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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Available data on tantalum, tantalum pentachloride and tantalum pentoxide indicate a lack of mutagenicity in all available in vitro assays (OECD 471, OECD 476, and OECD 487). The data is used in a read-across approach for the assessment of tantalum carbide.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2015-01-05 to 2015-06-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Tantalum pentachloride is used as read-across partner.
Qualifier:
according to guideline
Guideline:
other: OECD TG 487
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Type and identity of media:
MEM medium supplemented with: 10% fetal bovine serum (FBS), 100U/100 µg/mL, 2 mM L-glutamine, 2.5% µg/mL amphotericin, 25 mM Hepes
Treatment medium (short term exposure): Complete culture medium with 0% FBS
After treatment medium/ treatment medium (long term exposure): Complete culture medium with 10% FBS and 1.5 µg/ml cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
microsomal liver enzymes (S9)
Test concentrations with justification for top dose:
Pre-test for toxicity: 0, 0.0025, 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1 and 2 mM with and without S9 mix
Main test:
Experiment I: 0, 0.10, 0.25, 0.50 and 2.0 mM without S9 mix
0, 0.05, 0.10, 0.25, 0.50 and 2.0 mM with S9 mix
Experiment II: 0, 0.25, 0.50 and 1.0 mM without S9 mix
Vehicle / solvent:
- vehicle/solvent used: 1% Ethanol/9% Aqua ad injectabilia
- justification for choice of solvent: Solubility test
Untreated negative controls:
yes
Remarks:
Treatment medium (MEM Medium)
Negative solvent / vehicle controls:
yes
Remarks:
1% ethanol/9% Aqua ad injectabilia
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation, final concentration 300 µg/mL
Untreated negative controls:
yes
Remarks:
Treatment medium (MEM Medium) plus S9 mix
Negative solvent / vehicle controls:
yes
Remarks:
1 % ethanol/9% Aqua ad injectabilia plus S9 mix
Positive controls:
yes
Positive control substance:
other: Colcemid
Remarks:
without metabolic activation, final concentration0.16 and 2.0 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation, final concentration 2.5 µg/ml
Details on test system and experimental conditions:
Method of application: in medium
Experiment I: Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test group). Approx. 50 000 cells were seeded per cell culture flask, containing 5 ml complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 μg/mL cytochalasin B for 20 h at 37 °C.
Experiment II:
Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 μg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

DETERMINATION OF CYTOTOXICITY
- Methods: Cytokinesis Block Proliferation Index, % cytostasis
Evaluation criteria:
A mutation assay is considered acceptable if it meets the following criteria:
- the micronucleus induction in the V79 cells of the negative control and/or solvent control is within the range of the historical control data,
- the positive controls induced a detectable increase over the background, which demonstrates the sensitivity of the test system.
There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei,
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative control range.
Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response. A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric x² test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).
- Effects of osmolality: not examined
- Precipitation: Precipitation of the test item was noted in experiment I at concentrations of 0.25 mM (without metabolic activation) and at concentrations of 0.10 mM and higher (with metabolic activation). At experiment II precipitation of the test item was observed at concentrations of 1.0 mM and higher (without metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
Solubility Test:
A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test EtOH was used as solvent. After pre-dissolving the test item in EtOH (200 mM) a dilution series was prepared in EtOH. Then the 9fold volume of Aqua ad injectabilia was added to each concentration. After an initial reaction of approx. 10 minutes, this test item solution was added to cell culture medium (MEM without serum) at a ratio of 1:10, resulting in 1 % EtOH and 9% Aqua ad injectabilia in the final treatment medium. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). The solvent used is a composition of well-established solvents and is compatible with the survival of the cells and the activity of the S9 mix.
Pre-Experiment:
A pre-experiment was conducted under identical conditions as described for the main experiment I. According to results of the solubility test the highest concentration used was 2.0 mM. The following concentrations were tested with and without S9 mix:
- 0.0025, 0.005, 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0 and 2.0 mM

Cytotoxicity:
In experiment I without and with metabolic activation and in experiment II without metabolic activation no increase of the relative cytostasis above 30 % was noted.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I with and without metabolic activation and in experiment II without metabolic activation the solvent controls and the negative controls were within the range of the historical control data range. The number of micronucleated cells observed in the groups treated with the test item was within the range of the historical negative and solvent control data and did not show a biologically relevant increase compared to the corresponding solvent control.
EMS (900 and 1200 μg/ml) and CPA (2.5 μg/ml) were used as clastogenic controls and Colcemid
as aneugenic control (0.16 and 2.0 μg/ml). They induced distinct and biologically relevant increases
of the micronucleus frequency. This demonstrates the validity of the assay.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 1: Summary: Experiment I and II, without metabolic activation

 

Dose Group

Concentration [mM]

Number of cells evaluated

Cytotstasis
[%]

Relative CBPI
[%]

Micro-nucleated
Cells Frequency
[%]

Historical Laboratory Negative Control Range

Precipitation

Statistical Significant Increasea

Exp. I

4 h treatment, 24 h preparation interval

C

0

2000

0*

107

0.50

0.45% - 1.60%

aberrant cells

-

/

S

0

2000

0

100

0.80

-

/

4

0.10

2000

0*

105

0.85

-

-

5

0.25

2000

3

97

1.15

+

-

6

0.5

2000

2

98

0.80

+

-

8

2.0

2000

9

91

0.95

+

-

EMS

1200 µg/mL

2000

23

77

5.15

-

+

Colc

2.0 µg/mL

2000

13

87

2.85

-

+

 

 

 

 

Exp. II

24 h treatment, 24 h preparation interval

C

0

2000

0*

127

0.85

0.45% - 1.60%

aberrant cells

-

/

S

0

2000

0

100

0.85

-

/

6

0.25

2000

22

78

0.70

-

-

7

0.5

2000

12

88

0.75

-

-

8

1.0

2000

16

84

0.60

+

-

EMS

900 µg/mL

2000

53

47

2.25

-

+

Colc

0.16 µg/mL

2000

0*

101

6.00

-

+

C:       Negative Control (Culture medium)

S:       Solvent Control (EtOH 1% v/v and Aqua ad injectabilia 9% v/v in culture medium)

a:       statistical significant increase compared to negative/solvent controls (chi2- test , p< 0.05),  +: significant; -not significant

EMS:   Ethylmethanesulfonate, Positive Control (without metabolic activation) [900 and 1200 µg/mL]

Colc:   Colcemid, Positive Control (without metabolic activation) [0.16 and 2.0 µg/mL]

CBPI:  Cytokinesis Block Proliferation Index, CBPI = ((c1x 1) + (c2x 2) + (cxx 3))/n

c1:      mononucleate cells

c2:      binucleate cells

cx:      multinucleate cells

n:       total number of cells

*: the cytostasis is defined 0, when the relative CBPI exceeds 100%

Table 2: Summary: Experiment I, with metabolic activation

 

Dose Group

Concentration [mM]

Number of cells evaluated

Cyto stasis
[%]

Relative CBPI
[%]

Micro-

Nucleated
Cells
Frequency
[%]

Historical Laboratory Negative Control Range

Precipitation

Statistical Significant Increasea

 

 

Exp. I

4 h treatment,

24 h preparation interval

C

0

2000

0*

107

0.65

0.50% - 1.75%

aberrant cells

-

/

S

0

2000

0

100

1.00

-

/

3

0.05

2000

0*

120

0.95

-

-

4

0.10

2000

0*

115

1.40

+

-

5

0.25

2000

0*

108

1.55

+

-

6

0.5

2000

0*

118

1.05

+

-

8

2.0

2000

0*

113

1.00

+

-

CPA

2.5 µg/mL

2000

28

72

4.00

-

+

C:       Negative Control (Culture medium)

S:        Solvent Control (etOH 1% v/v and Aqua ad injectabilia 9% v/v in culture medium)

a:       statistical significant increase compared to negative/solvent controls (chi² test , p< 0.05), +: significant; -: not significant

CPA:  Cyclophosphamide, Positive Control (with metabolic activation)[2.5 µg/mL]

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1x 1) + (c2x 2) + (cxx 3))/n

c1:      mononucleate cells

c2:       binucleate cells

cx:       multinucleate cells

n:       total number of cells

*: the cytostasis is defined 0, when the relative CBPI exceeds 100%

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the study described and under the experimental conditions reported, tantalum pentachloride (decomposed) did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, tantalum pentachloride (decomposed) is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Executive summary:

In an in vitro mammalian micronucleus assay V79 cells cultured in vitro were exposed to tantalum pentachloride (decomposed) (99.9 %) in 1% ethanol / 9% Aqua ad injectabilia in experiment I (short term exposure, 4h) at concentrations of 0.10, 0.25, 0.50 and 2.0 mM (without metabolic activation) and at 0.05, 0.10, 0.25, 0.5 and 2.0 mM (with metabolic activation). In experiment II (long term exposure, 24 h) the cells were exposed at concentrations of 0.25, 0.50, and 1.0 mM (without metabolic activation).

In experiment I (with and without metabolic activation) and in experiment II (without metabolic activation) the micronucleated cell frequencies of the negative controls and solvent controls were within the range of the historical data.

In experiment I (with and without metabolic activation) and in experiment II (without metabolic activation) no biologically relevant and statistically significant increase of the micronucleus frequency was noted after treatment with the test item. Moreover, all values were within the historical control data range.The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency. This study is classified as acceptable.This study satisfies the requirement for Test Guideline OECD 487. Tantalum pentachloride is used as read-across partner to tantalum carbide.

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

Additional information

Additional information from genetic toxicity in vitro:

No data is available for tantalum carbide (target substance). Thus, data from tantalum, tantalum pentachloride and tantalum pentoxide were used in a read-across approach. Tantalum pentachloride was tested negative in an in vitro HPRT study (OECD 476) and in an in vitro micronucleus test (OECD 487). Tantalum and tantalum pentoxide were tested negative in a bacterial reverse mutation test (OECD 471). Additionally, tantalum pentoxide was tested negative in a chromosome aberration assay (OECD 473).

Due to lower transformation/dissolution results for tantalum carbide than for tantalum pentachloride the resulting toxicity potential would also be expected to be lower. Therefore, the read across to the source substance tantalum pentachloride is adequately protective. For more details refer to the attached description of the read-across approach.

As negative data is available for the higher tier mammalian mutagenicity tests (OECD 476 and OECD 487) with tantalum pentachloride, tantalum carbide is considered to be non-mutagenic.


Justification for selection of genetic toxicity endpoint
GLP guideline study.

Justification for classification or non-classification

No genotoxic effects were observed in OECD guideline tests with tantalum, tantalum pentachloride and tantalum pentoxide. Based on the available data from the read-across partners, tantalum carbide does not warrant classification for mutagenicity.