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

Genetic toxicity: in vitro

Administrative data

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: New OECD guideline GLP study without deficiencies that affect study validity. Study is read-across from a close structural analog.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

BARIUM BIS( DI C8-C10, BRANCHED, C9 RICH, ALKYLNAPHTHALENE SULPHONATE)

IUPAC Name:

BARIUM BIS( DI C8-C10, BRANCHED, C9 RICH, ALKYLNAPHTHALENE SULPHONATE)

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

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

NA

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

Controls

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.

Results and discussion

Test results

Key result

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 colireverse mutation assay (with independent repeat).

 

The test material was tested in the Salmonella typhimuriumreverse 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.