Reaction mass of Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]-N-methyl- & acetate

Endpoint summary

Description of key information

Short-term toxicity to fish:

Fish Acute Toxicity test according to OECD Guideline 203 was conducted forReaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetateon Zebra fish.

The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 100 mg of the test substance in 100 mL of potable water (passed through reverse osmosis system) with continuous 1 hour stirring for achieving test concentrations of 0.1562mg/L,0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/L,respectively. Stability was check with the help of spectrometer.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203.

The nominal concentration selected for the experiment was

0.1562mg/L,0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/Land Zebra fish were exposed to this concentration for 96 hours. The lethal concentrations LC50 was found to be 0.1562 mg/L.

After 96 hours of exposure to test itemReaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetateto various nominal test concentrations, LC50 was determine to be 0.1562 mg/l . Based on the LC50, it can be consider that the chemical was toxic and can be consider to be aq aquatic chronic 1 as per the CLP classification criteria.

Short-term toxicity to aquatic invertebrate:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic invertebrate of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.Determination of the inhibition of the mobility of daphnids was carried out with the substance according to OECD Guideline 202.The stock solution 20 mg/L was prepared by dissolving dark violet iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.

The test substance was tested at the concentrations 0, 0.006, 0.012, 0.025, 0.050, 0.100 mg/l. Effects on immobilisation were observed for 48 hours.

The median effective concentration (EC50) for the test substance, in Daphnia magna was determined to be 0.037 mg/L for immobilisation effects.

This value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified as aquatic acute 1/ Chronic 1 as per the CLP criteria.

2.Short term toxicity to aquatic invertebrate was performed in daphnia magna for 48 hrs. After the experiment, the EC 50 value for test substance was determined to be 0.13 mg/l. Thus, based on the EC50 value, it can be concluded that the test substance can be considered as toxic to aquatic organisms and can be consider to be classified as aquatic acute 1 category as per the CLP classification criteria.

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 0.037 - 0.13 mg/

L. Thus, comparing this value with the criteria of CLP regulation Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified for short term toxicity for aquatic invertebrate as "Aquatic chronic 1/ chronic 1" .

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae and cyanobacteria of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201.The stock solution (10 mg/L) was prepared by dissolving yellow powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

The test substance was dissolved in DMSO/ OECD growth medium and tested at the concentrations 0, 3, 6, 12, 24 and 50 mg/L.Determination of cell concentrations was performed by microscope with counting chamber Cyrus I or electronic particle counter.Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, test substance, in Desmodesmus subspicatus was determined to be 0.13 mg/L.

This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as Aquatic acute category 1 as per the CLP criteria.

2.The effect of test item was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.5mg/l, 1.25mg/l, 3.125mg/l, 7.813mg/l, 19.531mg/l and 48.828mg/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. EC50 calculated graphically through probit analysis was observed to be 0.610 mg/L.Thus, based on the EC50 value, test substancecan be considered astoxic to aquatic organisms and thus can be classified as aquatic acute category 1 as per the CLP criteria.

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 0.13 - 0.61 mg/

L. Thus, comparing this value with the criteria of CLP regulation Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified for short term toxicity for aquatic algae and cyanobacteria as "Aquatic chronic 1/ chronic 1" .

Toxicity to microorganisms:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate .The studies are as mentioned below:

1.Determination of toxicity of test material on the growth of 14 gram negative microorganisms. Inhibitory activity of test chemical was determined by the traditional agar gel method. The conc. of test chemical used for the study was 0.6918 mg/l (1 µM).

14 different gram negative bact. were used for the study was Agrobacterium radiobacter, Agrobacterium tumefaciens, Bradyrhizobium japonicum, Escherichia coli, Erwinia atroseptica,Erwinia herbicola, Erwinia uredovora, Pseudomonas fluorescence, Pseudomonas phaseolicola, Pseudomonas syringae , Rhizobium trifolii, Xanthomonas malvacearum, Xanthomonas phaseoli and X. stewartii.

 

These test organisms were taken from the collection of the Plant Protection Institute, Hungarian Academy of Sciences (Budapest, Hungary). The bacteria were maintained on Nutrient Agar (Oxoid CM3) completed with vitamins pyridoxine. HCl, thiamine. HCl, riboflavine and nicotinamide at 1, 10, 1 and 20 mg/l concentrations. Bacterial suspensions for screening were prepared by washing cells with sterile tap water containing 0.3% peptone from slants of 20 h old cultures grown at (21±1) °C.

 

A layer of 5 mm depth of inoculated medium was dispensed into Petri dishes of 90 mm diameter. Filter paper discs of 5 mm diameter were impregnated with 1 µM solution of test compounds and placed centrally on the surface of the agar plate. Growth inhibition zones were measured after 24 h incubation at 21 ± 1 °C. After incubation colony diameters were determined and growth inhibition was calculated in percentage of dye free colony growth. Each determination was run in quadruplicate.

 

Based on no effect on growth inhibition of gram negative test organisms, the NOEC value of test material was determine to be 0.6918 mg/l for Erwinia herbicola and Pseudomonas syringae.

Whereas the growth inhibition was observed on the other organisms so LOEC was 0.6918 mg/l for remaining 12 bacteria.

2.The effect of test material on leucine aminopeptidase, acid phosphatase, and y-glutamyl transpeptidase activity in P. caudatum was studied in order to investigate the mechanism of toxicity.

The effect of test material on the survival time of Paramaecium caudatum was measured microscopically for 20 min. Concentration of test material in the bathing fluid was 0.1 or 1 .0%.

During the experiment the 36.7% mortality observed of test organism i.e Paramecium caudatum after of 17.28 min.which is not consider to be very toxic.

Additional information

Short-term toxicity to fish:

Fish Acute Toxicity test according to OECD Guideline 203 was conducted forReaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetateon Zebra fish.

The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 100 mg of the test substance in 100 mL of potable water (passed through reverse osmosis system) with continuous 1 hour stirring for achieving test concentrations of 0.1562mg/L,0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/L,respectively. Stability was check with the help of spectrometer.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203.

The nominal concentration selected for the experiment was

0.1562mg/L,0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/Land Zebra fish were exposed to this concentration for 96 hours. The lethal concentrations LC50 was found to be 0.1562 mg/L.

After 96 hours of exposure to test itemReaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetateto various nominal test concentrations, LC50 was determine to be 0.1562 mg/l . Based on the LC50, it can be consider that the chemical was toxic and can be consider to be aq aquatic chronic 1 as per the CLP classification criteria.

Short-term toxicity to aquatic invertebrate:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic invertebrate of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.Determination of the inhibition of the mobility of daphnids was carried out with the substance according to OECD Guideline 202.The stock solution 20 mg/L was prepared by dissolving dark violet iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.

The test substance was tested at the concentrations 0, 0.006, 0.012, 0.025, 0.050, 0.100 mg/l. Effects on immobilisation were observed for 48 hours.

The median effective concentration (EC50) for the test substance, in Daphnia magna was determined to be 0.037 mg/L for immobilisation effects.

This value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified as aquatic acute 1/ Chronic 1 as per the CLP criteria.

2.Short term toxicity to aquatic invertebrate was performed in daphnia magna for 48 hrs. After the experiment, the EC 50 value for test substance was determined to be 0.13 mg/l. Thus, based on the EC50 value, it can be concluded that the test substance can be considered as toxic to aquatic organisms and can be consider to be classified as aquatic acute 1 category as per the CLP classification criteria.

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 0.037 - 0.13 mg/

L. Thus, comparing this value with the criteria of CLP regulation Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified for short term toxicity for aquatic invertebrate as "Aquatic chronic 1/ chronic 1" .

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae and cyanobacteria of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate.The studies are as mentioned below:

1.Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the test substance according to OECD Guideline 201.The stock solution (10 mg/L) was prepared by dissolving yellow powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

The test substance was dissolved in DMSO/ OECD growth medium and tested at the concentrations 0, 3, 6, 12, 24 and 50 mg/L.Determination of cell concentrations was performed by microscope with counting chamber Cyrus I or electronic particle counter.Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, test substance, in Desmodesmus subspicatus was determined to be 0.13 mg/L.

This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as Aquatic acute category 1 as per the CLP criteria.

2.The effect of test item was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.5mg/l, 1.25mg/l, 3.125mg/l, 7.813mg/l, 19.531mg/l and 48.828mg/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. EC50 calculated graphically through probit analysis was observed to be 0.610 mg/L.Thus, based on the EC50 value, test substancecan be considered astoxic to aquatic organisms and thus can be classified as aquatic acute category 1 as per the CLP criteria.

Thus, based on the above summarised studies, Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate and it’s structurally similar read across substance, it can be concluded that effect concetration value is in the range of 0.13 - 0.61 mg/

L. Thus, comparing this value with the criteria of CLP regulation Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate can be classified for short term toxicity for aquatic algae and cyanobacteria as "Aquatic chronic 1/ chronic 1" .

Toxicity to microorganisms:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical Reaction mass of Methanaminium, N-​[4-​[[4-​(dimethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​methyl-​ & acetate .The studies are as mentioned below:

1.Determination of toxicity of test material on the growth of 14 gram negative microorganisms. Inhibitory activity of test chemical was determined by the traditional agar gel method. The conc. of test chemical used for the study was 0.6918 mg/l (1 µM).

14 different gram negative bact. were used for the study was Agrobacterium radiobacter, Agrobacterium tumefaciens, Bradyrhizobium japonicum, Escherichia coli, Erwinia atroseptica,Erwinia herbicola, Erwinia uredovora, Pseudomonas fluorescence, Pseudomonas phaseolicola, Pseudomonas syringae , Rhizobium trifolii, Xanthomonas malvacearum, Xanthomonas phaseoli and X. stewartii.

 

These test organisms were taken from the collection of the Plant Protection Institute, Hungarian Academy of Sciences (Budapest, Hungary). The bacteria were maintained on Nutrient Agar (Oxoid CM3) completed with vitamins pyridoxine. HCl, thiamine. HCl, riboflavine and nicotinamide at 1, 10, 1 and 20 mg/l concentrations. Bacterial suspensions for screening were prepared by washing cells with sterile tap water containing 0.3% peptone from slants of 20 h old cultures grown at (21±1) °C.

 

A layer of 5 mm depth of inoculated medium was dispensed into Petri dishes of 90 mm diameter. Filter paper discs of 5 mm diameter were impregnated with 1 µM solution of test compounds and placed centrally on the surface of the agar plate. Growth inhibition zones were measured after 24 h incubation at 21 ± 1 °C. After incubation colony diameters were determined and growth inhibition was calculated in percentage of dye free colony growth. Each determination was run in quadruplicate.

 

Based on no effect on growth inhibition of gram negative test organisms, the NOEC value of test material was determine to be 0.6918 mg/l for Erwinia herbicola and Pseudomonas syringae.

Whereas the growth inhibition was observed on the other organisms so LOEC was 0.6918 mg/l for remaining 12 bacteria.

2.The effect of test material on leucine aminopeptidase, acid phosphatase, and y-glutamyl transpeptidase activity in P. caudatum was studied in order to investigate the mechanism of toxicity.

The effect of test material on the survival time of Paramaecium caudatum was measured microscopically for 20 min. Concentration of test material in the bathing fluid was 0.1 or 1 .0%.

During the experiment the 36.7% mortality observed of test organism i.e Paramecium caudatum after of 17.28 min.which is not consider to be very toxic.