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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
15 December 2009 till 11 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed by The Department of Health of the Government of the United Kingdom (2010-06-23)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Barium chloride dihydrate
- EC number: 233-788-1
- Molecular formula (if other than submission substance): BaCl2 * 2H2O
- Molecular weight (if other than submission substance): 244.2 g/mol
- Physical state: solid, white powder
- Storage condition of test material: stored at 15 to 25°C in the dark

Method

Target gene:
tk +/- locus
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 Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. The cells were 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.

- Type and identity of media: RPMI 1640 media supplemented with heat inactivated horse serum (0%, 10% or 20% (v/v)), 100 units/mL penicillin and 100 µg/mL streptomycin, 2.5 µg/mL Amphotericin B, 0.2 mg/mL pyruvic acid and 0.5 mg/mL pluronic (except fpr RPMI 20%).
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes; Each batch of frozen cells was checked that it was mycoplasma free.
- Periodically "cleansed" against high spontaneous background: yes; Each batch of frozen cells was checked for spontaneous mutant frequency.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Concentrations selected for Experiments I and II were based on the results of this cytotoxicity Range-Finder Experiment.
Range-Finder:
- with and without S9 mix, 3 hours treatment: 65.06, 130.1, 260.3, 520.5, 1041, 2082 µg/mL
- without S9 mix, 24 hours treatment: 8.133, 16.27, 32.53, 65.06, 130.1, 260.3, 520.5, 1041, 2082 µg/mL

Experiment I:
- with and without S9 mix, 3 hours treatment: 250, 500, 750, 1000, 1200, 1400, 1600, 1800, 1950, 2082 µg/mL

In Experiment I, cultures treated at 1000 and 1200 µg/mL in the absence of S9 mix gave 26% and 4% relative total growth, respectively, therefore Experiment II consisted of 3 hour treatments in the absence and presence of S9 mix and a 24 hour treatment in the absence of S9 mix in order to better define the toxicity profile.

Experiment II:
- without S9 mix, 3 hours treatment: 100, 200, 400, 600, 800, 900, 1000, 1100, 1200, 1400 µg/mL
- with S9 mix, 3 hours treatment: 200, 400, 600, 800, 900, 1000, 1100, 1200, 1300, 1400 µg/mL
- without S9 mix, 24 hours treatment: 100, 200, 400, 600, 800, 900, 1000, 1100, 1200, 1400 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
treatments with the vehicle purified water diluted 10 fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation

Migrated to IUCLID6: Experiment I: 15.0 and 20.0 µg/mL and Experiment II: 5.0 and 7.5 µg/mL; dissolved in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

For Experiments I and II in the absence and presence of S9 mix least 10^7 cells in a volume of 17 mL tissue culture medium were used.
For Experiment II in the absence of S9 mix at least 4 x 10^6 cells in a volume of 18 mL RPMI 10 were used.

DURATION
- Exposure duration: 3 hours (with and without S9 mix; Experiment I and II) and 24 hours (without S9 mix; Experiment II), at 37±1°C
After incubation, cultures were centrifuged, washed and resuspended fresh RPMI 10 medium. Cells were transferred to tissue culture flasks for growth throughout the expression period.
- Expression time (cells in growth medium): Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. Some cultures were selected for determination of viability and TFT resistance (see table below).
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 14 days; At the end of the expression period, the cells were placed into each well of four 96 well microtitre plates (384 wells at 2 x 10³ cells/well). Plates were incubated at 37±1°C with 5% v/v CO2 in air until scoreable and wells containing clones were identified.

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: duplicate cultures (single cultures only used for positive control treatments)

NUMBER OF CELLS EVALUATED: Number of cells per well is 2000 cells per well on average on all mutant plates.

EVALUATION: Mutant frequency (MF) was calculated as follows:
MF = -ln P(o) for mutant plates/Number of cells per well x (viability/100)

DETERMINATION OF CYTOTOXICITY
- Method: viability, cloning efficiency, relative total growth:
In the absence of S9 mix, 3 and 24 hours treatment incubation periods were used, in the presence of S9 mix a 3 hour treatment incubation was used.
Following treatment, cells were centrifuged, washed with tissue culture medium and resuspended in RPMI 10. Cell densities were determined. All cultures were incubated at 37±1°C for 1 day, recounted and where possible diluted to 2 x 10^5 cells/mL. Cultures were incubated for a further day, counted and adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96 well microtitre plate (incubation at 37±1°C with 5% CO2 in air for 7 to 11 days) for determination of viability (plating efficiency). Wells containing viable clones were identified by eye. Viability is the measure of the cells' ability to clone i.e. Cloning efficiency (CE).
Relative Total Growth (RTG) is the measure of cytotoxicity relative to the control, that takes into account all cell growth, cell loss during the treatment period and the 2 day expression period (RSG) and the cells' ability to clone 2 days after treatment (viability).
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutation frequency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus global evaluation factor (GEF)
2. The linear trend test was positive.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003. The control mutant frequency was compared with each test article treatment and the data were checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The mutant frequency of the concentrations plated were all less than the sum of the mean control mutant frequency plus the Global Evaluation Factor (GEF, 126 mutants per 10^6 viable cells), indicating a negative result. (For details see attached document)
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed in the 3 and 24 hours Range Finder Experiments at the highest concentration tested.
- Effects of osmolality: No marked changes in osmolality were observed in the 3 and 24 hours Range Finder Experiments at the highest concentration tested.
- Water solubility: Preliminary solubility data indicated that Barium chloride dihydrate was soluble in water for irrigation (purified water) at concentrations up to at least 23.85 mg/mL (anhydrous). The solubility limit in culture medium was below 2385 µg/mL (anhydrous), as indicated by the appearance of slight precipitate (haziness) at this concentration approximately 26 hours after test article addition. A maximum concentration of 2082 µg/mL (anhydrous) was selected for the cytotoxicity Range Finder Experiment in order that treatments were performed up to 10 mM.

RANGE-FINDING/SCREENING STUDIES: In the cytotoxicity Range-Finder Experiment, 3 hour treatment, 6 concentrations were tested, in the absence and presence of S 9, ranging from 65.06 to 2082 µg/mL (equivalent to 10 mM anhydrous Barium chloride dihydrate at the highest concentration tested). The highest concentration, 2082 µg/mL, gave 18% and 31% relative total growth (RTG) in the absence and presence of S9 mix, respectively.
In the cytotoxicity Range-Finder Experiment, 24 hour treatment, 9 concentrations were tested in the absence of S9 mix, ranging from 8.133 to 2082 µg/mL. The highest concentration to provide >10% RTG was 1041 µg/mL, which gave 24% RTG.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that barium chloride dihydrate did not induce mutation at the tk 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 and presence of a rat liver metabolic activation system (S9 mix).
Executive summary:

Barium chloride dihydrate was assayed for its ability to induce mutation at the tk locus (5‑trifluorothymidine [TFT] resistance) in mouse lymphoma cells.

Experiment I was performed using a 3 -hour treatment incubation and Experiment II was performed using 3- and 24 -hour treatment incubations.

In the cytotoxicity Range-Finder Experiment, concentrations were tested ranging from 65.06 to 2082 µg/mL (3 -hour treatment; -/+S9 mix) and from 8.133 to 2082 µg/mL (24 -hour treatment; -S9 mix).

Concentrations selected for Experiments I and II were based on the results of this cytotoxicity Range-Finder Experiment.

Two Experiments were performed with selected concentrations:

- In Experiment I concentrations, ranging from 250 to 2082 µg/mL,were tested in the absence and presence of S9 mix.

- In Experiment II (3-hour treatment) concentrations, ranging from 100 to 1400 µg/mL in the absence of S9 mix and from 200 to 1400 µg/mL in the presence of S9 mix, were tested. Additionally cells were tested in a 24 -hour treatment with concentrations ranging from 100 to 1400 µg/mL in the absence of S9 mix.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment. Mutant frequencies in negative control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S‑9) and Benzo[a]pyrene (with S‑9). Therefore the study was accepted as valid.

In Experiments I and II, the mutant frequency of the concentrations plated were all less than the sum of the mean control mutant frequency plus the Global Evaluation Factor, indicating a negative result. Statistically significant linear trends were observed in Experiment II in the presence of S9 mix (3-hour treatment) and in the absence of S9 mix (24-hour treatment). However, in the absence of any marked increases in mutant frequency under either treatment condition, these observations were not considered biologically relevant.