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Short term toxicity to fish:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the short term toxicity of fish of the test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one (522 -75 -8).The studies are as mentioned below:

1. Test material was subjected to evaluate its acute toxicity effect on Fathead Minnow (Pimephales promelas)10 Fathead Minnow (Pimephales promelas) were acclimatized in 5-gallon wide mouth glass jar for 10 days before the test in the test temperature 17°C. The test organisms were pre-treated with tetracycline . The nominal concentration were0 , 18 , 32 ,56 , 100 , 180 mg/l

 Test material was subjected in the test vessel upto the concentration of 180 mg/l . No toxic effect were observed after 96 hrs. Hence the effect concentration was considered to be >180g/l.

2. Short term toxicity test on fish was performed to evaluate potential of toxicity of test material on Oryzias latipes.The median lethel effect concentration (LC50) was noted at 24 , 72 , 96 h . The LC50 for test material was observed to be >1000 mg/l after 96 h. Based on this value it can be conluded that test material is not toxic to fish and can not be classified as per CLP criteria.

By considering LC50 value it is concluded that test chemical is not toxic tofish and can be considered as “Not classified” according to CLP classification criteria.

Thus, based on the above summarised studies, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one and it’s structurally and functionally similar read across substance, it can be concluded that effect concetration value is greater than 1000 mg/L. Thus, comparing this value with the criteria of CLP regulation, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one cannot be classified

for short term toxicity for fish .Hence,based on the data available for the structurally and functionally similar

read across, test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is not likely to be toxic atleast in the range concentration range of 1000mg/L .

Short term toxicity to aquatic invertebrates:

Short term toxicity study for aquatic invertebrates was summarized in the experimental report as follows:

In the acute immobilisation test with Daphnia magna (STRAUS) performed as per OECD Guideline 202 (Daphnia sp. Acute Immobilisation test in GLP certified lab . The effect of the of the saturated solution (DOC 3.5 mg/L) of the test item 2-(3-oxobenzo[b]thien -2(3H) -ylidene)benzo[b]thiophene-3(2H)-one was determined according to OECD 202 (2004) and Directive 92/69/EC Method The limit test was conducted under static conditions over 48 h . 20 test organisms were exposed to the saturated solution and the control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item.

Daphnia magna STRAUS (Clone 5) was cultured in Elendt M4 modified to a total hardness of 160 to 180 mg CaCO3/L . At least 5 per week ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an alga cell density of > 106cells/mL. The solution (100 mg/L test item was weighed out) was prepared with dilution water in brown glass flask one day day prior to application. The stock solution was shaken with 20 rpm for 24 h (rotating shaker 3040, GFL). Undissolved particles were removed by centrifugation (20 min at 3000). The test was conducted in Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used. 20 animals, divided into 4 parallel samples, each with 5 animals. The daphnids were not fed during the study . A preliminary range finding test was conducted under static conditions. EC10- and EC50-values were only calculated for the reference item by sigmoidal dose-response regression. Calculation of the confidence interval for EC50 were carried out using standard procedures according to CLOPPER and PEARSON (1934). The EC50 value of test material 2-(3-oxobenzo[b]thien-2(3H)-ylidene) benzo[b]thiophene-3(2H)-one after 48h  was observed to be >100 mg/l . There was no biologically significant effect neither in the tested saturated solution nor in the control group. Based on the above observation it can be considered that test material 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is not toxic to aquatic invertebrate and hence, can not be classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae and cyanobacteria of the test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one (522 -75 -8).The studies are as mentioned below:

1) The 72 h algal growth inhibition test with Raphidocelis subcapitata was done according to OECD 201 (2011) and ISO 8692 (2012) guidelines with slight modifications. Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The

culture was split twice a week, adding fresh medium. Algae were exposed to the treatment in sterile 24-well plates with 2 mL of each treatment solution to the respective microplate well, for three days.

Untreated ISO formulation freshwater and potassium dichromate (0.10 – 1.8 mg/L) were used as negative and positive controls respectively. Each treatment was replicated three times and randomized within the plate. Algal stock solution containing 1.01E6 cells/mL was added (20 μL) to each well to obtain a starting algal density of 1E4 cells/mL. Plates were incubated for 72 h at 25±1 °C on an orbital shaker (90 rpm) in a thermostatic chamber. Light was provided by a 2 W LED unit for each plate. Algal density at the beginning and end of treatment was measured with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc) . All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. The effect concentration on test material for the aquatic algae was observed to be 142 mg/l. Based on the above value it can be considered that the test material is not toxic to aquatic algae and can be considered to be not classified as per CLP criterion.

2) Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.

The solution 200.0 mg.l'1 was prepared by dissolving blue powder in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. The substance was declared as light sensitive. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs.

 

The median effective concentration (ErC50) for the test substance, in algae was determined to be 159.8 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as aquatic as per the CLP criteria.

Thus, based on the above summarised studies, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one and it’s structurally and functionally similar read across substance, it can be concluded that effect concentration value is greater than 1000 mg/L. Thus, comparing this value with the criteria of CLP regulation, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one cannot be classified

for toxicity for aquatic algae and cyanobacteria .Hence, based on the data available for the structurally and functionally similar

read across, test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is not likely to be toxic atleast in the range concentration range of 159 mg/L .

Toxicity to microorganism:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one .The studies are as mentioned below:

A screening method based on the measurement of the respiration rate of activated sludge for assessing the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria. The test principle involves measuring the respiration rate of an activated sludge and comparing it with the respiration rate of the same activated sludge under identical conditions, but in the presence of the chemical under test. The test was carried out in activated sludge respiration rate apparatus with constant 20 ± 2°C and pH about 7-8. The test concentration used was 100 mg/l. OECD recommended synthetic sewage was used as feed, while activated sludge was obtained from a sewage works treating predominantly domestic sewage or from a sewage works treating predominantly industrial waste water. The respiration rate of an activated sludge and the respiration rate of activated sludge with test chemical were noted down. In order to calculate the inhibitory effect of a particular chemical at 100 mg/l test concentration its respiration rate is expressed as a percentage of the mean of the two control respiration rates. Thus, IC50 value (concentration for 50% inhibition of respiration rate) for the test chemical on activated sludge (aerobic bacteria) is determined to be10 - 100 mg/1 after 3 hrs of exposure.

The test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is  likely to be toxic to microorganism  in the concentration  range of 10-100 mg/l

Additional information

Short term toxicity to fish:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the short term toxicity of fish of the test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one (522 -75 -8).The studies are as mentioned below:

1. Test material was subjected to evaluate its acute toxicity effect on Fathead Minnow (Pimephales promelas)10 Fathead Minnow (Pimephales promelas) were acclimatized in 5-gallon wide mouth glass jar for 10 days before the test in the test temperature 17°C. The test organisms were pre-treated with tetracycline . The nominal concentration were0 , 18 , 32 ,56 , 100 , 180 mg/l

 Test material was subjected in the test vessel upto the concentration of 180 mg/l . No toxic effect were observed after 96 hrs. Hence the effect concentration was considered to be >180g/l.

2. Short term toxicity test on fish was performed to evaluate potential of toxicity of test material on Oryzias latipes.The median lethel effect concentration (LC50) was noted at 24 , 72 , 96 h . The LC50 for test material was observed to be >1000 mg/l after 96 h. Based on this value it can be conluded that test material is not toxic to fish and can not be classified as per CLP criteria.

By considering LC50 value it is concluded that test chemical is not toxic tofish and can be considered as “Not classified” according to CLP classification criteria.

Thus, based on the above summarised studies, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one and it’s structurally and functionally similar read across substance, it can be concluded that effect concetration value is greater than 1000 mg/L. Thus, comparing this value with the criteria of CLP regulation, 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one cannot be classified

for short term toxicity for fish .Hence,based on the data available for the structurally and functionally similar

read across, test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is not likely to be toxic atleast in the range concentration range of 1000mg/L .

Short term toxicity to aquatic invertebrates:

Short term toxicity study for aquatic invertebrates was summarized in the experimental report as follows:

In the acute immobilisation test with Daphnia magna (STRAUS) performed as per OECD Guideline 202 (Daphnia sp. Acute Immobilisation test in GLP certified lab . The effect of the of the saturated solution (DOC 3.5 mg/L) of the test item 2-(3-oxobenzo[b]thien -2(3H) -ylidene)benzo[b]thiophene-3(2H)-one was determined according to OECD 202 (2004) and Directive 92/69/EC Method The limit test was conducted under static conditions over 48 h . 20 test organisms were exposed to the saturated solution and the control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item.

Daphnia magna STRAUS (Clone 5) was cultured in Elendt M4 modified to a total hardness of 160 to 180 mg CaCO3/L . At least 5 per week ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an alga cell density of > 106cells/mL. The solution (100 mg/L test item was weighed out) was prepared with dilution water in brown glass flask one day day prior to application. The stock solution was shaken with 20 rpm for 24 h (rotating shaker 3040, GFL). Undissolved particles were removed by centrifugation (20 min at 3000). The test was conducted in Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used. 20 animals, divided into 4 parallel samples, each with 5 animals. The daphnids were not fed during the study . A preliminary range finding test was conducted under static conditions. EC10- and EC50-values were only calculated for the reference item by sigmoidal dose-response regression. Calculation of the confidence interval for EC50 were carried out using standard procedures according to CLOPPER and PEARSON (1934). The EC50 value of test material 2-(3-oxobenzo[b]thien-2(3H)-ylidene) benzo[b]thiophene-3(2H)-one after 48h  was observed to be >100 mg/l . There was no biologically significant effect neither in the tested saturated solution nor in the control group. Based on the above observation it can be considered that test material 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is not toxic to aquatic invertebrate and hence, can not be classified as per CLP criteria.

Toxicity to microorganism:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one .The studies are as mentioned below:

A screening method based on the measurement of the respiration rate of activated sludge for assessing the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria. The test principle involves measuring the respiration rate of an activated sludge and comparing it with the respiration rate of the same activated sludge under identical conditions, but in the presence of the chemical under test. The test was carried out in activated sludge respiration rate apparatus with constant 20 ± 2°C and pH about 7-8. The test concentration used was 100 mg/l. OECD recommended synthetic sewage was used as feed, while activated sludge was obtained from a sewage works treating predominantly domestic sewage or from a sewage works treating predominantly industrial waste water. The respiration rate of an activated sludge and the respiration rate of activated sludge with test chemical were noted down. In order to calculate the inhibitory effect of a particular chemical at 100 mg/l test concentration its respiration rate is expressed as a percentage of the mean of the two control respiration rates. Thus, IC50 value (concentration for 50% inhibition of respiration rate) for the test chemical on activated sludge (aerobic bacteria) is determined to be10 - 100 mg/1 after 3 hrs of exposure.

The test chemical 2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one is  likely to be toxic to microorganism  in the concentration  range of 10-100 mg/l