Niobium pentachloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Niobium pentachloride 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 the read across partner niobium pentoxide did not induce mutagenicity. In a mammalian cell HPRT mutation assay according to OECD 476 and GLP niobium pentachloride was tested negative. Moreover, niobium pentachloride did not induce DNA damage tested by an in vitro comet assay. Based on the lack of mutagenicity in all in vitro assays, niobium pentachloride is considered to be non-mutagenic.

Link to relevant study records

Reference

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:

2015-01-07 to 2015-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media:
Short-term exposure: MEM medium, 100 U/100 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/ml amphotericin B
Long-term exposure: MEM Medium with 10 % fetal bovine serum (FBS), 100 U/100 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/ml amphotericin 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, 0.01, 0.05, 0.10, 0.25, 0.50, 1.0 mM without/with S9 mix
Main test:
Experiment I: 0, 0.025, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 mM without/with S9 mix
Experiment II: 0, 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 0.75, 1.0 mM without S9 mix
Experiment II: 0, 0.004, 0.007, 0.02, 0.04, 0.07, 0.2, 0.4, 0.7, 1.0 mM with S9 mix

Vehicle / solvent:

- vehicle/solvent used: 1% Ethanol/9% Aqua ad injectabilia
- justification for choice of solvent: Solubility test and pre-test for toxicity

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:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation, final concentration 0.8 and 1.0 µg/ml

Details on test system and experimental conditions:

Method of application: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (short time exposure, Experiment I with and without metabolic activation)
20 hours (long time exposure, Experiment II with and without metabolic activation)
- Expression time (cells in growth medium): 6 days (subcultered after 3 days)
- Selection time (if incubation with a selection agent): 7-11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-17 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: one culture per test group (expression period), 5 dishes per culture per test group (selection period)

DETERMINATION OF CYTOTOXICITY
- Methods: Relative Growth, Cloning efficiency

Evaluation criteria:

- Negative and/or solvent controls fall within the performing laboratories historical control data range: 2-44 mutants/10^6 cells
-The absolute cloning efficiency: ([number of positive cultures x 100] I total number of seeded cultures) of the negative and /or
solvent controls is > 50%
-The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.
- A test is considered to be negative if there is no biological relevant increase in the number of mutants.
- There are several criteria for determining a positive result: a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
- According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

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 without metabolic activation at concentrations of 0.5 mM and higher and with metabolic activation at concentrations of 0.25 mM and higher. In experiment II precipitation was detected at concentrations of 0.2 mM and higher with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
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 (100 mM) a dilution series was prepared in EtOH. First a 9fold volume of phosphate buffer was used adding it to each concentration. After noticing that in the pre-experiment without metabolic activation the buffer reacts with the cells the phosphate buffer was replaced for the main experiments by Aqua ad injectabilia. So 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 The toxicity of the test item was determined in pre-experiments. Eight concentrations [0.0025, 0.005, 0.01, 0.050, 0.10, 0.25, 0.50, 1.0 mM] were tested without and with metabolic activation. In the pre-experiment the test item concentrations were dissolved in 1% ethanol and 9% phosphate buffer. The experimental performance for the pre-experiment was the same as described below for the main experiments (excepting the solvent composition)

COMPARISON WITH HISTORICAL CONTROL DATA:
In all experiments the mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facility (without metabolic activation: 2-43 mutants per 10^6 cells, with metabolic activation: 5-44 mutants per 10^6 cells

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Table 4
Experiment I - Mutagenicity, without metabolic activation
Dose Group	Concentration [mM]	Number of mutant colonies per flask^a					Mean	SD	Mutant SD colonies per 10^6 cells^b	Mutation factor
		I	II	III	IV	V
NC1	0	6	6	9	10	6	7.4	1.74	20.11
NC2		4	6	6	*	8	6	1.41	18.29
S1	0	9	10	13	16	10	11	2.58	33.72
S2		7	6	9	8	10	11.6	1.41	23.46
2	0.025	6	6	5	14	9	8	3.29	20.05	0.69
3	0.05	5	3	6	2	8	4.8	2.14	14.08	0.48
4	0.1	4	4	6	5	12	6.2	2.99	18.34	0.63
5	0.25	9	10	10	7	5	8.2	1.94	25.39	0.87
6	0.5	6	11	5	5	7	6.8	2.23	18.28	0.63
7	0.75	15	14	11	15	18	14.6	2.24	40.67	1.4
8	1.0	9	6	6	12	12	9	2.68	25.86	0.89
9	1.5	3	5	5	4	7	4.8	1.33	14.72	0.51
10	2.0	9	13	6	9	6	8.6	2.58	25.83	0.89
EMS	300 µg/mL	84	72	80	89	90	83	6.57	233.15	8.01
NC:	negative control/ medium control
S:	solvent control
a:	number of mutant colonies in flask I to V
b:	mean mutant colonies x 10^6/ (400000 x Cloning Efficiency/100)
EMS:	Ethylmethanesulfonate [300 µg/mL]
*:	Contamination of cell culture in flask

Table 6
Experiment I - Mutagenicity, with metabolic activation
Dose Group	Concentration [mM]	Number of mutant colonies per flask^a					Mean	SD	Mutant SD colonies per 10^6 cells^b	Mutation factor
		I	II	III	IV	V
NC1	0	5	5	6	6	11	6.6	2.24	20.06
NC2		2	5	7	11	11	7.2	3.49	20.11
S1	0	3	5	6	7	10	6.2	2.32	18.62
S2		3	4	9	9	10	7.0	2.90	19.83
2	0.025	4	4	5	6	10	5.8	2.23	14.61	0.76
3	0.05	5	12	8	3	8	7.2	3.06	18.56	0.97
4	0.10	11	10	11	10	11	10.6	0.49	30.64	1.59
5	0.25	5	6	7	10	14	8.4	3.26	20.64	1.07
6	0.50	2	2	4	5	5	3.6	1.36	9.65	0.50
7	0.75	3	7	8	9	9	7.2	2.23	19.15	1.00
8	1.0	4	8	11	12	12	9.4	3.07	25.61	1.33
9	1.5	7	7	8	8	9	7.8	0.75	18.22	0.95
10	2.0	7	7	9	9	11	8.6	1.50	22.34	1.16
DMBA	0.8µg/mL	105	107	133	96	97	107.6	13.41	303.95	15.81
DMBA	1.0µg/mL	111	149	118	122	109	121.8	14.39	369.09	19.20
NC:	negative control/ medium control
S:	solvent control
a:	number of mutant colonies in flask I to V
b:	mean mutant colonies x 10^6/ (400000 x Cloning Efficiency/100)
DMBA:	7, 12-Dimethylbenz(a)anthracene [0.8 and 1.0 µg/mL]

Table 8
Experiment II - Mutagenicity, without metabolic activation
Dose Group	Concentration [mM]	Number of mutant colonies per flask^a					Mean	SD	Mutant SD colonies per 10^6 cells^b	Mutation factor
		I	II	III	IV	V
NC1	0	8	9	10	6	5	7.6	1.85	21.05
NC2		14	8	5	9	12	9.6	3.14	26.89
S1	0	15	7	8	9	12	10.2	2.93	29.74
S2		9	12	9	8	5	8.6	2.24	24.02
2	0.025	16	4	8	5	6	7.8	4.31	25.91	0.96
3	0.010	10	12	12	11	7	10.4	1.85	33.02	1.23
4	0.025	12	5	10	10	5	8.4	2.87	25.23	0.94
5	0.050	15	15	8	13	13	12.8	2.56	36.57	1.36
6	0.10	8	18	12	18	14	14.0	3.79	39.11	1.45
7	0.25	10	10	7	10	10	9.4	1.2	26.26	0.98
8	0.50	16	10	13	8	20	13.4	4.27	37.22	1.38
9	0.75	14	7	18	16	15	14.0	3.74	35.90	1.34
10	1.0	9	12	8	11	8	9.6	1.62	27.12	1.01
EMS	300µg/mL	160	175	179	169	177	172.0	6.87	607.77	22.61
NC:	negative control/ medium control
S:	solvent control
a:	number of mutant colonies in flask I to V
b:	mean mutant colonies x 10^6/ (400000 x Cloning Efficiency/100)
EMS:	Ethylmethanesulfonate [300 µg/mL]

Table 10
Experiment II - Mutagenicity, with metabolic activation
Dose Group	Concentration [mM]	Number of mutant colonies per flask^a					Mean	SD	Mutant SD colonies per 10^6 cells^b	Mutation factor
		I	II	III	IV	V
NC1	0	13	10	8	11	3	9.0	3.41	24.93
NC2		19	12	18	12	16	15.4	2.94	43.38
S1	0	3	8	6	5	3	5.0	1.90	13.7
S2		9	7	12	13	5	9.2	2.99	25.41
2	0.004	13	13	8	13	6	10.6	3.01	26.84	1.37
3	0.01	4	10	8	7	8	7.4	1.96	20.11	1.03
4	0.02	5	12	7	11	7	8.4	2.65	22.22	1.14
5	0.04	9	8	11	12	6	9.2	2.14	23.41	1.20
6	0.07	15	6	10	12	7	10.0	3.29	25.71	1.31
7	0.2	7	8	4	11	14	8.8	3.43	26.19	1.34
8	0.4	9	13	14	17	13	13.2	2.56	35.97	1.84
9	0.7	9	13	12	9	6	9.8	2.48	29.17	1.49
10	1.0	13	10	14	12	13	12.4	1.36	35.63	1.82
DMBA	0.8µg/mL	77	89	71	88	90	83.0	7.62	244.12	12.48
DMBA	1.0µg/mL	109	120	126	106	114	115.0	7.27	357.14	18.26
NC:	negative control/ medium control
S:	solvent control
a:	number of mutant colonies in flask I to V
b:	mean mutant colonies x 10^6/ (400000 x Cloning Efficiency/100)
DMBA:	7, 12-Dimethylbenz(a)anthracene [0.8 and 1.0 µg/mL]

Conclusions:

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

Under the experimental conditions, the test item niobium pentachloride (decomposed) is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell HPRT gene mutation assay, V79 cells cultured in vitro were exposed to niobium pentachloride (decomposed) (99.9 %) in 1% ethanol and 9% Aqua ad injectibilia at concentrations of 0.025, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 mM in the presence and absence of mammalian metabolic activation (experiment I) and for experiment II at concentrations of 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 0.75 and 1.0 mM without metabolic activation and 0.004, 0.007, 0.02, 0.04, 0.07, 0.2, 0.4, 0.7 and 1.0 mM with metabolic activation.

In experiment I and II with and without metabolic activation mutant values of the negative controls, the solvent controls and all mutant values of the test item concentrations found were within the historical control data.

For all tested treatment groups no dose-response relationship could be observed. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Niobium 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 niobium pentoxide, which was tested negative for mutagenicity in a bacterial reverse mutation assay (OECD 471, GLP). The water solubility of this substance is lower in comparison to the target substance. Nevertheless, niobium pentoxide is to a certain degree water soluble and thus able to release the toxicological relevant niobium ions. Moreover, niobium pentoxide are not classified within the C&L inventory for genotoxicity. Based on the results, no concern is identified, that soluble niobium ions will cause mutagenic effects in bacteria.

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, niobium pentachloride did not induce mutagenicity in Chinese hamster V79 cells, which further strengthen the non-mutagenic capability of the target substance. Moreover, niobium pentachloride did not induce genotoxic effects in an in vitro Comet assay.

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

Justification for selection of genetic toxicity endpoint
GLP guideline study conducted with the target substance niobium pentachloride

Justification for classification or non-classification

Based on the lack of mutagenicity in all in vitro assays, niobium pentachloride is considered to be non-mutagenic and does not warrant classification for genotoxicity.