Dirubidium oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A guideline and GLP compliant bacterial reverse mutation study is available for the submission substance

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:

Experimental start date: 29 November 2017. Experimental completion date: 15 December 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 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

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

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.

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.

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.

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	-	-

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

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.

Justification for classification or non-classification

A negative result was observed for the submission substance in the bacterial reverse mutation assay (Ames test).