μg/mL were chosen for evaluation in case of the experiment without metabolic activation
Concentrations of 75, 50, 25 and 12.5 μg/mL were chosen for evaluation in case of the experiment with metabolic activation.

Any other information on results incl. tables

  Summary table of Chromosome Aberration Assay 1 experiment without metabolic activation: 

Concentration (μg/mL)

Time of Treatment / Sampling

Relative Survival# (%)

Insolubility##

Mean % aberrant cells###

Tellurium dioxide without metabolic activation (-S9)

Untreated control

3h / 20h

124

2.5

Negative (vehicle) control

3h / 20h

100

3.0

200 μg/mL

3h / 20h

5

+a

NE

100 μg/mL

3h / 20h

30

+a

NE

75 μg/mL

3h / 20h

45

5.5

50 μg/mL

3h / 20h

62

3.5

25 μg/mL

3h / 20h

85

2.0

12.5 μg/mL

3h / 20h

113

NE

6.25 μg/mL

3h / 20h

117

NE

3.125 μg/mL

3h / 20h

113

NE

Positive control

3h / 20h

92

4.0

Negative (vehicle) control: 1% Methyl cellulose

Positive control (-S9): Ethyl methanesulfonate, 1μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate was observed

 

Summary table of Chromosome Aberration Assay 1 experiment with metabolic activation: 

Concentration (μg/mL)

Time of Treatment / Sampling

Relative Survival# (%)

Insolubility##

Mean % aberrant cells###

Tellurium dioxide with metabolic activation (+S9)

Untreated control

3h / 20h

90

4.0

Negative (vehicle) control

3h / 20h

100

3.0

200 μg/mL

3h / 20h

11

+a

NE

100 μg/mL

3h / 20h

30

+a

6.0

75 μg/mL

3h / 20h

58

4.5

50 μg/mL

3h / 20h

80

4.5

25 μg/mL

3h / 20h

81

NE

12.5 μg/mL

3h / 20h

99

NE

6.25 μg/mL

3h / 20h

94

NE

3.125 μg/mL

3h / 20h

104

NE

Positive control

3h / 20h

60

100.0***

Negative (vehicle) control: 1% Methyl cellulose

Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate was observed

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative (vehicle) control

Summary table of Chromosome Aberration Assay 2 experiment without metabolic activation:

Concentration (μg/mL)

Time of Treatment / Sampling

Relative Survival# (%)

Insolubility##

Mean % aberrant cells###

Tellurium dioxide without metabolic activation (-S9)

Untreated control

20h / 28h

99

2.5

Negative (vehicle) control

20h / 28h

100

3.5

60 μg/mL

20h / 28h

5

NE

40 μg/mL

20h / 28h

6

NE

30 μg/mL

20h / 28h

3

NE

20 μg/mL

20h / 28h

25

NE

15 μg/mL

20h / 28h

43

NE

10 μg/mL

20h / 28h

38

5.0

7.5 μg/mL

20h / 28h

51

3.5

5 μg/mL

20h / 28h

94

1.5

2.5 μg/mL

20h / 28h

93

NE

Positive control

20h / 28h

73

50.8***

Negative (vehicle) control: 1% Methyl cellulose

Positive control (-S9): Ethyl methanesulfonate, 0.4μL/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative (vehicle) control

 

Summary table of Chromosome Aberration Assay 2 experiment with metabolic activation:

Concentration (μg/mL)

Time of Treatment / Sampling

Relative Survival# (%)

Insolubility##

Mean % aberrant cells###

Tellurium dioxide with metabolic activation (+S9)

Untreated control

3h / 28h

103

1.5

Negative (vehicle) control

3h / 28h

100

4.0

200 μg/mL

3h / 28h

7

+a

NE

100 μg/mL

3h / 28h

19

+a

NE

75 μg/mL

3h / 28h

37

2.0

50 μg/mL

3h / 28h

52

4.0

25 μg/mL

3h / 28h

64

1.5

12.5 μg/mL

3h / 28h

100

4.0

6.25 μg/mL

3h / 28h

90

NE

3.125 μg/mL

3h / 28h

104

NE

Positive control

3h / 28h

70

75.0***

Negative (vehicle) control: 1% Methyl cellulose

Positive control (+S9): Cyclophosphamide, 6μg/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate was observed

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative (vehicle) control

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, Tellurium dioxide did not induced a significant level of chromosome aberrations in the performed experiments with and without metabolic activation. Therefore, Tellurium dioxide is considered not clastogenic in this test system.
Executive summary:

Tellurium dioxide was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was dissolved in 1% (w/v) Methyl cellulose solution and it was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, untreated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases.

Treatment with the test item did not result in a repeatable, statistically and biologically significant increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.