(benzothiazol-2-ylthio)succinic acid

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:

From May 18, 1984 until June 8, 1984 ; Report date July 6, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

no cytotoxicity test performed, only 4 strains used

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The tests were carried out in accordance with the method described by AMES et al.

Principles of method if other than guideline:

The tests were carried out in accordance with the method described
by AMES et al.

The bacteria on which the tests were performed were the following
histidine-auxotrophic strains of Salmonella typhimurium:
TA 98, TA 100, TA 1535 and TA 1537 (origin: Prof.B. Ames,
Berkeley, U.S.A.).

The tests were performed with the following concentrations of
the trial substance without and with microsomal activation: 20,
80, 320, 1280 and 5120 )ig/0.1 ml. The substance was dissolved in
acetone. Acetone alone was used for the negative controls (the
substances and vehicles used for the positive controls are indicated
below). Each Petri dish contained: 1) approx. 20 ml of
minimum agar (Agar, Difco Laboratories, Detroit, Michigan,
U.S.A., plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle
and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories,
Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in
2.0 ml of soft agar. The soft agar was composed of: 100 ml of
0.6% agar solution with 0.6% NaCl and 10 ml of a solution of
1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and -i-biotin
0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which
the substance was metabolically activated, 0.5 ml of an activation
mixture was added also (lit.1,2,3). 1 ml activation mixture
contained: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF))
induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut,
U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously
with the following substances: 1) for strain TA 98: daunorubi-
® cin-HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica
GmbH, Freiburg i.Br., Germany), 5 and 10 jag/O.l ml phosphate
buffer; 2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka,
Buchs, Switzerland), 0.125 and 0.25 vig/0.1 ml phosphate buffer;
3) for strain TA 1535: N-methy1-N'-nitro-N-nitrosoguanidine (Serva,
Heidelberg, Germany), 3 and 5 pg/O.l ml phosphate buffer;
4) for strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate
(Fluka, Buchs, Switzerland), 50 and 100 jig/O.l ml DMSO.
The activation mixture was tested with strain TA 1535 and cyclo-
phosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany),
250 pg/0.1 ml phosphate buffer.

In the experiments without and with the addition of microsomal
activation mixture three Petri dishes were prepared per strain
and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37 - 1.5 C in
darkness.

When the colonies had been counted, the arithmetic mean was calculated.
The test substance is generally considered to be nonmutagenic
if the colony count in relation to the negative control
is not doubled at any concentration.

GLP compliance:

no

Remarks:

This test has been put under QA surveillance by the QAU

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(benzothiazol-2-ylthio)succinic acid

EC Number:

401-450-4

EC Name:

(benzothiazol-2-ylthio)succinic acid

Cas Number:

95154-01-1

Molecular formula:

C11H9NO4S2

IUPAC Name:

2-(1,3-benzothiazol-2-ylsulfanyl)butanedioic acid

Test material form:

solid

Details on test material:

(benzothiazol-2-ylthio)succinic acid, Batch: 1

Method

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: E. coli: in silico prediction available. Please refer to“ Mutagenicity_QSAR_ Sarah Nexus v.3.1”.

Metabolic activation:

with and without

Metabolic activation system:

1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors

Test concentrations with justification for top dose:

20, 80, 320, 1280 and 5120 µg/0.1 ml.

According to OECD 471, the recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 µl/plate.

Vehicle / solvent:

The substance was dissolved in acetone (0.1 ml).

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Acetone alone was used for the negative controls

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: daunorubi- ® cin-HCl (DAUNOBLASTIN)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).

METHOD OF TREATMENT/ EXPOSURE:
Each Petri dish contained: 1) approx. 20 ml of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and -i-biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also (lit.1,2,3). 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for strain TA 98: daunorubi- ® cin-HCl (DAUNOBLASTIN , Farmitalia, Montedison FarmaceuticaGmbH, Freiburg i.Br., Germany), 5 and 10 jag/O.l ml phosphate buffer; 2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland), 0.125 and 0.25 vig/0.1 ml phosphate buffer; 3) for strain TA 1535: N-methy1-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 pg/O.l ml phosphate buffer; 4) for strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 50 and 100 jig/O.l ml DMSO. The activation mixture was tested with strain TA 1535 and cyclo- ® phosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 pg/0.1 ml phosphate buffer.

TREATMENT AND HARVEST SCHEDULE:
The plates were incubated for about 48 hours at 37 +/- 1.5 C in darkness. When the colonies had been counted, the arithmetic mean was calculated.

Evaluation criteria:

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without microsomal activation growthinhibiting effect of the compound at the upper concentrations; with microsomal activation no effect

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without microsomal activation growthinhibiting effect of the compound at the upper concentrations; with microsomal activation no effect

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without microsomal activation growthinhibiting effect of the compound at the upper concentrations; with microsomal activation no effect

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without microsomal activation growthinhibiting effect of the compound at the upper concentrations; with microsomal activation no effect

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In the experiments performed without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed a reduction in the colony count due to a growthinhibiting effect of the compound at the upper concentrations.

In the experiments performed with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed no marked deviations.

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, in the experiments performed without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed a reduction in the colony count due to a growthinhibiting effect of the compound at the upper concentrations.
In the experiments performed with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed no marked deviations.
No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments up to the highest concentration of 5120 µg/plate.

Executive summary:

The test item was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and

TA 1537 with the following concentrations of the trial substance with and without microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 ml. In order to confirm the results the experiments

were repeated.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed a reduction in the colony count due to a growthinhibiting effect of the compound at the upper concentrations.

In the experiments performed with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test item revealed no marked deviations.

No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.