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Genetic toxicity: in vitro

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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:
29 March 2012 to 10 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP without significant deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
CAS Number 25322-17-2
Description Brown highly viscous liquid (determined at NOTOX)
Batch 966018
Purity Provisional 84%

Method

Test concentrations with justification for top dose:

Test concentrations for the dose rangefinding: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

For experiment 1:
TA1535, TA1537 and TA98:
Without S9-mix: 3, 10, 33, 100, 200 and 333 µg/plate
With S9-mix: 10, 33, 100, 333 and 1000 µg/plate
TA100 and WP2uvrA:
Without S9-mix: 3, 10, 33, 100 µg/plate
With S9-mix: 10, 33, 100, 333 and 1000 µg/plate

The following dose ranges were selected for the second mutation experiment:
Without S9-mix: 3, 10, 33, 100, 200 and 333 µg/plate
With S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:
Dimethyl sulfoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
See details of positive controls in next section
Details on test system and experimental conditions:

Positive controls
Without metabolic activation (-S9-mix):
Strain Chemical
TA1535 : sodium azide (SA)
TA1537 : ICR-191
TA98 : 2-nitrofluorene (NF)
TA100 : methylmethanesulfonate (MMS)
WP2uvrA : 4-nitroquinoline N-oxide (4-NQO)

With metabolic activation (+S9-mix):
The positive control substance used for all tester strains was 2-aminoanthracene (2AA)
Evaluation criteria:
Data evaluation and statistical procedures

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.

Statistics:
None required

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA98 TA1535 TA1537 TA100 and WP2uvrA
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 333 and 1000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA98 TA1535 TA1537 TA100 and WP2uvrA
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Dose Range Finding

Di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid (DNNSA) was tested in the tester strains TA100 and WP2uvrA with concentrations of 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. This dose range finding test is reported as a part of the first experiment of the mutation test. 

 

Precipitate

Precipitation of DNNSA on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and at 1000 µg/plate and above at the end of the incubation period.

 

Toxicity

To determine the toxicity of di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated inAPPENDIX2.

 

Since DNNSA precipitated 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 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 test substance concentrations of 333 and 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.

Experiment 1

Based on the results of the dose range finding test, the following dose range was selected for the first mutation experiment in the absence and presence of 5% (v/v) S9-mix with theSalmonella typhimuriumstrains, TA1535, TA1537 and TA98:

Without S9-mix: 3, 10, 33, 100, 200 and 333 µg/plate

With S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Results are presented in detail in attached document (Results Table Ames Test DNNSA).

Experiment 2

To obtain more information about the possible mutagenicity of di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid, a second mutation experiment was performed in the absence of

S9-mix and in the presence of 10% (v/v) S9-mix. The following dose ranges were selected for the second mutation experiment:

Without S9-mix: 3, 10, 33, 100, 200 and 333 µg/plate

With S9-mix: 10, 33, 100, 333 and 1000 µg/plate.

Detailed results are presented in the attached document.

Applicant's summary and conclusion

Conclusions:
Di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid (DNNSA) is non-mutagenic in the Ames bacterial reverse mutation assay.
Executive summary:

Di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid (DNNSA) was evaluated for mutagenic activity in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat). DNNSA 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 coli reverse 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.

 

Batch 966018 of di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid was a brown highly viscous liquid. A correction factor of 1.19 was used to correct for the provisional purity of 84%. The test substance was dissolved in dimethyl sulfoxide.

 

In the dose range finding test, di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. DNNSA precipitated on the plates at dose levels of 1000 μg/plate and upwards. Toxicity was observed at dose levels of 333 and 1000 μg/plate in the absence of S9-mix in both tester strains. Since DNNSA precipitated 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, DNNSA was tested in the first mutation assay at a concentration range of 3 to 333 µg/plate in the absence of S9-mix and at 10 to 1000 µg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid 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 and in tester strain TA98 in the presence of S9-mix.

 

In an independent repeat of the assay with additional parameters, DNNSA was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. DNNSA precipitated on the plates at the top dose of 1000 μg/plate. Toxicity was only observed in the tester strains TA1535, TA1537, TA98, TA100 in the absence of S9-mix.

 

Di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid 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.

 

In this study, 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.

 

Based on the results of this study it is concluded that di C8-C10, branched, C9 rich, alkylnaphthalene sulfonic acid is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.