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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

read across:

in vitro: Gene mutation in bacteria: key study, Ames test, S. typhimurium, with and without metabolic activation: negative (GLP, OECD 471 conform, Anonymous, NTP 1994)

in vitro, Gene mutation in mammalian cells: key study, TK test, L5178 mouse lymphoma cells, with and without metabolic activation: negative (GLP, OECD 476, Lloyd, 2010)

Cytogenicity in mammalian cells: no data

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see attached justification.
Reason / purpose:
read-across source
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'.
Conclusions:
Interpretation of results: 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

There are reliable in vitro studies available to assess the potential of the test substance for gene mutations in bacteria and in mammalian cells.

For read-across barium chloride is adopted as it is also an inorganic barium salt whose relevant eco-/toxicological nature depends on the common cation barium whereas the toxicological nature of the anion is negligible. The substances differ in solubility. Barium chloride is soluble while barium titanium trioxide is slightly soluble. But this difference is considered as negligible as it is supported by the absence of any adverse findings in acute toxicity for the analogue substance. In conclusion, read-across for the endpoint genetic toxicity is justified.

Gene mutation in bacteria:

In a GLP conform study equivalent to OECD guideline 471 the potential of the analogous test substance barium chloride dihydrate (purity: > 99 weight-%) to induce gene mutations based on the ability to induce back mutations was carried out using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 97 (Anonymous, NTP 1994). The test was performed both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 100, 333, 1000, 3333 and 10.000 µg/plate dissolved in water. A weakly bacteriotoxic effect was occasionally observed in the strains TA1535, TA1537, TA98 and TA100 at doses ≥ 333 µg/plate and at the highest test concentration in strain TA 97.

An increase in the number of his+ revertants was not observed either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance barium chloride dihydrate and with it barium calcium zirconium titanate are not mutagenic in the Ames test under the experimental conditions chosen here.

Gene mutation in mammalian cells:

A GLP conform study was performed to investigate the potential of the analogous test substance barium dichloride dihydrate (purity: > 99.9 weight-%) to induce gene mutations at the TK locus in mouse lymphoma L5178 cells according to OECD guideline 476 (Lloyd, 2010).

In a cytotoxicity range-finding experiment concentrations from 8.133 up to 2082 µg/mL were applied. The concentrations selected for the experiments I and II were based on this range-finding experiment. Therefore, the concentrations chosen used in the main experiments were: 0, 250, 500, 750, 1000, 1200 and 1400 µg/mL in experiment I and 0, 100, 200, 400, 600, 800, 900, 1000, 1100, 1200, 1300 and 1400 µg/mL in experiment II.

In the first experiment the treatment period was 3 hours with and without metabolic activation. The second experiment was performed with a treatment time of 3 hours with and without metabolic activation and 24 hours without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item Barium dichloride dihydrate did not induce gene mutations at the TK locus in mouse lymphoma L5178 cells.

Therefore, barium dichloride dihydrate and with it barium calcium zirkonium titanate are considered to be non-mutagenic in this TK mammalian cell gene mutation assay.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

Based on the results, the classification of the test substance for genetic toxicity under Regulation 1272/2008 is not warranted.