Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

Short-term toxicity to fish:

Based on the experimental data for the target as well as read-across analogues which are extracted by using mechanistic approach and functionally and structurally similar to the target chemical, toxicity of test chemical were determined. The studies are summarized as below:

 Study was conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test) to assess the effect of test chemical on the mortality of fish Danio rerio. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 400 mg of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100mg/L, respectively and Zebra Fish Danio rerio were 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 item to 100 mg/l nominal test concentrations, LC50 was determine to be > 100 mg/l. Thus, based on the LC50, it was considered that the chemical was not toxic and cannot be classified as per the CLP classification criteria.

 In a supportive evidence, a 96 h study was conducted to determine the effect of test chemical on fish Danio rerio, at a test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L. Test was performed according to OECD Guideline 203 (Fish, Acute Toxicity Test). Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. Zebra Fish (Danio rerio) were exposed to this concentration for 96 hours. After 96 hours of exposure of test chemical at of 100mg/L, no mortality was observed. Therefore the LC50 was determined to be >100mg/L. Based on the LC50, the test chemical can be considered as non-toxic and can be concluded as not classified as per the CLP classification criteria.

 Further to support first and second study again, A Fish Acute Toxicity test was performed to evaluate the toxic nature of test substance. Test was performed according to OECD Guideline 203. Potable water (passed through reverse osmosis system) was used for the preparation of test solution The test was performed on the limit concentration i.e nominal concentration selected for the experiment were 100 mg/L and test fish were exposed to these concentration for 96 hours.

Effect on the symptoms and the normal activity was checked in the interval of 24rs. pH, tempterature and dissolved oxygen was also checked. After 96hrs of exposure, the lethal concentration LC50 was determined to be >100 mg/L. Thus based on the LC50 it can be concluded that the chemical was nontoxic and was considered to be not classified as per the CLP classification criteria.

 

Hence from the above data, the test chemical was considered as nontoxic and was not classified as per the CLP classification criteria.

Short-term toxicity to aquatic invertebrates:

Based on the experimental data for the target as well as read-across analogues which are extracted by using mechanistic approach and functionally and structurally similar to the target chemical, toxicity of test chemical were determined on the basis of immobility of daphnia magna. The studies are summarized as below:

 Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for determining toxicity of test material. The test substance was soluble in water. The test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 1 g/L, respectively. The nominal concentration selected for the experiment was 100 mg/l and test Daphnia magna were exposed to this concentration for 48 hours. The median lethal concentration (EC50) for test material on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be >100 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, does not exhibit short term toxicity to Daphnia and can be considered to be not classified as per the CLP classification criteria.

 Objective of second study was to determine the effect of test chemical on the mobility of daphnia magna. Test was conducted in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). A limit test at sample concentration of 100 mg/L was performed. Effects on immobilisation were observed for 48 hours. The solution 100.0 mg/L was prepared by dissolving dark blue powder in reconstituted water. The solution was kept 5 min in ultrasonic bath. The median effective concentration (EC50) for the test substance on water flea, Daphnia magna was determined to be >100 mg/L on the basis of mobiity inhibition effects in a 48 hour study as EC50 was observed at the limit test concentration 100 mg/l. Based on this EC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance does not exhibit short term toxicity to aquatic invertebrate (Daphnia Magna) and thus not classified as per the CLP classification criteria.

 Again to support both the studies, another study was performed to determine the toxicity of test chemical on D. magna for 48 hours in a static system. Daphnids were exposed to test chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The nominal concentrations used were 100 mg/L (limit test).

The EC50 was defined as a concentration that immobilizes 50% of the exposed D. magna. Eight percent immobilization in D. magna was observed after 48 hours of exposure to 100 mg/L of test chemical. The EC50 was therefore estimated to be >100 mg/L.

 

Based on the EC50 value, it can be concluded that the chemical was nontoxic and was considered to be not classified as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Based on the predicted data for the target and experimental data for the read-across analogues, toxicity of test chemical were determined on the basis of growth inhibition of green algae. The studies are summarized as below:

The short-term toxicity of the test substance to green algae was predicted using EPI Suite ECOSAR version 1.10. On the basis of effects observed in a static freshwater system during a 96 hr exposure, the effect concentration (EC50) for the substance was estimated to be 149.940 mg/L. Based on this value, it can be concluded that the test chemical can be considered as non-toxic to green algae at environmentally relevant concentrations and can be considered as not-classified as per the CLP classification criteria.

To support the above value, freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 5x10(3) cells /ml algal culture was use in the study for total exposure period of 72hrs. Test concentrations used were 0, 0, 20, 30, 45, 67, 100 mg/l. Effects on the growth rate of the organism were studied.

Thus, the median effective concentration (ErC50) for the test substance after 72h, in Desmodesmus subspicatus was determined to be 285.8 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP criteria.

 Again as a supporting study, algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 200 mg/L was prepared by dissolving dark blue 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.Test concentrations 12.5, 25, 50, 100 and 200 mg/L were prepared by diluting stock solution with OECD medium. Along with these concentrations control was also run simultaneously. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, on Desmodesmus subspicatus after 72h was determined to be 276.1 mg/L.

 

Thus, based on the above ErC50 values and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the test substance does not exhibit toxicity to aquatic algae and hence cannot be classified.

Toxicity to microorganisms:

Various studies available for the test chemical were reviewed to determine the toxic nature of test chemical on the growth and other activity of microorganisms. The studies are as mentioned below:

 To measure the effects of test substance on microorganisms a study was conducted on Paramecium caudatum (PC), a unicellular animal, which can be observed more readily and in far less time than that of small animals. P. Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Chemical was tested in 0.1% and 1% concentration. 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 organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.

 First study was supported by the second study from peer reviewed journal which was conducted to determine the toxicity of test substance on microorganisms. The organisms (Vibrio fisheri) were exposed for 30 s and 30 min to test substance, and the peak luminescence value was obtained during the first 5s after adding the bacterial suspension to the sample. The test concentrations were 500–5000 mg/l with RO16. The results were calculated as the inhibition % of light production and expressed using the corresponding EC50 values. With 30-min exposure time, EC50 value was determined to be 1375 mg/L for the test chemical. Thus based on the EC50 value, chemical was considered to be nontoxic to the growth of microorganism.

Thus based on the overall studies for the test chemical was considered to be nontoxic and the toxicity ranges from the concentration 1375 mg/l - 10000 mg/l.

Additional information

Short-term toxicity to fish:

Based on the experimental data for the target as well as read-across analogues which are extracted by using mechanistic approach and functionally and structurally similar to the target chemical, toxicity of test chemical were determined. The studies are summarized as below:

 Study was conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test) to assess the effect of test chemical on the mortality of fish Danio rerio. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 400 mg of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100mg/L, respectively and Zebra Fish Danio rerio were 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 item to 100 mg/l nominal test concentrations, LC50 was determine to be > 100 mg/l. Thus, based on the LC50, it was considered that the chemical was not toxic and cannot be classified as per the CLP classification criteria.

 In a supportive evidence, a 96 h study was conducted to determine the effect of test chemical on fish Danio rerio, at a test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L. Test was performed according to OECD Guideline 203 (Fish, Acute Toxicity Test). Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. Zebra Fish (Danio rerio) were exposed to this concentration for 96 hours. After 96 hours of exposure of test chemical at of 100mg/L, no mortality was observed. Therefore the LC50 was determined to be >100mg/L. Based on the LC50, the test chemical can be considered as non-toxic and can be concluded as not classified as per the CLP classification criteria.

 Further to support first and second study again, A Fish Acute Toxicity test was performed to evaluate the toxic nature of test substance. Test was performed according to OECD Guideline 203. Potable water (passed through reverse osmosis system) was used for the preparation of test solution The test was performed on the limit concentration i.e nominal concentration selected for the experiment were 100 mg/L and test fish were exposed to these concentration for 96 hours.

Effect on the symptoms and the normal activity was checked in the interval of 24rs. pH, tempterature and dissolved oxygen was also checked. After 96hrs of exposure, the lethal concentration LC50 was determined to be >100 mg/L. Thus based on the LC50 it can be concluded that the chemical was nontoxic and was considered to be not classified as per the CLP classification criteria.

 

Hence from the above data, the test chemical was considered as nontoxic and was not classified as per the CLP classification criteria.

Short-term toxicity to aquatic invertebrates:

Based on the experimental data for the target as well as read-across analogues which are extracted by using mechanistic approach and functionally and structurally similar to the target chemical, toxicity of test chemical were determined on the basis of immobility of daphnia magna. The studies are summarized as below:

 Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for determining toxicity of test material. The test substance was soluble in water. The test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media. Achieving test concentrations of 1 g/L, respectively. The nominal concentration selected for the experiment was 100 mg/l and test Daphnia magna were exposed to this concentration for 48 hours. The median lethal concentration (EC50) for test material on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be >100 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, does not exhibit short term toxicity to Daphnia and can be considered to be not classified as per the CLP classification criteria.

 Objective of second study was to determine the effect of test chemical on the mobility of daphnia magna. Test was conducted in accordance with OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). A limit test at sample concentration of 100 mg/L was performed. Effects on immobilisation were observed for 48 hours. The solution 100.0 mg/L was prepared by dissolving dark blue powder in reconstituted water. The solution was kept 5 min in ultrasonic bath. The median effective concentration (EC50) for the test substance on water flea, Daphnia magna was determined to be >100 mg/L on the basis of mobiity inhibition effects in a 48 hour study as EC50 was observed at the limit test concentration 100 mg/l. Based on this EC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance does not exhibit short term toxicity to aquatic invertebrate (Daphnia Magna) and thus not classified as per the CLP classification criteria.

 Again to support both the studies, another study was performed to determine the toxicity of test chemical on D. magna for 48 hours in a static system. Daphnids were exposed to test chemical in 50 ml beakers in a volume of 25 ml of liquid solution containing both the chemical and media as specified in OECD 202. The nominal concentrations used were 100 mg/L (limit test).

The EC50 was defined as a concentration that immobilizes 50% of the exposed D. magna. Eight percent immobilization in D. magna was observed after 48 hours of exposure to 100 mg/L of test chemical. The EC50 was therefore estimated to be >100 mg/L.

 

Based on the EC50 value, it can be concluded that the chemical was nontoxic and was considered to be not classified as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Based on the predicted data for the target and experimental data for the read-across analogues, toxicity of test chemical were determined on the basis of growth inhibition of green algae. The studies are summarized as below:

The short-term toxicity of the test substance to green algae was predicted using EPI Suite ECOSAR version 1.10. On the basis of effects observed in a static freshwater system during a 96 hr exposure, the effect concentration (EC50) for the substance was estimated to be 149.940 mg/L. Based on this value, it can be concluded that the test chemical can be considered as non-toxic to green algae at environmentally relevant concentrations and can be considered as not-classified as per the CLP classification criteria.

To support the above value, freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 5x10(3) cells /ml algal culture was use in the study for total exposure period of 72hrs. Test concentrations used were 0, 0, 20, 30, 45, 67, 100 mg/l. Effects on the growth rate of the organism were studied.

Thus, the median effective concentration (ErC50) for the test substance after 72h, in Desmodesmus subspicatus was determined to be 285.8 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic as per the CLP criteria.

 Again as a supporting study, algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201. The stock solution 200 mg/L was prepared by dissolving dark blue 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.Test concentrations 12.5, 25, 50, 100 and 200 mg/L were prepared by diluting stock solution with OECD medium. Along with these concentrations control was also run simultaneously. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, on Desmodesmus subspicatus after 72h was determined to be 276.1 mg/L.

Thus, based on the above ErC50 values and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the test substance does not exhibit toxicity to aquatic algae and hence cannot be classified.

Toxicity to microorganisms:

Various studies available for the test chemical were reviewed to determine the toxic nature of test chemical on the growth and other activity of microorganisms. The studies are as mentioned below:

 To measure the effects of test substance on microorganisms a study was conducted on Paramecium caudatum (PC), a unicellular animal, which can be observed more readily and in far less time than that of small animals. P. Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Chemical was tested in 0.1% and 1% concentration. 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 organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.

 First study was supported by the second study from peer reviewed journal which was conducted to determine the toxicity of test substance on microorganisms. The organisms (Vibrio fisheri) were exposed for 30 s and 30 min to test substance, and the peak luminescence value was obtained during the first 5s after adding the bacterial suspension to the sample. The test concentrations were 500–5000 mg/l with RO16. The results were calculated as the inhibition % of light production and expressed using the corresponding EC50 values. With 30-min exposure time, EC50 value was determined to be 1375 mg/L for the test chemical. Thus based on the EC50 value, chemical was considered to be nontoxic to the growth of microorganism.

Thus based on the overall studies for the test chemical was considered to be nontoxic and the toxicity ranges from the concentration 1375 mg/l - 10000 mg/l.

 

Thus, based on the above ErC50 values and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the test substance does not exhibit toxicity to aquatic algae and hence cannot be classified.