7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl]azo]naphthalene-1,3,6-trisulphonic acid

Endpoint summary

Description of key information

Short term toxicity to fish:

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on fish was predicted for 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (CAS:35642-64-9). LC50 value was estimated to be 165.307 mg/l for Danio rerio (previous name: Brachydanio rerio) for 96hrs of duration. 

Based on this value it can be concluded that the substance 7-[[2-[(aminocarbonyl) amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid is considered to not toxic to aquatic environment as per the criteria mentioned in CLP regulation. 

Short term toxicity to aq. invertebrates:

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the closest read across substances, the toxicity to Daphnia magna was predicted 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9). Intoxication value was estimated to be 113.58 mg/l for Daphnia magna for 48 hrs duration. It was concluded that 7-[(E)-2-{4-[(4 -amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9) was likely to be not toxic to aquatic invertebrate.

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 closest read across substances, the toxicity to algae was predicted 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9). Growth rate inhibition value was estimated to be 131.678 mg/l for Desmodesmus subspicatus for 72  hrs duration. It was concluded that 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9) was likely to be not toxic to aquatic algae.

Toxicity to microorganisms:

Based on the prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the six closest read across substances, the toxicity on Tetrahymena pyriformis was predicted for 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (CAS:35642-64-9). IGC50 value was estimated to be 1508.952 mg/l for Tetrahymena pyriformis for 48 hrs of duration. Based on this value it can be concluded that the substance 7-[[2-[(aminocarbonyl) amino]-4- [(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid is considered to not toxic to microorganisms.

Additional information

Summarized result for the determination of nature of chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) on the growth and other biological and physical activity of fishes, aquatic invertebrates, algae and cyanobacteria and microorganisms when chemical comes in contact with test organisms, by considering the data for target as well as structurally and functionally similar read across chemicals are as follows: 

 

Short term toxicity to fish:

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 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) on the fishes. The studies are as mentioned below:

In the first study for the target chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) from OECD QSAR toolbox 2017, prediction done using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on fish was predicted for 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (CAS:35642-64-9). LC50 value was estimated to be 165.307 mg/l for Danio rerio (previous name: Brachydanio rerio) for 96hrs of duration. Based on this value it can be concluded that the substance 7-[[2-[(aminocarbonyl) amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid is considered to not toxic to aquatic environment 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 aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex (15790-07-5) from UERL report. Fish Acute Toxicity test according to OECD Guideline 203 was conducted for (test item name) aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex. 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 2 hrs continuous stirring for achieving test concentrations of 100mg/L, respectively. 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. After 96hrs of exposure LC0, LC50 and LC100 was observed. Effect on the symptoms and the normal activity was checked in the interval of 24rs. pH, temperature and dissolved oxygen was also checked. The lethal concentrations LC50 was determine to be >100 mg/L. LC0 (96 hours) (highest loading at which no mortality was observed) = 100 mg/L. LC50 (96 hours) Experimental = >100 mg/L. LC100 (96 hours) (lowest loading at which 100% mortality was observed) =No mortality was observed. Thus based on the LC50 it can be concluded that the chemical aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

In the third weight of evidence study for the read across chemical (68583-95-9) from UERL report 2017, study on was conducted. Fish Acute Toxicity test according to OECD Guideline 203 was conducted for (test item name) aluminium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex. The stock solution prepared as 400mg/4L, with the concentration of 100mg/L, and was kept for 24 hr stirring. After this filter the stock, give it for analytical detection and then stock taken for experiment. 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. After 96hrs of exposure LC0, LC50 and LC100 was observed. Effect on the symptoms and the normal activity was checked in the interval of 24rs. pH, temperature and dissolved oxygen was also checked.

The lethal concentrations LC50 was determine to be >100 mg/L

LC0 (96 hours) (highest loading at which no mortality was observed) = 100 mg/L

LC50 (96 hours) Experimental = >100 mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) =No mortality was observed Thus based on the LC50 it can be concluded that the chemical aluminium 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Similarly in the fourth study for another read across chemical (3734-67-6) from UERL lab supports the classification and nontoxic behavior of chemical. Fish Acute Toxicity test was performed to evaluate the toxic nature of Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7- disulphonate (3734 -67 -6). Test was performed according to OECD Guideline 203. The test substance was poorly 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 48 hr stirring for achieving test concentrations of 100mg/L , respectively. 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. After 96hrs of exposure LC0, LC50 and LC100 was observed. Effect on the symptoms and the normal activity was checked in the interval of 24rs. pH, temperature and dissolved oxygen was also checked. The lethal concentrations LC50 was determine to be >100 mg/L

LC0 (96 hours) (highest loading at which no mortality was observed) = 100 mg/L

LC50 (96 hours) Experimental = >100 mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) =No mortality was observed Thus based on the LC50 it can be concluded that the chemical Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7- disulphonate (3734 -67 -6) was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

The fifth study form peer reviewed journal (The Journal of Toxicological Sciences, 1978) for the read across chemical (915-67-3) study was conducted. Different food dyes were subjected to median tolerance limit (TLm) test by use of Himedaka (Oryzias Latipes) for the comparison of the toxicity. Himedaka (Oryzias Latipes) was used. The same age fish (about 2 cm long, weight ca 0.2 g) were chosen and acclimated for 10 days in the tap water before experiment. One liter of deionized water was placed in a 2 liter tank. The food dyes were obtained from National Institute of Hygienic Sciences, Japan. Survival rate test: In one liter of pH 7.0 containing 3000mg/l of dye solution, 10 fishes were kept in the tank without direct sunlight for 48 hours. Water temperature was 20C. Aeration was performed with bubbler. TLm test: TLm was determined according to the procedure in JIS K0102 (Japanese Industrial Standards Committee, 1971). Amaranth was tested at 4 – 6 steps of concentration utilizing ten fish per one group and the number of survivals were counted after 24 and 48 hours. The values were mean of 3 trials. 100% survival rate was determined after 48 hours exposure of fishes to Amaranth. Based on these observations, the test chemical can be considered non-toxic to fishes.

 

Based on the predicted data for the target chemical (from OECD QSAR 2017) and for the read across chemical from experimental lab reports and peer reviewed journal (The Journal of Toxicological Sciences, 1978 ), it can be concluded that the substance 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) is considered to be not toxic to aquatic environment (fishes) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

 

Short term toxicity to aq. 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 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) on the mobility of aquatic invertebrates. The studies are as mentioned below:

 

In the first study for the target chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) from OECD QSAR toolbox 2017, prediction was done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the closest read across substances, the toxicity to Daphnia magna was predicted 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9). Intoxication value was estimated to be 113.58 mg/l for Daphnia magna for 48 hrs duration. It was concluded that 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9) was likely to be not toxic to aquatic invertebrate.

 

First predicted study was supported by the second experimental weight of evidence study for the read across chemical Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate (3734-67-6) from ABITEC report. An acute immobilisation test was used to test how a range of concentrations of Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate exerts different degrees of toxic effects on the swimming capability of Daphnia magna under otherwise identical test conditions. The test was performed in close resemblance to OECD guideline 202 by ABITEC in Prague. The testing aim was to determine a EC50 after 48 hours of exposure to D. magna. Daphnids were exposed to Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate 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 beakers were placed in a temperature controlled room at 20±1 degrees Celsius. The D. magna (age ≤24) used for the test had been breed at ABITEC. The breeding stock of D. Magna originated from University of Technology in Prague. The animals were exposed to medium (i.e. a beaker containing only medium) and/or the tested chemical during 48 hours (±1 hour). None of the exposed animal’s immobilization were affected by exposure to only medium. The nominal concentrations used were: 100 mg/L (limit test). There were 5 Daphnia per test vessels and 5 replicates per concentration. The pH in test vessels were 7.7-7.8 mg/L. The positive control/reference substance used in the tested showed an expected result and gave an EC50 that corresponded to previous exposures with this chemical in D. magna. The IC50 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 Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate. The IC8 was therefore estimated to be 100 mg/L. Based on the IC8, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per CLP classification criteria.

 

In the third weight of evidence study for the read across chemical (915-67-3) from UERL report 2017, study on was conducted. Determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test was performed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobility inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and cannot be classified as toxic as per the CLP criteria.

 

Similarly in the fourth study for another read across chemical (4548-53-2) from ABITEC lab supports the classification and nontoxic behavior of chemical. Determination of the inhibition of the mobility of daphnids was carried out with the substance Disodium 3-[(2,4-dimethyl- 5-sulpho- natophenyl) azo]-4-hydroxy naphthalene-1-sulphonateaccording to OECD Guideline 202. A limit test at sample concentration of 100 mg/L was performed. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene -1-sulphonate, in Daphnia magna was determined to be >100 mg/L for immobilisation effects. Based on this EC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance, Disodium 3-[(2,4-dimethyl -5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate does not exhibit short term toxicity to aquatic invertebrate (Daphnia Magna).

 

The fifth study form ABITEC lab report 2016 for the read across chemical (68583-95-9) was carried out. Determination of the inhibition of the mobility of daphnids was carried out with the substance Aluminium,6-hydroxy-5- [(2-methoxy-5-methyl-4-sulfophenyl)azo ]-2-naphthalenesulfonic acid complex according to OECD Guideline 202. A limit test at sample concentration of 100 mg/L was performed. Effects on immobilisation were observed for 48 hours. At 100 mg/l only 8% inhibition was observed, thus it can be concluded that the EC50 was >100 mg/l. The median effective concentration (EC50) for the test substance, Aluminium, 6-hydroxy-5- [(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex, in Daphnia magna was determined to be >100 mg/L for immobilisation effects. Based on this EC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance, Aluminium, 6-hydroxy-5- [(2-methoxy-5-methyl-4-sulfophenyl)azo ]-2-naphthalenesulfonic acid complex does not exhibit short term toxicity to aquatic invertebrate (Daphnia Magna).

 

Similarly the sixth study was used for the read across chemical (915-67-3) from peer reviewed journal (The Journal of Toxicological Sciences, 1977). The toxic effects of Amaranth were studied on Artemia salina larvae. Artemia salina (A. salina eggs) a crustacean, commonly known as brine shrimp eggs, are commercially available, and are easily cultured in the laboratory because they are resistant to environmental stresses. Active larvae can be obtained within 1 to 2 days and no live culture is required for a few days thereafter. A. salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -200°C. Eggs used in experiments were washed and stored at room temperature in a desiccators over anhydrous granular CaCl2. 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 towards a light source. Amaranth at concentrations of 6044.7mg/l and 604.47 mg/l 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. 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. 100% death rate was noted after 48 hours when 6044.7 mg/l of Amaranth was exposed to the test organism and 0% death rate after 24 hours in case of exposure to 604.47 mg/l of test chemical.

 

Based on the predicted data for the target chemical (from OECD QSAR 2017) and for the read across chemical from experimental lab reports (ABITEC reports), it can be concluded that the substance 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) is considered to be not toxic to aquatic environment (aquatic invertebrates) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

 

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 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) on the growth of aquatic algae. The studies are as mentioned below:

 

In the first study for the target chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) from OECD QSAR toolbox 2017, prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the closest read across substances, the toxicity to algae was predicted 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9). Growth rate inhibition value was estimated to be 131.678 mg/l for Desmodesmus subspicatus for 72 hrs duration. It was concluded that 7-[(E)-2-{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-(carbamoylamino)phenyl}diazen-1-yl]naphth.(CAS: 35642-64-9) was likely to be not toxic to aquatic algae.

 

First predicted study was supported by the second experimental weight of evidence study for the read across chemical aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex (15790-07-5) from UERL report. 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-naphthalenesulfonic 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.

 

In the third supporting weight of evidence study for the read across chemical from UERL lab, 2016 toxicity was measured on algae. The effect of test item aluminium, 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex, CAS No. 68583-95-9 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-[(2-methoxy-5-methyl-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 for 24 hours and filter it 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. 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 0.1 units. 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. EC50 calculated graphically through probit analysis was observed to be 136.57 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.

 

 

Similarly in the fourth study for RA chemical 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt (Amaranth dye) (915-67-3), ABITEC lab report, 2016. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance 2,7-Naphthalenedi- sulfonic acid, 3-hydroxy-4-[(4-sulfo- 1-naphthal - enyl), sodium salt (Amaranth dye) according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 12.5, 25, 50, 100, 200 mg/L. Effects on the growth rate of the organism were studied. The effective concentration (ErC50) for the test substance, 2,7-Naphthalen -edisulfonic acid, 3-hydroxy-4-[(4-sulfo-1- -naphthalenyl), sodium salt, in Desmodesmus subspicatus was determined to be 356.2 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.

 

The fifth study for RA chemical 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt (Amaranth dye) (915-67-3),UERL report, 2016 test conducted. The effect of test substance Trisodium 3-hydroxy-4-(4’-sulphonatonaphthylazo) naphthalene-2,7-disulphonate (CAS No. 915-67-3) 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 6.25 mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/l were prepared using stock solution of the test substance using de-ionized water. 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 test substance. EC50 calculated from equation through probit analysis was observed to be > 200 mg/L. Thus based on the EC50 it was concluded that the chemical was nontoxic and can be consider to be not classified as toxic to aquatic environment as per the CLP classification criteria.

 

For further supporting the classification of target chemical read across chemical (3734-67-6) was used. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7- disulphonate according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving red powder in OECD growth medium. The solution was kept 30 min in ultrasonic bath. 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 were use in the study for total exposure period of 72hrs. Test conducted in 50 ml glass vessel filled with 50 ml of sample volume and tested at the concentrations 0, 0, 20, 30, 45, 67, 100 mg/l. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate, 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.

 

Similarly in the seventh study for another read across chemical (4548-53-2) from ABITEC lab supports the classification and nontoxic behavior of chemical. Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance Disodium 3-[(2,4-dimethyl -5-sulphonatophenyl) azo]-4-hydroxynaphthalene-1-sulphonate according to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations 0, 0, 12.5, 25, 50, 100 and 200 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]- 4-hydro xynaphthalene-1-sulphonate, in Desmodesmus subspicatus was determined to be 276.1 mg/L. Based on this ErC50 value and after comparing with CLP criteria for aquatic classification of the substance it is concluded that the substance Disodium 3-[(2,4-dimethyl-5-sulphonatophenyl)azo]-4-hydroxynaphthalene-1-sulphonate does not exhibit toxicity to aquatic algae (Desmodesmus subspicatus).

 

Based on the predicted data for the target chemical (from OECD QSAR 2017) and for the read across chemical from experimental lab reports (ABITEC reports and UERL lab reports), it can be concluded that the substance 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (35642-64-9) is considered to be not toxic to aquatic environment (aquatic algae and cyanobacteria) and cannot be classified as toxic as per the criteria mentioned in CLP regulation.

 

Toxicity to microorganisms:

 

Based on the various experimental data for the target chemical and read across chemicals study have been reviewed to determine the toxic nature of target chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl]azo]naphthalene-1,3,6-trisulphonic acid (35642 -64 -9). The studies are as mentioned below:

 

In the first weight of evidence study for the target chemical (35642 -64 -9) from OECD QSAR toolbox version 3.4 with log kow as the primary descriptor prediction was done and considering the six closest read across substances, the toxicity on Tetrahymena pyriformis was predicted for 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid (CAS:35642-64-9). IGC50 value was estimated to be 1508.952 mg/l for Tetrahymena pyriformis for 48 hrs of duration. Based on this value it can be concluded that the substance 7-[[2-[(aminocarbonyl) amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl] azo]naphthalene -1,3,6-trisulphonic acid is considered to not toxic to microorganisms.

 

In the second study for read across chemical (915-67-3) Biotechnology Letters, 2002. The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri. 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 70μl 1 M NaOH. Colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of a modified 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/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis. The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20) was reported instead. The following rating was adapted from Coleman & Qureshi (1985) – EC20: >100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic. The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similarly in the third weight of evidence study for the same read across chemical from (Indian J Microbiol 2011). This investigation was aimed at identifying the effects of the Amaranth dye and its degradation products on microbial growth. Amaranth dye was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India. Aspergillus ochraceus NCIM 1146 was obtained from National Chemical Laboratory, Pune, India. E. coli MTCC 452, B. subtilis MTCC 6910 and Penicillium ochrochloron MTCC 517 were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. It was regularly maintained and preserved at 4°C on nutrient agar slants contained in (g/l); bacteriological peptone 10.0, beef extract 10.0 and NaCl 5.0 Microbial toxicity of control dye amaranth and metabolites obtained after its decolorization (final concentration 1,000 ppm) was carried out in relation to E. coli, Bacillus substilis, Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 and zone of inhibition (diameter in mm) was recorded. The diameter of the discs used was 10mm. Amaranth and its degradation products were not toxic to Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 at 1,000 ppm concentration. Amaranth inhibited growth of E. coli and Bacillus substilis.

 

 

Similarly in the fourth study for the read across chemical (915-67-3) (from, TOXICOLOGY AND APPLIED PHARMACOLOGY, 1977). The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. Hence a bioassay was conducted to study the toxic effect of Amaranth. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Amaranth 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.

 

Thus based on the overall studies for the target chemical 7-[[2-[(aminocarbonyl)amino]-4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]phenyl]azo]naphthalene-1,3,6-trisulphonic acid (35642 -64 -9) including the toxicity results on the growth and other biological activity of fishes, aquatic invertebrates and algae from various lab reports and peer reviewed journals for target as well as read across chemicals, 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.