Naphthalenesulfonic acid, di-C9-rich C8-10-branched alkyl derivs., barium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was non-mutagenic in the Ames assay, is not clastogenic in human lymphocytes and is not mutagenic in the TK mutation test system (mouse lymphoma test).

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Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 29, 2012 to April 10, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: New OECD guideline GLP study without deficiencies that affect study validity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

Rat liver S9

Test concentrations with justification for top dose:

In preliminary trials, 8 doses were used: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in triplicate.
In definitive experiments 1 and 2, doses were 3 to 1000 ug/plate.

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:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see details section

Details on test system and experimental conditions:

Positive Controls
Strain Chemical
TA1535 sodium azide (SA)
TA1537 ICR-191
TA98 2-nitrofluorene (NF)
TA100 methylmethanesulfonate (MMS)
WP2uvrA 4-nitroquinoline N-oxide (4-NQO)


Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 36.0 – 39.0°C) in the dark.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 333 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at 1000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 333 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at 1000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at 333 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at 1000 ug/plate (exp 1 only)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 666 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at 1000 ug/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 333 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at 1000 ug/plate (exp 2 only)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Precipitate
Precipitation of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) on the plates was observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate. The test substance precipitated on the plates at dose levels of 1000 μg/plate and upwards in tester strain TA100 and at 3330 and 5000 µg/plate in tester strain WP2uvrA at the end of the incubation period.

Toxicity
To determine the toxicity of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate), the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Since Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated moderately to heavily on the plates at test substance concentrations of 3330 and 5000 μg/plate, neither the bacterial background lawn nor the number of revertants of these dose levels could be determined.

In tester strain TA100 in the absence of S9-mix, a moderate reduction of the revertant colonies was observed at the test substance concentration of 333 μg/plate and an extreme reduction of the bacterial background lawn and an increase in the size of the micro-colonies compared to the solvent control plate was observed at the test substance concentration of 1000 μg/plate.

In tester strain WP2uvrA in the absence of S9-mix, a slight reduction of the bacterial background lawn was observed at the test substance concentration of 333 μg/plate and a moderate to extreme reduction of the bacterial background lawn and an increase in the size of the micro-colonies compared to the solvent control plate was observed at the test substance concentration of 1000 μg/plate.

In the presence of S9-mix, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

See attached document for results tables for the definitive and repeat (confirmatory) assays.

Conclusions:

Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was non-mutagenic in the Ames assay.

Executive summary:

The mutagenic activity of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was evaluated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

 

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia colireverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines. A correction factor of 1.11 was used to correct for the estimated purity of 90%. The test substance was dissolved in ethanol.

 

In the dose range finding test, Barium bis ( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The test substance precipitated on the plates at dose levels of 1000 μg/plate and upwards in tester strain TA100 and at 3330 and 5000 µg/plate in tester strain WP₂uvrA. Toxicity was observed at dose levels of 333 and 1000 μg/plate in the absence of S9-mix in both tester strains. Since Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated moderately to heavily on the plates at test substance concentrations of 3330 and 5000 μg/plate, neither the bacterial background lawn nor the number of revertants of these dose levels could be determined. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested in the first mutation assay up to the dose level of 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated on the plates at the top dose of 1000 μg/plate. Toxicity was observed in all three tester strains in the absence of S9-mix.

 

In an independent repeat of the assay with additional parameters, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to the dose level of 1000 µg/plate in the tester strains TA98, TA100 and WP₂uvrA, up to 666 µg/plate in tester strain TA1535 and up to 333 µg/plate in tester strain TA1537 in the absence of S9-mix. The test substance was tested up to the dose level of 1000 µg/plate in all tester strains in the presence of 10% (v/v) S9-mix. Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) precipitated on the plates at the top dose of 1000 μg/plate. Except in tester strain TA1537 where no precipitate was observed at the top dose of 1000 μg/plate. Toxicity was observed in all tester strains in the absence of S9-mix.

Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosomal aberrations. No target gene.

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

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.

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start : 18 July 2012 Completion : 17 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study without deficiencies that affect study integrity

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Test concentrations with justification for top dose:

In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 10 to 1000 μg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
Based on the results of the dose range finding test, the following dose range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.1, 0.3, 1, 3, 10, 30, 40, 50, 60, 70, 80 and 90 μg/ml exposure medium.
During the performance of the study Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) used in this experiment was handled as if it had an estimated purity of 90% (as initially given by the sponsor) and a correction factor of 1.11 was used in this assay. In the course of the development it has been identified that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is UVCB and the purity is 100%. Therefore, the actual concentrations were 11% higher than stated in the report.

Vehicle / solvent:

dimethyl sulfoxide

Details on test system and experimental conditions:

In the dose range finding test, at the concentration of 120 mg/ml the test substance was translucent in dimethyl sulfoxide. At concentrations of 40 mg/ml and lower the test substance was dissolved in dimethyl sulfoxide. In the mutation experiments, at the concentration of 12 mg/ml the test substance formed a translucent solution in dimethyl sulfoxide whereas at 10.8 mg/ml and lower it was fully soluble.
In the first experiment, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to concentrations of 50 and 90 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 93 and 85% in the absence and presence of S9-mix, respectively.
In the second experiment, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to concentrations of 60 and 90 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix The test substance was tested up to cytotoxic levels of 54 and 92% in the absence and presence of S9-mix, respectively.
Positive controls:
Without metabolic activation (-S9-mix)
Methyl methanesulfonate (MMS); CAS no. 66-27-3 (purity 98%, Sigma, Zwijndrecht, The Netherlands). MMS was used as a direct acting mutagen at a concentration of 15 and 5 μg/ml for a 3 and 24 hours treatment period, respectively. MMS was dissolved in dimethyl sulfoxide. The stock solutions of MMS were prepared immediately before use.
With metabolic activation (+S9-mix):
Cyclophosphamide (CP); CAS no. 50-18-0 (purity 100%, Endoxan, Asta-Werke, Germany). CP was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μg/ml. CP was dissolved in Hanks’ balanced salt solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands) without calcium and magnesium. The stock solutions of CP were stored in aliquots at
≤-15°C in the dark and one sample was thawed immediately before use.
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 43 – 96%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.9 – 38.0°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 34.9 - 36.0°C), humidity (with a maximum of 20%) and CO2 percentage (with a maximum of 1%) that occurred were caused by opening and closing of the incubator door, the duration of these deviations did not exceed 4 hours. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Evaluation criteria:

Determination of the mutation frequency
Eight doses of the test substance were selected for the mutation assay, both in the absence and presence of S9-mix.
For determination of the cloning efficiency (CE-day2) the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the MF a total number of 9.6 x 10^5 cells/concentration were plated in five
96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CE-day2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE-day2 and MF were scored with the naked eye or with the microscope.

Statistics:

Analysis of results

Determination of the mutant colonies:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
Calculation of the survival or viability:
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. This value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) is therefore:
CE = -ln P(0)/number of cells plated per well
The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures:
RS = [CE(test)/CE(controls)] x 100
The Relative Total Growth (RTG) was calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture:
RTG = RSG x RSday2/100
Suspension Growth (SG) =
[Day 0 cell count/ (1.25 x 105) *] x [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count]
Relative Suspension Growth (RSG) = SG (test) / SG (controls)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test substance was tested up to cytotoxic levels of 93 and 85% in the absence and presence of S9-mix, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range (See Attached Tables).
The growth rate over the two-day expression period for cultures treated with DMSO was between 9 and 30 (3 hours treatment) and 45 and 46 (24 hours treatment) (See Attached Tables, Table 5, Table 6, Table 8 and Table 9).
Mutation frequencies in cultures treated with positive control chemicals were increased by 8.3- and
14-fold for MMS in the absence of S9-mix, and by 18- and 21-fold for CP in the presence of S9-mix, in the first and second experiment respectively (See Attached Tables, Table 3 and Table 4). It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay (See Attached Tables, Table 12).
In the absence of S9-mix, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Evaluation of Cytotoxicity

In the absence of S9-mix, the dose levels of 0.1 and 50 μg/ml showed no cytotoxicity. Therefore, the dose level of 0.1 μg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 70 to 100 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the presence of S9-mix, the dose levels of 0.1 to 3 μg/ml showed no cytotoxicity. Therefore, the dose levels of 0.3 and 3 μg/ml were not regarded relevant for mutation frequency measurement. The dose levels of 50 to 70 μg/ml showed similar cytotoxicity. Therefore, the dose level of 60 μg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 80 and 100 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Conclusions:

Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is not mutagenic in the TK mutation test system under the experimental conditions of this experiment.

Executive summary:

Evaluation of the mutagenic activity of Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was conducted in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat). The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures 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. During the performance of the study Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) used in this experiment was handled as if it had an estimated purity of 90% (as initially given by the sponsor) and a correction factor of 1.11 was used in this assay. In the course of the development it has been identified that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is UVCB and the purity is 100%. Therefore, the actual concentrations were 11% higher than stated in the report. The conclusion of this report remains unchanged. In the dose range finding test, at the concentration of 120 mg/ml the test substance was translucent in dimethyl sulfoxide. At concentrations of 40 mg/ml and lower the test substance was dissolved in dimethyl sulfoxide. In the mutation experiments, at the concentration of 12 mg/ml the test substance formed a translucent solution in dimethyl sulfoxide whereas at 10.8 mg/ml and lower it was fully soluble. In the first experiment, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to concentrations of 50 and 90 μg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. The test substance was tested up to cytotoxic levels of 93 and 85% in the absence and presence of S9-mix, respectively. In the second experiment, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) was tested up to concentrations of 60 and 90 μg/ml in the absence and presence of 12% (v/v) S9-mix, respectively. The incubation times were 24 hours for incubations in the absence of S9-mix and 3 hours for incubations in the presence of S9-mix.

The test substance was tested up to cytotoxic levels of 54 and 92% in the absence and presence of S9-mix, respectively. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased 8.3- and 14-fold for MMS in the absence of S9-mix, and 18- and 21-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate) is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As the substance is not genotoxic based on the results of the Ames bacterial reverse mutation assay, the mouse L5178Y lymphoma test (MLA) and human peripheral lymphocytes test, no classification according to CLP (Regulation EC No 1272/2008) for mutagenicity is required.