3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1)

Endpoint summary

Description of key information

Short term toxicity to fish:

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 fish was predicted for the 96 h LC50 for 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) (CAS:63589-47-9). Based on the mortality rate, LC50 value was estimated to be 0.9281 mg/l for Oncorhynchus mykiss (previous name: Salmo gairdneri) for 96hrs of duration. Based on this value it can be concluded that the substance 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) (CAS:63589-47-9) is considered to be toxic to aquatic environment and classified in aquatic acute category 1 and chronic 1 as per the criteria mentioned in CLP regulation. 

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 five closest read across substances, toxicity on daphnia magna was predicted for 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9). The EC50 value was estimated to be 0.1632 mg/l when 3,7-bis(diethylamino)phenoxazin-5- iumtetrachloro- zincate (2:1) exposed to daphnia magna for 48hrs. Thus, based on this value, it can be concluded that the test chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) can be considered as toxic to aquatic organisms and thus can be classified as aquatic acute category 1 and chronic 1 as per the CLP criteria.

 

Short term toxicity to aquatic algae:

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 Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was predicted for 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589 -47 -9). The EC50 value was estimated to be 0.2410 mg/l when 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) exposed to Pseudokirchneriella subcapitata for 72hrs. Thus, based on this value, it can be concluded that the test chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) can be considered as toxic to aquatic organisms and thus can be classified as aquatic acute 1 and chronic 1 as per the CLP criteria.

Additional information

Summarized result of toxicity of 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) 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 structure similarity are as follows: 

Short term toxicity to fish:

Based on the various experimental data for the target chemical and prediction data for RA chemical which was selected on the basis of structure similarity study have been reviewed to determine the toxic nature of 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) on the mortality of fish. The studies are as mentioned below:  

In the first predicted weight of evidence report for the 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from SSS (QSAR, 2017) toxicity study was carried out. 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 fish was predicted for the 96 h LC50 for 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) (CAS:63589-47-9). Based on the mortality rate, LC50 value was estimated to be 0.9281 mg/l for Oncorhynchus mykiss (previous name: Salmo gairdneri) for 96hrs of duration. Based on this value it can be concluded that the substance 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) (CAS:63589-47-9)is considered to be toxic to aquatic environment and classified in aquatic acute 1 and chronic 1 as per the criteria mentioned in CLP regulation. 

 

Similarly the second prediction study was performed based on the prediction done by EPI suite, ECOSAR version 1.11, 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 fish was predicted. On the basis of this programe, the LC 50 value for short term toxicity to fish was predicted to be 0.289 mg/l for 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) in 96 hrs. Based on this value it can be concluded that the substance 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) is considered to be toxic to aquatic environment and can be classified as aquatic acute 1 and chronic 1 category as per the criteria mentioned in CLP regulation.

 

Similarly in the third weight of evidence study for the read across (548-62-9) (from Haskell Laboratory for Toxicology and Industrial Medicine, 1977) toxicity was conducted on fish to determine the toxic nature of chemical. Study was conducted to determine the effect of [4-[4,4'-bis(dimethylamino) benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride on aquatic environment, [4-[4,4'-bis(dimethylamino)benzhydrylidene]cyclohexa -2,5-dien-1-ylidene]dimethylammonium chloride was exposed to Pimephales promel as for 96hr. Test performed according to the standard static system. 0.1 mg/ ml solution in distilled water was introduced into all glass vessels which contained 10 fathead minnows in 15 liters of water. Fishes were not fed for 48 hours prior to testing nor during exposure. No aeration was provided during the test and temperature was maintained at 20 ± 0.2 degC. After the exposure no distinct signs of toxicity were observed. Its 96-hour LC-50  is 0.082 mg/l with 95% confidence limits of 0.065 mg/l and 0.11 mg/l. The slope of the dose response curve is 4.55 with 95% confidence limits of 2.16 and 6.94. It ca be concluded that the 4-{bis[4 (dimethylamino) phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1 and chronic 1" as per CLP regulation.

 

Similarly in the fourth weight of evidence study for the same read across chemical selected on the basis of structural and functional similarity basis (548-62-9) from The Journal of Toxicological Sciences, 1982. TLm (median tolerance limit) test was carried out according to the procedure of Japan Industrial Standards (Japanese Industrial Standards Committee, 1971). Ten fish of Himedaka per one trial were kept in 2 liter of deionized water at 25 degree C and, after 24 or 48 hours, lethal concentration of 50% fish was determined. The Lethal concentration to 50% of Oryzias latipes for 24 Hour under static condition is 0.2 mg/L and for 48 hr is 0.1 mg/L. It can be concluded that the [4-[4,4'-bis (dimethylamino) benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium chloride is toxic to the aquatic environment and can be considered as “Aquatic Acute 1 and chronic 1” as per CLP regulation.

 

Thus based on the above data sources for target chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from various experimental studies and predicted studies from QSAR, EPIsuite, Haskell Laboratory for Toxicology and Industrial Medicine, 1977 and The Journal of Toxicological Sciences, 1982, it was summarized that the chemical was toxic to aquatic organisms and thus can be classified as aquatic acute category 1 and chronic 1 as per the CLP criteria. 

Short term toxicity to aquatic invertebrates:

Based on the various experimental data and prediction data for the target chemical as well as RA chemical which was selected on the basis of structure similarity study have been reviewed to determine the toxic nature of 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) on the mobility of daphnia magna. The studies are as mentioned below: 

In the first predicted weight of evidence report for the 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from SSS (QSAR, 2017) toxicity study was carried out. 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 daphnia magna was predicted for 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9). The EC50 value was estimated to be 0.1632 mg/l when 3,7-bis(diethylamino)phenoxazin-5- iumtetrachloro- zincate (2:1) exposed to daphnia magna for 48hrs. Thus, based on this value, it can be concluded that the test chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) can be considered as toxic to aquatic organisms and thus can be classified as aquatic acute category 1 and chronic 1 as per the CLP criteria.

 

Similarly in the second prediction study was performed based on 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 0.222 mg/l for 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) in 48 hrs. Based on this value it can be concluded that the substance 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) is considered to be toxic to aquatic environment and can be classified as aquatic acute 1 and chronic 1 category as per the criteria mentioned in CLP regulation.

 

Similarly in the third weight of evidence study for the read across (548-62-9) (from Intox Pvt. Ltd. 2010) toxicity was conducted on daphnia magna to determine the toxic nature of chemical. Acute Immobilisation test in Daphnia magna was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 202, "Daphnia Sp., Acute Immobilisation Test" . N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride was tested for acute toxic effects on the swimming capabilities of Daphnia magna employing a static test procedure. In the main study, six groups of daphnids, each comprising 5 daphnids per replicate, were exposed to the test article at concentration levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L. Observations were made for immobilisation at 24 hours and 48 hours of treatment. A concurrent blank control was also run simultaneously. Potassium Dichromate was employed as a positive control and tested at the concentration of   0.4 mg, 0.63 mg and 1 mg/L. The animals were observed for immobility after 24 hours and 48 hours of exposure. Those daphnids which were unable to swim within 15 seconds after gentle agitation of the test container were considered to be immobile. The rates of immobilisation observed at 24 hours were 0%, 0%, 0%, 10%, 25% and 75% at the test concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. The rates of immobilisation observed at 48 hours were 0%, 0%, 10%, 25%, 55% and 85% at the test concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. The Positive control, Potassium Dichromate induced immobilisation in 20%, 50% and 80% of the exposed daphnids at 24 hours and 25%, 65% and 100% immobilization at 48 hours at the concentrations of 0.4 mg, 0.63 mg and 1 mg/L respectively. No immobilisation was observed in blank control group. The acute immobilisation EC50value of N-(4-{bis[4-(dimethylamino) - phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 24 hour exposure period was determine to be 0.53 mg/L with fiducial limits between 0.41 to 0.69 mg/L at 95% confidence level. The acute immobilisation EC50 value of N-(4-{bis[4-(dimethylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride to Daphnia magna at 48 hour exposure period was determine to be 0.35 mg/L with fiducial limits between 0.24 to 0.50 mg/L at 95% confidence level. On the basis of EC50 value obtained it can be concluded that the substance N-(4-{bis[4-(dimethylamino) phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1 and chronic 1" as per CLP regulation.

Similarly in the fourth weight of evidence study for the read across (569 -64 -2) from j-check, 2017. Short term toxicity to aquatic invertebrate was performed in daphnia magna for 48 hrs. After the experiment, the EC 50 value for N-(4-{[4-(dimethylamino)phenyl](phenyl)methylene}cyclohexa -2,5-dien-1-ylidene)-N-methylmethanaminium chloride was determined to be 0.13 mg/l. Thus, based on the EC50 value, it can be concluded that the substance N-(4 -{[4 -(dimethylamino)phenyl](phenyl)methylene}cyclohexa-2,5 -dien-1 -ylidene)-N- methylmethanaminium chloride can be considered as toxic to aquatic organisms and can be consider to be classified as aquatic acute 1 and chronic 1 category as per the CLP classification criteria.

 

Thus based on the above data sources for target chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from various experimental studies and predicted studies from QSAR, EPIsuite, Intox 2010, it was summarized that the chemical was toxic to aquatic organisms and thus can be classified as aquatic acute category 1 as per the CLP criteria.

 

Short term toxicity to aquatic algae:

Based on the various experimental data and prediction data for the target chemical as well as RA chemical which was selected on the basis of structure similarity study have been reviewed to determine the toxic nature of 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) on the growth of algae and cyanobacteria. The studies are as mentioned below: 

In the first predicted weight of evidence report for the 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from SSS (QSAR, 2017) toxicity study was carried out. 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 Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was predicted for 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589 -47 -9). The EC50 value was estimated to be 0.2410 mg/l when 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) exposed to Pseudokirchneriella subcapitata for 72hrs. Thus, based on this value, it can be concluded that the test chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) can be considered as toxic to aquatic organisms and thus can be classified as aquatic acute category 1 and chronic 1 as per the CLP criteria.

Similarly in the second prediction study which was performed based on the prediction done using the EPI Suite ECOSAR version 1.10, the short term toxicity on green algae was predicted for test substance 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589 -47 -9). On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) is estimated to be 0.589 mg/l for green algae for 96 hrs duration. Based on this value, it can be concluded that the test chemical 3,7-bis(diethylamino)phenoxazin-5-ium tetrachlorozincate (2:1) can be considered as toxic to green algae at environmentally relevant concentrations and can be considered as classified as aquatic acute category 1 and chronic 1 as per the CLP classification criteria.

 

Similarly in the third weight of evidence study for the read across (548-62-9) (from Intox Pvt. Ltd. 2010) toxicity was conducted on Pseudokirchneriella subcapitata to determine the toxic nature of chemical. Freshwater Alga, Growth Inhibition Test of 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride in fresh water Unicellular green alga, Pseudokirchneriella subcapitata (Selenastrum capricornutum) was performed in compliance with the OECD Guidelines for Testing of Chemicals, Section 2, No. 201, "Fresh water Alga and Cyanobacteria, Growth Inhibition Test". 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride was tested for its possible inhibitory effect on growth of alga, Pseudokirchneriella subcapitata. Exponentially growing cultures of the algae were exposed to test article at concentrations levels of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L for a period of 72 hours, along with a concurrent blank control. Potassium Dichromate, serving as a positive control, was also concurrently tested at concentrations levels of 0.4 mg, 0.8 mg and 1.6 mg/L. The cell concentration, i.e. the number of cells per ml, for all the concentration levels, was counted at 24, 48 and 72 hours of start of the exposure by counting them under microscope in a haemocytometer counting chamber and the inhibition of growth in relation to the control culture was determined. The endpoint was inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period. From the average specific growth rates recorded in a series of test solutions, the concentration bringing about 50% inhibition of growth rate was determined and expressed as the ErC50. The additional endpoint was inhibition of yield, expressed as the biomass at the end of exposure period minus the biomass at the start of the exposure period. From the yield recorded in a series of test solutions, the concentration bringing about 50% inhibition of yield was determined and expressed as the EyC50. In the present study, percent inhibition of growth in fresh water unicellular green alga, Pseudokirchneriella subcapitata at 72 h, was 3.62%, 5.82%, 9.82%, 20.88%, 47.95 and 72.83 % recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 and 0.8 mg/L respectively. In positive control group percent inhibition of growth in fresh water unicellular green alga, Pseudokirchneriella subcapitata at 72 h, was 21.01%, 65.18% and 90.97% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively. In the present study, percent inhibition in yield in fresh water unicellular green alga, Pseudokirchneriella subcapitata at 72 h, was 10.45%, 16.37%, 25.64%, 47.73%, 79.29% and 92.90% recorded at the concentrations of 0.025 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.4 mg and 0.8 mg/L respectively. In positive control group percent yield in fresh water unicellular green alga, Pseudokirchneriella subcapitata at 72 h, was 48.13%, 89.74% and 98.22% recorded at the concentrations of 0.4 mg, 0.8 mg and 1.6 mg/L respectively. On the basis of observations made during this test, the ErC50(72h) of 4-{bis[4-(dimethylamino) phenyl]methylidene}-N,N-dimethylcyclohexa-2,5 dien-1-iminium chloride inducing inhibition of the growth of fresh water unicellular green algae Pseudokirchneriella subcapitata was determine to be 0.42 mg/L with fiducial limits between 0.19 to 0.93 mg/L at 95% confidence level. On the basis of observations made during this test, the EyC50(72h) of 4-{bis[4-(dimethylamino) phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride inducing inhibition of the yield of fresh water unicellular green algae Pseudokirchneriella subcapitata was determine to be 0.21 mg/L with fiducial limits between 0.11 to 0.40 mg/L at 95% confidence level. Thus on the basis of EcC50 and EyC50 value it can be concluded that the 4-{bis[4-(dimethylamino) phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium chloride is toxic to the aquatic environment and can be considered as "Aquatic Acute 1 and chronic 1" as per CLP regulation.

Thus based on the above data sources for target chemical 3,7-bis(diethylamino)phenoxazin-5-iumtetrachloro- zincate (2:1) (63589-47-9) from various experimental studies and predicted studies from QSAR, EPIsuite, Intox 2010, it was summarized that the chemical was toxic to aquatic organisms and thus can be classified as aquatic acute 1 and chronic category 1 as per the CLP criteria.

 

Thus based on the toxicity data for the fish, invertebrates and algae, it can be concluded that the chemical was toxic and and can be consider to be classified in aquatic acute 1 and chronic 1 category as per CLP classification criteria.