Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 29 November 2017. Experimental completion date: 15 December 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997 version
Deviations:
no
GLP compliance:
yes
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being applied to the test system and therefore study integrity is not compromised
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dirubidium oxide
EC Number:
241-993-2
EC Name:
Dirubidium oxide
Cas Number:
18088-11-4
Molecular formula:
ORb2
IUPAC Name:
dirubidium(1+) ion hydroxide
Details on test material:
EC 241-993-2
Specific details on test material used for the study:
The test substance was Rubidium Oxide 45.8 wt% in water. Dirubidium oxide is hygroscopic, highly basic and reacts strongly with water to produce rubidium hydroxide. As dirubidium oxide is chemically unstable and not used in its isolated form, testing has been performed on an aqueous solution of rubidium hydroxide because test material was applied as aqueous solutions.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: S19D031
- Expiration date of the lot/batch: 20 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Method

Species / strainopen allclose all
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:
S9 Microsomal fractions (CD Sprague-Dawley)
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Method): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Experiment 2 (Pre-Incubation Method): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Vehicle / solvent:
Sterile distilled water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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:
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot Number PB/βNF S9 01 October 2017 was used in this study.
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test: S9 5.0 mL, 1.65 M KCl/0.4 M MgCl2 1.0 mL, 0.1 M Glucose-6-phosphate 2.5 mL, 0.1 M NADP 2.0 mL, 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL, Sterile distilled water 14.5 mL.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Bacto agar (lot number 6221620 06/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 45930 12/2017 and 46055 01/2018).
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Rationale for test conditions:
As specified in the guidelines
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 (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate in the absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The mean number of revertant colonies, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 for Experiment 1 (plate incorporation method) and Experiment 2 (pre-incubation method).
In the first experiment (plate incorporation method), the maximum dose level of the test item was 5000 μg/plate (the maximum concentration). Weakened bacterial background lawns were noted in one strain only (TA100 dosed in the absence of S9-mix) at 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawns noted to any of the remaining tester strains in either the absence or presence of S9-mix at any test item dose level.
These results from Experiment 1 were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate. In the second experiment (pre-incubation method), the test item induced a slightly stronger toxic response with weakened bacterial background lawns noted at 5000 μg/plate to TA100 and TA1535 in the absence of S9-mix and to all of the tester strains at the same concentration in the presence of S9-mix. There was no visible reduction in the growth of the bacterial background lawns noted to any of the remaining tester strains (WP2uvrA, TA98 and TA1537 dosed in the absence of S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically meaningful increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix at 5 μg/plate in the second mutation test. However, this response was within the in-house historical vehicle/untreated control values for the bacterial strain and was, therefore, considered of no biological relevance.

Any other information on results incl. tables

Table 1.Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1 (PLATE INCORPORATION)

TA100

TA1535

WP2uvrA

TA98

TA1537

106

15

28

21

8

111

14

18

24

5

102

15

26

25

8

EXPERIMENT 2 (PREINCUBATION)

TA100

TA1535

WP2uvrA

TA98

TA1537

127

21

25

23

13

128

19

10

25

8

128

11

15

23

7

Table 2. Test Results

Mean revertant numbers

Treatment

EXPERIMENT 1 (PLATE INCORPORATION)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Solvent

114

107

15

12

31

26

21

31

10

13

1.5 µg

114

90

11

16

35

22

22

24

13

10

5 µg

111

103

12

10

28

25

16

23

8

8

15 µg

106

104

15

14

30

21

20

23

8

11

50 µg

110

102

13

12

25

22

18

19

13

12

150 µg

107

92

13

11

29

24

19

25

7

5

500 µg

107

97

14

12

31

19

18

29

10

6

1500 µg

103

98

14

11

31

21

20

29

12

9

5000 µg

83

80

12

12

26

25

18

24

10

10

ENNG

523

-

276

-

931

-

-

-

-

-

4NQO

-

-

-

-

-

-

201

-

-

-

9AA

-

-

-

-

-

-

-

-

384

-

2AA

-

1574

-

302

-

216

-

-

-

402

BP

-

-

-

-

-

-

-

226

-

-

Treatment

EXPERIMENT 2 (PREINCUBATION)

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Solvent

102

107

14

11

25

29

18

25

8

10

5 µg

107

96

13

10

21

33

23

26

9

20

15 µg

106

89

19

12

22

28

27

21

15

17

50 µg

110

94

18

9

24

32

24

25

12

15

150 µg

114

91

17

15

25

25

22

31

13

16

500 µg

97

97

14

12

22

24

27

29

16

11

1500 µg

104

104

13

12

25

29

23

22

14

7

5000 µg

104

42

12

4

23

31

18

17

10

6

ENNG

879

-

688

-

800

-

-

-

-

-

4NQO

-

-

-

-

-

-

147

-

-

-

9AA

-

-

-

-

-

-

-

-

132

-

2AA

-

1223

-

244

-

172

-

-

-

258

BP

-

-

-

-

-

-

-

117

-

-

Applicant's summary and conclusion

Conclusions:
Rubidium Oxide was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The Ames test was conducted according to OECD (1997 version) guideline 471 and GLP (1997 version). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation method), the maximum dose level of the test item was 5000 μg/plate (the maximum concentration). Weakened bacterial background lawns were noted in one strain only (TA100 dosed in the absence of S9-mix) at 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawns noted to any of the remaining tester strains in either the absence or presence of S9-mix at any test item dose level.

These results from Experiment 1 were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate. In the second experiment (pre-incubation method), the test item induced a slightly stronger toxic response with weakened bacterial background lawns noted at 5000 μg/plate to TA100 and TA1535 in the absence of S9-mix and to all of the tester strains at the same concentration in the presence of S9-mix. There was no visible reduction in the growth of the bacterial background lawns noted to any of the remaining tester strains (WP2uvrA, TA98 and TA1537 dosed in the absence of S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically meaningful increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix at 5 μg/plate in the second mutation test. However, this response was within the in-house historical vehicle/untreated control values for the bacterial strain and was, therefore, considered of no biological relevance.