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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 six closest read across substances, toxicity on daphnia magna was predicted for 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl) -4,5- dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5). Based on the immobility of test organism the EC50 value was estimated to be 257.1 mg/l when 4-[(E)-2-(5- {[(2Z,4E)-6-chloro-4- [(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid exposed to daphnia magna for 48 hrs. Based on this EC50 value it can be concluded that the substance 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino} -2-sulfophenyl) diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid is considered to be nontoxic to aquatic environment and cannot classified as per the criteria mentioned in CLP regulation.         

 

Toxicity to aquatic 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 Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) predicted for 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2- ylidene] amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5).The EC50 value was estimated to be 192.7 mg/l when 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl) imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid exposed to Desmodesmus subspicatus for 72 hrs. Based on this value it can be concluded that the substance4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) is considered to be not toxic to aquatic environment and not classified as per the criteria mentioned in CLP regulation.   

Additional information

Summarized result of toxicity of 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) on the growth and other activity of aquatic invertebrates, algae and cyanobacteria by considering the data for target as well as RA chemical which was selected on the basis of structural similarity and log Kow as the primary descriptor are as follows:  

Short term toxicity to aquatic invertebrates:

Various predicted data for the target compound 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) and supporting weight of evidence studies for its closest read across substance with log Kow as the primary descriptor were reviewed for the toxicity on the invertebrates end point which are summarized as below: 

 

In a prediction done by SSS (2018), based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the six closest read across substances, toxicity on daphnia magna was predicted for 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5). Based on the immobility of test organism the EC50 value was estimated to be 257.1 mg/l when 4-[(E)-2-(5- {[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid exposed to daphnia magna for 48 hrs. Based on this EC50 value it can be concluded that the substance4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl) diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid is considered to be nontoxic to aquatic environment and cannot classified as per the criteria mentioned in CLP regulation.         

 

In second prediction using the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to aquatic invertebrates was predicted. On the basis of this programe, the LC 50 value for short term toxicity to aquatic invertebrates was predicted to be 2475.6 mg/l for 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl) imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1 -(4-sulfophenyl)- 4,5-dihydro-1H-pyrazole-3-carboxylic acid in 48 hrs. Based on this value it can be concluded that the substance 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1 -(4-sulfophenyl)- 4,5-dihydro-1H-pyrazole-3-carboxylic acid is considered to be not toxic to aquatic environment and cannot be classified as per the criteria mentioned in CLP regulation.

 

 

Similarly in the third weight of evidence study for the read across chemical (70616-89-6) from ABITEC study was carried out. Short term toxicity of trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonato-2-naphthyl}diazenyl)naphthalene-1,5-disulfonate to aquatic invertebrates was performed according to the OECD guideline in a static system for the total exposure period of 48 hr. The stock solution 250 mg/l was prepared by dissolving redbrown powder in reconstituted water. Test solutions of required concentration as were prepared by mixing the stock solution of the test sample with reconstituted test water. Chemical was tested at 0 , 45, 67, 100, 150, 225 mg/l nominal concentrations. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance trisodium2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonato-2-naphthyl}diazenyl) naphthalene-1,5-disulfonate, in Daphnia magna was determined to be 229 mg/l. On the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, indicates that the substance is likely to be non-hazardous to aquatic invertebrates and cannot be classified as per the CLP criteria.

 

Similarly in a supporting weight of evidence study from Ecotoxicology and Environmental Safety, 1999 for read across chemical (1934-21-0) study were to determine the toxicity of a chemical Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate (Tartrazine; Food yellow 4) on the growth of water flea Ceriodaphnia cf. dubia. Stock solutions were made by dissolving the appropriate amount in 1 or 2 L of the water described previously and then gently stirred for 12 h in the dark at 23 ± 1°C using Teflon magnetic stirrers. Light was excluded during the stirring to minimize photodegradation of the chemicals. Stock solutions were diluted to the appropriate concentrations immediately prior to the commencement of a test. Test was conducted on the < 24hrs old water flea. 250-ml glass beakers which held 200 ml of the test solution or control solution having headspace of 50 ml were used. The absence of visible movement by the cladocera within 15 s of gentle agitation of the test solution was observed at 5706.55 mg/l. Based on the immobilization of Ceriodaphnia cf. dubia due to the exposure of chemical Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo) pyrazole-3-carboxylate, the EC50 was 5706.55 mg/l. Thus based on the EC50, the chemical was consider as nontoxic and can be consider to be not classified.

 

Similarly fourth study was conducted on the read across chemical selected on the basis of structure similarity (1934-21-0) from The Journal of Toxicological Sciences, 1977. Short term toxicity study to Artemia Salina was carried out for 24-48 hrs. The test chemical conc. used for the study was 5343.68 mg/l and 534.368 mg/l, respectively. A. salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -20°C, Eggs used in experiments were washed and stored at room temperature in a desiccator over anhydrous granular CaCl, . Larvae were obtained by incubating eggs in petri dishes containing muslin-filtered sea water at 30°C for 24 hours. The larvae were separated from shells, dead larvae and unhatched eggs by their phototactic movements toward a light source. Food dyes of various concentrations were placed in a petri dish, and sea water containing 20 to 30 larvae was added. After this was incubated at 30°C for 24 hours and 48 hours, larvae surviving were measured by direct count. The same method was tested from 5 to 6 times for each concentration, and the death rate was calculated. The study was performed under static conditions for 24 – 48 hrs at 30°C. Death was assumed to have occurred when there was no movement. The death rate was defined as the average of the percentage of deaths observed for 24 hours and 48 hours. Based on death rate or mortality of test organism, the LC83.8 value was determine to be 5343.68 mg/l. The chemical was considered as not classified according to the CLP regulation.

 

On the basis of above results for target chemical 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) (from OECD QSAR toolbox version 3.3, and EPIsuite, 2017) and for its read across substance from ABITEC report, peer reviewed journal Ecotoxicology and Environmental Safety, 1999 and The Journal of Toxicological Sciences, 1977, it can be concluded that the test substance 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid is not toxic and can be consider to be not classified as toxic as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

Various predicted data for the target compound 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) and supporting weight of evidence studies for its closest read across substance with log Kow as the primary descriptor were reviewed for the toxicity on the algae end point which are summarized as below:  

 

In a prediction done by SSS (2018), 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 Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) predicted for 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5).The EC50 value was estimated to be 192.7 mg/l when 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl) imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid exposed to Desmodesmus subspicatus for 72 hrs. Based on this value it can be concluded that the substance4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen -1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) is considered to be not toxic to aquatic environment and not classified as per the criteria mentioned in CLP regulation.     

 

In second prediction using the prediction done by EPI suite ECOSAR version 1.10, the short term toxicity on green algae was predicted for test substance  4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1 -(4-sulfophenyl)- 4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858 -25 -4) . On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 1269.3 mg/l for green algae for 96 duration. Based on this value, it can be concluded that the test chemical 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl) imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1 -(4-sulfophenyl)- 4,5-dihydro-1H-pyrazole -3-carboxylic acid can be considered as non-toxic to green algae at environmentally relevant concentrations and can be considered not-classified as per the CLP classification criteria.

 

Similarly in the third weight of evidence study for the read across chemical (70616-89-6) from ABITEC report was carried out. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl) amino]-1-hydroxy- 3-sulfonato-2-naphthyl}diazenyl)naphthalene-1,5-disulfonate according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving redbrown powder in OECD growth medium and tested at the limit concentration i.e 100 mg/l. Effects on the growth rate of the organism were studied after the exposure period of 72hrs with the positive reference control Potassium dichromate (K2Cr2O7). Test performed in the six replicates. The inhibitory concentration for the test substance, trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl) amino]-1-hydroxy-3-sulfonato-2-naphthyl}diazenyl) naphthalene-1,5- disulfonate, in Desmodesmus subspicatus was determined. At 100 mg/l inhibition was not observed in any replicates (0% inhibition was observed). This value indicates that the substance is likely to be non-hazardous to aquatic algae and can be consider as not classified as per the CLP criteria.

 

 

Similarly in the third weight of evidence study for the read across chemical (15790-07-5) from ABITEC study was carried out. The effect of test item aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex (15790-07-5) 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, 200mg/L. The test solution was prepared in aseptic condition. The test item aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring/sonication 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 concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment. Test was performed in static manner at proper requirement of pH and temperature. The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0 units. After 72 hours of exposure to aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalene sulfonic acid complex (15790-07-5) to various nominal test concentration, EC50 calculated from equation through probit analysis was determine to be > 200 mg/l. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as toxic as per the CLP classification criteria.

 

On the basis of above results for target chemical 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (93858-24-5) (from OECD QSAR toolbox version 3.3, and EPIsuite, 2017) and for its read across substance from ABITEC report, it can be concluded that the test substance 4-[(E)-2-(5-{[(2Z,4E)-6-chloro-4-[(3-sulfophenyl)imino]-1,2,3,4-tetrahydro-1,3,5-triazin-2-ylidene]amino}-2-sulfophenyl)diazen-1-yl]-5-oxo-1- (4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid is not toxic and can be consider to be not classified as toxic as per the CLP classification criteria.