Cesium tungsten oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative, OECD 471, Bowles, 2004

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug-24 Nov 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EC Commissiion Directive 2000/32/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United kingdom, London, England

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium) and trp operon (for E. coli)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ β-naphthoflavone

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was non-soluble in distilled water, dimethyl sulfoxide, acetone, dimethyl formamide, ethanol and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks. It formed the best doseable suspension in dimethyl sulfoxide, and this was selected as the vehicle of choice.
- Prior to use, the solvent was dried using molecular seives.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA) (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation)

DURATION
- Exposure duration: 37 °C for 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Rationale for test conditions:

The test material caused no reduction in the growth of the bacterial background lawn at any dose level. The test material therefore, was tested up to the maximum recommended dose level of 5000 µg/plate

Evaluation criteria:

The test material may be considered positive in this test if the following criteria are met:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: A grey colour was observed at 5000 µg/plate with an associated black precipitate visible only with the use of a light microscope at 5000 µg/plate. These observations did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The test material dose range was the same for both the range-finding and the main tests.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Negative (solvent/vehicle) historical control data:
- Positive historical control data:
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Combined Vehicle and Untreated Control Values 2002
TA100 TA1535 WP2uvrA- TA98 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 91 100 20 17 23 27 22 35 12 17
SD 16.6 18.0 5.5 4.3 4.8 5.3 5.2 6.8 4.0 4.7
Min 61 68 8 8 10 14 11 13 4 5
Max 160 162 38 37 47 45 44 66 29 38
Values 939 742 912 718 685 521 946 749 918 718
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Positive Control Values 2002

TA100 TA1535 WP2uvrA- TA98 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 460 1880 347 310 621 866 136 239 1953 453
SD 118.2 594.3 204.0 119.7 235.5 350.5 38.2 74.7 803.1 145.9
Min 235 499 80 91 185 210 66 91 486 140
Max 952 3397 1985 810 1295 3406 323 507 4622 1365
Values 190 190 188 186 169 168 192 192 188 184
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Combined Vehicle and Untreated Control Values 2003

TA100 TA1535 WP2uvrA- TA98 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 98 106 20 15 23 28 20 30 10 14
SD 20.7 23.4 8.5 3.9 5.6 7.0 5.1 7.1 3.9 5.0
Min 61 64 8 7 11 13 10 15 3 3
Max 165 191 45 39 44 57 52 53 26 32
Values 882 735 838 690 686 539 877 721 838 678
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Positive Control Values 2003

TA100 TA1535 WP2uvrA- TA98 TA1537
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 429 1529 297 305 649 656 174 249 1273 370
SD 108.1 493.1 156.8 82.6 175.7 264.4 64.4 72.2 623.1 109.9
Min 197 485 98 162 273 136 64 87 262 148
Max 849 3662 1099 625 1198 1373 410 465 3704 759
Values 160 160 156 156 150 150 162 162 155 155
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 Test Results: Range-Finding Test

EXPERIMENT 1 (Standard Plate Test, SPT)
S9-Mix	Without
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA-	TA98	TA1537
VC	85±13.0	19±12.4	22±5.5	22±5.5	13±4.5
50	84±9.0	20±1.5	14±2.1	27±5.2	18±2.0
150	81±7.5	20±7.0	13±5.5	20±2.0	15±3.0
500	71±11.8	21±6.4	17±4.0	22±8.4	11±5.0
1500	79±7.2	23±2.0	17±4.5	24±7.5	14±4.0
5000	85±5.6	21±4.0	20±4.2	20±3.8	16±4.0
PC (mg/plate)	ENNG (3)	ENNG (5)	EENG (2)	4NQO (0.2)	9-AA (80)
	586±25.6	526±91.0	899±32.5	180±24.7	574±53.3
S9-Mix	With
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA-	TA98	TA1537
VC	98±6.5	13±4.5	24±1.0	36±1.5	14±5.3
50	97±6.9	12±1.0	22±0.6	27±2.0	14±4.2
150	96±10.2	9±3.1	17±7.1	27±6.1	16±3.6
500	87±15.5	8±1.5	21±1.5	21±4.6	11±5.3
1500	89±13.2	10±1.5	20±6.2	29±2.6	15±2.9
5000	104±12.3	12±0.6	25±6.7	28±4.9	20±4.7
PC (mg/plate)	2-AA (1)	2-AA (2)	2-AA (10)	BP (5)	2-AA (2)
	637±37.2	424±16.1	403±6.9	239±20.0	278±36.3

VC = Vehicle control; PC = Positive control

EENG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9-AA = 9-Aminoacridine

2-AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

 

 

Test Results: Main Test

EXPERIMENT 2 (Standard Plate Test, SPT)
S9-Mix	Without
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA-	TA98	TA1537
VC	71±6.4	33±4.5	27±7.2	21±2.1	18±5.5
50	72±8.7	18±2.5	26±6.1	19±4.9	13±4.2
150	72±7.6	38±9.7	22±2.5	18±4.4	16±7.4
500	60±6.7	31±9.5	25±5.2	18±6.1	17±1.0
1500	69±5.0	42±9.2	30±8.7	15±4.5	17±2.5
5000	74±6.5	39±13.7	21±2.1	20±2.0	18±1.0
PC (mg/plate)	ENNG (3)	ENNG (5)	EENG (2)	4NQO (0.2)	9-AA (80)
	402±22.4	386±84.1	1133±67.5	215±16.7	612±120.1
S9-Mix	With
Test Item (mg/plate)	Base-pair substitution type			Frameshift type
	TA100	TA1535	WP2uvrA-	TA98	TA1537
VC	89±25.0	11±1.5	17±4.5	27±8.5	21±1.5
50	76±9.5	8±1.2	25±3.6	21±2.0	18±5.2
150	73±7.1	12±5.5	25±3.6	27±10.1	27±3.5
500	70±7.0	15±2.9	14±1.7	24±1.2	19±6.9
1500	70±4.4	8±0.6	18±6.8	29±3.5	20±3.8
5000	72±10.5	12±6.1	20±2.3	27±7.5	17±1.5
PC (mg/plate)	2-AA (1)	2-AA (2)	2-AA (10)	BP (5)	2-AA (2)
	967±74.1	215±27.0	430±61.3	178±17.0	329±51.0

VC = Vehicle control; PC = Positive control

EENG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9-AA = 9-Aminoacridine

2-AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

Conclusions:

Interpretation of results: negative -- the test material was considered to be non-mutagenic under the conditions of this study.

Executive summary:

In a reverse gene mutation assay in bacterial strains were exposed to the test itme in DMSO (as an undissolved suspension) at concentrations of 50, 150, 500, 1500 and 5000 µg/plate the test was conducted in the presence and absence of S9 mix.

The test item was tested up to maximum recommended concentration of 5000 µg/plate.

It was concluded that the test item did not have mutagenic potential. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not relevant.

Additional information

AMES study 2004 - In a reverse gene mutation assay in bacterial strains were exposed to the test itme in DMSO (as an undissolved suspension) at concentrations of 50, 150, 500, 1500 and 5000 µg/plate the test was conducted in the presence and absence of S9 mix.

The test item was tested up to maximum recommended concentration of 5000 µg/plate.

It was concluded that the test item did not have mutagenic potential. 

 

The study was conducted on the particulate nanoform (i.e. not the dissolved form). Based on dissolution study results (Rosenfeldt, 2021, Section 4) this aligns with the draft ECHA guidance (Appendix R7-1 for nanoforms applicable to Chapter R7a and R7c Endpoint specific guidance, draft v3.0, 2021), whereby testing on the nanoform should be conducted if the nanoform is not highly soluble in water (>33,3 g/L) and/or does not have a half-life of water dissolution ≤ 10 min (Appendix R7-1 for nanoforms applicable to Chapter R7a and R7c Endpoint specific guidance, draft v3.0, 2021; Figure 1).  This is further supported by the lack of dissolution seen in any vehicle and at pH 4 (relevant gut pH) seen during the dissolution study (Section 4, Rosenfeldt, 2021). 

In accordance with the updated legislation (Regualtion (EC) No. 1907/2006) Section for nanoforms "The [AMES] study does not need to be conducted for nanoforms where it is not appropriate. In this case other studies involving one or more in vitro mutagenicity study(ies) in mammalian cells (Annex VIII, sections 8.4.2. and 8.4.3 or other internationally recognised in vitro methods) shall be provided."  In order to further support the results of the OECD 471 (AMES test), a confirmatory study is being conducted, that of an OECD 490 is ongoing, this test is considered appropriate for nanoforms and in line with ECHA guidance may provide evidence that the results from the AMES were appropriate (ECHA Appendix R7-1 for nanomaterials applicable to Chapter R7a and R7c Endpoint specific guidance - draft v3.0 2021).

Justification for classification or non-classification

Interpretation of results: negative -- the test material was considered to be non-mutagenic under the conditions of this study. No requirement to classify under Regulation (EC) No. 1272/2008.