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EC number: 232-122-7 | CAS number: 7787-59-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria (OECD 471, GLP): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and in E.coli WP2 uvrA with and without metabolic activation (reference 7.61 -5), additionally (similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation RA from source substance 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9), reference (7.6.1 -4)
In vitro mammalian chromosome aberration test (OECD 473, GLP): negative in Chinese hamster lung fibroblasts (V79)
RA from source substances 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9, reference 7.6.1 -1) and bismuth hydroxide nitrate oxide (CAS 1304-85-4, reference 7.6.1 -2)
In vitro mammalian cell gene mutation assay (OECD 476, GLP): negative in L5178Y mouse lymphoma cells (reference 7.6.1 -3)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- / AT reversion site not covered, no historical data provided, no concentrations of positive controls provided, no data on dose-finding study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21 July 1997
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats and male hamsters, treated with Aroclor 1254.
- Test concentrations with justification for top dose:
- main experiment: 3.3, 10, 33, 100, 333, 666 µg/plate with and without metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 4-nitro-o-phenylenediamine (4-NPD), 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: The dose-finding assay and main assay were carried out in triplicates.
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number his+ colonies, clearing of the bacterial background lawn - Evaluation criteria:
- The test article is judged to be mutagenic or weakly mutagenic, if it produced a reproducible dose-related increase in his+ revertants compared to the corresponding solvent control.
- Statistics:
- Mean values and standard error of means were calculated.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- other: no data on historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no data on historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- other: no data on historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no data on historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control at 333.3 and 666.6 μg/ plate without S9 mix (93 and 100%, respectively) and with S9 mix (hamster) and with S9 mix (rat) (60, 100, 91 and 100%, respectively)
- Vehicle controls validity:
- other: no data on historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no data on historical control data given
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- number of revertant colonies decreased to less than half compared to solvent control at 333.3 and 666.6 μg/ plate without S9 mix (67 and 100%, respectively) and at 666.6 μg/ plate with S9 mix (hamster) and S9 mix (rat) (84 and 100%, respectively)
- Vehicle controls validity:
- other: no data on historical control data given
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- other: no data on historical control data given
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 23 May 2012 - 29 April 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- 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.
- Key result
- 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
- Conclusions:
- Interpretation of results:
negative with and 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 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. - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 June 2012 - 09 October 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- / No purity of test substance given.
- 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. No purity of test substance given.
- 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 37C 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.
- Key result
- 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
- Conclusions:
- Interpretation of results:
negative with metabolic activation
negative without metabolic activation
In conclusion, Bismuth subsalicylate test item did not induce 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. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 03 Nov - 05 Dec 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted Jul 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: In Vitro Mammalian Cell Gene Mutation Test using the Hprt gene
- Target gene:
- HPRT locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
MEDIA USED
- Type and identity of media: RPMI 1640 HEPES buffered containing pen/strep, amphotericin B, sodium pyruvate acid and pluronic with and without horse serum (10 or 20%).
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- With and without S9 mix: 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL (3 h)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 13 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: Duplicates each in 1 independent experiment
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival - Evaluation criteria:
- For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF 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. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/- 2 standard deviations).
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.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Remarks:
- at 1000 µg/mL
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed in the range-finding test at the highest concentration analysed (1000 μg/mL) as compared to the concurrent vehicle controls.
- Effects of osmolality: No marked changes in osmolality were observed in the range-finding test at the highest concentration analysed (1000 μg/mL) as compared to the concurrent vehicle controls.
- Precipitation: The test substance precipitated at concentrations ≥ 1000 µg/mL in the cell culture medium.
RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity range-finding test, ten concentrations (3.906, 7.813, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL) were tested in the absence and presence of S9 mix. Upon addition of the test substance to the cultures no evidence of precipitation was observed. However, following the 3 hour treatment period precipitate was observed at the two highest concentrations, therefore the highest concentration analysed was 1000 µg/mL, which gave a relative survival of 134 and 55% with and without metabolic activation, respectively.
HISTORICAL CONTROL DATA
- Positive historical control data:
4-nitroquinoline 1-oxide (0.15 µg/mL), -S9: Mean MF = 40.17; Range: 0.52 - 79.82
4-nitroquinoline 1-oxide (0.15 µg/mL), -S9: Mean MF = 59.96; Range: 15.44 - 104.48
Benzo[a]pyrene (2 µg/mL, +S9): Mean MF = 19.03; Range: 0 - 42.28
Benzo[a]pyrene (3 µg/mL, +S9): Mean MF = 31.75; Range: 2.27 - 61.23
- Negative (solvent/vehicle) historical control data:
-S9: Mean MF = 3.54; Range: 0.33 - 6.75
+S9: Mean MF = 4.22; Range: 1.83 - 6.60 - Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- January 08, 2018 - January 19, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- COUNCIL REGULATION (EC) No 440/2008 of 30 May 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon, trp operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix from Phenobarbital/ßNaphthoflavone- pretreated rats
- Test concentrations with justification for top dose:
- 1st series: 15.8, 50, 158, 500, 1580, and 5000 µg/plate (recommended top dose according to OECD Guideline)
2nd series: 15.8, 50, 158, 500, and 1580 µg/plate (Starting at 500 µg/plate precipitation occurred until end of experiment) - Vehicle / solvent:
- - Vehicle/solvent used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- sodium azide
- other: Daunomycin, 2-Aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3 (test item), 5 (solvent control)
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies; background lawn
- Rationale for test conditions:
- according to Guideline
- Evaluation criteria:
- A test material was to be defined as positive or mutagenic in this assay if:
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if:
- the assay is considered valid and
- none of the above-mentioned criteria are met - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material is completely insoluble in any commonly used solvent. Therefore, each test material concentration was prepared separately directly prior to assay conduct and ultra-pure water was used as vehicle (100 μL/plate).
- Precipitation: Yes, at concentration >= 500 µg/plate
RANGE-FINDING/SCREENING STUDIES: The test material is completely insoluble in any commonly used solvent. Therefore, each test material concentration was prepared separately directly prior to assay conduct and ultra-pure water was used as vehicle (100 μL/plate) and thus negative control for this study. The maximum test concentrations applied were 5000 μg/plate in the 1 st, and 1580 μg/plate in the 2nd experimental series.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA98: -S9 mix: range = 68-779, mean = 243, SD = 134.2; +S9 mix: range = 112 -3015, mean = 738, SD = 508.4
TA100: -S9 mix: range = 821 - 2376, mean = 1550, SD = 213.6; +S9 mix: range = 437 - 3429, mean = 1386, SD = 724.3
TA1535: -S9 mix: range = 351 - 2149, mean = 867, SD = 194.8; +S9 mix: range = 43 - 758, mean = 168, SD = 92.1
TA1537: -S9 mix: range = 247 - 1485, mean = 736, SD = 284.9; +S9 mix: range = 72 - 705, mean = 293, SD = 161.7
WP2 uvrA: -S9 mix: range = 317 - 2275, mean = 1677, SD = 381.9; + S9 mix: range = 154 - 696, mean = 353, SD = 129.2
- Negative (solvent/vehicle) historical control data:
TA98: -S9 mix: range = 19 - 52, mean = 36, SD = 7.1; +S9 mix: range = 20 - 58, mean = 42, SD = 7.3
TA100: -S9 mix: range = 94 - 159, mean = 118, SD = 12.1; +S9 mix: range = 90 - 172, mean = 123, SD = 13.4
TA1535: -S9 mix: range = 15 - 38, mean = 24, SD = 4.9; +S9 mix: range = 6 - 28, mean = 20, SD = 4.0
TA1537: -S9 mix: range = 4 - 11, mean = 8, SD = 1.5; +S9 mix: range = 6 - 16, mean = 10, SD = 2.3
WP2 uvrA: -S9 mix: range = 19 - 44, mean = 30, SD = 4.9; + S9 mix: range = 28 - 47, mean = 37, SD = 4.3 - Conclusions:
- The test item was not mutagenic in the absence and presence of a metabolizing system in the bacterial reverse mutation assay according to OECD Guideline 471.
- Executive summary:
The present study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix).
The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with ß‑Naphthoflavone/Phenobarbital was used.
Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
Following treatment of all bacteria tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.
Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.
Referenceopen allclose all
Table 1: Summary of test results (main assay)
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates) |
||||
Frameshift type |
Base-pair |
||||
TA1537 |
TA98 |
TA100 |
TA1535 |
||
– S9 |
Solvent control (DMSO) |
6 ± 0.3 |
17 ± 1.2 |
130 ± 4.6 |
27 ± 5.1 |
3.3 |
- |
- |
105 ± 2.1 |
- |
|
10 |
6 ± 0.9 |
14 ± 2.7 |
132 ± 9.3 |
26 ± 4.8 |
|
33.3 |
7 ± 3.5 |
17 ± 1.2 |
111 ± 5.8 |
21 ± 2.8 |
|
100 |
4 ± 1.2 |
13 ± 2.1 |
123 ± 9.0 |
18 ± 2.3 |
|
333.3 |
2 ± 0.3 |
12 ± 3.5 |
84 ± 2.9 |
2 ± 1.0 |
|
666.6 |
0 ± 0.0 |
9 ± 0.9 |
- |
0 ± 0.0 |
|
Positive controls |
9AA |
4-NPD |
SAZ |
SAZ |
|
Mean No. of colonies/plate (average of 3 plates) |
614 ± 162.9 |
1085 ± 2.6 |
1615 ± 43.6 |
1320 ± 45.1 |
|
+ 10% S9 (hamster) |
Solvent control (DMSO) |
6 ± 0.9 |
24 ± 6.8 |
108 ± 4.4 |
10 ± 2.7 |
3.3 |
- |
- |
96 ± 2.4 |
- |
|
10 |
6 ± 0.9 |
27 ± 3.5 |
92 ± 2.0 |
31 ± 1.2 |
|
33.3 |
6 ± 0.7 |
28 ± 2.1 |
113 ± 5.8 |
30 ± 1.2 |
|
100 |
6 ± 0.7 |
22 ± 2.1 |
101 ± 8.3 |
5 ± 0.9 |
|
333.3 |
3 ± 0.9 |
20 ± 4.0 |
90 ± 2.0 |
4 ± 1.3 |
|
666.6 |
1 ± 0.3 |
21 ± 1.9 |
- |
0 ± 0.0 |
|
Positive control |
2AA |
||||
Mean No. of colonies/plate (average of 3 plates) |
120 ± 18.2 |
1449 ± 83.9 |
1382 ± 127.5 |
184 ± 8.4 |
|
+ 10% S9 (rat) |
Solvent control (DMSO) |
8 ± 0.7 |
30 ± 3.7 |
107 ± 4.8 |
11 ± 1.0 |
3.3 |
- |
- |
103 ± 2.7 |
- |
|
10 |
7 ± 1.5 |
24 ± 1.9 |
100 ± 0.6 |
9 ± 2.0 |
|
33.3 |
8 ± 1.7 |
23 ± 1.8 |
108 ± 4.5 |
7 ± 1.8 |
|
100 |
8 ± 0.9 |
24 ± 0.6 |
100 ± 8.4 |
7 ± 2.6 |
|
333.3 |
4 ± 1.2 |
18 ± 2.7 |
83 ± 4.3 |
1 ± 0.6 |
|
666.6 |
0 ± 0.0 |
18 ± 3.8 |
- |
0 ± 0.0 |
|
Positive control |
2AA |
||||
Mean No. of colonies/plate (average of 3 plates) |
133 ± 2.7 |
1503 ± 9.5 |
1700 ± 55.2 |
182 ± 6.4 |
|
2AA = 2-aminoanthracene
4-NPD = 4-nitro-o-phenylenediamine
SAZ = sodium azide
9AA = 9-aminoacridine
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.
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.
Table 1: Results of the mutation experiment
| 3 h treatment without metabolic activation | ||
| Concentration (µg/mL) | Relative survival (%) | Mutant frequency |
| 0 | 100 | 5.13 |
| 15.63 | 117 | 4.47 |
| 31.25 | 118 | 4.47 |
| 62.5 | 108 | 4.65 |
| 125 | 99 | 6.56 |
| 250 | 103 | 6.06 |
| 500 | 108 | 3.69 |
| 1000 P | 101 | 6.11 |
| 4-nitroquinoline 1-oxide (0.15 µg/mL) | 31 | 63.61 |
| 4-nitroquinoline 1-oxide (0.2 µg/mL) | 9 | 79.38 |
| 3 h treatment with metabolic activation | ||
| Concentration (µg/mL) | Relative survival (%) | Mutant frequency |
| 0 | 100 | 4.95 |
| 15.63 | 75 | 7.78 |
| 31.25 | 97 | 4.55 |
| 62.5 | 97 | 5.86 |
| 125 | 134 | 7.39 |
| 250 | 99 | 5.32 |
| 500 | 90 | 4.97 |
| 1000 P | 85 | 7.38 |
| Benzo[a]pyrene (0.15 µg/mL) | 68 | 22.30 |
| Benzo[a]pyrene 1-oxide (0.2 µg/mL) | 55 | 39.16 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for read-across
There are data available regarding genetic toxicity (mutagenicity) in mammalian cells for bismuth chloride oxide (CAS 7787-59-9). In addition, read-across from appropriate substances has been conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements, defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).
As no data on genetic toxicity in bacteria and on genetic toxicity (cytogenicity) in mammalian cells are available for bismuth chloride oxide (CAS 7787-59-9), information available for the analogue substances 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9) and bismuth hydroxide nitrate oxide (CAS 1304-85-4) are taken into account to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4.
Genetic toxicity in bacteria (Ames)
CAS 7787-59-9
The study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse mutation test in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with ß‑Naphthoflavone/Phenobarbital was used. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test (reference 7.6.1 -5).
CAS 14882-18-9
The test substance was investigated for mutagenicity to bacteria (Ames test) in a study similar to OECD guideline 471 (Zeiger et al., 1987). The Salmonella typhimurium strains TA1535, TA1537, TA100, TA98 were exposed to concentrations ranging from 3.3 - 666 µg/plate (in DMSO) with and without the addition of a metabolic activation system (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats and male hamsters, treated with Aroclor 1254) in two independent assays with triplicates each (dose finding study and main experiment). The experiments were conducted according to the preincubation methodology. But only the results of the main assay were depicted in the literature. Cytotoxicity was observed in tester strain TA1535 in the absence of S9-mix, where the number of revertant colonies decreased to less than half compared to solvent control at 333.3 and 666.6 μg/plate (93 and 100%, respectively) and in the presence of the hamster S9-mix at 333.3 and 666.6 μg/plate (60 and 100%, respectively) and in the presence of the rat S9-mix at 333.3 and 666.6 μg/plate (91 and 100%, respectively). Cytotoxicity was in addition observed in tester strain TA1537 in the absence of S9-mix at 333.3 and 666.6 μg/plate (67 and 100%, respectively) and in the presence of the hamster or rat S9-mix at 666.6 μg/plate (84 and 100%, respectively). None of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. The reference mutagens showed the expected increase in the number of revertant colonies. A strong deviation in this study is the missing AT site, which makes it impossible to allow a definite prediction on the mutagenic potential of the test substance towards bacteria (reference 7.6.1 -4).
Genetic toxicity (cytogenicity) in mammalian cells in-vitro
CAS 14882-18-9
An in vitro mammalian chromosome aberration test was performed with the source substance 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9) in Chinese hamster lung fibroblasts (V79) according to OECD guideline 473 and GLP in the presence and absence of metabolic activation (reference 7.6.1-1). In the first experiment cells were exposed for 3 h to test substance concentrations of 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL without metabolic activation and to test substance concentrations of 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL with metabolic activation (prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone). In the second experiment cells were exposed for 20 hours to 1.96, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL without metabolic activation and to 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL for 3 h with metabolic activation. Ethylmethanesulphonate and cyclophosphamide were used as positive control substances and increased statistically significant the mean number of aberrant cells indicating the validity of the test and of the activity of the metabolizing system. 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 at 31.25 µg/mL in the second 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 (reference 7.6.1 -1).
CAS 1304-85-4
A second in vitro mammalian chromosome aberration test was performed with the source substance bismuth hydroxide nitrate oxide (CAS 1304-85-4) in Chinese hamster lung fibroblasts (V79) according to OECD guideline 473 and GLP in the presence and absence of metabolic activation (reference 7.6.1-2). In the first experiment cells were exposed for 3 h to test substance concentrations of 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL without metabolic activation and to test substance concentrations of 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL with metabolic activation (prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone). In the second experiment cells were exposed for 20 hours to 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 µg/mL without metabolic activation and to 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL for 3 h with metabolic activation. Ethylmethanesulphonate and cyclophosphamide were used as positive control substances and increased statistically significant the mean number of aberrant cells indicating the validity of the test and of the activity of the metabolizing system. Treatment with the test substance 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. Therefore, with no repeatability, including between replicates, and no dose response, bismuth hydroxide nitrate oxide was not considered to show genotoxic activity (reference 7.6.1 -2).
Genetic toxicity (mutagenicity) in mammalian cells in-vitro
CAS 7787-59-9
An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and GLP was performed with bismuth chloride oxide (CAS 7787-59-9) in L5178Y mouse lymphoma cells (reference 7.6.1-3). The cells were treated for 3 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254). The test substance was tested up to precipitation, the following concentrations were tested: 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. Benzo[a]pyrene and 4-nitroquinoline 1-oxide were used as positive controls with and without S9 mix, respectively. Relative survival was not influenced by the test substance within the concentration range. No statistically significant increases in mutation factor were observed following treatment with the test substance at any concentration tested in the absence and presence of S9 mix and there were no statistically significant linear trends, indicating a clear negative result. Positive and negative controls were valid and within the range of historical control data. Thus, it was concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility under the experimental conditions described (reference 7.6.1 -3).
Conclusion for genetic toxicity
The available data show that bismuth chloride oxide (CAS 7787-59-9) is not mutagenic to L5178Y mouse lymphoma cells. The analogue substance 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882 -18 -9) did not show mutagenic properties in the Ames test. In addition, the available data of the analogue substances 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9) and bismuth hydroxide nitrate oxide (CAS 1304-85-4) revealed no clastogenic properties in Chinese hamster lung fibroblasts in vitro. Therefore, based on common functional groups and structural similarities, bismuth chloride oxide (CAS 7787-59-9) is also considered not to be clastogenic in vitro.
Justification for classification or non-classification
The available data on genetic toxicity with bismuth chloride oxide (CAS 7787-59-9) and the appropriate read-across substances 2-hydroxy-4H-1,3,2-benzodioxabismin-4-one (CAS 14882-18-9) and bismuth hydroxide nitrate oxide (CAS 1304-85-4) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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