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Short-term toxicity to fish:

The toxic effects of the test compound trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS no. 50662-99-2) were predicted using QSAR toolbox version 3.3. The prediction was based on the effects of the test compound on Danio rerio (previous name: Brachydanio rerio) in a static fresh water system during a 96 hour exposure. The lethal concentration (LC50) for the test compound was estimated to be 253.57 mg/l on the basis of mortality effect. Thus, based on this value, test chemical compound trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate can be considered as non-toxic to fish and thus can be considered to be not classified as per the CLP classification criteria.

Short term toxicity on 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, the short term toxicity on aquatic invertebrates was predicted for trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl- 5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS:50662-99-2). EC50 value was estimated to be 140.69 mg/l for Daphnia magna for 48 hrs duration.

Toxicity to aqautic 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, the short term toxicity on aquatic algae and cyanobacteria was predicted for target substance trisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate (CAS no. 50662 -99 -2) (2017). EC50 value was estimated to be 244.37 mg/l for Desmodesmus subspicatus for 72 h duration. Based on this value it can be concluded that the substance trisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate is considered to be non-toxic to aquatic environment and can be considered to be not classified as per the CLP classification criteria.

Toxicity to microorganisms

From Microtox assay the test substance  trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate i.e Cibacron Brilliant Yellow 3B-A was moderately non-toxic to micro organism Vibrio fischeri  on the basis of the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC20).

Additional information

Short-term toxicity to fish:

Three studies from prediction and experimental study using peer reviewed journal for short term fish toxicity endpoint of target chemical trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (Cas no. 50662-99-2) with relevant read across which is close to the target chemical using log Kow as primary descriptor are summarized as follows:

 

In first study the short-term toxicity of the trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (Cas no. 50662-99-2) to Danio rerio (previous name: Brachydanio rerio) was predicted using QSAR toolbox version. 3.3 (2017), based on the effects observed in a static freshwater system during 96 hr exposure. The lethal concentration (LC50) for the test compound was estimated to be 253.57 mg/L. Thus, based on this value, test chemical trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate can be considered as non-toxic to fish and thus can be considered to be not classified as per the CLP classification criteria.

Above study is supported by experimental study of read across chemical 4-amino-3,5,6-trichloropyridine-2-carboxylate i.e Picloram K salt (Cas no. 2545-60-0) which suggest the toxicity of test substance Picloram K salt to aquatic organisms was evaluated in static acute toxicity tests. Fathead minnows were from laboratory reared stock. Prior to testing fish were held in either 180 L stainless steel or 56 L glass aquaria at a water temperature of 17 deg.C and provided a 16 h light9 h dark photoperiod.Fish were not fed during testing.Tests were conducted in round glass vessels,measuring 22 cm deep and 24.5 cm in diameter,held in constant temperature water troughs set to maintain the appropriate test temperatures 17 +- 1 deg.C.Illumination was provided by flourescent lights with an intensity of 860 -1180 lux set to provide a 16 h light9 h dark photoperiod. And test concentration used as control, 25, 40, 63,100,158 and 251 mg/l respectively. The LC50 and 95% confidence intervals were determined by using either Finney’s method of probit analyses or Thompson’s method of moving averages. Static fish test indicate the lethal concentration (LC50) of chemical 4-Amino3,5,6trichloro2pyridinecarboxylic acid, Potassium salt (1:1)i.e Picloram K salt on the basis of mortality effect was observed to be 201 mg/l for exposure period of 96 hrs.

An experimental study for another read across chemical 4 Amino 3,5,6trichloro2pyridinecarboxylic acid (Cas no. 1918-02-1) indicate in short term toxicity to fish study for 4 Amino3, 5, 6-trichloro2-pyridinecarboxylic acid in Oncorhynchus mykiss(Rainbow Trout) was observed for 96 hrs. 50% mortality was observed at concentration of 310 mg/l. As available lethal concentration (LC50) indicate the test substance 4 Amino3, 5, 6-trichloro2-pyridinecarboxylic acid (Cas no.1918-02-1) was considered to be non-toxic to fish and thus considered to be not classified as per the CLP regulations.(ECOTOX database;2017)

All available median lethal concentration (LC50) values for fish toxicity in acute exposure is in the range of 201 -310 mg/l which gives the common conclusion as the test chemical trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (Cas no. 50662-99-2) is likely to be non-toxic to fish and thus can be considered to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

Short term toxicity to aquatic invertebrates for trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl- 5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS:50662-99-2) was summarised with one prediction data and two experimental studies with two read across substances as follows:

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, the short term toxicity on aquatic invertebrates was predicted for trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl- 5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS:50662-99-2). EC50 value was estimated to be 140.69 mg/l for Daphnia magna for 48 hrs duration.

Study was performed in read across chemical 4-amino-3,5,6-trichloropyridine-2-carboxylic acid - 1,1',1''-nitrilotripropan-2-ol (1:1) (6753-47-5) from Ecotox database,2016 in aquatic invertebrates as 5 Daphnia were used at static test type for 48 hrs. After the 48 hrs, the EC 50 value for 4-amino-3,5,6-trichloropyridine-2-carboxylic acid - 1,1',1''-nitrilotripropan-2-ol (1:1) (6753-47-5) for daphnia magna was determined to be 226 mg/l on the basis of intoxication.

From the above Ecotox database,2016 short term toxicity was performed in another read across [(3,5,6-trichloropyridin-2-yl)oxy]acetic acid (55335-06-3) in 5 Daphnia at static test type for 48 hrs. After the 48 hrs, the EC 50 value for [(3,5,6-trichloropyridin-2-yl)oxy]acetic acid (55335-06-3) for daphnia magna was determined to be 132.9 mg/l on the basis of intoxication.

Based on the above predicted data and studies of read across chemicals it can be considered that all the above studies supported the target data. Based on the results it was considered that all weight of evidences supported the target classification and on the basis of which trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl- 5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate (CAS:50662-99-2) was not considered to be non-hazardous to aquatic invertebrates and can be considered to be not classified as per the CLP regulations.

Toxicity to aqautic algae and cyanobacteria

1 predicted data for the target chemical trisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate (CAS no. 50662 -99 -2) which is supported further by total 2 weight of evidence studies (from peer reviewed journal and authoritative database) for its closest read across substance with logKow as the primary descriptor were reviewed for the toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

Short term toxicity on aquatic algae and cyanobacteria of target chemical trisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate (CAS no. 50662 -99 -2) is predicted using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances (2017). EC50 value was estimated to be 244.37 mg/l for Desmodesmus subspicatus for 72 h duration.

 

In a supporting weight of evidence study of read across chemical 4 -amino-3,5,6 -trichloropyridine-2 -carboxylic acid (CAS no. 1918 -02 -1) from peer reviewed journal (V. M. Thomas et. al; 1973), short term toxicity to Chlorella pyrenoidosa study was carried out for 48 hrs. Paper disc agar diffusion method was used for determining the sensitivity of the test organism to 4 -amino-3,5,6 -trichloropyridine-2 -carboxylic acid. The study was based on the effects of the read across compound 4 -amino-3,5,6 -trichloropyridine-2 -carboxylic acid on green algae in a static fresh water system. Chlorella pyrenoidosa was used as a test organism. Test chemical conc. used for the study was 1000 mg/l (1 mg/l). Test solution of appropriate conc. was prepared using the ethanol as a solvent. Chlorella seeded agar plates were prepared using the buffered medium. Sterile paper discs (6 mm) containing 20µl of test solution with the appropriate solvent was used. Growth inhibition of test organism was determined by measuring the diameter of inhibition zone around the paper discs. Based on effect on growth inhibition of the test organism Chlorella pyrenoidosa, the 48 hr EC0 value was determined to be 1000 mg/l, respectively.

 

Another short term toxicity study to Pseudokirchneriella subcapitata (green algae) of the same read across chemical 4 -amino-3,5,6 -trichloropyridine-2 -carboxylic acid (CAS no. 1918 -02 -1) was carried out for 24 hrs (HSDB authoritative database, 2016). The study was based on the effects of the read across compound 4 -amino-3,5,6 -trichloropyridine-2 -carboxylic acid on green algae in a static fresh water system at a pH 7.7. Based on effect on physiology and photosynthesis of the test organism Pseudokirchneriella subcapitata (green algae), the 24 hr EC50 value was determined to be 115 mg/l.

 

Thus, based on the overall reported results for target chemical trisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate (OECD QSAR toolbox version 3.3, 2017) and for its read across substance (from peer reviewed journal and authoritative database), it can be concluded that the test substancetrisodium 2,5 -dichloro-4 -(4 -{[5 -({4 -chloro-6 -[(4 -sulfonatophenyl)amino]-1,3,5 -triazin-2 -yl}amino)-2 -sulfonatophenyl]diazenyl}-3 -methyl-5 -oxo-4,5 -dihydro-1H-pyrazol-1 -yl)benzenesulfonate can be considered as non-toxic to aquatic environment and thus can be considered to be not classified as per the CLP classification criteria.

Toxicity to microorganisms

In the study effect on toxicity on Vibrio fischeri are evaluated by Microtox assay for textile dye Cibacron Brilliant Yellow 3B-A (IUPAC name- trisodium 2,5-dichloro-4-(4-{[5-({4-chloro-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonate).

 

Frozen samples were brought to room temperature,and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples according to the test protocols defined by the manufacturer (Azur Environmental, Newark, Del.).From eight serial dilutions,the percent concentration to decrease 20% of the luminescence of amodified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software (1999) Azur Environmental, Newark, Del.]. A solution of 1 g ZnSO4 · 7H2O l−1 was used as the positive control and 1 g glucose l−1 as the negative control. Each EC20 reported is the average of triplicate analysis.

 

As per the criteria (the percent concentration to decrease 20% of the luminescence of Vibrio fischeri EC20))

>100% = not toxic

100%> EC20 > 75% = moderately non-toxic

75% > EC20 > 50% = toxic

50% > EC20 > 25% = moderately toxic

 

And test chemical Cibacron Brilliant Yellow 3B-A gives the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC20) was 100%> EC20 > 75% thus it is concluded that the textile dye Cibacron Brilliant Yellow 3B-A was observed to be moderately non-toxic to micro organism Vibrio fischeri.