1-(3-sulphonatopropyl)-2-vinylpyridinium

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:

2016-09-13 - 2016-09-22 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by the Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-sulphonatopropyl)-2-vinylpyridinium

EC Number:

229-565-3

EC Name:

1-(3-sulphonatopropyl)-2-vinylpyridinium

Cas Number:

6613-64-5

Molecular formula:

C10H13NO3S

IUPAC Name:

3-(2-vinylpyridinium-1-yl)propane-1-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), protected from light and humidity.
- Solubility and stability of the test substance in the solvent/vehicle: H2O: >1 g/L; EtOH: >1 g/L

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix produced from the livers of male Sprague-Dawley rats, induced with Aroclor 1254

Test concentrations with justification for top dose:

1st experiment: 5000 / 1500 / 500 / 150 / 50 / 0 µg/plate
2nd experiment: 5000 / 2500 / 1250 / 625 / 313 / 156 / 0µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Demin. H2O was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

Water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-Anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (1st experiment); preincubation (2nd experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
His-negative plates

NUMBER OF REPLICATIONS:
Per strain and dose, three plates with and three plates without S9 mix were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

as described in OECD TG 471

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A spreadsheet soft-ware (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA97a

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:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 2

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:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 3

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:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

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:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Test results 5

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:

valid

Remarks:

vehicle controls

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: none stated
- Water solubility: test item completely solved
- Precipitation: In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 µg/plate.

Remarks on result:

other:

Remarks:

determined in both experiments

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 1-(3-Sulfopropyl)-2-vinyl-pyridinumbetain to induce reverse mutations in bacteria. The test item 1-(3-Sulfopropyl)-2-vinyl-pyridinumbetain (SPV) showed no increase in the number of revertants in all bacteria strains in both experiments. Based on the results of this study it is concluded that 1-(3-Sulfopropyl)-2-vinyl-pyridinumbetain (SPV) is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), strains TA97a, TA98, TA100, TA102 and TA1535 of S. typhimurium were exposed to 1-(3-Sulfopropyl)-2-vinyl-pyridinumbetain (SPV) at concentrations of 5000, 1500, 500, 150, 50, 0 µg/plate (1st experiment, plate incorporation) and 5000, 2500, 1250, 625, 313, 156, 0 µg/plate (2nd experiment, preincubation) in the presence and absence of mammalian metabolic activation (Aroclor-induced rat liver S9).

1-(3-Sulfopropyl)-2-vinyl-pyridinumbetain (SPV) was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.