[4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate

Endpoint summary

Description of key information

Short term toxicity to fish:

Fish Acute Toxicity test according to OECD Guideline 203 was conducted for (test item name) [4-[[4 -(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate The nominal concentration selected for the experiment were 0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/L,5mg/Land test fish were exposed to these concentration for 96 hours. The lethal concentrations LC50 was found to be >0.3125 mg/L but <0.625 mg/L,LC0 (96 hours) (highest loading at which no mortality was observed) = 0.3125 mg/L LC50 (96 hours) ,Experimental = >0.3125 mg/L but <0.625 mg/L and LC100 (96 hours) (lowest loading at which 100% mortality was observed) = 0.625 mg/L.

Short term toxicity to aquatic invertebrates:

Based on the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the four closest read across substances, the short term toxicity on aquatic invertebrates was predicted for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9). EC50 value was estimated to be 0.505 mg/l for Daphnia magna for 48 hrs duration. Thus, based on the EC50 value, it can be concluded that the substance N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9) can be considered as toxic to aquatic organisms and can be classified in aquatic acute 1 category as per the CLP regulations.

 

Short term toxicity to aquatic algae:

Short term toxicity on aquatic algae and cyanobacteria of target chemicalN-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (CAS No. 84434 -47 -9) 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 0.135 mg/l for Pseudokirchneriella subcapitata for 72 h duration. Based on this value it can be concluded that the substance N-(4 -{[4 -(dimethylamino)phenyl] [4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate is considered to be toxic to aquatic environment and can be considered to be classified in aquatic acute 1 as per the CLP classification criteria.

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 five closest read across substances, toxicity on microorganisms was predicted forN-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino) phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate (84434-47-9),IGC50 value was estimated to be 91.765 mg/l whenN-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate wasexposed to Tetrahymena pyriformis for 48 hours.

Additional information

Short term toxicity to fish:

Three studies including predicted data from experimental data and from authorative database and also from validated tools for short term fish toxicity endpoint of target chemical N-(4-{[4 -(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (Cas no. 84434-47-9) with relevant read across chemical which is structurally similar to target were summarized as follows:

 

First key experimental study suggest Fish Acute Toxicity test according to OECD Guideline 203 was conducted for (test item name) [4-[[4 -(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate The nominal concentration selected for the experiment were 0.3125mg/L,0.625mg/L,1.25mg/L,2.5mg/L,5mg/Land test fish were exposed to these concentration for 96 hours. The lethal concentrations LC50 was found to be >0.3125 mg/L but <0.625 mg/L,LC0 (96 hours) (highest loading at which no mortality was observed) = 0.3125 mg/L LC50 (96 hours) ,Experimental = >0.3125 mg/L but <0.625 mg/L and LC100 (96 hours) (lowest loading at which 100% mortality was observed) = 0.625 mg/L 

 

Similarly above experimental result supported by prediction using the aquatic toxicity module i.e ACD/LabsI-Lab 2.0; Algorithm Version: v5.0.0.184, the lethal concentration (LC50) of the test substance N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate was predicted to be 0.12 mg/l on fish species Fathead minnow (Pimephales promelas).

The Reliability Index (RI) of the prediction was reported to be 0.6(As per model substance have moderate and high reliability if RI > 0.5). Thus reported RII value indicate it has moderate reliability. Based on the LC50 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 thus classified in aquatic category 1 as per the CLP criteria.

 

In the third supportingstudy experiment data of read across chemical N-(4-{[4-(dimethylamino)phenyl](phenyl)methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium chloride (Cas no. 569 -64 -2) wherein acute fish toxicity study was carried out to determine the lethal concentration (LC50) of test substance on fish for exposure period of 96 hrs. Test organism used was Morone saxatilis (Striped Bass) The Lethal concentration to 50% of test organisms (LC50) on the basis of mortality effect was observed to be 0.138 mg/l for exposure period of 96 hrs. (Ecotox database by US EPA)

 

Thus available studies for short term fish toxicity give common conclusion as test chemical N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)- N-methylmethanaminium acetate (Cas no. 84434-47-9) was toxic to fish and thus classified in aquatic category 1 as per the CLP criteria.

 

Short term toxicity to aquatic invertebrates:

Short term toxicity to aquatic invertebrates for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9) was summaries with predicted data for target and experimental study of structural and functional read across are summaries as follows:

 

In the first weight of evidence study using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the four closest read across substances, the short term toxicity on aquatic invertebrates was predicted for N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9). EC50 value was estimated to be 0.505 mg/l for Daphnia magna for 48 hrs duration. Thus, based on the EC50 value, it can be concluded that the substance N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9) can be considered as toxic to aquatic organisms and can be classified in aquatic acute 1 category as per the CLP regulations.

 

 

Study was carried out on the read across chemical (548-62-9), Intox report which was structurally similar to the target 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" as per CLP regulation.

 

 

Similarly in the third weight of evidence study for read across chemical N-(4 -{[4 -(dimethylamino)phenyl](phenyl)methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium chloride (569-64-2) from J-check, study was performed in daphnia magna for 48 hrs. After the experiment, the EC50 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.

 

Another fourth weight of evidence study on read across diphenylarsinous cyanide (23525-22-6) from Ecotox database was performed in static salt water condition for 48 hrs in daphnia magna. 5 daphnia magna of <24 hrs were used. After 48 hrs, the EC 50 value for diphenylarsinous cyanide (23525-22-6) was determined to be 0.072 mg/l.

 

Based on the experimental studies of target and study of structural and functional read across, it was considered that N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene} cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (84434-47-9) was considered to be toxic to aquatic invertebrates and can be considered to be classified in aquatic acute 1 category as per the CLP regulations.

 

Short term toxicity to aquatic algae:

Predicted data for the target chemical N-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methyl methanaminium acetate (CAS No. 84434 -47 -9) and various weight of evidence study for its closest read across substancewere reviewed to summarize the following information:

 

Short term toxicity on aquatic algae and cyanobacteria of target chemicalN-(4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methylmethanaminium acetate (CAS No. 84434 -47 -9) 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 0.135 mg/l for Pseudokirchneriella subcapitata for 72 h duration. Based on this value it can be concluded that the substance N-(4 -{[4 -(dimethylamino)phenyl] [4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate is considered to be toxic to aquatic environment and can be considered to be classified in aquatic acute 1 as per the CLP classification criteria.

 

In a supporting weight of evidence study from peer reviewed journal (E Kasai and S. Hatakeyama, 1993) for the read across chemical N,N'-diethyl-6 -(methylsulfanyl)-1,3,5 -triazine-2,4 -diamine (CAS no. 1014 -70 -6), short term toxicity to Chlorella vulgaris (CCAP 211/11b and NIES-227) study was carried out for 72 hrs. The study was based on the effects of the read across compound N,N'-diethyl-6 -(methylsulfanyl)-1,3,5 -triazine-2,4 -diamine on Chlorella vulgaris in a static fresh water system at a temperature of 25 -28⁰C.Two hundred-fold stock solutions in 75 % DMSO (dimethyl sulfoxide) were prepared to make six final concentrations in the media, respectively. Chlorella vulgaris (CCAP 211/11b and NIES-227) was used as a test organism obtained as axenic unialgal cultures. To obtain log-phase materials in population growth, a few drops of stock cultures were inoculated into 100 ml flasks, each containing 50 ml Medium C. These flasks were incubated for three days. An equal amount (0.5 ml) of respective stock solutions of the herbicides and 1 to 4.5 ml of the precultured algae were added to a freshly prepared Medium C. The final volume was adjusted to 100 ml, which contained 1 x 10(4) algal cells/ml. The untreated control received only 0.5 ml of 75 % DMSO. A final concentration of DMSO used for the herbicide solvent was 0.375 %. A preliminary experiment showed no adverse effects at this concentration on the population growth of the algae. For the assay, 50 ml conical flasks each containing 30 ml of the test solution, covered with a silicon cap was used. Three replicates were used at each herbicide concentration. Preculture and the assays were conducted at 25-28⁰C under continuous illumination at 100-150 µmol photon/m2/s. The flasks were shaken manually twice a day. After 72 hours the cell density was determined using a Coulter Counter ZM (Coulter Electronics Ltd.). Percentage growth was calculated comparing cell number in the culture containing the respective herbicides to that of untreated control culture. EC50 values were estimated by the linear regression of probit of percentage growth on log dose of herbicides. Comparison of regression lines was performed statistically. Based on effect on growth rate and cell density of the test organism Chlorella vulgaris(CCAP 211/11b and NIES-227),the 72 hr EC50 value was determined to be 0.494 and 0.464 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance N,N'-diethyl-6-(methylsulfanyl)-1,3,5-triazine-2,4-diaminecan be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic acute category 1 as per the CLP classification criteria.

 

For the same read across chemical N,N'-diethyl-6 -(methylsulfanyl)-1,3,5 -triazine-2,4 -diamine (CAS no. 1014 -70 -6), short term toxicity to green algae study was carried out for 72 hrs (J-CHECK, 2017). The study was based on the effects of the read across compound N,N'-diethyl-6 -(methylsulfanyl)-1,3,5 -triazine-2,4 -diamine on green algae in a static fresh water system. Based on effect on growth rate of the test organism green algae, the 72 hr EC50 and NOEC value was determined to be 0.028 and 0.0046 mg/l, respectively and on the basis of biomass and area under the growth curve, the 72 hr EC50 and NOEC value was determined to be 0.012 and 0.0046 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance N,N'-diethyl-6 -(methylsulfanyl)-1,3,5 -triazine-2,4 -diamine can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic acute category 1 as per the CLP classification criteria.

 

Another short term toxicity to green algae study was carried out for 72 hrs(J-CHECK, 2017). The study was based on the effects of the read across compound 4,4'-carbonimidoylbis (N,N-dimethylaniline) hydrochloride (CAS no. 2465 -27 -2) on green algae in a static fresh water system. Based on effect on growth rate of the test organism green algae, the 72 hr EC50 and NOEC value was determined to be 0.34 and 0.026 mg/l, respectively and on the basis of AUG, the 72 hr EC50 and NOEC value was determined to be 0.093 and 0.026 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance 4,4'-carbonimidoylbis(N,N-dimethylaniline) hydrochloride can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic acute category 1 as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical N-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate (from 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 substance N-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate can be considered as toxic to aquatic environment and can be considered to be classified in aquatic acute category 1 as per the CLP classification criteria.

 

 Toxicity to microorganisms:

Based on the various experimental data and prediction data for the target chemical study have been reviewed to determine the toxic nature of N-(4-{[4-(dimethylamino)phenyl][4-(methylamino) phenyl]methylene}cyclohexa-2,5-dien-1-ylidene)-N-methyl methanaminium acetate (CAS No. 84434 -47 -9) on the growth of microorganisms. The studies are as mentioned below:

 

In the first weight of evidence study for target chemical (SSS, 2017) 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, toxicity on microorganisms was predicted forN-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino) phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate (84434-47-9),IGC50 value was estimated to be 91.765 mg/l whenN-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate wasexposed to Tetrahymena pyriformis for 48 hours.

 

 

In the second weight of evidence study for the read across chemical (1694-09-4) (From Toxicology and applied pharmacology, 1977) toxicity to micro-organisms study was conducted on Paramecium caudatum for 20 mins. The test chemical conc. used for the study was 10000 mg/l (0.1%) and 1000 mg/l (1%). The test organism Paramecium caudatum was maintained at 22°C on 0.15% dried lettuce infusion and fed with Aerobacter aerogenes. Food dye (test chemical) of 0.1% conc. was 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 Paramecium caudatum were added, their survival times were measured microscopically. 30 to 40 test organism for each conc. were tested and the mean survival time and the death rate was calculated after 20 mins. 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 min. The mean survival time of test organism Paramecium caudatum at 1% was 298 sec and at 0.1% was 865 sec. Based on the mean survival time and death rate of organism due to the exposure of chemical acid violet, the EC100 was 10,000 mg/l and EC83.3 was 1000 mg/l.

 

Similarly study was conducted for the third weight of evidence study for the read across chemical methyl violet (8004-87-3) (From Chemosphere, 2003) Determination of toxicity of methyl violet on the growth of 14 gram negative microorganisms. Inhibitory activity of test chemical (methyl violet) 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 ongrowth inhibition of gram negative test organisms, the NOEC value of methyl violet 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.

 

 

Similarly study was conducted for the fourth weight of evidence study for the read across chemical methyl violet (8004-87-3) from chemosphere 2003, Determination of toxicity of methyl violet on the growth of 8 gram positive microorganisms. Inhibitory activity of test chemical (methyl violet) was determined by the traditional agar gel method. The conc. of test chemical used for the study was 0.6918 mg/l (1 µM). 8 different gram positive bact. were used for the study was Corynebacterium fascians, Corynebacterium flaccumfaciens, Corynebacterium michiganense, Corynebacterium oortii, Stapylococcus aureus, Micrococcus luteus, Bacillus subtilis and Bacillus thuringiensis Berliner subp. Kurstaki. 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 growth inhibition of gram positive test organisms, the LOEC value of methyl violet was determine to be 0.6918 mg/l.

On the basis of above mentioned available information for short term toxicity to aquatic fish, invertebrates and toxicity to aquatic algae and cyanobacteria, test substance N-(4 -{[4 -(dimethylamino)phenyl][4 -(methylamino)phenyl]methylene}cyclohexa-2,5 -dien-1 -ylidene)-N-methylmethanaminium acetate can be considered as toxic to aquatic environment and can be considered to be classified in aquatic acute category 1 as per the CLP classification criteria.