1-Propanesulfonic acid, 2-methyl-2-[[1-oxo-3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)thio]propyl]amino]-, sodium salt (1:1)

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:

04 December, 2014 - 15 December, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-methyl-2-{3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propanamido}propane-1-sulfonate

EC Number:

811-522-0

Cas Number:

62880-93-7

Molecular formula:

C15H17F13NNaO4S2

IUPAC Name:

sodium 2-methyl-2-{3-[(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)sulfanyl]propanamido}propane-1-sulfonate

Test material form:

other: lumps

Details on test material:

- Name of test material (as cited in study report): NS-3000
- Appearance: White to off-white lumps (determined by WIL Research Europe)
- Storage condition of test material: At room temperature desiccated

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254 (500 mg/kg).

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2: All 5 strains
Without and with S9-mix: 492, 878, 1568, 2800 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines. Test substance concentrations were used within 2.5 hours of preparation.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene 10 µg/plate in DMSO for TA98

Remarks:

without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: ICR-191 2.5 µg/plate in DMSO for TA1537

Remarks:

without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the 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 follow-up experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the 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 follow up 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 follow up experiment.

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Oily droplets of NS-3000 on the plates were observed at the start of the incubation period at concentrations of 878 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA98, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 1600 μg/plate (presence of S9-mix). Since no dose-relationship was observed and no indication of toxicity was observed in any of the other tester strains in the first and second experiments, this reduction is not considered to be caused by toxicity of the test substance, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY/MUTAGENICITY:
In experiment 1, in tester strain TA1537, NS-3000 induced an up to 6.6-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix at the lowest dose level of 52 μg/plate. In tester strain TA98, NS-3000 induced an up to 3.8-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix at the lowest dose level of 52 μg/plate.
In the other tester strains, no biologically relevant increase in the number of revertants was observed upon treatment with NS-3000 under all conditions tested.
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. No increase in the number of revertants was observed upon treatment with NS-3000 under all conditions tested.

DISCUSSION:
In experiment 1 in the presence of S9-mix, NS-3000 induced increases in two tester strains (TA1537 and TA98). Although, the observed increases were more than 3-fold compared to the concurrent solvent control, the increases were observed at the lowest dose level only and could not be repeated in the follow-up study. Furthermore, in tester strain TA1537 the increase was caused by only two out of three plates (111, 111 and 14 revertant colonies/plate) and in tester strain TA98 by only one out of three plates (18, 154 and 8 revertant colonies/plate). Therefore, these increases are considered to be not biologically relevant and NS-3000 is considered to be not mutagenic
In the absence of S9-mix, all bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two experiments.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In an AMES test, performed according to OECD 471 guideline and GLP principles, NS-3000 was found not to be mutagenic up to and including 5000 μg/plate with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate in two experiments, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. 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 NS-3000 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.