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Ecotoxicological information

Endpoint summary

Administrative data

Description of key 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 .The studies are as mentioned below:

Study was conducted to assess the effect of test chemical on the mortality of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 450 mg of the test substance in 4.5 liters of potable water (passed through reverse osmosis system) with 1 hr stirring for achieving test concentrations of 100mg/L ,respectively.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 item to 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:

Data available for the read across chemicals has been reviewed to determine the short term toxicity of aquatic invertebrate of the test chemical .The studies are as mentioned below:

Aim of this study was to assess the short term toxicity of test material 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 stock solution 200 mg/l was prepared by dissolving light grey powder in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.5.0 , 10.0 ,25.0, 50.0 , 100.0 , 200.0 mg/lconcentrations were used in the study. 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.

 The median effective concentration (EC50) for the test substance , in Daphnia magna was determined to be 500 mg/L on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be non-hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

In another study , daphnia sp., Acute Immobilization Test according to OECD Guideline 202 was conducted for test material to assess the toxic effects of the test compound on the test Daphnids. Beaker containing 20ml of media with 10 Daphinds. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 50 mg of the test substance in 500 ml of ADaM’s media. Achieving test concentrations of 100 mg/L, respectively.

A semi-static procedure was used for the study. The nominal concentration selected for the experiment was 100 mg/L and test Daphnids were exposed to this concentration for 48hours. The Effective concentrations EC50 was found to be >100 mg/L.

Thus, according to the CLP Criteria for aquatic classification of thesubstance, it is concluded that test material does not exhibit toxicity to aquatic invertebrate (daphnia magna) andtherefore it cannot be classified as hazardous substance. The above study was further supported by data from peer reviewed journal for another read across substance,The 96 h toxicity test of test chemical with Daphnia magna were conducted in accordance with the recommended procedure outlined in peer reviewed journal.During experiment test material used as vehicle which is not used in excess of 0.5% in the final exposure solution.test condition maintained as 20 deg.C test temp. in static freshwater and Water quality parameters including hardness, alkalinity, pH, conductivity, and dissolved oxygen were routinely monitored following the standard methods by American Public Health Association, American Water Works Association, and Water Pollution Control Federation (1992).

The acute toxicity results were reasonably predicted by a quantitative structure activity relationship model using pH-dependent distribution coefficient and molecular orbital energy parameters of the test.

In experiment theEffective concentration to 50% of test organisms (EC50) on the basis of immobility effect was observed to be 174.4 mg/l with 95% confidence interval-158.4–191.9 for 48 hrs exposure period and 158.8 mg/l with 95% confidence interval-133.0–184.4 for 96 hrs exposure period for test chemical.

Based on the EC50 value, substance is likely to be non-hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the read across chemicals has been reviewed to determine the toxicity of aquatic algae and cyanobacteria.The studies are as mentioned below:

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).The stock solution 200 mg/l was prepared by dissolving light grey 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, 2.2 , 11 , 25 , 55 , 120 mg/lconcentration 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 non linear 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 (EC50) for the test substance in algae was determined to be 129.4 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be non-hazardous to aquatic algae and can not be classified as per the CLP classification criteria.

Data from another study report for read across substance ,

The study was designed to assess the toxic effects of the test compound 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  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 of the culture was kept 1 X 10E4cells/ml.

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

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Above data was further supported by data from peer reviewed journal for read across substance,short term toxicityto Chlorella pyrenoidosa(green algae) study was carried out for 72 hrs. Emerson strain of bacteria free, experimentally reproducible cultures ofChlorella pyrenoidosawas used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs andchlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l.Thus, based on this value, it can be concluded that the substancecan be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

This study was examined to assess the toxic effects of the test compound on Pseudomonas fluorescens in a 24 hours of exposure. The effective concentration (EC0) value of test material in bacteria in a 24 hours of exposure on the basis of mortality effect was observed to be in the range 500- 1000 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 .The studies are as mentioned below:

Study was conducted to assess the effect of test chemical on the mortality of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 450 mg of the test substance in 4.5 liters of potable water (passed through reverse osmosis system) with 1 hr stirring for achieving test concentrations of 100mg/L ,respectively.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 item to 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:

Data available for the read across chemicals has been reviewed to determine the short term toxicity of aquatic invertebrate of the test chemical .The studies are as mentioned below:

Aim of this study was to assess the short term toxicity of test material 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 stock solution 200 mg/l was prepared by dissolving light grey powder in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.5.0 , 10.0 ,25.0, 50.0 , 100.0 , 200.0 mg/lconcentrations were used in the study. 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.

 The median effective concentration (EC50) for the test substance , in Daphnia magna was determined to be 500 mg/L on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be non-hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

In another study , daphnia sp., Acute Immobilization Test according to OECD Guideline 202 was conducted for test material to assess the toxic effects of the test compound on the test Daphnids. Beaker containing 20ml of media with 10 Daphinds. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 50 mg of the test substance in 500 ml of ADaM’s media. Achieving test concentrations of 100 mg/L, respectively.

A semi-static procedure was used for the study. The nominal concentration selected for the experiment was 100 mg/L and test Daphnids were exposed to this concentration for 48hours. The Effective concentrations EC50 was found to be >100 mg/L.

Thus, according to the CLP Criteria for aquatic classification of thesubstance, it is concluded that test material does not exhibit toxicity to aquatic invertebrate (daphnia magna) andtherefore it cannot be classified as hazardous substance. The above study was further supported by data from peer reviewed journal for another read across substance,The 96 h toxicity test of test chemical with Daphnia magna were conducted in accordance with the recommended procedure outlined in peer reviewed journal.During experiment test material used as vehicle which is not used in excess of 0.5% in the final exposure solution.test condition maintained as 20 deg.C test temp. in static freshwater and Water quality parameters including hardness, alkalinity, pH, conductivity, and dissolved oxygen were routinely monitored following the standard methods by American Public Health Association, American Water Works Association, and Water Pollution Control Federation (1992).

The acute toxicity results were reasonably predicted by a quantitative structure activity relationship model using pH-dependent distribution coefficient and molecular orbital energy parameters of the test.

In experiment theEffective concentration to 50% of test organisms (EC50) on the basis of immobility effect was observed to be 174.4 mg/l with 95% confidence interval-158.4–191.9 for 48 hrs exposure period and 158.8 mg/l with 95% confidence interval-133.0–184.4 for 96 hrs exposure period for test chemical.

Based on the EC50 value, substance is likely to be non-hazardous to aquatic invertebrate and cannot be classified as aquatic as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the read across chemicals has been reviewed to determine the toxicity of aquatic algae and cyanobacteria.The studies are as mentioned below:

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).The stock solution 200 mg/l was prepared by dissolving light grey 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, 2.2 , 11 , 25 , 55 , 120 mg/lconcentration 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 non linear 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 (EC50) for the test substance in algae was determined to be 129.4 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be non-hazardous to aquatic algae and can not be classified as per the CLP classification criteria.

Data from another study report for read across substance ,

The study was designed to assess the toxic effects of the test compound 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  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 of the culture was kept 1 X 10E4cells/ml.

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

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Above data was further supported by data from peer reviewed journal for read across substance,short term toxicityto Chlorella pyrenoidosa(green algae) study was carried out for 72 hrs. Emerson strain of bacteria free, experimentally reproducible cultures ofChlorella pyrenoidosawas used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs andchlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l.Thus, based on this value, it can be concluded that the substancecan be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

This study was examined to assess the toxic effects of the test compound on Pseudomonas fluorescens in a 24 hours of exposure. The effective concentration (EC0) value of test material in bacteria in a 24 hours of exposure on the basis of mortality effect was observed to be in the range 500- 1000 mg/L.