(benzothiazol-2-ylthio)succinic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, under the present test conditions, the test item tested up to a concentration of 5120 µg/plate, in the experiments performed without microsomal activation the test item revealed a reduction in the colony count due to a growthinhibiting effect at the upper concentrations but not in experiments performed with microsomal activation.

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 (CIBA-GEIGY, Ltd., 1985).

 

Additionally, a QSAR prediction for the mutagenicity potential in the 5th strain (E. coli) has been performed  for the test item using Sarah Nexus v. 3.1 in order to fulfil the data gap according to REACH Regulation, Annex VII. In line with the results from Ciba Geigy, 1985, the in silico assessment confirmed the overall negative mutagenicity result for the test substance and furthermore, revealed also a negative result in E. coli.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Remarks:

Sarah Nexus v. 3.1 (provided by Lhasa)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

An AMES test according to OECD 471 is available indicating 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. Based on the earlier requirements, the investigations were performed on four strains TA 98, TA 100, TA 1535 and TA 1537. During that time, in 1985, the E. coli strain was not yet mandatory.
However, in order to fulfil the Annex VII information requirements of today, the mutagenicity potential of the test item needs also to be assessed in the E. coli strain (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102).
Therefore , an in silico assessment by using Sarah Nexus v. 3.1 (provided by Lhasa) seems to be appropriate as the (quantitative) structure-activity relationship ((Q)SAR) methodology gives an overall transparent prediction about Ames Mutagenicity of a structure, and a confidence rating in the prediction. The confidence score is based on each fragment’s contribution to the overall prediction and the weight placed upon each fragment. Furthermore, Sarah Nexus is able to predict individual results for the different bacterial stains.
A QSAR prediction of the mutagenicity potential in the 5th strain (E. coli) is provided for the test item by Sarah Nexus . These in silico results can supplement and support the results of the Ames test (Ciba Geigy, 1985) based on a weight-of-evidence.

Principles of method if other than guideline:

Sarah Nexus (provided by Lhasa limited) is an in silico tool. The (quantitative) structure-activity relationship ((Q)SAR) methodology gives an overall transparent prediction about Ames Mutagenicity of a structure, and a confidence rating in the prediction. The confidence score is based on each fragment’s contribution to the overall prediction and the weight placed upon each fragment. Furthermore, Sarah Nexus is able to predict individual results for the different bacterial stains.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Key result

Species / strain:

E. coli WP2

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

The compound is predicted to be negative with 32% confidence for the 'Mutagenicity in vitro' endpoint in
the model: 'Sarah Model - 2020.1'. Supporting hypotheses containing similar examples from the training
set have been found.

In line with the results from Ciba Geigy, 1985, the in silico assessment by Sarah Nexus v. 3.1 confirmed the overall negative mutagenicity result for the test substance and furthermore, revealed an also negative result in E. coli.

Results	Number of Hypotheses
Positive	0
Negative	3
Positive (Overruled by Training Set Example)	0
Negative (Overruled by Training Set Example)	0
Total Count	3

Conclusions:

In line with the results from Ciba Geigy, 1985, the in silico assessment by Sarah Nexus v. 3.1 confirmed the overall negative mutagenicity result for the test substance and furthermore, revealed an also negative result in E. coli.

Executive summary:

In line with the results from Ciba Geigy, 1985, the in silico assessment by Sarah Nexus v. 3.1 confirmed the overall negative mutagenicity result for the test substance and furthermore, revealed an also negative result in E. coli.

Reference 2

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

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

Target gene:

mutated gene loci responsible for histidine auxotrophy

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).

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

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

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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the in vitro genotoxicity study the substance must not be classifed according to the criteria of EC Regulation 1272/2008.