Ethylenediamine, salt with phosphoric acid

Endpoint summary

Description of key information

Short-term toxicity to fish:

Study was conducted to access the effect of test chemical on the growth 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 1g of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100 mg/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 various nominal test concentrations, LC50 was determine to be >100 mg/l.The fishes were freely swimming without showing any abnormal symptoms. Based on the LC50, it can be consider that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

Long-term toxicity to fish:

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

For a functionally similar read across substance, long term toxicity test of test material was performed for 14 days , The LC 50 for test material observed after 14 days was 250 mg/l and NOEC was noted at 35 mg/l.Based on the above effect concentration it can be considered that test material has no long term toxicity effect on fish , and can be considered to be not classified as per CLP criteria.

For another functionally similar read across substance , long term toxicity test of test material was performed for 14 days , The LC 50 for test material observed after 14 days was 440 mg/l and NOEC was noted at 74 mg/l.Based on the above effect concentration it can be considered that test material has no long term toxicity effect on fish and can be considered to be not classified as per CLP criteria.

Short-term toxicity to aquatic invertebrates:

Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for disodium butanedioate. The test substance was soluble in ADaMs’ media. The in-house water solubility of test substance in milli Q water was found to be 5509.96 mg/L. Therefore, the stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of ADaMs’’ media with continuous 15 minutes of stirring and the concentration of stock solution was analytically detected and then the test solution was prepared foe achieving test concentrations of 1.5625mg/L,3.125mg/L,6.25mg/L,12.5mg/L,25mg/L, respectively.

 The median lethal concentration (EC50) for test substance on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be > 12.5 mg/L but < 25 mg/L mg/L.Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the substance, exhibit short term toxicity to Daphnia and can be classified as aquatic chronic 3. But as the chemical is readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Long-term toxicity to aquatic invertebrates:

The study was conducted to evaluated long term toxicity of test material on aquatic invertebrate Daphnia magna in accordance with OECD guideline 211 in semi-static condition.The test substance is soluble in ADaMs’ media. Therefore, the stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of ADaMs’media with continuous 15 minutes stirring and the concentration of stock solution was analytically detected and then the test solution was prepared for achieving the test concentrations of 0.625 mg/L,1.25 mg/L,2.5 mg/L,5 mg/L,10 mg/L ,respectively.The test was condected in 100 ml beaker with 50 ml fill volume one organism was used in each replicates. The daphids were fed with Selenestrum capricornutum algae. First brood observed on 13th day of experiment. Based on nominal concentrations, experimental LOEC (21 days)] for test substance  on Daphnia magna was found to be 0.625 mg/L. The EC50 was 1.1 mg/l.On the basis of EC50 value it can be concluded that test material had no toxic effect on aquatic invertebrate and hence, it is concluded that test substance is not classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

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).The test substance , was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 mg/L. 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 104 cells/ml.Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/lwere 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. 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 material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively.Based on the EC50, it can be concluded that the chemical was non-hazardous and can be consider to be not classified as per the CLP classification 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:

In a study effect of test material was observed on microorganism . The EC0 after 48 h for test organism Pseudomonas fluorescens was observed to be 500 mg/l.

In another study , The effect of test material was tested on activated sludge . The EC50 observed after 1 h for activated sludge was 1600 mg/l.

The test chemical  is not likely to be toxic to microorganism atleast in the concentartion range of 500-1600 mg/l

Additional information

Short-term toxicity to fish:

Study was conducted to access the effect of test chemical on the growth 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 1g of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100 mg/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 various nominal test concentrations, LC50 was determine to be >100 mg/l.The fishes were freely swimming without showing any abnormal symptoms. Based on the LC50, it can be consider that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

Long-term toxicity to fish:

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

For a functionally similar read across substance, long term toxicity test of test material was performed for 14 days , The LC 50 for test material observed after 14 days was 250 mg/l and NOEC was noted at 35 mg/l.Based on the above effect concentration it can be considered that test material has no long term toxicity effect on fish , and can be considered to be not classified as per CLP criteria.

For another functionally similar read across substance , long term toxicity test of test material was performed for 14 days , The LC 50 for test material observed after 14 days was 440 mg/l and NOEC was noted at 74 mg/l.Based on the above effect concentration it can be considered that test material has no long term toxicity effect on fish , and can be considered to be not classified as per CLP criteria.

Short-term toxicity to aquatic invertebrates:

Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for disodium butanedioate. The test substance was soluble in ADaMs’ media. The in-house water solubility of test substance in milli Q water was found to be 5509.96 mg/L. Therefore, the stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of ADaMs’’ media with continuous 15 minutes of stirring and the concentration of stock solution was analytically detected and then the test solution was prepared foe achieving test concentrations of 1.5625mg/L,3.125mg/L,6.25mg/L,12.5mg/L,25mg/L, respectively.

 The median lethal concentration (EC50) for test substance on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be > 12.5 mg/L but < 25 mg/L mg/L.Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the substance, exhibit short term toxicity to Daphnia and can be classified as aquatic chronic 3.

But as the chemical was readily biodegradable in water, thus on the basis of readily biodegradable nature of chemical, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Long-term toxicity to aquatic invertebrates:

The study was conducted to evaluated long term toxicity of test material on aquatic invertebrate Daphnia magna in accordance with OECD guideline 211 in semi-static condition.The test substance is soluble in ADaMs’ media. Therefore, the stock solution was prepared by dissolving 200 mg of the test substance in 200 ml of ADaMs’media with continuous 15 minutes stirring and the concentration of stock solution was analytically detected and then the test solution was prepared for achieving the test concentrations of 0.625 mg/L,1.25 mg/L,2.5 mg/L,5 mg/L,10 mg/L ,respectively.The test was condected in 100 ml beaker with 50 ml fill volume one organism was used in each replicates. The daphids were fed with Selenestrum capricornutum algae. First brood observed on 13th day of experiment. Based on nominal concentrations, experimental LOEC (21 days)] for test substance  on Daphnia magna was found to be 0.625 mg/L. The EC50 was 1.1 mg/l.On the basis of EC50 value it can be concluded that test material had no toxic effect on aquatic invertebrate and hence, it is concluded that test substance is not classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

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). The test substance , was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 mg/L. 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 104 cells/ml. Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/lwere 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. 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 material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively.Based on the EC50, it can be concluded that the chemical was non-hazardous and can be consider to be not classified as per the CLP classification 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:

In a study effect of test material was observed on microorganism . The EC0 after 48 h for test organism Pseudomonas fluorescens was observed to be 500 mg/l.

In another study , The effect of test material was tested on activated sludge . The EC50 observed after 1 h for activated sludge was 1600 mg/l.

The test chemical  is not likely to be toxic to microorganism atleast in the concentartion range of 500-1600 mg/l