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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-08 to 2012-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix; phenobarbital/β-naphthoflavoneinduced rat liver
Test concentrations with justification for top dose:
Range finding test: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate (TA 98 and TA 100 with and without metabolic activation).
Initial mutation test: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.
Confirmatory mutation test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate.
Complementary confirmatory mutation test: 158.1; 50; 15.81; 5; 1.581, 0.5, 0.1581 and 0.05 μg test item/plate.
Vehicle / solvent:
1 % (v/v) methyl cellulose solution was used as vehicle to prepare the stock formulation of the test material.
Test formulations were freshly prepared at the beginning of the experiments in the testing laboratory by
diluting the stock formulation using the selected vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preliminary and initial experiment: in agar (plate incorporation)
For confirmatory and complementary confirmatory tests preincubation method was used

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 3 plates/concentration
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic
activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the vehicle control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Based on the results of the Preliminary Solubility Test, 1 % (v/v) methyl cellulose solution was selected for vehicle of the test item in the study. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Preliminary Range Finding Test. Based on the results of the Preliminary Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test were 158.1; 50; 15.81; 5; 1.581, 0.5, 0.1581 and 0.05 μg test item/plate.

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.

Slight inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in Salmonella typhimurium TA98 bacterial strain at 5000 μg/plate concentration with metabolic activation and in Escherichia coli WP2 uvrA bacterial strain at 5000 μg/plate concentration without metabolic activation. Reduced numbers of revertant colonies compared to the vehicle control plates were also observed in Salmonella typhimurium TA100 strain at 5000 μg/plate concentration without metabolic activation, and in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains at 5000 μg/plate concentration with metabolic activation, although no effect on the background lawn was observed.

Similar, but stronger cytotoxic effect was observed using the pre-incubation method (Confirmatory Mutation Test and Complementary Confirmatory Mutation Test) in Salmonella typhimurium TA100, TA1535 and TA1537 strains at 158.1 and 50 μg/plate concentrations without metabolic activation; and in Escherichia coli WP2 uvrA strain at 5000, 1581 and 500 μg/plate concentrations without metabolic activation.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item Tellurium had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 1535, TA 1537, TA98 and TA100 of S. typhimurium and E. coli WP2 uvrA were exposed to Tellurium, (powder 99.95 % a.i.), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; phenobarbital/β-naphthoflavone induced rat liver).

The initial Mutation Test was performed as plate incorporation method; the confirmatory and complementary assays were performed according to the pre-incubation method.

 

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls and were within the historical control range and were within the normal biological variability of the test system.

Slight inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in Salmonella typhimurium TA98 bacterial strain at 5000 μg/plate concentration with metabolic activation and in Escherichia coli WP2 uvrA bacterial strain at 5000 μg/plate concentration without metabolic activation. Reduced numbers of revertant colonies compared to the vehicle control plates were also observed in Salmonella typhimurium TA100 strain at 5000 μg/plate concentration without metabolic activation, and in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains at 5000 μg/plate concentration with metabolic activation, although no effect on the background lawn was observed.

Similar, but stronger cytotoxic effect was observed using the pre-incubation method (Confirmatory Mutation Test and Complementary Confirmatory Mutation Test) in Salmonella typhimurium TA100, TA1535 and TA1537 strains at 158.1 and 50 μg/plate concentrations without metabolic activation; and in Escherichia coli WP2 uvrA strain at 5000, 1581 and 500 μg/plate concentrations without metabolic activation.

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was confirmed by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

 

 

In conclusion there was no evidence of induced mutant colonies over background.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-09-17 to 2012-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see section 13.1 for read-across justification
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.
Additional strain / cell type 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-hour treatment with metabolic activation: 100; 75; 50; 25; 20; 15; 10; 7.5; 5; 2.5; 1.25 and 0.625 µg/mL
Assay 1: 3-hour treatment without metabolic activation: 80; 70; 60; 50; 40; 30; 20; 10; 5; 2.5; 1.25 and 0.625 µg/mL
Assay 2: 3-hour treatment with metabolic activation: 20; 17.5; 15; 12.5; 10; 7.5; 5; 2.5; 1.25 and 0,625 µg/mL
Assay 2: 24-hour treatment without metabolic activation: 15; 12.5; 10; 9; 8; 7; 6; 5; 4; 2; 1; 0.5 and 0.25 µg/mL
Vehicle / solvent:
1 % (w/v) methyl cellulose solution
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): two weeks


SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other: smal and large colonies




Evaluation criteria:
The assay was considered valid if all of the following criteria were met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (50-170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative (vehicle) controls is within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations are present, where the highest concentration produces approximately 80-90% toxicity (measured by %RS or RTG), results in precipitation, or it is 5 mg/mL, 5 µL/mL or 0.01 M (whichever is the lowest), or it is the highest practical concentration.

The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006 [6]):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the
corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells
(GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no large changes in pH
- Effects of osmolality: no large changes in osmolarity
- Water solubility: insoluble
- Precipitation: insolubility was detected in the final treatment medium at the end of the treatment at some concentrations


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 100, 75, 50, 25 and 20 µg/mL concentrations, cells of these samples did not survive the expression period. Marked cytotoxicity was observed at 15 µg/mL concentration (relative total growth of 4%); therefore, this concentration was excluded from the evaluation. An evaluation was made using data of following concentration of 10 µg/mL (relative total growth of 16%) and the next five concentrations (a total of six concentrations). Statistically significant increases in the mutation frequency were observed at 10, 7.5, 5, 2.5, 1.25 µg/mLconcentrations. However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor in case of the 7.5, 5 and 2.5 µg/mLconcentrations, thus they were considered as biologically non relevant increases. In case of the 10 and 1.25 µg/mLconcentrations, the values were over the limit of the biological relevance (the difference was higher than the global evaluation factor) but the results did not follow a clear dose response and the increases were not reproduced in Assay 2.

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 80, 70, 60, 50, 40 and 30 µg/mL concentrations, cells of these samples did not survive the expression period. Therefore, an evaluation was made using data of the first surviving concentration of 20 µg/mL (relative total growth of 18%) and the next five examined concentrations (a total of six concentrations).

A statistically significant increase in the mutation frequency was observed at 20 µg/mL concentration. However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

In Assay 2, following a3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity was observed at 20 µg/mL concentration, no cells of this sample survived the expression period. Marked cytotoxicity was observed at 17.5, 15, 12.5, 10 and 7.5 µg/mL concentrations (relative total growth values of 2, 3, 6, 5, and 5%, respectively); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of 5 µg/mL concentration (relative total growth of 12%) and additional three treatment concentrations (a total of four concentrations). Statistically significantincrease in the mutation frequency was observed at 5 µg/mL concentration.However, similarly to the other experiments, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at 15 µg/mL concentration, no cells of this sample survived the expression period. Marked cytotoxicity was observed at 12.5, 10 and 9 µg/mL concentrations (relative total growth values were less than 1%); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of 8 µg/mL concentration (relative total growth of 27%) and additional seven treatment concentrations (a total of eight concentrations).A statistically significant increase in the mutation frequency was observed at 8 µg/mL concentration. However, similarly to the other experiments, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate, as they covered the range from cytotoxicity to no or little cytotoxicity. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.


Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, no clear mutagenic effect of Tellurium dioxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, it was considered being not positive with regard to classification.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential of Tellurium dioxide to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

In conclusion, no clear mutagenic effect of Tellurium dioxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, it was considered being not positive with regard to classification.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of 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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see section 13.1 for read-across justification
Reason / purpose:
read-across source
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
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (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 / cell type 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 / solvent:
- Vehicle(s)/solvent(s) used: 1% methyl cellulose
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental 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.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
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.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

  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

 

 

 

 

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-17 to 2012-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.
Additional strain / cell type 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-hour treatment with metabolic activation: 100; 75; 50; 25; 20; 15; 10; 7.5; 5; 2.5; 1.25 and 0.625 µg/mL
Assay 1: 3-hour treatment without metabolic activation: 80; 70; 60; 50; 40; 30; 20; 10; 5; 2.5; 1.25 and 0.625 µg/mL
Assay 2: 3-hour treatment with metabolic activation: 20; 17.5; 15; 12.5; 10; 7.5; 5; 2.5; 1.25 and 0,625 µg/mL
Assay 2: 24-hour treatment without metabolic activation: 15; 12.5; 10; 9; 8; 7; 6; 5; 4; 2; 1; 0.5 and 0.25 µg/mL
Vehicle / solvent:
1 % (w/v) methyl cellulose solution
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): two weeks


SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other: smal and large colonies




Evaluation criteria:
The assay was considered valid if all of the following criteria were met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (50-170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative (vehicle) controls is within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations are present, where the highest concentration produces approximately 80-90% toxicity (measured by %RS or RTG), results in precipitation, or it is 5 mg/mL, 5 µL/mL or 0.01 M (whichever is the lowest), or it is the highest practical concentration.

The test item was considered to be mutagenic in this assay if all the following criteria were met (based on M. Moore 2006 [6]):
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the
corresponding negative (vehicle) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells
(GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no large changes in pH
- Effects of osmolality: no large changes in osmolarity
- Water solubility: insoluble
- Precipitation: insolubility was detected in the final treatment medium at the end of the treatment at some concentrations


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 100, 75, 50, 25 and 20 µg/mL concentrations, cells of these samples did not survive the expression period. Marked cytotoxicity was observed at 15 µg/mL concentration (relative total growth of 4%); therefore, this concentration was excluded from the evaluation. An evaluation was made using data of following concentration of 10 µg/mL (relative total growth of 16%) and the next five concentrations (a total of six concentrations). Statistically significant increases in the mutation frequency were observed at 10, 7.5, 5, 2.5, 1.25 µg/mLconcentrations. However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor in case of the 7.5, 5 and 2.5 µg/mLconcentrations, thus they were considered as biologically non relevant increases. In case of the 10 and 1.25 µg/mLconcentrations, the values were over the limit of the biological relevance (the difference was higher than the global evaluation factor) but the results did not follow a clear dose response and the increases were not reproduced in Assay 2.

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 80, 70, 60, 50, 40 and 30 µg/mL concentrations, cells of these samples did not survive the expression period. Therefore, an evaluation was made using data of the first surviving concentration of 20 µg/mL (relative total growth of 18%) and the next five examined concentrations (a total of six concentrations).

A statistically significant increase in the mutation frequency was observed at 20 µg/mL concentration. However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

In Assay 2, following a3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity was observed at 20 µg/mL concentration, no cells of this sample survived the expression period. Marked cytotoxicity was observed at 17.5, 15, 12.5, 10 and 7.5 µg/mL concentrations (relative total growth values of 2, 3, 6, 5, and 5%, respectively); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of 5 µg/mL concentration (relative total growth of 12%) and additional three treatment concentrations (a total of four concentrations). Statistically significantincrease in the mutation frequency was observed at 5 µg/mL concentration.However, similarly to the other experiments, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at 15 µg/mL concentration, no cells of this sample survived the expression period. Marked cytotoxicity was observed at 12.5, 10 and 9 µg/mL concentrations (relative total growth values were less than 1%); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of 8 µg/mL concentration (relative total growth of 27%) and additional seven treatment concentrations (a total of eight concentrations).A statistically significant increase in the mutation frequency was observed at 8 µg/mL concentration. However, similarly to the other experiments, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus it was considered as biologically non relevant increase.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate, as they covered the range from cytotoxicity to no or little cytotoxicity. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.


Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, no clear mutagenic effect of Tellurium dioxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, it was considered being not positive with regard to classification.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential of Tellurium dioxide to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

In conclusion, no clear mutagenic effect of Tellurium dioxide was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, it was considered being not positive with regard to classification.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-26 to 2012-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (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 / cell type 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 / solvent:
- Vehicle(s)/solvent(s) used: 1% methyl cellulose
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental 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.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
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.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

  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

 

 

 

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 1535, TA 1537, TA98 and TA100 of S. typhimurium and E. coli WP2uvrA were exposed to Tellurium, (powder 99.95 % a.i.), at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation, (S9 mix; phenobarbital/β-naphthoflavone induced rat liver).

Tellurium had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

Chromosomal Aberration Test with Tellurium dioxide

The read-across substance Tellurium dioxide was tested according to OECD guideline 479 in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells . The test item was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity.

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.

Mammalian Cell Gene Mutation Test with Tellurium dioxide:

An in vitro mammalian cell assay according to OECD Guideline 476 was performed in mouse lymphoma L5178Y TK+/- cells at the tk locus to test the potential of Tellurium dioxide to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation. The evaluated concentration ranges covered the range from cytotoxicity to no or little cytotoxicity.

 

Statistical significant increase was observed, however taking into account the global evaluation factor no biological relevant mutagenic effect of tellurium dioxid, was found either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Justification for read-across is outlined in the attached document.

 


Justification for selection of genetic toxicity endpoint
Reliable data are available from the full in-vitro test set required under REACH regulation.

Short description of key information:
Negative data are available from a gene mutation assay in bacteria with tellurium. As well negative data were obtained from a chromosomal aberration test and a mammalian cell gene mutation assay with the read-across substance tellurium dioxide.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on all available information the substance is devoid of any relevant genotoxic potency.