Cerium(4+) disulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria (reference 7.6.1 -1).

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:

07 February 2017 - 22 May 2017

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats

Test concentrations with justification for top dose:

1st serie: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate
2nd serie: 158, 500, 1580, 2810, 5000 µg/plate
top dose: maximum recommended concentration (according to OECD 471)

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

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

sodium azide

other: 2-aminoanthracene, daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 to 3 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

OTHER:
- S9 concentration: 1st serie: 10%; 2nd serie: 20%

Evaluation criteria:

A test material was to be defined as negative or non-mutagenic in this assay if
- The assay was to be considered valid, and
- "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
- a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
- "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Statistics:

Not performed as not mandatory for this test system.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

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:

cytotoxicity

Remarks:

at 1580 µg/plate

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:

cytotoxicity

Remarks:

at 5000 µg/plate

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:

cytotoxicity

Remarks:

at 5000 µg/plate

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:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material precipitate was observed on the plates at the concentration 5000 µg/plate (1st serie) and at concentrations ≥ 2810 µg/plate (2nd serie) until the end of the experiment.

Table 1: Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		49 ± 15	120 ± 10	20 ± 4	6 ± 2	26 ± 7
	Test item	5.00	44 ± 11	118 ± 7	29 ± 5	7 ± 2	20 ± 4
		15.8	38 ± 7	131 ± 4	26 ± 11	7 ± 1	21 ± 2
		50.0	42 ± 8	121 ± 17	22 ± 9	5 ± 4	32 ± 4
		158	47 ± 4	126 ± 13	24 ± 6	8 ± 1	20 ± 5
		500	44 ± 13	123 ± 12	20 ± 4	4 ± 2	26 ± 6
		1580	36 ± 6	122 ± 17	22 ± 6	1 ± 0	25 ± 10
		5000	16 ± 3^E	82 ± 11^E	12 ± 2^E	0 ± 0^E	7 ± 5^E
	DAUN	1.00	240 ± 12
	NaN3	2.00		1539 ± 35	891 ± 31
	9-AA	50.0				514 ± 45
	NQO	2.00					114 ± 204
With Activation	H2O		47 ± 9	120 ± 9	17 ± 5	8 ± 1	41 ± 9
	Test item	5.00	52 ± 7	124 ± 8	20 ± 7	7 ± 1	47 ± 10
		15.8	41 ± 15	128 ± 9	24 ± 5	9 ± 9	46 ± 9
		50.0	53 ± 5	115 ± 7	22 ± 1	5 ± 5	29 ± 3
		158	41 ± 7	118 ± 15	19 ± 3	4 ± 3	30 ± 8
		500	43 ± 10	110 ± 8	25 ± 5	6 ± 1	44 ± 4
		1580	42 ± 10	113 ± 13	18 ± 3	9 ± 4	34 ± 7
		5000	14 ± 4^E	66 ± 2^E	17 ± 4^E	0 ± 0^E	16 ± 1^E
	2-AA	2.00	840 ± 41	1280 ± 69
	2-AA	5.00			230 ± 30	354 ± 19
	2-AA	10.0					459 ± 34

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide,E: Precipitation until end of experiment

Table 2: Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		37 ± 8	124 ± 14	31 ± 9	10 ± 4	36 ± 6
	Test Item	158	38 ± 5	133 ± 10	37 ± 6	5 ± 2	34 ± 10
		500	33 ± 7	131 ± 16	37 ± 6	12 ± 3	32 ± 3
		1580	35 ± 1	117 ± 19	29 ± 14	9 ± 3	36 ± 8
		2810	20 ± 5^E	111 ± 10^E	18 ± 9^E	0 ± 0^E	29 ± 3^E
		5000	16 ± 7^E	51 ± 14^E	15 ± 2^E	1 ± 1^E	18 ± 5^E
	DAUN	1.00	271 ± 62
	NaN3	2.00		1606 ± 30	871 ± 26
	9-AA	50.0				823 ± 155
	NQO	2.00					1347 ± 30
With Activation	H2O		53 ± 9	117 ± 11	25 ± 4	10 ± 4	35 ± 11^C
	Test Item	158	40 ± 10	112 ± 19	22 ± 9	10 ± 4	37 ± 12
		500	47 ± 9	113 ± 5	24 ± 7	12 ± 6	34 ± 6
		1580	36 ± 11	102 ± 7	22 ± 2	9 ± 3	37 ± 7
		2810	29 ± 6^E	95 ± 23^E	20 ± 1^E	1 ± 1^E	24 ± 4^E
		5000	0 ± 1^E	47 ± 18^E	14 ± 2^E	1 ± 1^E	16 ± 3^E
	2-AA	2.00	304 ± 14	467 ± 15
	2-AA	5.00			148 ± 19	89 ± 7
	2-AA	10.0					198 ± 14

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide,E: Precipitation until end of experiment, C: Contaminated

Table 3: Historical Data - Negative Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	402	410	397	413	270	282	276	287	385	398
Number of Values	78	79	77	80	45	47	46	48	74	76
Minimum	23	25	82	101	20	18	23	20	16	17
Maximum	45	59	138	157	34	40	36	43	39	48
Mean	34	40	110	126	27	26	28	30	30	36
Standard Deviation	5.2	7.0	11.7	11.9	3.2	3.9	3.5	4.4	4.9	5.9

Table 4: Historical Data - Positive Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3	2-AA	NaN3	2-AA	9-AA	2-AA	NQO	2-AA
Total Plates	201	205	199	207	135	141	138	144	193	199
Number of Values	79	79	77	80	45	47	46	48	74	76
Minimum	117	105	412	512	583	92	122	125	395	112
Maximum	887	1632	2075	3337	1847	390	2882	1103	2286	1313
Mean	352	529	1337	1262	900	200	1175	418	1613	347
Standard Deviation	146.3	296.1	316.8	435.4	193.4	64.2	564.2	201.3	438.7	192.4

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

The test item was examined for its mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from phenobarbital/p-naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.

Treatments of all tester strains were performed using test item formulations prepared in ultra-pure water in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquin-oline-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active.

Precipitation of the test material on the agar plates occurred at concentrations ≥ 2810 µg/plate. Toxicity to the bacteria (drop of revertant numbers) was observed beginning at 1580 µg/plate and in all strains at 5000 µg/plate. The individual and summarized data are presented in the tables section.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix. Therefore, the test item was not mutagenic under the described experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Test

The test item was examined for its mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from phenobarbital/p-naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used.

Treatments of all tester strains were performed using test item formulations prepared in ultra-pure water in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls.

The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquin-oline-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene, which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active.

Precipitation of the test material on the agar plates occurred at concentrations ≥ 2810 µg/plate. Toxicity to the bacteria (drop of revertant numbers) was observed beginning at 1580 µg/plate and in all strains at 5000 µg/plate. The individual and summarized data are presented in the tables section.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix. Therefore, the test item was not mutagenic under the described experimental conditions.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.