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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Start : 16 March 2012 Completion : 04 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: Beige-brown crystalline powder with lumps
Batch: 278-123
Purity: UVCB (treated as 100% pure)

Method

Target gene:
Chromosomal aberrations. No target gene.
Species / strain
Species / strain / cell type:
other: Peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
(phenobarbital and ß-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 3, 10, 33, 100 and 333 μg Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate)/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure time blood cultures were treated with 3, 10, 33, 100, 333 and 1000 μg Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate)/ml culture medium without S9-mix. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested beyond the limit of solubility to obtain adequate toxicity data.
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 33, 100 and 150 μg/ml culture medium
(3 h exposure time, 24 h fixation time).

Batch 278-123 of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was initially determined to have a purity of 90% . Based on this all concentrations reported were corrected for the purity with a correction factor of 1.11. After setting doses and running the assay the test substance was designated as UVCB with purity of 100%. Therefore, the test doses reported here are 10% lower than the doses when considered as UVCB.
Vehicle / solvent:
dimethyl sulfoxide
Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC).
Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was dissolved in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany). The stock solution was treated with ultrasonic waves until Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) had completely dissolved. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) concentrations were used within 1.5 hour after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).
Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 90%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.8 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.3 - 36.0°C), humidity (with a maximum of 13%) and CO2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
Chi-square statistics

Results and discussion

Test results
Species / strain:
mammalian cell line, other: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cell lysis at 333 and 1000 ug/ml
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first cytogenetic assay, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 150 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated in the culture medium at this dose level.
In the second cytogenetic assay, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 130 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 100 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix Barium bis
( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 250 μg/ml for a 3 h exposure time with a 48 h fixation time. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated in the culture medium at this dose level.
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.
No effects of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
Finally, it is concluded that this test is valid and that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:
Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is not clastogenic in human lymphocytes.
Executive summary:

Evaluation of the ability of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment). This study evaluates the effect of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 278-123 of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was a beige-brown crystalline powder with lumps with an estimated purity of 90% . All concentrations reported were corrected for the purity. A correction factor of 1.11 was used (after setting doses and running the assay the test substance was designated as UVCB with purity of 100%. Therefore, the test doses reported here are 10% lower than the doses when considered as UVCB. This change does not affect the integrity of the study or the validity of the results). Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 150 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 130 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 100 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to 250 μg/ml for a 3 h exposure time with a 48 h fixation time. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated in the culture medium at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is not clastogenic in human lymphocytes under the experimental conditions described in this report.