Aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to access the effect of test chemicalaluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene) -2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complexon the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).

The test substance was soluble in water; In house solubility was found to be 339.2 mg/L. Therefore the stock solution prepared as 400mg/4L, with the concentration of 100mg/L, and was kept for 48 hr. stirring.After this filter the stock, gave it for analytical detection and then stock taken for experiment.This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L and Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test itemaluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex.to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

Aim of this study was to access the short term toxicity of test chemical C.I. Food Blue 1 to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs. The solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The solution was kept 5 min in ultrasonic bath. Limit test at 100 mg/l were performed. Effects on immobilisation were observed for 48 hours. 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. After the incubation period of 48 hrs in which daphnids exposed with test chemical C.I. Food Blue 1, only 8 % inhibition were observed at 100 mg/l. Thus on the basis of only 8 % inhibition it was consider that the IC50 was > 100 mg/l and the chemical was nontoxic and non-hazardous to aquatic invertebrates and cannot be classified as per the CLP criteria.

Toxicity to algae and cyanobacteria:

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 stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.0, 12, 20, 35, 60 and 100 mg/l concentration were used. 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. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical  C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l. Based on the ErC50 value, it was concluded that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

Additional information

Short term toxicity to fish:

Study was conducted to access the effect of test chemicalaluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene) -2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complexon the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).

The test substance was soluble in water; In house solubility was found to be 339.2 mg/L. Therefore the stock solution prepared as 400mg/4L, with the concentration of 100mg/L, and was kept for 48 hr. stirring.After this filter the stock, gave it for analytical detection and then stock taken for experiment.This test solution was then added to the remaining three liters of water for achieving test concentrations of 100 mg/L and Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test itemaluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex.to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

Summarized result for the toxicity of test chemical (Cas no. 16521 -38 -3) on the growth of aquatic invertebrates are as follows:

In the first key study from experimental report toxicity was measured. Aim of this study was to access the short term toxicity of test chemical C.I. Food Blue 1 to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs. The solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The solution was kept 5 min in ultrasonic bath. Limit test at 100 mg/l were performed. Effects on immobilisation were observed for 48 hours. 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. After the incubation period of 48 hrs in which daphnids exposed with test chemical C.I. Food Blue 1, only 8 % inhibition were observed at 100 mg/l. Thus on the basis of only 8 % inhibition it was consider that the IC50 was > 100 mg/l and the chemical was nontoxic and non-hazardous to aquatic invertebrates and cannot be classified as per the CLP criteria.

Similarly in the second supporting study from peer reviewed journal, acute toxicity study for test chemical in Daphnia similis when they were exposed for 48 hr. 20 % immobilization was observed at test concentrations of 30mg/l. Test conducted under the static system. Daphnia fed during the test on algae Ankistrodesmus falcatus. After the exposure of test chemical for 48 hrs, EC20 were observed at 30 mg/l on the basis of immobility of daphnia similis. Thus on that basis chemical was not consider as toxic and not classified as per the CLP classification criteria.

Based on the above experimental studies, it was concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.

Toxicity to algae and cyanobacteria:

Summarized result for the toxicity of test chemical (Cas no. 16521 -38 -3) on the growth and other biological activity of aquatic algae and cyanobacteria are as follows:

Aim of 1st experimental study 2018, 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 stock solution 100 mg/l was prepared by dissolving blue powder in OECD growth medium. The stock solution was kept in ultrasonic bath for 5 min. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.0, 12, 20, 35, 60 and 100 mg/l concentration were used. 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. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical  C.I. Food Blue 1 the ErC50 was determine to be 187.8 mg/l. Based on the ErC50 value, it was concluded that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as per the CLP criteria.

First study was supported by second experimental report 2016. The study was designed to assess the toxic effects of the test compound aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3- oxo-1H-indole-5- sulfonic acid complex on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution was prepared in aseptic condition. The test item aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201. Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical aluminium, 2-(1,3-dihydro-3-oxo-5-sulfo-2H-indol-2-ylidene)-2,3-dihydro-3-oxo-1H-indole-5-sulfonic acid complex, the EC50 was determine to be > 200 mg/l.. Based on the EC50, it can be concluded that the test chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Similar Short term toxicity study were carried out for 72hr to check the toxicity of test chemical on algae Pseudokirchneriella subcapitata. Algae was exposed the concentration of 30mg/L under static condition. Test chemical strongly stimulate the algal growth indicating the nutritional effects . Pseudokirchneriella subcapitata when exposed for 72 hr, 164% growth was observed at the test concentration of 30mg/L .Therefore the  NOEC for test chemical can be considered as 30mg/L. Based on the NOEC value the substance may not be classified.

Thus based on the overall studies it was concluded that the chemical was nontoxic and not classified as per the CLP classification criteria.