2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt

Endpoint summary

Description of key information

Short term toxicity to fish

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, the 96 hr LC50 value of the test chemical on test fish can be expected to be > 100 mg/l. 

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, the 48 hr EC50 value of the test chemical on test organism can be expected to be in the range of >100 to 203.2 mg/l.

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 Pseudokirchneriella subcapitata (Experimental study report, 2019). 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 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. 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 7%, 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 200 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) value was determined to be 185.4 mg/l. On the basis of the EC50 value, chemical was considered as 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 read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 30 mins EC50 value can be expected to be 1375 mg/l.

Additional information

Short term toxicity to fish

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

 

An acute toxicity study was conducted for 96 hrs for assessing the effect of test chemical on fish. The test was performed following the OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average weight 0.066 g and average length of 1.7 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 7.0 mg/l, pH 7.4, water temperature 24°C and under a photoperiod of 16:8 hr light: dark conditions, respectively. Test concentrations selected for the study was were 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively. 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°C, pH 7.2, hardness of water 150.5 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 and test vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) value was determined to be > 100 mg/l.

 

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”. Zebra fish (Danio rerio) of average weight 0.335 g and average length of 3.42 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 7.06 mg/l, pH 7.23, water temperature 24°C and under a photoperiod of 12:12 hr light: dark conditions, respectively. Test concentrations selected for the study was 100 mg/L. Thus, limit test was performed using 100 mg/l of test chemical concentration. 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 25°C, pH 7.14, hardness of water 140.5 mg of CaCO3 and under a photoperiod of 12:12 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 and test vessel. On the basis of effect of test chemical on mortality of the test organism, the median lethal concentration (LC50 (96 h)) value was determined to be > 100 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, the 96 hr LC50 value of the test chemical on test fish can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

Data available for its structurally and functionally similar read across chemicals has been reviewed to determine the toxic 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 stock solution 150 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted test water. Nominal test chemical concentrations used for the study were 0,10, 45, 67.5, 100 and 150 mg/l, respectively. 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 setup 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. EC50 was calculated using non-linear regression by the software Prism 4. EC50 value of the reference substance was determined to be 0.79 mg/l. 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 203.2 mg/l (95% C. I. - 140.9 to 293.2 mg/l).

 

Another 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 100 mg/l was prepared by dissolving test chemical in reconstituted water. The solution was kept 5 min in ultrasonic bath. Thus, limit test was performed using a nominal test conc. of 100 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 simulatenously 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 >100 mg/l (with percent immobility as 0 at 100 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, the 48 hr EC50 value of the test chemical on test organism can be expected to be in the range of >100 to 203.2 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.

Toxicity to aquatic algae and cyanobacteria

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

 

In an experimental study from study report (2019), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. 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 300 mg of test chemical in 300 ml of OECD medium. The remaining test solutions were prepared by dilution from the above stock solution. 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 7%, 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 200 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) value was determined to be 185.4 mg/l.

 

In a supporting weight of evidence study, another freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae (Experimental study report, 2019). 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. Sterile, unicellular, suspension cultures of algae Pseudokirchneriella subcapitata of length 8 – 14 μm and width 2 - 3 μm was used as a test organism. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD medium to get the final concentration of 1000 mg/L. Test chemical concentrations were verified analytically by UV-VIS spectrophometer. Green algae were exposed to six different nominal concentrations of test chemical (0, 6.25, 12.5, 25, 50, 100 and 200 mg/L) in 100 ml conical flasks. The study was performed under the same test conditions as mentioned in the above study. The cell count of each test vessel was also noted with the help of a microscope. Cell densities were recorded in section by section growth rate at 24 hr intervals, which was calculated as specific growth rate. Potassium dichromate (K2Cr2O7) was used as a reference substance for the study. Since the concentration of the test chemical being tested has been satisfactorily maintained within ±20% of the nominal concentration throughout the test, all concentrations will be reported as nominal concentration. 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 17 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. The 72 hr EC50 value of reference substance was determined to be 0.809 mg/l. On the basis of growth rate of the test organism Pseudokirchneriella subcapitata, the 72 hrs median effect concentration (ErC50) was determined to be > 200 (nominal concentration).

 

For the test chemical, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae (Study report, 2016). The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 200 mg/l was prepared by dissolving test chemical in OECD growth medium. The stock solution was kept 5 min in ultrasonic bath. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Nominal test chemical conc. used for the study were 0, 0, 12.5, 25.0, 50, 100 and 200 mg/l, respectively. Study was performed in a static system for 72 hrs. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessels containing OECD medium without the test chemical were also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr median effect concentration (ErC50) value was determined to be 276.1 mg/l (95 % C. I. 149.8 to 508.9 mg/l).

 

On the basis of the above results, it can be concluded that the test chemicalwas 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 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 micro-organism study was carried out for 30 mins. For assessing the effect of test chemical on test organism, Kinetic bioluminescence test ‘‘flash test’’ was carried out. Vibrio fisheri was used as a test organism. Test chemical concentrations used for the study was in the range of 500–5000 mg/L (nominal concentration). During the first 5 sec after adding the batcerial suspension to the test chemical, the peak luminescence value was obtained. Kinetic measurement was performed with a 125 l Luminometer at 20°C. The results were calculated as the inhibition percentage of light productiom and expressed using the corresponding EC50 values. On the basis of the effect of test chemical on the inhibition in percentage of light production of the test organism Vibrio fisheri, the 0.5 min and 30 min EC50 value was determined to be 2403 ± 17 mg/l and 1375 ± 46 mg/l , respectively.

 

For the test chemical, toxicity to micro-organism study was carried out for 20 mins. Paramaecium caudatum was used as a test organism. The test organismParamecium caudatumwas maintained at 22°C on 0.15% dried lettuce infusion and fed withAerobacter aerogenes.Test chemical conc. used for the study was 1000 mg/l (0.1%) and 10000 mg/l (1%). The test concentrations were put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After 5 to 10 test organisms were added, their survival times were measured microscopically. Thirty to forty test organisms for each concentration were tested by the same method, and 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 minutes. The mean survival time (in sec) of test organismParamecium caudatumwas determined to be 695 seconds.  The death rate of the test organism at 10000 mg/l was 77.4%. Thus, based on effect on mortality of the test organism Paramecium caudatum, the EC77.4 value was determined to be 100000 mg/l.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 30 mins EC50 value can be expected to be 1375 mg/l.

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