Dihydrogen (ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, aluminium salt

Endpoint summary

Description of key information

Short term toxicity to fish

Short term toxicity to fish was conducted for 96 hrs for assessing the effect of test chemical (Experimental study report). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.1204g and average length of 1.79 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.8 mg/l, pH 7.4, water temperature 23.5°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was 100 mg/L, respectively. Thus, limit test was perfromed. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24.3°C, pH 7.4, hardness of water 148.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. No mortalities were observed in the control vessel. Test fishes were moving slowly as compared to control. On the basis of effect of test chemical on mortality of the test organism, the LC0 and median lethal concentration (LC50 (96 h)) was determined to be 100 mg/l and >100 mg/L, respectively. Thus, based on the LC50 value, test chemical was considered to be non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be classified 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by  evaluating the effect of test chemical on test organism, the 48 hr EC50/LC50 value can be expected to be in the range 200 to >1000 mg/l, respectively.

Toxicity to aquatic algae and cyanobacteria

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris (Experimental report, 2017). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 50 mg of test chemical in 250 ml of BBM to get the final concentration of 200 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 6.25, 12.5, 25, 50, 100 and 200 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 16 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value was determined to be 125.46 mg/l. On the basis of the EC50 value, chemical was considered to be non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 20 mins NOEC value can be expected to be 1000 mg/l and EC50 value can be expected to be in the range 1000 to 10000 mg/l, respectively.

Additional information

Short term toxicity to fish

Experimental study of the test chemical and various supporting studies of its structurally and functionally similar read across chemical has been reviewed for short term toxicity to fish endpoint. The studies were mentioned below:

 

In an experimental study from study report, short term toxicity to fish was conducted for 96 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.1204g and average length of 1.79 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.8 mg/l, pH 7.4, water temperature 23.5°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was 100 mg/L, respectively. Thus, limit test was perfromed. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24.3°C, pH 7.4, hardness of water 148.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. No mortalities were observed in the control vessel. Test fishes were moving slowly as compared to control. On the basis of effect of test chemical on mortality of the test organism, the LC0 and median lethal concentration (LC50 (96 h)) was determined to be 100 mg/l and >100 mg/L, respectively.

 

Another short term toxicity to fish was conducted for 96 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test” (Study report). Zebra fish (Danio rerio) of average weight 0.18 g and average length of 3.18 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. The test conditions during the housing of the test organisms were oxygen content of 7.2 mg/l, pH 8.12, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was 100 mg/L, respectively. Thus, limit test was performed. Total 8 fishes were exposed to test chemical in a 5 lit bowl aquaria containing 4 liters of potable water. The test vessels were placed in a room at a temperature of 24.6°C, pH 7.98, hardness of water 142.5 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions, respectively. Aeration in test vessels was provided 1 day before the start of the experiment. No mortalities were observed in the control vessel. Test fishes were moving slowly as compared to control. On the basis of effect of test chemical on mortality of the test organism, the LC0 and median lethal concentration (LC50 (96 h)) was determined to be 100 mg/l and >100 mg/L, respectively.

 

For the test chemical from authoritative databases and secondary source, short term toxicity to fish study was conducted for 96 hrs for assessing the effect of test chemical. Oncorhynchus mykiss (Rainbow trout) (Juvenile) of 3.5 months old, 4.2 cm in length and 0.7 gm of weight was used as a test organism. The study was performed under static conditions at a temperature of 14°C for 96 hrs. On the basis of the effect of test chemical on mortality of test fishes, the 96 hr median lethal concentration (LC50) value was determined to be 412 mg/l.

 

On the basis of the above results, it can be concluded that the test chemicalwas considered to be non-toxic to aquatic fishesand hence, considered to be classified ‘not classified’ as per the CLP classification criteria. 

Short term toxicity to aquatic invertebrates

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic invertebrates. The studies are as mentioned below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The solution 200 mg/l was prepared by dissolving test chemical in reconstituted water. Thus, limit test was performed using a nominal test conc. of 200 mg/l. Study was performed using 5 organisms per vessel/replicates in a static system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also run simultaneously during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be >200 mg/l (with percent immobility as 4.0 at 100 mg/l). Thus, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The study was performed under static conditions using Daphnia spp. (Water flea) as a test organism. On the basis of the toxic effect of the chemical on mobility of the test organism Daphnia magna, the 48 hr median lethal concentration (LC50) value was determined to be >1000 mg/l. Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr EC50/LC50 value can be expected to be in the range 200 to >1000 mg/l, respectively.

Toxicity to aquatic algae and cyanobacteria

Experimental study of the test chemical and various supporting studies of its structurally and functionally similar read across chemicalwere reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental study from study report (2017), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 50 mg of test chemical in 250 ml of BBM to get the final concentration of 200 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 6.25, 12.5, 25, 50, 100 and 200 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental flask also no significant changes were observed up to the concentration of 16 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value was determined to be 125.46 mg/l.

 

Another toxicity to aquatic algae test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test chemical was prepared by dissolving 50 mg of test chemical in 250 ml of BBM to get the final concentration of 200 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 6.25, 12.5, 25, 50, 100 and 200 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10%, thus, fulfilling the validity of the criteria. All the cells appeared healthy, sickle shaped cells and green throughout the test duration in the control and in the experimental vessels black dotted rings were observed inside the cells in concentrations of 200mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value was determined to be 213.816 mg/l.

 

On the basis of the above results, it can be concluded that the test chemicalcan be considered as non-toxic to aquatic algae and hence, considered to be ‘not classified’ as per the CLP classification criteria. 

Toxicity to microorganisms

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

 

Toxicity to microorganism study was carried out for 20 mins. Paramaecium caudatum (Pc) was used as a test organism. Test organism was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Test organisms were exposed to chemical conc. (0.1% (1000 mg/l) and 1% (10000 mg/l)) in a hollow glass slide. An equal volume of 0.04 M phosphate buffer (pH 7.0) was added. in the test vessel. After 5 to 10 Pc were added, their survival time were measured microscopically. 30 to 40 Paramaecium caudatum/conc. were tested by the same method. After an exposure period of 20 mins, the mean survival time and the death rate were calculated. The survival time was defined as the time required until death was observed for each concentration. Death was assumed to have occurred when there was no movement. The death rate was defined as the percentage of deaths observed during 20 min. On the basis of the effect of test chemical on mortality of the test organism Paramaecium caudatum, the 20 mins NOEC and EC value was determined to be 1000 and 10000 mg/l, respectively.

 

Another toxicity to microorganism study was carried out for 20 mins. Paramaecium caudatum (Pc) was used as a test organism. Test organism was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Study was performed using the same method as mentioned in above study. On the basis of the effect of test chemical on mortality of the test organism Paramaecium caudatum, the 20 mins EC value was determined to be 1000 mg/l.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 20 mins NOEC value can be expected to be 1000 mg/l and EC50 value can be expected to be in the range 1000 to 10000 mg/l, respectively.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemicalwas considered as non-toxic to aquatic organisms at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.