2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test;

In genetic toxicity study test substance was assessed for its possible mutagenic potential by In vitro bacterial gene mutation assay. For this purpose the AMES assay was performed as per OECD guideline 471. The test was performed in Salmonella typhemurium strain TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA by Plate incorporation and Pre incubation method.. The test substance was exposed at the concentration of 0,33,100,333,1000,2500 and 5000 µg/plate in the presence and absence of metabolic activation.No mutagenic effects were observed in both the assay in the presence and absence of S9. Therefore test substance was considered to be non-mutagenic with and without metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 ,TA 100 and E. coli WP2uvrA .Hence the substance cannot be classified as gene mutant in vitro.

In vitro Mammalian cell gene mutation assay

Test chemical tested for mutagenicity in mouse lymphoma TK⁺′⁻ assay. toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml and positive control is Ethyl methanesulfonate at 0.5 µl/ml. With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml concentration of test chemical and positive control is 3-methylcholanthrene at 5.0 or 10.0 µg/ml. There was no dose-related increase in the mutant frequency above the spontaneous control frequency, no 2-fold increase at more than 1 dose and not relative total growth greater than 10% at any exposed concentration. Hencethe substance is considered to be not mutagenic inL5178Y TK +/- mouse lymphoma cells with and without metabolic activation.

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature ofthe test chemical.  The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from1642-3680µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase.The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 )did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental reports

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

To evaluate the mutagenic potential of test chemical in Salmonella thyphemurium starin TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA by Bacterial reverse mutation assay.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella Thyphemurium and Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 prepared from Phenobarbital/Beta -Naphthoflavone induced male Wistar Hanlbm rats.

Test concentrations with justification for top dose:

0,33,100,333,1000,2500 and 5000 µg/plate

Vehicle / solvent:

Vehicle
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosenfor solubility properties and its relative non toxicity to bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9;Sodium azide,4-Nitro-o-phenylene-diamine,4-NOPD,Methyl methane sulfonate +S9;2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
in agar (plate incorporation); Experiment I
preincubation; ExperimentII

DURATION
- Preincubation period: 1hour
- Exposure duration: 48 hours
- Expression time (cells in growth medium):

NUMBER OF REPLICATIONS: Triplicate


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A dose dependent increase in the number of revertent colonies below the threshold was regarded as indication of mutagenic potential.

Statistics:

No statistical evaluation of the data is required .

Species / strain:

bacteria, other: S. typhimurium strains TA98, TA100, TA1535, TA1537 and EColi WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Yes , Range finding study was performed.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Remarks on result:

other: No mutagenic effect were observed.

AMES assay result for test substance

Test strain : TA 1535:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	71.4
33	1.2
100	1.2
333	1.2
1000	1.4
2500	1.4
5000	1.1

 

 With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	11.8
33	0.7
100	0.8
333	0.9
1000	0.8
2500	0.8
5000	0.4

 

Test strain: TA 1537:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	4.8
33	1.0
100	1.0
333	0.8
1000	0.5
2500	0.1
5000	0.1

 

 

 With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	2.7
33	1.1
100	1.1
333	0.9
1000	0.9
2500	0.9
5000	0.7

 

Test strain : TA 98:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	9.1
33	0.7
100	0.9
333	1.2
1000	1.2
2500	1.2
5000	1.2

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	9.5
33	1.0
100	1.0
333	1.2
1000	1.2
2500	1.1
5000	1.1

 

 

 

Test strain : TA 100:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	4.5
33	1.1
100	1.2
333	1.0
1000	0.9
2500	1.1
5000	1.0

 

 With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	2.4
33	1.1
100	1.1
333	0.9
1000	1.1
2500	0.9
5000	0.8

 

 

Test strain : WP2uvrA:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	20.5
33	0.8
100	1.0
333	0.9
1000	0.7
2500	0.9
5000	0.9

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	3.6
33	0.7
100	0.8
333	0.7
1000	0.9
2500	0.9
5000	0.8

 

 Experiment II: Pre-Incubation test

Test strain : TA1535:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	52.6
33	1.1
100	1.0
333	0.9
1000	1.2
2500	1.1
5000	0.9

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	9.1
33	1.0
100	0.9
333	1.0
1000	0.7
2500	0.6
5000	0.6

 

 

 

 

 

 

 

 

 

Test strain: TA1537:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	9.0
33	1.1
100	1.1
333	1.0
1000	0.8
2500	0.5
5000	0.4

 

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	4.2
33	0.9
100	0.8
333	0.9
1000	1.0
2500	1.1
5000	1.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test strain : TA 98:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	15.1
33	1.0
100	1.0
333	1.2
1000	1.3
2500	1.3
5000	1.4

 

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	11.7
33	1.0
100	1.0
333	1.3
1000	1.4
2500	1.0
5000	0.9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test strain : TA 100:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	5.8
33	1.0
100	1.0
333	0.9
1000	0.9
2500	1.1
5000	1.1

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	3.4
33	1.1
100	1.1
333	1.0
1000	1.1
2500	1.2
5000	1.1

 

 

 

 

 

 

 

 

 

 

Test strain : WP2uvrA:

Without S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^#	7.9
33	1.1
100	0.9
333	1.1
1000	1.2
2500	0.8
5000	0.9

 

 

 

 

 

 

With S9 mix-

Concentration µg/plate	Factor*
Negative Control
Solvent Control	1.0
Positive Control^##	3.6
33	0.9
100	0.9
333	0.9
1000	1.0
2500	1.1
5000	0.9

 

Conclusions:

The test substance was evaluated for its mutagenic potential in Salmonella thyphemurium starin TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA by Bacterial reverse mutation assay.The test result was considered to be non mutagenic in the presence and absence of metabolic activation.

Executive summary:

In genetic toxicity study test substance was assessed for its possible mutagenic potential by In vitro bacterial gene mutation assay. For this purpose the AMES assay was performed as per OECD guideline 471. The test was performed in Salmonella typhemurium strain TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA by Plate incorporation and Pre incubation method.. The test substance was exposed at the concentration of 0,33,100,333,1000,2500 and 5000 µg/plate in the presence and absence of metabolic activation.No mutagenic effects were observed in both the assayin the presence and absence of S9.Therefore test substance was considered to be non-mutagenic with and without metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 ,TA 100and E. coli WP2uvrA.Hence the substance cannot be classified as gene mutant in vitro.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as per mentioned below

Principles of method if other than guideline:

Weight of evidence prepared from In vitro Gene mammalian assay
1.Evaluate mutagenicity of test substance by the mouse lymphoma TK assay.
2.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media: The cells were grown in Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY) supplemented with 10% horse serum (Gibco, Grand Island, NY), antibiotics (50 U
penicillin/mi and 50 /µg streptomycin/ml), and 0.02% Pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). All serum lots were prescreened for their ability to support optimal growth.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: : No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml
With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml

Vehicle / solvent:

Vehicle
- Vehicle(s)/solvent(s) used: Yes
- Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Ethyl methanesulfonate at 0.5 µl/ml with metabolic activation: 3-methylcholanthrene at 5.0 or 10.0 µg/ml.

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration: 4 hr
- Expression time (cells in growth medium): 48 hr
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

Evaluation criteria:

Mutant frequencies were expressed as mutants per 10⁴ surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.

Statistics:

No data available

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Aroclor-1254-induced male Fischer 344 rats

Concentration µl/ml	S9	Relative suspension growth (%)	Relative cloning efficiency (%)	Relative total growth (%)	Average no. colonies TFT/viable	Mutant frequency (per 10⁴)
0.0	-					x0.61
971	-	100	113	112	82/230	0.71
971	-	103	108	111	71/221	0.64
1229	-	45	95	43	59/194	0.61
1229	-	102	84	86	66/172	0.7
1486	-	102	91	93	62/185	0.67
1486	-	100	96	95	49/195	0.50
1743	-	101	88	88	47/179	0.53
1743	-	93	92	86	47/187	0.50
2000	-	96	86	83	44/175	0.50
2000	-	108	94	101	55/192	0.57
Positive control	-	47	29	13	306/58	10.55
Solventᵃ	-					x0.56

 

 

 

 

Concentration µl/ml	S9	Relative suspension growth (%)	Relative cloning efficiency (%)	Relative total growth (%)	Average no. colonies TFT/viable	Mutant frequency (per 10⁴)
0.0	+					X0.53
92	+	51	45	23	131/106	2.47
206	+	37	44	16	131/102	2.57
332	+	37	49	18	137/114	2.40
439	+	34	40	14	130/94	2.77
439	+	31	46	14	124/108	2.30
556	+	26	50	13	134/116	2.31
556	+	25	40	10	125/93	2.69
Positive control	+	65	56	36	191/96	3.98
Solvent^b	+					X0.61

 

a: Solvent control for test chemical.

b: Solvent for positive control.

Conclusions:

The substance is considered to be not mutagenic in L5178Y TK +/- mouse lymphoma cells with and without metabolic activation.

Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 ) . The studies are as mentioned below:

Test chemical tested for mutagenicity in mouse lymphoma TK⁺′⁻ assay. toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml and positive control is Ethyl methanesulfonate at 0.5 µl/ml. With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml concentration of test chemical and positive control is 3-methylcholanthrene at 5.0 or 10.0 µg/ml. There was no dose-related increase in the mutant frequency above the spontaneous control frequency, no 2-fold increase at more than 1 dose and not relative total growth greater than 10% at any exposed concentration. Hencethe substance is considered to be not mutagenic inL5178Y TK +/- mouse lymphoma cells with and without metabolic activation.

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature ofthe test chemical.  The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from1642-3680µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase.The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 )did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various test chemicals was reviewed to determine the mutagenic nature of 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 ) . The studies are as mentioned below:

 

AMES test;

In genetic toxicity study test substance was assessed for its possible mutagenic potential by In vitro bacterial gene mutation assay. For this purpose the AMES assay was performed as per OECD guideline 471. The test was performed in Salmonella typhemurium strain TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA by Plate incorporation and Pre incubation method.. The test substance was exposed at the concentration of 0,33,100,333,1000,2500 and 5000 µg/plate in the presence and absence of metabolic activation.No mutagenic effects were observed in both the assay in the presence and absence of S9. Therefore test substance was considered to be non-mutagenic with and without metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 ,TA 100 and E. coli WP2uvrA .Hence the substance cannot be classified as gene mutant in vitro.

Plate-incorporation was carried out to observe genetic effect of test substance in S.typhimurium strainTA1535, TA1537, TA1538, TA98 and TA100. All strain were tested at dose 3333, 1000, 6666 and 10000 µg/plate both with and without metabolic activation (Aroclor 1254-induced male Fischer). Positive control and solvent control as used. There is no dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.Hence, the test substance is considered to be not mutagenic inSalmonella typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance . The study was performed using Salmonella typhimurium strains TA97, TA98, TA100 with and without PCB induced S9 metabolic activation system. The plates were incubated for 48 hrs and the number of dose dependent increase in the revertants was counted. Test substance did not induce reversion of histidine gene mutation in Salmonella typhimurium strains TA97, TA98, TA100 both in the presence and absence of PCB induced rat liver S9 fraction and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro Mammalian cell gene mutation assay

Test chemical tested for mutagenicity in mouse lymphoma TK⁺′⁻ assay. toxicity of test chemical was determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. L5178Y TK +/- mouse lymphoma cells was exposed to Without S9: 971, 971, 1229, 1229, 1486, 1486, 1743, 1743, 2000 and 2000 µg/ml and positive control is Ethyl methanesulfonate at 0.5 µl/ml. With S9: 92, 206, 332, 439, 439, 556 and 556 µg/ml concentration of test chemical and positive control is 3-methylcholanthrene at 5.0 or 10.0 µg/ml. There was no dose-related increase in the mutant frequency above the spontaneous control frequency, no 2-fold increase at more than 1 dose and not relative total growth greater than 10% at any exposed concentration. Hencethe substance is considered to be not mutagenic inL5178Y TK +/- mouse lymphoma cells with and without metabolic activation.

The gene mutation study was conducted according toL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature ofthe test chemical.  The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from1642-3680µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase.The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 )did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Thus based on the above annotation and CLP criteria for target substance 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 )did not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

 

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria for target substance 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo( 522-75-8 )did not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.