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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Experimental work started on 17 March 2010 and was completed on 31 May 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Bismuth hydroxide nitrate oxide
EC Number:
215-136-8
EC Name:
Bismuth hydroxide nitrate oxide
Cas Number:
1304-85-4
IUPAC Name:
1304-85-4
Details on test material:
- Name of test material (as cited in study report): Bismuth hydroxide nitrate oxide (also known as Bismuth subnitrate)
- Molecular formula (if other than submission substance): Bi5O(OH)9(NO3)4
- Molecular weight (if other than submission substance): 1461.99 g/mol
- Physical state: solid, white powder
- Storage condition of test material: stored at 15 to 25C under desiccant, in the dark

Method

Target gene:
hprt locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 media supplemented with 100 units/mL Penicillin, 100 µg/mL Streptomycin, 2.5 µg/mL Amphotericin B, 0.5 mg/mL Pluronic (except for RPMI 20 medium) and with 0%, 10% and 20% v/v heat inactivated horse serum for RPMI A, RPMI 10 and RPMI 20, respectively.

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of frozen cells was purged of HPRT- mutants, checked for spontaneous mutant frequency and confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Range-finder (with and without S9-mix): 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL;
Experiment I (with and without S9-mix): 20, 40, 60, 80, 100, 110, 120, 130 and 140 µg/mL;
Experiment II (with and without S9-mix): 10, 20, 40, 60, 80, 100, 110, 120, 130 and 140 µg/mL.





Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Initial solubility tests with the test item in purified water.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline 1 oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
purified water diluted 10-fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: 2 and 3 µg/mL (dissolved in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours at 37°C with gentle agitation
- Expression time (cells in growth medium): 7 days; cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. At the end of the expression period, cell cultures were plated for 6TG resistance.
- Selection time (if incubation with a selection agent): 12 days; cells were placed into 96 well microtitre plates that were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Each treatment, in the absence or presence of S9-mix, was in duplicate (single cultures only used for positive control treatments).

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment of cell cultures for the cytotoxicity Range Finder Experiment was as described for the Mutation Experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were centrifuged, washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER: From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance:
Experiment I (with and without S9-mix): 0, 40, 80, 100, 110, 120, 130 and 140 µg/mL;
Experiment II (with and without S9-mix): 0, 20, 40, 60, 80, 100, 120, 130 and 140 µg/mL.

Analysis of results:
- Plating efficiency (PE) = P/No of cells plated per well;
- Percentage relative survival (% RS) = [PE (test)/PE (control)] x 100;
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No statistically significant increases in mutant frequency were observed following treatment with bismuth hydroxide nitrate oxide at any concentration tested.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment and Experiment I.
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range Finder Experiment and Experiment I at the highest concentrations tested (100 and 140 µg/mL, respectively), compared to the concurrent vehicle controls (individual data not reported).
- Water solubility: Initial solubility data indicated that bismuth hydroxide nitrate oxide was soluble in water at a concentration of 1 mg/mL. The maximum practicable concentration was 100 µg/mL, based on 10% v/v additions of the test article stock solution into culture medium. A maximum concentration of 100 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to the maximum practicable concentration. However, prior to the Mutation Experiments, further solubility measurements indicated that the test item was soluble in purified water at an increased solubility level of 1.4 mg/mL. The maximum concentration selected for the Mutation Experiments was therefore 140 µg/mL, based on these additional data.

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the (with and without S9-mix) The highest concentration tested was 100 µg/mL, which gave 135% (without S9-mix) and 106% RS (with S9-mix).

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison of controls with historical means.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I concentrations, ranging from 20 to 140 µg/mL, were tested in the absence and presence of S9. Seven days after treatment, concentrations of 20 and 60 µg/mL in the absence and presence of S9 were not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. The highest concentration analysed was 140 µg/mL in the absence and presence of S9, which gave 97% and 74% RS, respectively.
In Experiment II concentrations, ranging from 10 to 140 µg/mL, were tested in the absence and presence of S9. Seven days after treatment, concentrations of 10 and 110 µg/mL were not selected to determine viability and 6TG resistance as there were sufficient non-toxic concentrations. The highest concentration selected was 140 µg/mL in the absence and presence of S9, which gave 96% and 82% RS, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that bismuth hydroxide nitrate oxide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to the maximum practicable concentration, 140 µg/mL (limited by solubility in the primary vehicle), in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).
Executive summary:

Bismuth hydroxide nitrate oxide was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation (S9). A 3-hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 3.125 to 100 µg/mL (limited by solubility in the primary vehicle, purified water). The highest concentration tested, 100 µg/mL in the absence and presence of S9, gave 135% and 106% RS, respectively.

Prior to the Mutation Experiments, further solubility measurements gave an increased solubility of bismuth hydroxide nitrate oxide in purified water at 1.4 mg/mL. Accordingly, for Experiment I 9 concentrations, ranging from 20 to 140 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 140 µg/mL in the absence and presence of S9, which gave 97% and 74% RS, respectively.

In Experiment II, 10 concentrations, ranging from 10 to 140 µg/mL, were tested in the absence and presence of S9. 7 days after treatment, the highest concentrations selected to determine viability and 6TG resistance was 140 µg/mL in the absence and presence of S9, which gave 96% and 82% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals.

In Experiments I and II, no statistically significant increases in mutant frequency were observed following treatment with bismuth hydroxide nitrate oxide at any concentration tested in the absence and presence of S9 and there were no significant linear trends.

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