Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September 2012 - 6 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.
Qualifier:
according to guideline
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Lanthanum trihydroxide
EC Number:
238-510-2
EC Name:
Lanthanum trihydroxide
Cas Number:
14507-19-8
Molecular formula:
H3LaO3
IUPAC Name:
lanthanum(3+) trihydroxide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: Yellow powder
Storage Conditions: Room temperature in the dark

Method

Target gene:
Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia coli strain.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (10 % liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiment 1: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the purity of the test material. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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
Details on test system and experimental conditions:
- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0 mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli), 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES
A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was toxic only at 5000 μg/plate in TA100 both in the presence and absence of metabolic activation.

DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 2 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 and Table 4 for Experiments 1 and 2, respectively.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material precipitate (fine in appearance) was noted at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Any other information on results incl. tables

Table 2: Spontaneous Mutation Rates (Concurrent Vehicle Controls)

Experiment

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

1

108

18

30

19

11

2

108

26

34

13

10

 

Table 3: Experiment 1

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

-

Solvent

5

15

50

150

500

1500

5000

90

96

104

99

107

100

76

53*

20

20

20

19

17

14

11

9*

29

28

30

26

24

27

22

18*

21

22

18

24

22

15

19

10*

12

13

12

11

11

11

10

7*

+

+

+

+

+

+

+

+

Solvent

5

15

50

150

500

1500

5000

113

111

110

125

110

92

90

63*

15

13

13

12

12

13

9

10*

33

40

32

30

30

33

27

16*

21

21

24

20

25

28

24

14*

16

15

15

13

12

12

12

13*

                                                     Positive Controls

 

 

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

479

367

584

163

598

 

 

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

1883

315

374

161

387

*Precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

 

Table 4: Experiment 2

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

-

Solvent

5

15

50

150

500

1500

5000

107

95

91

81

88

98

102

89*

26

25

21

19

15

22

23

27*

32

28

27

25

28

31

30

25*

17

16

20

14

16

13

16

17*

10

11

10

12

9

12

10

5

+

+

+

+

+

+

+

+

Solvent

5

15

50

150

500

1500

5000

102

103

102

94

96

99

93

92*

13

15

13

13

13

14

14

13*

35

36

36

31

38

34

33

29*

25

25

27

25

24

23

21

21*

13

11

12

13

14

15

12

14*

                                                     Positive Controls

 

 

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

385

259

758

168

725

 

 

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

527

161

242

111

190

*Precipitate

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at seven dose levels, both with and without metabolic activation. The dose levels assessed were 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. However, slight reductions in revertant colony frequency were noted in Experiment 1 (plate incorporation method) at 5000 μg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material precipitate (fine in appearance) was noted at this dose level, though it did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.