Tungsten hexachloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no data available for the target substance on genetic toxicity. Thus, available data from Tungsten trioxide and Sodium tungstate dihydrate (source substances) were used to assess in a read-across approach the genotoxicity of Tungsten hexachloride.

Tungsten trioxide was tested negative for mutagenicity in an in vitro bacterial reverse mutation assay conducted according to OECD 471. Moreover, in an in vitro chromosome aberration assay conducted according to OECD 473 Tungsten trioxide was tested also negative.

Sodium tungstate dihydrate was tested negative in an in vitro Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476/OECD 490.

Based on the lack of mutagenicity reported in all in vitro assays reported above, the target substance Tungsten hexachloride is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: The test substance formed a transparent, colourless solution in water at 35 mg/mL, the highest concentration prepared for use in the assay. The test substance remained soluble at all concentrations tested.

RANGE-FINDING/SCREENING STUDIES: Cells were treated with the test substance for approximately 4 hours in the presence and absence of S9 activation at concentrations ranging from 6.90-3500 ug/mL. Test substance concentration for the main gene mutation assay were chosen to cover a toxicity range from 10 % to 20 % survival to no apparent effect on growth compared to the vehicle control. If little or no toxicity was observed and solubility was maintained, the mutation experiment was initiated with a maximum concentration of 5 mg/mL or 10 mM (whichever was lowest). If precipitation of the test substance occurred in the culture medium, the maximum applied dose was at least twice the solubility limit in culture medium. In this assay, the high dose was slightly higher than the OECD recommended guideline of 10 mM.
In the non-activation and activation assays, the test substance induced no cytotoxicity to weak cytotoxicity up to and including 1750 ug/mL and moderate cytotoxicity at 3500 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: One of the vehicle control cultures for the initial assay in the absence of metabolic activation (144.8 x 10^-6) was slightly above historical data range (36.4 to 135.7 x 10^-6) for mutant frequency. In this same assay one of the positive control cultures (522.0 x 10^-6) was also above the historical data range (227.0 to 487.4 x 10^-6) for mutant frequency. In addition, one vehicle control culture (139.1 x 10^-6) in the initial mutation assay with activation was slightly above the historical data range (34.0 to 123.9 x 10^-6) for mutant frequency.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Initial Non-activation Mutation Assay-Concentrations at 62.5 and 125 ug/mL were discarded because a sufficient number of higher concentrations were available. The remaining eight treatments induced no cytotoxicity to moderate cytotoxicity (88.3 % (250 ug/mL) to 34.0 % (3000 ug/mL) relative growths).
2. Confirmatory Non-activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to high cytotoxicity (120.3 % (250 ug/mL) to 13 % (3500 ug/mL) relative growth).
3. Initial Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity (117.2 % to 81.8 % relative growths).
4. Confirmatory Activation Mutation Assay- Concentrations at 62.5 and 125 ug/mL were terminated because there were sufficient higher concentrations available for analysis. The remaining eight concentrations induced no cytotoxicity to moderate cytotoxicity (95.5 % (250 ug/mL) to 39.9 % (3500 ug/mL) relative growths).


OTHER: The average cloning efficiencies for the vehicle control were 91.4 % and 110.9 % without metabolic activation and 96.6 % and 109.5 % with metabolic activation, which demonstrated acceptable cloning conditions for the assays. The positive control cultures induced large increases in mutant frequencies that were greatly in excess of the minimum criteria.
Mutant colonies from all the cultures showed the expected bimodal distribution, and mutant colonies from the positive control cultures showed both small and large colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of this assay, the test substance was reported as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells (OECD 490) in the presence and absence of Aroclor 1254 induced rat liver S9.

Executive summary:

In a mammalian cell gene mutation assay (Mouse Lymphoma Forward Mutation Assay) cells cultured in vitro were exposed to Sodium tungstate dihydrate at concentrations of 62.5, 125, 250, 500, 1000, 1500, 2000, 2500, 3000 and 3500 µg/mL in the presence and absence of mammalian metabolic activation (S9). 

Sodium tungstate dihydrate was tested up to cytotoxicity. 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 OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate was visible when the test substance was mixed with the agar at the 556 ug/plate samples and above. The precipitate was still visible at concentrations of 1667 and 5000 ug/plate when the colonies were counted, but did not impede the counting.

RANGE-FINDING/SCREENING STUDIES: The test substance was evaluated in a preliminary toxicity test using the Plate Incorporation Assay method. The test substance was tested at concentrations ranging from 21-5000 ug/plate in tester strain TA100 in the absence of metabolic activation. The test substance was not toxic up to 5000 ug/plate. It was therefore decided to use 5000 ug/plate as the highest concentration for the Ames Assay, which is the limit concentration according to the guidelines. Each of the other 4 concentrations was 1/3 of the preceding one.

COMPARISON WITH HISTORICAL CONTROL DATA: All solvent control values fell within historical range.

OTHER: The test substance formed a suspension in water which was obviously homogeneous. All positive control substances increased the mutation frequency to more than the above mentioned threshold values. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene required metabolic activation for mutagenicity, the results of these substances demonstrated the efficiency of the metabolizing system.

Table 2: Mean number of revertants per plate for strain TA97a

Without S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	77,3	26,8	3	5000	89,3	23,6	3
1667	77	13,9	3	1667	89,7	9,8	3
556	90,3	10,3	3	556	74,7	9,6	3
185	88,3	11,6	3	185	84,3	3,1	3
62	74	12,1	3	62	90	3	3
solvent	70	18,2	6	solvent	79	15,8	6
positive	201,7	34,3	3	positive	216,3	44,6	3

With S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	96,7	2,3	3	5000	100,7	3,8	3
1667	112	15,6	3	1667	116,3	2,5	3
556	101	14,2	3	556	98	7	3
185	113	18	3	185	120,3	9,5	3
62	92	6,6	3	62	120,3	16	3
solvent	105,2	13,9	6	solvent	99,8	6,6	6
positive	347,3	37,6	3	positive	324,3	24,8	3

solvent: water

positive: without S9 mix: 4 -Nitro-o-phenylene-diamine, 10 µg/plate; with S9 mix: 7,12 -Dimethylbenz[a]anthracene, 10 µg/plate

Table 3: Mean number of revertants per plate for strain TA98

Without S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	9,7	3,2	3	5000	13,7	2,1	3
1667	10	5,2	3	1667	9	5,3	3
556	6,3	2,1	3	556	8	4	3
185	7	3,6	3	185	8,3	1,5	3
62	9,3	4	3	62	10,3	4,6	3
solvent	8,5	1,5	6	solvent	11	3,9	6
positive	177,3	27,1	3	positive	283,3	23,9	3

With S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	13	0	3	5000	14,3	4	3
1667	13,3	1,5	3	1667	12,7	4,6	3
556	15	1,7	3	556	14	2,6	3
185	12,7	2,5	3	185	12,7	3,1	3
62	11,7	6,4	3	62	16,7	3,2	3
solvent	14,2	1,6	6	solvent	16,2	1,3	6
positive	262,7	14,6	3	positive	272,7	8,6	3

solvent: water

positive: without S9 mix: 2 -Nitrofluorene, 2 µg/plate; with S9 mix: 2 -Amino-anthracene, 1 µg/plate

Table 4: Mean number of revertants per plate for strain TA100

Without S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	67	5,6	3	5000	96,7	22,8	3
1667	63	2,6	3	1667	111	8,7	3
556	56,3	7,1	3	556	110,7	27	3
185	65	14,4	3	185	115	3,5	3
62	65,3	16	3	62	120	19,7	3
solvent	63,3	10,7	6	solvent	99,2	36,5	6
positive	418	16,8	3	positive	413,7	48,1	3

With S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	76,3	9,3	3	5000	122	8,5	3
1667	71,7	13,6	3	1667	99	7	3
556	84,7	11,9	3	556	96,7	12,7	3
185	97,7	17,6	3	185	116,3	18,6	3
62	83	7,5	3	62	89,7	10,8	3
solvent	87,8	21,2	6	solvent	98	12,6	6
positive	1006,7	74,6	3	positive	950	8,7	3

solvent: water

positive: without S9 mix: Sodium azide, 2 µg/plate; with S9 mix: 2-Amino-anthracene, 2 µg/plate

Table 5: Mean number of revertants per plate for strain TA102

Without S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	103	11,1	3	5000	94	12,5	3
1667	81	10,8	3	1667	83,7	3,8	3
556	110,3	2,5	3	556	102	6	3
185	97	21,8	3	185	130,7	31,9	3
62	89,7	4,2	3	62	124,3	27,1	3
solvent	105,7	7,7	6	solvent	106,3	27,5	6
positive	281,7	17,9	3	positive	287	47,1	3

With S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	170	29,8	3	5000	208	15,7	3
1667	157	5	3	1667	144,3	9,9	3
556	180	25,1	3	556	189	22,6	3
185	150,3	12,7	3	185	192,3	19,2	3
62	163,3	7,4	3	62	201,7	9,7	3
solvent	183,3	19	6	solvent	176,5	8,1	6
positive	571	118,3	3	positive	855,7	18,9	3

solvent: water

positive: without S9 mix: t-Butyl-hydroxyperoxide, 50 µg/plate; with S9 mix: 1,8 -Dihydroxy-anthraquinone, 50 µg/plate

Table 6: Mean number of revertants per plate for strain TA1535

Without S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	21	7	3	5000	13,7	4,9	3
1667	17,7	3,5	3	1667	13,7	4	3
556	23,3	5,7	3	556	11,3	3,8	3
185	21	4,6	3	185	12,3	3,2	3
62	19,7	1,5	3	62	14,7	2,9	3
solvent	24,5	4,8	6	solvent	18	1,4	6
positive	150,3	34,5	3	positive	357	75	3

With S9 mix

first experiment				second experiment
	revertants/ plate				revertants/ plate
conc. µg/plate	mean	SD	N	conc. µg/plate	mean	SD	N
5000	12,7	3,2	3	5000	11,3	1,5	3
1667	16,7	3,8	3	1667	8	1,7	3
556	15,7	1,2	3	556	12,3	2,5	3
185	15,7	0,6	3	185	10,3	3,8	3
62	16,7	1,2	3	62	13,7	2,1	3
solvent	15	5,4	6	solvent	18,3	1,4	6
positive	138	21,5	3	positive	178	17,6	3

solvent: water

positive: without S9 mix: Sodium azide, 1 µg/plate; with S9 mix: 2 -Amino-anthracene, 2 µg/plate

Conclusions:

In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), strains of S. typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were exposed to Tungsten oxide (WO3) (99.88% purity) suspended in deionized water up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance formed a yellow precipitate at most of the concentrations tested and was formulated as a suspension.

RANGE-FINDING/SCREENING STUDIES:
A cytotoxicity experiment was performed. Since acceptable cytotoxicity criteria were met by at least three of the dose levels of the test substance within an exposure time group, dose levels were selected for analysis of chromosome aberrations. This cytotoxicity experiment was reported as experiment 1 described above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
1. Experiment 1 with metabolic activation:
-The lowest concentration that approximated at 50% reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 58 %. The remaining concentrations selected for analysis were 0.078 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 0 % and 51 %, (relative to the vehicle control) were observed at these dose levels, respectively.

2. Experiment 1 without metabolic activation:
-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 2.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 2.5 mg/mL concentration was approximately 45 % . The remaining concentrations selected for analysis were 0.156 and 1.25 mg/mL. Percent reductions in cell numbers of approximately 5 % and 25 %, (relative to the vehicle control) were observed at these dose levels, respectively.

3. Experiment 2 with metabolic activation:
-The lowest concentration that approximated a 50 % reduction in cell number relative to the vehicle control (i.e., 1.5 mg/mL) and did not have an obscuring level of precipitation was selected as the highest dose level for the chromosome aberration analysis. The cytotoxicity at the 1.5 mg/mL concentration was approximately 39 %. The remaining concentrations selected for analysis were 0.75 and 1.0 mg/mL. Percent reductions in cell numbers of approximately 39 % and 18 %, (relative to the vehicle control) were observed at these dose levels, respectively.

4. Experiment 2 without metabolic activation
-Chromosome aberrations were evaluated from the cultures treated with concentrations of 1.0, 1.5 and 2.0 mg/mL. The reduction in cell numbers at these dose levels, relative to the vehicle control were 38 %, 40 % and 70 %, respectively.

Dosing Formulation Analysis:

Analysis of the dose formulations showed that the target concentrations for the test substance were not verified. This was most likely due to the nature of the suspension of the test substance in culture media when samples for analytical chemistry were collected. Since the target concentrations could not be verified other factors were used to select dosing levels for analysis (cytotoxicity and test substance precipitation).

 

Chromosome Aberration Induction:

1. Experiment 1 with metabolic activation

- No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was slightly elevated relative to the vehicle control in the presence of metabolic activation but these elevations were not statistically significant. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p <=0.01).

 

2. Experiment 1 without metabolic activation:

- No significant increase in cells with structural chromosomal aberrations was observed in the cultures treated with the test substance relative to the vehicle control. The percent of polyploid and endoreduplicated cells was not significantly elevated over the vehicle control vales at any of the doses tested in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p <0.01).

 

3. Experiment 2 with metabolic activation:

-No significant increases in cells with structural chromosomal aberrations were observed in the test substance treated cultures relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p <0.01).

 

4. Experiment 2 without metabolic activation:

- No significant increases in cells with structural chromosomal aberrations were observed in the cultures treated with the test substance relative to the vehicle control. The percentage of polyploid and endoreduplicated cells was not elevated relative to the vehicle control in the absence of metabolic activation. The positive control chromosome aberration response was elevated relative to the vehicle and the response was statistically significant (t-test, p <0.01).

Conclusions:

The test substance was evaluated for potential clastogenic effects using the structural chromosome aberration assay in Chinese hamster ovary cells, with and without metabolic activation. The test substance failed to induce any clastogenic responses in the CHO cells when tested in the presence and absence of metabolic activation up to dose levels that resulted in cytotoxicity and/or precipitation.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration, OECD 473), CHO cell cultures were exposed to Tungsten trioxide at concentrations up to 3000 µg/mL with and/or without metabolic activation. The highest concentration chosen in the main study was based on the results from a preliminary cytotoxicity test.

Tungsten trioxide was tested up to cytotoxicity. Positive controls induced did induce the appropriate response. There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no data available for the target substance on genetic toxicity. Thus, available data from Tungsten trioxide and Sodium tungstate dihydrate (source substances) were used to assess in a read-across approach the genotoxicity of Tungsten hexachloride. Due to a lower water solubility of Tungsten hexachloride compared to both source substances the resulting bioavailability (toxicity potential) would also be expected to be lower. Therefore, the read across to the source substances Tungsten trioxide and Sodium tungstate dihydrate is adequately protective. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Tungsten trioxide was tested negative for mutagenicity in an in vitro bacterial reverse mutation assay conducted according to OECD 471. Moreover, in an in vitro chromosome aberration assay conducted according to OECD 473 Tungsten trioxide was tested also negative.

Sodium tungstate dihydrate was tested negative in an in vitro Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476/OECD 490.

Based on the lack of mutagenicity reported in all in vitro assays reported above, the target substance Tungsten hexachloride is considered to be non-mutagenic.

Justification for classification or non-classification

Based on available data from suitable read-across partners, the target substance Tungsten hexachloride does not warrant classification for mutagenicity.