Bismuth hydroxide nitrate oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publication.

Justification for type of information:

Please see read-across justification attached below.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The test substance was tested initially at half-log doses up to a dose that elicited toxicity.
Doses: 0, 3.3, 10, 33, 100, 333, 666 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
A maximum of 0.05 mL solvent was added to each plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation; strains TA1535 and TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation; strains TA97 and TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without metabolic activation; strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation; all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Expression time: The histidine-revertant (his+) colonies arising on these plates were counted following 2 days incubation at 37°C.

NUMBER OF REPLICATIONS: At least 5 doses were tested in triplicate. Experiments were repeated at least 1 week following the initial trial.

EVALUATION: The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.

DETERMINATION OF CYTOTOXICITY
- Method: The test substance was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA 100. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn.

No further details are given.

Evaluation criteria:

An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weekly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable if a dose-related increase was judged insufficiently high to justify a call of "+W", if only a single dose was elevated over the control, or if a non-dose-related increase was seen.
A chemical was judged to be mutagenic, or weakly mutagenic, if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable if a reproducible increase of his+ revertants did not meet the criteria for either a mutagenic or weakly mutagenic, or if only single doses produced an increase in his+ revertants in repeat trials.

Statistics:

According to the guideline for a bacterial reverse mutation assay (e.g. Ames test), statistical analysis is not mandatory.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No further details are reported.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Bismuth subsalicylate is non mutagenic in the reverse mutation assay with Salmonella typhimurium, tested with doses up to 666µg/plate.

Executive summary:

Bismuth subsalicylate is non mutagenic in the reverse mutation assay with Salmonella typhimurium, tested with doses up to 666µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publication.

Justification for type of information:

Please see read-across justification attached below.

Qualifier:

no guideline followed

Principles of method if other than guideline:

After incubation of bacteria with the test substance for 2 hours at 37 °C, the cells were lysed and the activity of the ß galactose enzymes and the phosphatase alkaline enzymes were determined.

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Target gene:

not applicable

Species / strain / cell type:

E. coli, other: K12

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Lac Z gene

Metabolic activation:

not specified

Test concentrations with justification for top dose:

0.03 - 100 nmol/tube

Vehicle / solvent:

no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours at 37°C

NUMBER OF REPLICATIONS: no data

EVALUATION: After incubation the bacteria were lysed and the activity of the ß galactose enzymes and the phosphatase alkaline enzymes were determined.

DETERMINATION OF CYTOTOXICITY
no data

Evaluation criteria:

no data

Statistics:

no data

Species / strain:

E. coli, other: K12

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic dose: 3 nmol/tube

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No further details are given.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

According to the reference, bismuth (III) trinitrate is not mutagenic in an in vitro mutation test in E. coli bacteria, tested up to 100 nmol/tube.

Executive summary:

According to the reference, bismuth (III) trinitrate is not mutagenic in an in vitro mutation test in E. coli bacteria, tested up to 100 nmol/tube.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2012 to 29 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline with GLP certificate

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

These deviations were considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 ug/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically "cleansed" against high spontaneous background: no information

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced Rat Liver Homogenate S9 Fraction

Test concentrations with justification for top dose:

In Chromosome Aberration Assay 1, The examined concentrations of the test item were 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL in the experiment without metabolic activation; and 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.
In Chromosome Aberration Assay 2, The examined concentrations of the test item were 1250, 625, 312.5, 156.25, 78.13, 39.06, 19.53 and 9.77 µg/mL in the experiment without metabolic activation; and 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Based on the preliminary solubility test, the test item was insoluble in Distilled water; however, the formulation at 500 mg/mL concentration using was suitable for the test (continuous stirring was applied). As Distilled water is compatible to the test system, it was selected as vehicle for the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

without metabolic activation: ethylmethanesulphonate, with metabolic activation: cyclophosphamide monohydrate

Details on test system and experimental conditions:

Formulation

Stock formulations of the test item (500 mg/mL in the preliminary experiment; 500, 250 or 100 mg/mL in the main tests) were prepared as follows. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of vehicle (Distilled water) was added and the formulation was stirred until homogeneity was reached, then the volume was adjusted to the final volume.

From the stock formulation (continuous stirring was applied) several dilutions were prepared using the selected vehicle filtered sterile using a 0.22 µm syringe filter (Supplier: Millipore, Lot No.: R1SA57172 / R1NA26913 / R1JA31763, Expiry date: December 2014 / October 2014 / July 2015). The dosing solutions were prepared right before the treatment of the cells and were kept protected from light.

Analytical determination of the test item concentration, stability and homogeneity was not performed because of the nature and duration of the study.
NEGATIVE AND POSITIVE CONTROLS

Negative (vehicle) and positive controls were included in the experiments. Furthermore, untreated controls were also included in the preliminary experiments. Routine safety precautions (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.

Negative Control

Negative (vehicle) control was run concurrently with treatment groups.

Name: Distilled water
Supplier: TEVA Co.
Lot No.: 3450611
Appearance: Colourless liquid
Expiry date: 30 June 2014
Storage conditions: Room temperature

Positive Controls

Without metabolic activation

Name (Abbreviation): Ethyl methanesulfonate (EMS)
Synonym: Ethyl methanesulphonate
Supplier: Sigma-Aldrich Co.
Lot No.: BCBF0736V / BCBG1395V
Appearance: Colourless liquid
Expiry date: 22 November 2012 / 17 July 2013*
Storage conditions: Room temperature, under N2

*Note: Lot BCBF0736V was used in Assays 1-2; lot BCBG1395V was used in Assay 3.

Ethyl methanesulfonate, a known mutagen and clastogen, was dissolved in DMEM and was used as a positive control for the non-activation experiments at a final concentration of 0.4 µL/mL (28-hour harvesting time) or 1.0 µL/mL (20-hour harvesting time).

With metabolic activation

Name (Abbreviation): Cyclophosphamide monohydrate (CP)
Supplier: Sigma-Aldrich Co.
Lot No.: 079K1509 / 120M1253V*
Appearance: White powder
Expiry date: 31 July 2012 / 31 December 2013
Storage conditions:Refrigerated (2-8°C)

*Note: Lot 079K1509 was used in Assay 1; lot 120M1253V was used in Assay 2.

Cyclophosphamide, a clastogenic agent that requires metabolic transformation by microsomal enzymes, was dissolved in sterile physiological saline solution (0.9% NaCl infusion) for treatment and was used as a positive control item for the activation experiments at a final concentration of 6.0 µg/mL.

Positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 µm syringe filter before use.

INDICATOR CELLS

V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 05F013 / 10H016
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 degrees C. The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.

Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37  0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 ug/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).

EXTERNAL METABOLIC ACTIVATION SYSTEM

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).

In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

Induction of Rat Liver Enzymes

Male Wistar rats (298-354 g, animals were 8 weeks old at the initiation) were treated with Phenobarbital (PB) and beta-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hour before sacrifice when food was removed. Initiations of the induction of liver enzymes used for preparation S9 used in this study were 05 December 2011.

Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.

Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 +/- 10ºC.

The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 34.4 g/L. The date of preparation of S9 fraction used in this study 08 December 2011 (CiToxLAB code: E11250). The sterility of the preparation was confirmed.

The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9-mix

The complete S9-mix was freshly prepared on the day of use according to the following ratio:

S9 fraction 3 mL
HEPES 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL

Prior to addition to the culture medium the S9-mix was kept in an ice bath.

For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5%.

TEST PROCEDURE

Toxicity and Concentration Selection

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test. In this Preliminary Toxicity Test, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time. The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.

At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as % relative survival.

Chromosome Aberration Assays

The Chromosome Aberration Assays were conducted as three independent experiments (Assay 1, Assay 2 and Assay 3). Assays 1 and 2 were performed in the presence and in the absence of metabolic activation; the complementary experiment of Assay 3 was performed in the absence of metabolic activation.

Treatment of the Cells

For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37 degrees C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls. After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.

Cells were treated with different concentration test item solutions, negative (vehicle) or positive control solution (treatment volume: 100 µL/dish in all cases) for the given period of time at 37 degrees C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.

Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.

Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality can was also performed at the end of the treatment period in both main tests.

For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as % relative survival.

For an additional measurement of cytotoxicity, mitotic index (MI) was determined on the evaluated slides of Assay 3.

Preparation of Chromosomes

2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 µg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells* was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted. At least two slides (Assays 1 and 2) or four slides (Assay 3) were prepared for each culture.

*Note. Fixed cells were stored frozen in case if any additional slide dropping is required (as documented in the raw data and reported).

Examination of Slides

The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.

The metaphase analysis was conducted under the control of the Principal Investigator. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.

At least one hundred metaphases* with 22+/-2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Where there were insufficient metaphases in one replicate, the total was made up to 200 cells examined per concentration using the other replicate. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

*Note: The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defined in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.

Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.

The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.

Mitotic index (which evaluates the proportion of the cells in mitosis and is a measure of the actively proliferating cells in a cell culture) was also measured by counting a total of 1000 cells in interphase and mitosis for each code assessed for chromosome aberrations in Assay 3. For this step, two slides of a culture were used and 500 cells were evaluated from each slide.

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below for details

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL in the experiment without metabolic activation; and 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-625 µg/mL concentration range without metabolic activation and in the 2500-312.5 µg/mL concentration range with metabolic activation. There were no large changes in the pH or osmolality. Cytotoxicity was observed at 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL concentrations without metabolic activation (relative survival values were 6, 4, 10, 9, 18 and 32 %, respectively); and at 2500, 1250 and 625 µg/mL concentrations with metabolic activation (relative survival values were 33, 34 and 50 %, respectively).Therefore, concentrations of 156.25, 78.13 and 39.06 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 1250, 625, 312.5 and 156.25 µg/mL (a total of four) were chosen for evaluation in case of the experiment with metabolic activation. Based on the observed results (low number of analyzed cells at 156.25 µg/mL without metabolic activation), the slides of 312.5 µg/mL without metabolic activation were also scored, but no cells were available for metaphase analysis at this concentration. Furthermore, two additional concentrations (78.13 and 39.06 µg/mL) were scored for the experiment with metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1250, 625, 312.5, 156.25, 78.13, 39.06, 19.53 and 9.77 µg/mL in the experiment without metabolic activation; and 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium at 1250 and 625 µg/mL concentrations without metabolic activation and in the 2500-312.5 µg/mL concentration range with metabolic activation, and no large changes in the pH and osmolality were observed. Cytotoxicity was also observed at 1250, 625, 312.5, 156.25 and 78.13 µg/mL concentrations without metabolic activation (relative survival values were 2, 9, 15, 30 and 46%, respectively); and at 2500, 1250 and 625 µg/mL concentrations with metabolic activation (relative survival values were 22, 28 and 39 %, respectively). Therefore, concentrations of 78.13, 39.06 and 19.53 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 625, 312.5, 156.25, 78.13 and 39.06 µg/mL (a total of five) were chosen for evaluation in case of the experiment with metabolic activation.

In Assay 1 in experiment without metabolic activation, insufficient numbers of cells were available for metaphase analysis at the highest test item treated concentrations. An additional experiment (Assay 3) was performed without metabolic activation to complete data and provide valid results for the study. Experimental conditions of Assay 3 were the same as in Assay 1, but more closely spaced concentration range was used in this complementary experiment.

In Chromosome Aberration Assay 3, a 3-hour treatment without metabolic activation (in the absence of S9-mix) was performed. Sampling was performed 20 hours after the beginning of the treatment. The examined concentrations of the test item were 300, 200, 150, 100, 75, 50, 25, 12.5 and 6.25 µg/mL without metabolic activation.

In Assay 3, no insolubility was detected at the end of the treatment period in the final treatment medium, and no large changes in the pH and osmolality were observed. Cytotoxicity was observed at 300 µg/mL concentration without metabolic activation (relative survival value: 39%).Therefore, concentrations of 300, 200, 100, 75 and 50 µg/mL (a total of five) were chosen for evaluation. Based on the observed results (low number of analyzed cells at 300 and 200 µg/mL without metabolic activation), the slides of 150 µg/mL without metabolic activation were also scored.

None of the examined treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in Assay 1, Assay 2 and Assay 3 with or without metabolic activation.

The occurrence of polyploid and endoreduplicated metaphases is presented in Tables 10-12 of Appendix 7. Polyploid metaphases (1-5) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were found in the examined samples except of one endoreduplicated metaphase in Assay 1 at 78.13 µg/mL with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

VALIDITY OF THE STUDY

 

The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility and cytotoxicity was detected in the performed main experiments. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered a concentration range from cytotoxicity (less than 50 % relative survival) to no or little cytotoxicity as measured by relative survival or mitotic index*. The lowest examined concentration showed no insolubility either.

*Note: In Assay 3, insufficient number of cells were available for metaphase analysis at 300 and 200 µg/mL concentrations (15 and 54 cells in total, respectively) The relative survival value (measured by cell counting) of the first analyzed concentration of 150 µg/mL was 69%, higher than the required 50% survival for acceptable toxicity, although the number of analysable metaphases on the slides was clearly reduced. An additional measure of toxicity, mitotic index, which evaluates actively proliferating cells, was assessed for this assay. The mitotic index at 150 µg/mL was 50.6% that of the vehicle control, indicating that an acceptable level of toxicity was induced at this concentration.

 

The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the historical control range of the testing laboratory.

 In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected biologically relevant and statistically significant increase in the number of cells with structural chromosome aberrations except in one case*.

 

*Note: In Assay 3 in the experiment without metabolic activation, the observed increase in the aberration frequency of thepositive control substance compared to the negative control value was statistically not significant. However, the pooled data of Assay 1 and 3 when the experimental conditions were identical (3-hr treatment without metabolic activation) showed a statistically significant increase in the chromosome aberrations for. Furthermore,there was a good response using this positive control substance in Assay 2 using 20-hour treatment without metabolic activation. Therefore, the observed values were considered to be acceptable.

 

At least three test item concentrations were examined in each assay.

 

The study was considered to be valid.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item Bismuth subnitrate was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and three Chromosome Aberration Assays.

The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.

Treatment with the test item did not result in a statistically and biologically significant, increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/beta-naphthoflavone induced rats.

In conclusion, Bismuth subnitrate test item did not induced a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, Bismuth subnitrate is considered not clastogenic in this test system.

Executive summary:

Bismuth subnitrate was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Distilled water and it was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (vehicle) and positive control sample if possible.

 

In this study higher concentrations were associated with frank cytotoxicity (low survival of cells in media) but additionally at some concentrations below this, there was no or low mitotic activity. Only concentrations below these effects could be scored for mitotic damage.

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test itemwere 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 µg/mL in the experiment without metabolic activation; and2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-625 µg/mL concentration range without metabolic activation and in the 2500-312.5 µg/mL concentration range with metabolic activation. There were no large changes in the pH or osmolality. Cytotoxicity was observed at 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL concentrations without metabolic activation (relative survival values were 6, 4, 10, 9, 18 and 32 %, respectively with no metaphases at 312.5 µg/mL); and at 2500, 1250 and 625 µg/mL concentrations with metabolic activation (relative survival values were 33, 34 and 50 %, respectively with insufficient number metaphases at 1250 or 625 µg/mL). Therefore, concentrations of 156.25, 78.13 and 39.06 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 312.5, 156.25, 78.13 and 39.06 µg/mL (a total of four) were chosen for evaluation in case of the experiment with metabolic activation.

In Chromosome Aberration Assay 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test itemwere 1250, 625, 312.5, 156.25, 78.13, 39.06, 19.53 and 9.77 µg/mL in the experiment without metabolic activation; and2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 experiment with metabolic activation.

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium at 1250 and 625 µg/mL concentrations without metabolic activation and in the 2500-312.5 µg/mL concentration range with metabolic activation, and no large changes in the pH and osmolality were observed.

Cytotoxicity was also observed at 1250, 625, 312.5, 156.25 and 78.13 µg/mL concentrations without metabolic activation (relative survival values were 2, 9, 15, 30 and 46%, respectively); and at 2500, 1250 and 625 µg/mL concentrations with metabolic activation (relative survival values were 22, 28 and 39 %, respectively). Therefore, concentrations of 78.13, 39.06 and 19.53 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 625, 312.5, 156.25, 78.13 and 39.06 µg/mL (a total of five) were chosen for evaluation in case of the experiment with metabolic activation.

In Assay 1 in experiment without metabolic activation, insufficient numbers of cells were available for metaphase analysis at the highest test item treated concentrations
(due to toxicity of the test item). An additional experiment (Assay 3) was performed without metabolic activation to complete data and provide valid results for the study.

In Chromosome Aberration Assay 3, a 3-hour treatment without metabolic activation (in the absence of S9-mix) was performed. Sampling was performed 20 hours after the beginning of the treatment. The examined concentrations of the test itemwere 300, 200, 150, 100, 75, 50, 25, 12.5 and 6.25 µg/mL without metabolic activation.

In Assay 3, no insolubility was detected at the end of the treatment period in the final treatment medium, and no large changes in the pH and osmolality were observed. Cytotoxicity was observed at 300 µg/mL concentration without metabolic activation (relative survival value: 39%) and insufficient mitotic activity was found at 300 and 200 µg/mL. Therefore, concentrations of 150, 100, 75 and 50 µg/mL (a total of four) were evaluated.

None of the examined treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in Assays 1-3 with or without metabolic activation.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-5) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were found in the examined samples except of one endoreduplicated metaphase in Assay 1 at 78.13 µg/mL concentrationwith metabolic activation.

The negative (vehicle) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. At least three test item treated concentrations which were maximal without excessive cytotoxic effects were evaluated in each assay. The tests were considered to be valid.

In conclusion, Bismuth subnitrate test item did not induced a significant level of chromosome aberrations in the performed experiments with or without metabolic activation.Therefore, Bismuth subnitrate is considered not clastogenic in this test system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

28 June 2012 to 09 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline with GLP certificate. Rated as Klimisch 2 because it is a read-across study.

Justification for type of information:

Please see read-across justification attached below.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Due to technical reason, cells were maintained on 150 cm2 flask instead of 75 cm2 flasks before the experiments.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically "cleansed" against high spontaneous background: no information

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced Rat Liver Homogenate S9 Fraction

Test concentrations with justification for top dose:

In Chromosome Aberration Assay 1, the examined concentrations of the test item were 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL in the experiment with metabolic activation; and 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment without metabolic activation.
In Chromosome Aberration Assay 2, the examined concentrations of the test item were 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment with metabolic activation; and 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 and 1.96 µg/mL in the experiment without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the preliminary solubility test, the test item was insoluble in Distilled water; however, the formulation at 125 mg/mL concentration using DMSO as vehicle was suitable for the test (continuous stirring was applied). As DMSO is compatible with the test system, it was selected for vehicle of the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

without metabolic activation: ethylmethanesulphonate, with metabolic activation: cyclophosphamide monohydrate

Details on test system and experimental conditions:

Formulation

Stock formulations of the test item (125 mg/mL in the preliminary experiment, 100 mg/mL in the main tests) were prepared as follows. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of vehicle (DMSO) was added and the formulation was stirred until homogeneity was reached, then the volume was adjusted to the final volume.

From the stock formulation (continuous stirring was applied) several dilutions were prepared using the selected vehicle filtered sterile using a 0.22 µm syringe filter (Supplier: Millipore, Lot No.: R1SA57172, Expiry date: December 2014; and Lot No.: R1NA26913, Expiry date: October 2014). The dosing solutions were prepared right before the treatment of the cells and were kept protected from light.

NEGATIVE AND POSITIVE CONTROLS

Negative (vehicle/solvent) and positive controls were included in the experiments. Furthermore, untreated controls were also included in the preliminary experiments. Routine safety precautions (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety

Negative Control

Negative (solvent) control was run concurrently with treatment groups.

Name (Abbreviation): Dimethyl sulfoxide (DMSO)
Supplier: Sigma-Aldrich Co.
Lot No.: BCBF1944V
Appearance: Colourless liquid
Expiry date: January 2017
Storage conditions: Room temperature, under N2

Positive Controls

Without metabolic activation

Name (Abbreviation): Ethyl methanesulfonate (EMS)
Synonym: Ethyl methanesulphonate
Supplier: Sigma-Aldrich Co.
Lot No.: BCBF0736V
Appearance: Colourless liquid
Expiry date: 22 November 2012
Storage conditions: Room temperature, under N2

Ethyl methanesulfonate, a known mutagen and clastogen, was dissolved in DMEM and was used as a positive control for the non-activation experiments at a final concentration of 0.4 µL/mL (28-hour harvesting time) or 1.0 µL/mL (20-hour harvesting time).

With metabolic activation

Name (Abbreviation): Cyclophosphamide monohydrate (CP)
Supplier: Sigma-Aldrich Co.
Lot No.: 120M1253V /
Appearance: White powder
Expiry date: 31 December 2013
Storage conditions: Refrigerated (2-8°C)

Cyclophosphamide, a clastogenic agent that requires metabolic transformation by microsomal enzymes, was dissolved in sterile physiological saline solution (0.9% NaCl infusion) for treatment and was used as a positive control item for the activation experiments at a final concentration of 6.0 µg/mL.

INDICATOR CELLS

V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 05F013
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 degrees C. The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.

Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 150 cm2 plastic flasks at 37  0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).

EXTERNAL METABOLIC ACTIVATION SYSTEM

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).

In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.

The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

The supplier, batch number and expiry date of the used chemicals described in this section are summarized in Table 1 (Section 5.7. Chemicals Used in the Experiments). The composition of solution refers to 1000 mL.

Induction of Rat Liver Enzymes

Male Wistar rats (298-354 g, animals were 8 weeks old at the initiation) were treated with Phenobarbital (PB) and -naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hour before sacrifice when food was removed. Initiations of the induction of liver enzymes used for preparation S9 used in this study were 05 December 2011.

Preparation of Rat Liver Homogenate S9 Fraction

On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.

Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 +/- 10ºC.

The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 34.4 g/L. The date of preparation of S9 fraction used in this study 08 December 2011 (CiToxLAB code: E11250). The sterility of the preparation was confirmed.

The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9-mix

The complete S9-mix was freshly prepared on the day of use according to the following ratio:

S9 fraction 3 mL
HEPES 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL

Prior to addition to the culture medium the S9-mix was kept in an ice bath.

For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5%.

TEST PROCEDURE

Toxicity and Concentration Selection

Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test. In this Preliminary Toxicity Test, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time. The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.

At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.

Chromosome Aberration Assays

The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL in the experiment with metabolic activation; and 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment without metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment with metabolic activation; and 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 and 1.96 µg/mL in the experiment without metabolic activation.

Treatment of the Cells

For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37 degrees C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls. After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.

Cells were treated with different concentration test item solutions, negative (solvent) or positive control solution (treatment volume: 100 µL/dish in all cases) for the given period of time at 37C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.

Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.

Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality can was also performed at the end of the treatment period in both main tests.

For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.

Preparation of Chromosomes

2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 µg/mL). The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.

Examination of Slides

The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.

When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.

At least one hundred metaphases* with 22+/-2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

*Note: The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

The aberrations are defined in the following way:

Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage.

Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.

The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium at some of the highest concentrations, and there were no large changes in the pH or osmolality. Cytotoxicity was observed at 500, 250, 125, 62.5 and 31.25 µg/mL concentrations without metabolic activation (relative survival values were 6, 10, 11, 17 and 46 %, respectively); and at 1000, 500, 250, 125 and 62.5 µg/mL concentrations with metabolic activation (relative survival values were 20, 29, 34, 34 and 44 %, respectively).Therefore, concentrations of 31.25, 15.63 and 7.81 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 62.5, 31.25 and 15.63 µg/mL (a total of three) were chosen for evaluation in case of the experiment with metabolic activation.

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium at some of the highest concentrations, and no large changes in the pH and osmolality were observed. Cytotoxicity was also observed at 250, 125, 62.5 and 31.25 µg/mL concentrations without metabolic activation (relative survival values were 3, 7, 21 and 36%, respectively); and at 500, 250 and 125 µg/mL concentrations with metabolic activation (relative survival values were 19, 36 and 45 %, respectively). Therefore, concentrations of 31.25, 15.63 and 7.81 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 125, 62.5, 31.25 and 7.81 µg/mL (a total of four) were chosen for evaluation in case of the experiment with metabolic activation.

None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation, except a weak response in one replicate of 31.25 µg/mL in Assay 2 in the experiment with metabolic activation. Higher concentrations (62.5 and 125 µg/mL) showed no effect and no dose response was observed. Therefore, with no repeatability, including between replicates, and no dose response, bismuth subsalicylate was not considered to show genotoxic activity.

Polyploid metaphases* were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. Endoreduplicated metaphases (1-2) were found in Assay 1 in some cases, no endoreduplicated metaphases were found in the samples of Assay 2.

*Note: Increased numbers of polyploid metaphases (up to 20 metaphases) were detected in Assay 2 (using the longer harvesting period) for some test item treated samples. Data suggest a treatment related effect especially in the experiment without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

VALIDITY OF THE STUDY

 

The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility and cytotoxicity was detected in the performed main experiments. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered a concentration range from cytotoxicity (less than 50 % relative survival) to no or little cytotoxicity, and the lowest examined concentration showed no insolubility either.

 

The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the historical control range of the testing laboratory.

 

In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected biologically relevant and statistically significant increase in the number of cells with structural chromosome aberrations in each assay demonstrating the sensitivity of the test system.

 

At least three test item concentrations were examined in each assay.

 

The study was considered to be valid.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, Bismuth subsalicylate test item did not induced a significant level of chromosome aberrations on a repeatable, dose-dependent way in the performed experiments with or without metabolic activation. Therefore, Bismuth subsalicylate is considered not clastogenic in this test system.

Executive summary:

Bismuth subsalicylate was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was dissolved in DMSO and it was examined up to the cytotoxic concentrations according to the relevant OECD guideline covering the range from cytotoxicity to no or little cytotoxicity. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (solvent) and positive control sample.

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test itemwere 1000, 500, 250, 125, 62.5, 31.25, 15.63 and 7.81 µg/mL in the experiment with metabolic activation; and500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment without metabolic activation.

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium at some of the highest concentrations, and there were no large changes in the pH or osmolality. Cytotoxicity was observed at 500, 250, 125, 62.5 and 31.25 µg/mL concentrations without metabolic activation (relative survival values were 6, 10, 11, 17 and 46 %, respectively); and at 1000, 500, 250, 125 and 62.5 µg/mL concentrations with metabolic activation (relative survival values were 20, 29, 34, 34 and 44 %, respectively). Therefore, concentrations of 31.25, 15.63 and 7.81 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 62.5, 31.25 and 15.63 µg/mL (a total of three) were chosen for evaluation in case of the experiment with metabolic activation.

In Chromosome Aberration Assay 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 µg/mL in the experiment with metabolic activation; and250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 and 1.96 µg/mL in the experiment without metabolic activation.

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium at some of the highest concentrations, and no large changes in the pH and osmolality were observed. Cytotoxicity was also observed at 250, 125, 62.5 and 31.25 µg/mL concentrations without metabolic activation (relative survival values were 3, 7, 21 and 36%, respectively); and at 500, 250 and 125 µg/mL concentrations with metabolic activation (relative survival values were 19, 36 and 45 %, respectively). Therefore, concentrations of 31.25, 15.63 and 7.81 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 125, 62.5, 31.25 and 7.81 µg/mL (a total of four) were chosen for evaluation in case of the experiment with metabolic activation.

None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation, except a weak response in one replicate of 31.25 µg/mL in Assay 2 in the experiment with metabolic activation. Higher concentrations (62.5 and 125 µg/mL) showed no effect and no dose response was observed. Therefore, with no repeatability, including between replicates, and no dose response, bismuth subsalicylate was not considered to show genotoxic activity.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Treatment related increase in the number of polyploid cells was seen under the conditions of this in vitro study (particularly without metabolic activation using the longer harvesting period). No effect in the number of endoreduplicated metaphases was observed.

 

The solvent control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. At least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, Bismuth subsalicylate test item did not induced a repeatable, significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, Bismuth subsalicylate is considered not clastogenic in this test system.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

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

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

Target gene:

hprt locus

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.

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.

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'.

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 oxideat any concentration tested in the absence and presence of S9 and there were no significant linear trends.

Additional information

In a mammalian gene mutation test, bismuth subnitrate 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).

In addition, bismuth subnitrate was tested in vitro in a chromosome aberration assay using Chinese hamster V79 lung cells according to OECD Guideline 473. Bismuth subnitrate did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation.Therefore, bismuth subnitrate is considered not clastogenic in this test system.

Read-across substance, bismuth subsalicylate was not mutagenic in the reverse mutation assay (Ames test) with Salmonella typhimurium, tested at doses up to 666 µg/plate.

Read-across substance, bismuth (III) trinitrate, was not mutagenic in an in vitro mutation test in E. coli bacteria, tested up to 100 nmol/tube.

Read-across substance, bismuth subsalicylate was tested in vitro in a chromosome aberration assay using Chinese hamster V79 lung cells according to OECD Guideline 473. Bismuth subsalicylate did not induce a significant level of chromosome aberrations in a repeatable, dose-dependent way in the performed experiments with or without metabolic activation. Therefore, bismuth subsalicylate is considered not clastogenic in this test system.

Justification for selection of genetic toxicity endpoint
No one study is selected as 5 studies are available with negative results.

Short description of key information:
Two studies have been conducted with the test substance. A mammalian gene mutation study with bismuth subnitrate was negative and bismuth subnitrate did not induce a significant level of chromosome aberrations in vitro in a chromosome aberration assay using chinese hamster V79 lung cells conducted in accordance with OECD Guideline 473.

In addition, the results of a bacterial gene mutation test with read-across substance, bismuth trinitrate, and an Ames test with read-across substance, bismuth subsalicylate, were negative. Read-across substance, bismuth subsalicylate did not induce a significant level of chromosome aberrations in a chromosome aberration assay using chinese hamster V79 lung cells conducted in accordance with OECD Guideline 473. Bismuth subsalicylate is therefore considered not to be clastogenic in this test system. A read-across approach to these bismuth substances is considered acceptable due to the presence of the common bismuth component.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Bismuth subnitrate is not classified for genetic toxicity based on the negative results of a mammalian gene mutation study and a chromosome aberration test with bismuth subnitrate and on negative results of tests with closely related read-across substances (Ames test (OECD Guideline 471) and a chromosome aberration test (OECD Guideline 473) with bismuth subsalicylate and a bacterial gene mutation test with bismuth trinitrate).