3-((3,4-dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between the 5th May 2010 and 28th May 2010. The final report was issued 18th August 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-((3,4-Dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Cas Number:

569316-81-0

Molecular formula:

C14H17N3O5S2

IUPAC Name:

3-((3,4-Dicyanophenyl)sulfonyl)-N-(2-hydroxypropyl)propane-1-sulfonamide

Test material form:

solid

Method

Target gene:

Histidine for Salmonella typhimurium
Tryptophan for Escherichia coli

Species / strain

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 homogenate metabolising system ( liver S9)

Test concentrations with justification for top dose:

The test concentrations were determined from a preliminary toxicity assay and were 0, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dimethyl sulphoxide
Justification for choice of solvent/vehicle: The test material wasinsoluble in sterile distilled water at 50 mg/l but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks. Dimethyl sulphoxide was thereforre selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA 1535

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA1537

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 6

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA 100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 9

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

spontaneous mutation rate of 1537

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 11

Untreated negative controls:

yes

Remarks:

spontaneous mutation of TA100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar, direct plate incorporation method for experiment 1 and pre-incubation for experiment 2

DURATION
- Preincubation period: 10 hours
- Exposure duration: Approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plating

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance is considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance wasl not the only determining factor for a positive response.A test material is considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Yes, as recommened by UKEMS (Kirkland D J, (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data UKEMS sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small, statistically significant increase in Escherichia coli strain WP2uvrA- revertant colony frequency was observed in the presence of S9 at 5000 ug/plate in the second Experiment. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 5000 ug/plate were within the in-house historical untreatedtvehicle control range for the tester strain and the fold increase was only 1.47 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test item was tested using a protocol designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test" and Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.The method conforms to the guidelines published by the major Japanese regulatory regulatory authorities including METI, MHLW and MAFF.

Methods

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA were treated in two separate experiments with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the experiments was 50 to 5000 µg/plate.

 

 Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was therefore tested upto the maixmum recomended 5000 ug/plate.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 mix.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation.

 

 

Conclusion

 The test item was considered to be non-mutagenic under the conditions of this test.