Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

EC Number:

256-401-8

EC Name:

Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

Cas Number:

49625-94-7

Molecular formula:

C10H11NO3S3.Na

IUPAC Name:

sodium 3-(1,3-benzothiazol-2-ylsulfanyl)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 humidity.

Method

Target gene:

his

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: 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 produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.

Test concentrations with justification for top dose:

5000 / 1500 / 500 / 150 / 50 µg/plate (plate incorporation method)
5000 / 2500 / 1250 / 625 / 313/ 156 µg/plate (pre-incubation method)
Top dose was selected according to the guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralised H2O, dimethyl sulfoxide (DMSO) and ethanol.
DMSO 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:

H2O

Negative solvent / vehicle controls:

yes

Remarks:

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) and preincubation

DURATION
- Preincubation period: 20 min (Pre-incubation method)
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Minimal histidine agar

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

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

Two independent experiments with variations in methodology should be performed.

Evaluation criteria:

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.
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.

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

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

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

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

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

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: based on the substance' vapour pressure, evaporation is highly unlikely
- Water solubility: substance is sufficiently soluble
- Precipitation: none stated

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
COMPARISON WITH HISTORICAL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 30. Aug. 2016 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.
Table Historical Data of Spontaneous Revertants
Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
H2O Mean 97 99 15 18 95 99 271 291 16 16
Min 61 63 6 11 62 66 85 67 6 7
Max 144 138 35 41 141 141 425 511 30 30
SD 20 16 5 6 15 15 73 90 6 5
Exp 1 73 108 14 15 99 100 339 344 21 16
Exp 2 91 99 15 20 118 99 408 355 24 19
DMSO Mean 97 105 14 16 93 95 268 278 17 16
Min 58 70 7 10 60 63 79 80 8 6
Max 135 144 46 36 136 199 381 405 33 29
SD 19 16 6 5 17 21 65 70 7 6
Exp 1 77 83 9 14 90 107 315 320 14 20
Exp 2 95 83 15 18 120 126 379 459 19 20
Positive Controls* Mean 595 530 413 76 525 696 1119 1258 244 111
Min 325 241 112 39 223 325 491 408 55 45
Max 1152 1181 793 217 984 1912 2331 6083 484 284
SD 185 152 150 36 172 259 434 837 96 48
Exp 1 497 888 265 78 563 627 2021 1296 369 176
Exp 2 553 412 383 61 508 571 1307 1013 217 78
* Different positive controls were used

The value 459, DMSO, exp.2, TA102 +S9 lies outside the range of the historical data.
Bacteria strains are living biological systems, therefore variations in behaviour are not unusual.
Each bacteria strain in this study has its own characteristic spontaneous revertant colony number. Day to day variations in the number of spontaneous revertant colonies are usual.
The variations of the values of all bacteria strains lie in an acceptable range.
Evaluation of the mutagenicity of the test item and validity of the study was not affected by these variations.
No critical impact on the outcome of the study is expected.

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 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) to induce reverse mutations in bacteria.
The results of this experiments showed that the test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.
Based on the results of this study it is concluded that 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Executive summary:

The study was performed according to the most recent OECD and EC guidelines, i.e. OECD 471 and EU method B.13/14.

The test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

Two valid experiments were performed.

 

In the first experiment, 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

The f(I) (twofold higher than the solvent control) of the strain TA98 in concentration 50 µg/plate can be seen as uncritical, because no dose-response relationship was observed.

 

On the base of the first experiment, 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strains using the pre-incubation method. 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.