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Description of key information

Short term toxicity to aquatic invertebrates:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the seven closest read across substances, toxicity on daphnia magna was predicted for [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1). Based on the intoxication of test organism the EC50 value was estimated to be 25.5 mg/l when [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]] bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt exposed to daphnia magna for 48 hrs. Based on this value it can be concluded that the substance [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment and can be classified as chronic category 3 as per the criteria mentioned in CLP regulation.    

Toxicity to aq. algae and cyanobacteria:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, toxicity on Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was predicted for [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt (75173-68-1). Based on the growth rate inhibition of algae Pseudokirchneriella subcapitata by the chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt, the EC50 was estimated to be 38.68 mg/l. Based on this value it can be concluded that the substance [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment and can be classified as aquatic chronic 3 as per the criteria mentioned in CLP regulation.    

Additional information

Summarized result for the determination of nature of chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) on the growth and other biological and physical activity of aquatic invertebrates, algae and cyanobacteria when chemical comes in contact with test organisms, by considering the data for target as well as structurally and functionally similar read across chemicals which are as follows: 

 

Short term toxicity to aquatic invertebrates:

Based on the various predicted data for the target chemical and experimental data for structurally and functionally similar read across chemicals study have been reviewed to determine the toxic nature of target chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) on the behavior and growth rate of algae. The studies are as mentioned below:  

 

The first predicted study for the target chemical (100-07-2) done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the seven closest read across substances, toxicity on daphnia magna was predicted for [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1). Based on the intoxication of test organism the EC50 value was estimated to be 25.5 mg/l when [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]] bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt exposed to daphnia magna for 48 hrs. Based on this value it can be concluded that the substance [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment and can be classified as chronic category 3 as per the criteria mentioned in CLP regulation.    

 

First predicted study was supported by the second experimental weight of evidence study for the structurally closest read across chemical (17095-24-8) from peer reviewed journal (Journal of Hazardous Materials (2014)). Aim of this study was to evaluate the toxic nature of chemical Tetrasodium 4-amino-5-hydroxy-3,6-bis[[4-[[2(sulphonatooxy)ethyl]sulphonyl] phenyl]azo]naphthalene-2,7-disulphonate (Reactive Black 5) on the growth of microcrustacea D. Similis. Neonates of less than 24 h old isolated from Daphnia culture were used for the study. Screw cap tubes, filled with 10 mL solution were used in the study in which 5 test organisms added in each vessel. 4 replicates per concentration was used. After 48 hrs of exposure, the number of immobile organisms was recorded and EC50 was calculated. Based on the immobility of daphnia similis by the chemical Tetrasodium 4-amino-5-hydroxy-3,6-bis[[4-[[2(sulphonatooxy)ethyl] sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate (Reactive Black 5) for 48 hrs, the EC50 was determine to be 36 mg/l. Based on the EC50, chemical was consider as toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

 

Similarly for another read across chemical (633-96-5) from peer reviewed journal (Journal of Hazardous Materials (2014)) short term toxicity was studied. Short term toxicity study to Daphnia similis was carried out for 48 hrs. Neonates of less than 24 h old isolated from Daphnia culture were used for the study. Five test organisms were placed in each screw cap tubes, filled with 10 mL solution, and then incubated at 21±0.3°C in the dark. After 48 h of exposure, the number of immobile organisms was recorded. Jspear software was used to calculate the median 50% immobilization concentration (EC50). Based on immobilization of test organism by test chemical Acid Orange 7, the EC50 value was determine to be 87 mg/l along with confidence interval (p – 0.05) of 76-99 mg/l.

 

Based on the predicted data for the target chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]] bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) and for the read across chemical from peer reviewed journal (Journal of Hazardous Materials (2014)), it can be concluded that the [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment (aquatic algae and cyanobacteria) and can be classified as chronic 3 category as per the CLP classification criteria.

 

Toxicity to aq. algae and cyanobacteria:

Based on the various predicted data for the target chemical and experimental data for structurally and functionally similar read across chemicals study have been reviewed to determine the toxic nature of target chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) on the growth of algae. The studies are as mentioned below:  

 

The first predicted study for the target chemical (100-07-2) done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, toxicity on Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was predicted for [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt (75173-68-1). Based on the growth rate inhibition of algae Pseudokirchneriella subcapitata by the chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt, the EC50 was estimated to be 38.68 mg/l. Based on this value it can be concluded that the substance [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]] dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment and can be classified as aquatic chronic 3 as per the criteria mentioned in CLP regulation.    

 

First predicted study was supported by the second experimental weight of evidence study for the read across chemical Brilliant black 1 (2159-30-4) from UERL lab report. Aim of this study was to assess the effect of test item tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate, CAS No. 2519-30-4 on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The chemical was analytically evaluated by using 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. The test solution was prepared in aseptic condition. The test item tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate was prepared by adding 21 mg of test item in 200ml of BBM to get the final concentration of 105 mg/L. This stock solution was kept for stirring for 30 minutes to obtain a homogenous solution for the experiment. The concentrations were chosen according to the available data of the test item. 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment. Test organism green alga Chlorella vulgaris were collected from National Environmental Engineering Research Institute (NEERI), Nagpur and cultivate in Bold’s Basal Medium (BBM). For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates. After the exposure of chemical effect were measured. 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) was determined. The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration in the control and in the test vessels cells appeared shrinked and black dotted rings observed in concentrations of 0.96mg/l, 3.072mg/l, 9.830mg/l, 31.457mg/l & 100.663mg/l. After 72 hours of exposure to test item tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate (CAS No. 2519-30-4) to various nominal test concentrations, EC50 was determine to be 24.342 mg/l graphically and through probit analysis. Thus based on the EC50, chemical was concluded as toxic and can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.

 

Similarly for another read across chemical (3567-69-9) from UERL lab study was conducted. The objective of the test was to determine the effects of Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl) azo]naphthalene sulphonate CAS No. 3567-69-9 on the growth of freshwater microalgae Chlorella vulgaris. The algae were exposed to various concentrations of the test item under static conditions over a period of 72h. The growth rate relative to the control in a 72h test was taken as a parameter to observe the algal growth inhibition. Sample analytically motorized by spectrophotometer. The absorbance values of each test vessel and control vessel was noted at 680nm.TheBBM 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. The test solution was prepared in aseptic condition. The test item Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo] naphthalene sulphonate 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 for 30 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment. Test organism green alga Chlorella vulgaris were collected from National Environmental Engineering Research Institute (NEERI), Nagpur and cultivate in Bold’s Basal Medium (BBM). For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations 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) was determined. After 72 hours of exposure to test item Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalene sulphonate (CAS No. 3567-69-9) to various nominal test concentrations, EC50 was determine to be 34.823 mg/l graphically and through probit analysis. Thus based on the EC50, chemical was concluded as toxic and can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.

 

The fourth study was used from the UERL report for the structurally and functionally similar read across chemicals (4548-53-2) which supports the classification of target chemical. The effect of the test item disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate, CAS No. 4548-53-2 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/l. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. After 72 hours of exposure to test item disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]- 4-hydroxynaphthalene-1-sulphonate (CAS No. 4548-53-2) to various nominal test concentrations, EC50 was determine to be 17.321mg/l graphically and through probit analysis. Based on the EC50, chemical was consider as toxic and can be consider to be classified as aquatic chronic 3 category as per the CLP classification criteria.

 

Based on the predicted data for the target chemical [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]] bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) and for the read across chemical from experimental lab reports (UERL lab), it can be concluded that the [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment (aquatic algae and cyanobacteria) and can be classified as chronic 3 category as per the CLP classification criteria.

 

Thus based on the overall studies for the toxicity to invertebrates and algae, it can be concluded that the [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicopper, tetrasodium salt (75173-68-1) is considered to be toxic to aquatic environment and can be classified as aquatic chronic 3 category as per the CLP classification criteria.