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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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

Constituent 1
Chemical structure
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:
[mu-ethanedioato(2-)-kappaO~1~:kappaO~2~][bis(ethanedioato-kappaO~1~)]dilutetium(2+)
Constituent 2
Chemical structure
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 resultsopen allclose all
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
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.