Tris[oxalato(2-)]dilutetium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 March 2017 to 13 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22 September 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris[oxalato(2-)]dilutetium

EC Number:

222-328-5

EC Name:

Tris[oxalato(2-)]dilutetium

Cas Number:

3426-45-7

Molecular formula:

C6Lu2O12

IUPAC Name:

4,4'',5,5''-tetraoxo-1',4'-dihydro-1λ³,1''λ³,3λ³,3''λ³-dispiro[1λ³,3λ³-dioxa-2-lutetacyclopentane-2,5'-1λ³-oxa-3λ³-oxa-2-lutetacyclopenta[4,5-d]1λ³-oxa-3λ³-oxa-2-lutetacyclopentane-2',2''-[1λ³,3λ³]dioxa-[2]lutetacyclopentane]-2',2',2',2,2,2-hexakis(ylium)-1',4',1,1'',3,3''-hexaide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

solid: particulate/powder

Details on test material:

Name: Tris[oxalate(2-)]dilutetium

Specific details on test material used for the study:

Correction factor: 1.16

Method

Species / strain

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:

cofactor-supplemented post-mitochondrial fraction (S9 fraction) obtained from the liver of male Wistar rats treated with with phenobarbital and B-naphthoflavone at 80 mg/kg/ day by oral gavage for three consecutive days.

Test concentrations with justification for top dose:

- Preliminary concentration range finding test (Informatory toxicity test): Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
- Test item concentrations in the Mutagenicity tests (Initial Mutation test and Confirmatory Mutation test): Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO and distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). At 100 mg/mL concentration suspension with quick sedimentation was observed in each case. After approximately 4 minutes incubation in an ultrasonic water bath no changes were observed using Distilled water and DMF, however white suspension with slower sedimentation was observed using DMSO. Therefore DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

other:

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine [1] and 2-aminoanthracene [2]

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation for Preliminary Concentration Range Finding Test and Initial Mutation Test); preincubation (Confirmatory Mutation test)

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours
- Incubation: 37°C

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertants per plates are counted manually.

OTHER EXAMINATIONS:
- Other: Visual examination of the plates was performed : precipitation or signs of growth inhibition were recorded and reported.

Rationale for test conditions:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used.

Evaluation criteria:

CRITERIA FOR VALIDITY:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

CRITERIA FOR A NEGATIVE RESPONSE:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Additional information on results:

In the Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.79). There was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5000 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.30).
There was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

Precipitate/slight precipitate was detected on the plates at 5000 and 1581 μg/plate concentrations in the main tests in all examined strains with and without metabolic activation.

Any other information on results incl. tables

Table 1:

Concentration (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated control	Mean	20.7	26.3	88.0	92.3	11.0	12.0	12.3	14.3	49.3	52.0
	MF	1.02	0.89	1.04	1.00	0.94	1.29	1.00	0.88	1.07	1.20
DMSO control	Mean	20.3	29.7	84.3	92.0	11.7	9.3	12.3	16.3	46.0	43.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	91.3	--	14.7	--	--	--	49.3	--
	MF	--	--	1.08	--	1.26	--	--	--	1.07	--
5000	Mean	20.3 P	24.3 P	85.7 P	97.0 P	16.3 P	13.7 P	13.7 P	13.7 P	40.0 P	50.0 P
	MF	1.00	0.82	1.02	1.05	1.40	1.46	1.11	0.84	0.87	1.15
1581	Mean	22.7 SP	30.7 SP	96.0 SP	95.0 SP	16.0 SP	14.3 SP	16.0 SP	17.3 SP	50.7 SP	50.7 SP
	MF	1.11	1.03	1.14	1.03	1.37	1.54	1.30	1.06	1.10	1.17
500	Mean	21.7	33.0	103.3	98.0	16.0	16.0	14.7	14.7	44.7	48.3
	MF	1.07	1.11	1.23	1.07	1.37	1.71	1.19	0.90	0.97	1.12
158.1	Mean	19.3	23.3	100.3	94.3	17.0	15.7	14.3	15.0	44.0	47.0
	MF	0.95	0.79	1.19	1.03	1.46	1.68	1.16	0.92	0.96	1.08
50	Mean	21.3	28.3	102.7	99.7	17.3	15.0	16.0	16.3	36.3	45.3
	MF	1.05	0.96	1.22	1.08	1.49	1.61	1.30	1.00	0.79	1.05
15.81	Mean	21.7	23.0	94.3	95.3	17.3	16.7	14.3	13.3	36.0	47.0
	MF	1.07	0.78	1.12	1.04	1.49	1.79	1.16	0.82	0.78	1.08
NDP (4 µg)	Mean	373.3
	MF	18.36
2AA (2 µg)	Mean		2440.0		2396.0		202.3		202.3
	MF		82.25		26.04		21.68		12.39
2AA (50 µg)	Mean										264.7
	MF										6.11
SAZ (2 µg)	Mean			1193.3		1198.7
	MF			13.07		81.73
9AA (50 µg)	Mean							400.7
	MF							32.49
MMS (2 µL)	Mean									959.3
	MF									19.45

P: Precipitate

SP: Slight precipitate

Table 2:

Concentration (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated control	Mean	20.3	20.7	99.0	99.3	11.0	10.3	10.0	12.7	39.0	44.0
	MF	0.97	1.02	1.11	1.03	0.89	1.03	0.86	1.19	1.06	1.09
DMSO control	Mean	21.0	20.3	89.3	96.0	12.3	10.0	11.7	10.7	36.7	40.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	95.0	--	11.0	--	--	--	39.3	--
	MF	--	--	1.06	--	0.89	--	--	--	1.07	--
5000	Mean	19.0 P	21.7 P	58.0 P	61.7 P	11.0 P	13.0 P	9.3 P	11.3 P	43.3 P	44.0 P
	MF	0.90	1.07	0.65	0.64	0.98	1.30	0.80	1.06	1.18	1.09
1581	Mean	18.3 P	22.7 P	77.7 P	81.0 P	12.3 P	11.7 P	9.7 P	8.3 P	39.3 P	43.0 P
	MF	0.87	1.11	0.87	0.84	1.00	1.17	0.83	0.78	1.07	1.07
500	Mean	20.0	21.3	87.0	78.0	12.0	9.7	9.0	11.0	38.0	44.3
	MF	0.95	1.05	0.97	0.81	0.97	0.97	0.77	1.03	1.04	1.10
158.1	Mean	19.0	21.3	93.7	88.3	13.0	10.0	10.0	12.3	37.7	44.3
	MF	0.90	1.05	1.05	0.92	1.05	1.00	0.86	1.16	1.03	1.10
50	Mean	16.3	24.3	86.7	89.7	11.7	10.7	8.0	10.3	41.7	42.0
	MF	0.78	1.20	0.97	0.93	0.95	1.07	0.69	0.97	1.14	1.04
15.81	Mean	20.3	22.0	86.0	86.7	10.7	10.3	9.0	12.0	39.0	44.0
	MF	0.97	1.08	0.96	0.90	0.86	1.03	0.77	1.13	1.06	1.09
5	Mean	16.7	22.0	87.3	90.0	12.7	11.0	8.0	11.0	40.3	42.07
	MF	0.79	1.08	0.98	0.94	1.03	1.10	0.69	1.03	1.10	1.06
NDP (4 µg)	Mean	404.0
	MF	19.24
2AA (2 µg)	Mean		2390.0		2394.7		210.0		201.3
	MF		117.54		24.94		21.00		18.88
2AA (50 µg)	Mean										216.7
	MF										5.37
SAZ (2 µg)	Mean			1175.3		1210.0
	MF			12.37		110.00
9AA (50 µg)	Mean							392.2
	MF							33.60
MMS (2 µL)	Mean									988.0
	MF									25.12

P: Precipitate

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item tris[oxalate(2-)]dilutetium (Batch Number: 1534498) had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item tris[oxalate(2-)]dilutetium was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli

(Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

Precipitate/slight precipitate was detected on the plates in the main tests in all examined strains with and without metabolic activation at higher concentrations.

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item tris[oxalate(2-)]dilutetium (Batch Number: 1534498) had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.