Slags, steelmaking, vanadium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2009-11-04 to 2010-01-29.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1314-34-7

Cas Number:

1314-34-7

IUPAC Name:

1314-34-7

Constituent 2

Reference substance name:

Divanadium trioxide

EC Number:

215-230-9

EC Name:

Divanadium trioxide

IUPAC Name:

215-230-9

Details on test material:

- Name of test material (as cited in study report): Divanadium trioxide
- Analytical purity: > 99 %
- Composition of test material, percentage of components: 67.93 % vanadium content
- Lot/batch No.: MO82409
- Expiration date of the lot/batch: January 2012
- Physical state: solid, black powder
- Storage condition of test material: stored at room temperature (nominally 15-25ºC) in the dark, under desiccant conditions

Method

Target gene:

hprt locus (i.e. hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk +/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance are stored as frozen stocks in liquid nitrogen.
- Type and identity of media: RPMI 1640 medium containing 100 units/mL Penicillin, 100 µg/mL Streptomycin, 2.5 µg/mL Amphotericin B, 0.5 mg/mL Pluronic (except for RPMI 20 with no Pluronic) and heat inactivated horse serum (0%, 10% or 20% (v/v) for RPMI A, RPMI 10 or RPMI 20, respectively).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of cells was checked that it was mycoplasma free.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes; each batch of cells was checked for spontaneous mutant frequency.
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. When the cells were growing well, subcultures were established in an appropriate number of flasks.

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): 46.88, 93.75, 187.5, 375, 750 and 1500 µg/mL.
Experiment I (with and without S9-mix): 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL;
Experiment II (with and without S9-mix): 0.25, 0.50, 1, 2, 4, 6, 8, 15, 25 and 50 µg/mL.

Cultures selected for mutation assessment:
Experiment I (with and without S9-mix): 0, 0.1953, 0.3906, 0.7813, 1.563, 3.125 and 6.25 µg/mL;
Experiment II (with and without S9-mix): 0, 1, 2, 4, 6, 8, 15 and 25 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that divanadium trioxide was not soluble in dimethyl sulphoxide but formed a homogeneous suspension in purified water with warming to approximately 60ºC at 5 to 14 mg/mL which was considered suitable for dosing.

Controls

Controls 1

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: 2.0 and 3.0 µg/mL (dissolved in DMSO)

Controls 2

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours’ incubation at 37±1ºC with gentle agitation
After exposure, cells were centrifuged, washed and resuspended in 20 mL RPMI 10 medium. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival (scored after 7 days incubation).
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. From observations on recovery and growth of the cultures during the expression period, the cultures were selected to be plated for
viability and 6TG resistance.
- Selection time (if incubation with a selection agent): 13 to 15 days; At the end of the expression period, the cells were placed into each well of 4 x 96-well microtitre plates. Plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (13 to 15 days) and wells containing clones were identified and counted.

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

NUMBER OF REPLICATIONS: Cultures were tested in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative survival:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described above for the Mutation Experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were washed with tissue culture medium and then resuspended in 20 mL tissue culture medium. Cells were plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. Wells containing viable clones were identified by microscope and counted.

OTHER:
Probable number of clones/well (P) = -ln (EW/TW);
Plating efficiency (PE) = P/No of cells plated per well;
PE = P/1.6;
Percentage relative survival (%RS) = [PE (test)/PE (control)] x 100;
Adjusted %RS = Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control;
Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article would be 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 satisfy 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. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Test results 1

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 divanadium trioxide at any concentration tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

for details see below in the field "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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 divanadium trioxide at any concentration tested.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below in the field "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder Experiment at the highest concentration analysed (93.75 μg/mL), compared to the concurrent vehicle controls.
- Water solubility: Preliminary solubility data indicated that divanadium trioxide was soluble in purified water with warming to approximately 60ºC at 5 to 14 mg/mL.
- Precipitation: Due to the intense colouration of the test article, it was difficult to determine the presence of precipitate accurately, therefore all concentrations cited in this report may be considered nominal.
In Experiment I: After the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence and presence of S9 (6.25 to 100 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and higher concentrations were discarded.
In Experiment II: After 3 hour treatment incubation period, precipitate was observed at the highest 2 concentrations tested in the absence and presence of S9 (25 and 50 μg/mL). The lower concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 was retained and the higher concentration was discarded.

RANGE-FINDING/SCREENING STUDIES: 6 concentrations were tested in the absence and presence of S9 ranging from 46.88 to 1500 μg/mL (equivalent to approximately 10 mM at the highest concentration tested). No precipitate was observed at the time of treatment but, after the 3 hour treatment incubation period, precipitate was observed at all concentrations tested. The lowest two concentrations at which precipitation was observed at the end of the treatment incubation period in the absence and presence of S9 were retained (to provide an estimate of toxicity, even though post-treatment precipitate was observed at all concentrations) and all higher concentrations were discarded. The highest concentrations to give >10% RS were 46.88 μg/mL in the absence of S9 and 93.75 μg/mL in the presence of S9, which gave 56% and 69% RS, respectively.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I 10 concentrations, ranging from 0.1953 to 100 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, concentrations were selected to determine viability and 6TG resistance. The highest concentration selected, 6.25 μg/mL, gave 83% and 81% RS in the absence and presence of S9, respectively.
In Experiment II 10 concentrations, ranging from 0.25 to 50 μg/mL, were tested in the absence and presence of S9. 7 days after treatment, concentrations were selected to determine viability and 6TG resistance. The highest concentration selected was 25 μg/mL, which gave 12% and 2% RS in the absence and presence of S9, respectively. In the presence of S9, no concentration gave 10-20% RS (cultures treated at 15 and 25 μg/mL gave 65% and 2% RS, respectively, therefore both concentrations were analysed).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment I

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Divanadium trioxide 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 toxic and/or precipitating concentrations in two independent experiments in the absence or presence of a rat liver metabolic activation system (S9).

Executive summary:

Divanadium trioxide was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. 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).

In the cytotoxicity Range-Finder Experiment, 6 concentrations were tested in the absence and presence of S9, ranging from 46.88 to 1500 μg/mL with a treatment period of 3 hours. The highest concentrations to give >10% relative survival (RS) were 46.88 μg/mL in the absence of S9 and 93.75 μg/mL in the presence of S9, which gave 56% and 69% RS, respectively.

Accordingly, in Experiment I 10 concentrations, ranging from 0.1953 to 100 μg/mL, were tested in the absence and presence of S9. After the 3 hour treatment incubation period, precipitate was observed at the highest 5 concentrations tested in the absence and presence of S9 (6.25 to 100 μg/mL). 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 6.25 μg/mL, which gave 83% and 81% RS in the absence and presence of S9, respectively.

In Experiment II 10 concentrations, ranging from 0.25 to 50 μg/mL, were tested in the absence and presence of S9. After the 3 hour treatment incubation period, precipitate was observed at the highest 2 concentrations tested in the absence and presence of S9 (25 and 50 μg/mL). 7 days after treatment, the highest concentration selected to determine viability and 6TG resistance was 25 μg/mL, which gave 12% and 2% RS in the absence and presence of S9, respectively. In the presence of S9, no concentration gave 10 -20% RS.

Vehicle and positive control treatments were included in each Mutation Experiment in the absence and presence of S9.

In Experiments I and II, no statistically significant increases in mutant frequency were observed following treatment with divanadium trioxide at any concentration tested in the absence and presence of S9. A statistically significant linear trend was observed in the presence of S9 in Experiment II but, in the absence of any marked increases in mutant frequency at any concentration tested in this experiment, this observation was not considered biologically relevant.