Tantalum pentachloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tantalum pentachloride (TaCl5) was tested negative in the Bacillus subtilis Rec assay. This test is a screening test for DNA damage. Furthermore, in a bacterial reverse mutation test according to OECD 471 and GLP tantalum and tantalum pentoxide did not induce mutagenicity. In a mammalian cell HPRT mutation assay according to OECD 476 and GLP tantalum pentachloride was tested negative. Moreover, tantalum pentachloride did not induce structural and/or numerical chromosomal damage tested by an in vitro micronucleus test in Chinese hamster V79 cells (OECD 487, GLP). Based on the lack of mutagenicity in all in vitro assays, tantalum pentachloride is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-01-05 to 2015-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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

Additional strain / cell type characteristics:

not applicable

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

True negative controls:

no

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)

Negative solvent / vehicle controls:

yes

Remarks:

1 % ethanol/9% Aqua ad injectabilia

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colcemid

Remarks:

without metabolic activation, final concentration0.16 and 2.0 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

True negative controls:

no

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 Increase^a
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) + (c_xx 3))/n

c₁:      mononucleate cells

c₂:      binucleate cells

c_x:      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 Increase^a
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) + (c_xx 3))/n

c₁:      mononucleate cells

c₂:       binucleate cells

c_x:       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 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 (OECD 487), V79 cells cultured in vitro were exposed to Tantalum pentachloride (decomposed) (99.9 %) in 1% ethanol/9% Aqua ad iniectabilia 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, 24h) 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) no biologically relevant and statistically significant increase of the micronucleus frequency was noted after treatment with the test item. The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency. This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Tantalum pentachloride (target substance) was tested negative in the Bacillus subtilis Rec mutagenicity screening assay. This assay is detecting DNA damage, which is subjected to cellular recombination repair. Supporting data of the non-mutagenic potential of the target substance in bacteria was received by read-across to tantalum and tantalum pentoxide, which were tested negative for mutagenicity in a bacterial reverse mutation assay (OECD 471, GLP). The water solubility of both substances is lower in comparison to the target substance. Nevertheless, both substances are to a certain degree water soluble and thus able to release the toxicological relevant tantalum ion. Based on the results, no concern is identified, that soluble tantalum ions will cause mutagenic effects in bacteria. Moreover, tantalum and tantalum pentoxide are not classified within the C&L inventory for genotoxicity.

To test for mutagenicity in mammalian cells an in vitro HPRT gene mutation assay was conducted according to OECD guideline 476 and GLP with the target substance. In this test, tantalum pentachloride did not induce mutagenicity in Chinese hamster V79 cells. Also, an in vitro micronucleus test was conducted according to OECD guideline 487 and GLP with the target substance. No induction of micronuclei were observed after treatment with tantalum pentachloride.

Based on the results and the high quality of the higher tier mammalian cell assays for mutagenicity (OECD 476 & OECD 487) tantalum pentachloride is considered to be non-mutagenic.

Justification for selection of genetic toxicity endpoint
in vitro GLP guideline study

Justification for classification or non-classification

Based on the available data, tantalum pentachloride does not warrant classification for mutagenicity.