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Ecotoxicological information

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Description of key information

Short term toxicity to fish

Short term toxicity test to rainbow trout, bluegill and sea lamprey was carried out for 24 hrs (Vernon C. Applegate et. al; 1957). Test organisms Sea lamprey (Petromyzon marinus), Rainbow trout (Salmo gairdnerii) and Bluegill (Lepomis macrochius) were used for the study. The study was performed under static conditions for 24 hrs. The test chemical2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS no. 6441-82-3) conc. used for the study was 5 mg/l. Larval lampreys were collected by means of an electric shocker in the Ocqueoc River, Presque Isle County, Michigan, and were held in running water in aquaria and small “races” under conditions which simulated their natural stream habitat.Test fishes (Rainbow trout and Bluegill) were obtained fromthe stocks of local state and Federal fish hatcheriesand were held in large raceways. These specimens were maintained in the best possible physical condition until used in the laboratory.

The aggregate test animals available, usually six in number, were placed together in a 10-literglass battery jar containing 5 liters of water. The jars were provided with aeration through standard stone air-breakersand were maintained at a constant temperature by immersion in specially constructed constant temperature troughs. Water temperature was maintained within the limits of ± 1.0 °F.Twelve of these test jars (each containing a substance being assayed) were included with one control jar in each trough. Fish and larvae in the control jar were exposed only to the water and physical conditions of the typical test container.Test condition contains dissolved oxygen from 8.6 to 13.7 ppm and free CO2from 5.0 to 9.0 ppm. Observations of each test were measured approx. six times, at various intervals, during the 24 hr test period. At each observation, the condition of every test specimen was determined and recorded. Chronological histories were thus obtained of any symptoms of illness and the occurrence of death.No effects were observed when the test organisms sea lamprey, bluegill and rainbow trout were exposed to the test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride for 24 hrs. Thus, the EC0 value was determined to be 5 mg/l.

Short term toxicity to aquatic invertebrates

Using the OECD QSAR toolbox version 3.4 (2018) with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic invertebrates was predicted for target substance2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride(CAS no. 6441 -82 -3). EC50 value was estimated to be 10.5 mg/l for Daphnia magna for 48 h duration. Based on this value it can be concluded that the substance2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chlorideis considered to be toxic to aquatic environment and can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Using the OECD QSAR toolbox version 3.4 (2018) with log kow as the primary descriptor and considering the five closest read across substances, the short term toxicity on aquatic algae and cyanobacteria was predicted for target substance 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS no. 6441 -82 -3). EC50 value was estimated to be 11.6 mg/l for Desmodesmus subspicatus for 72 h duration. Based on this value it can be concluded that the substance 2 -[2 -[4 -[(2 -chloroethyl)ethylamino] -o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride is considered to be toxic to aquatic environment and can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs (F. Malpei et. al; 1998). Anaerobic granular sludge or methanogens were used as a test organism during the study. Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively. Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study. The basal medium M9 was used with laboratory grade yeast extract (1 g/l), trypticase (1 g/l), sodium acetate, a microelements solution. All manipulations were carried out in an anaerobic chamber. The autoclaved medium was aliquoted in the vials (50 ml/each), to which was added: approximately 0.5 ml of whole granules of the sludge; concentrated dye solutions to obtain final test dye concentrations of 0, 300, 600, 1200 and 2400 mg/l, respectively. For test chemical, four replicate vials were prepared.Methane production were monitored in each vial every 24 h for 3 or 4 days. The final biomass concentration in each vial was determined at the end of the test.Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino] -o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens,the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.

Additional information

Short term toxicity to fish

Experimental study for the target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS No. 6441-82-3) and various supporting weight of evidence studies for its structurally similar read across substanceswere reviewed to summarize the following information:

 

In an experiment key study from peer reviewed journal (Vernon C. Applegate et. al; 1957), Short term toxicity test to rainbow trout, bluegill and sea lamprey was carried out for 24 hrs. Test organisms Sea lamprey (Petromyzon marinus), Rainbow trout (Salmo gairdnerii) and Bluegill (Lepomis macrochius) were used for the study. The study was performed under static conditions for 24 hrs. The test chemical2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS no. 6441-82-3) conc. used for the study was 5 mg/l. Larval lampreys were collected by means of an electric shocker in the Ocqueoc River, Presque Isle County, Michigan, and were held in running water in aquaria and small “races” under conditions which simulated their natural stream habitat.Test fishes (Rainbow trout and Bluegill) were obtained fromthe stocks of local state and Federal fish hatcheriesand were held in large raceways. These specimens were maintained in the best possible physical condition until used in the laboratory.

The aggregate test animals available, usually six in number, were placed together in a 10-literglass battery jar containing 5 liters of water. The jars were provided with aeration through standard stone air-breakersand were maintained at a constant temperature by immersion in specially constructed constant temperature troughs. Water temperature was maintained within the limits of ± 1.0 °F.Twelve of these test jars (each containing a substance being assayed) were included with one control jar in each trough. Fish and larvae in the control jar were exposed only to the water and physical conditions of the typical test container.Test condition contains dissolved oxygen from 8.6 to 13.7 ppm and free CO2from 5.0 to 9.0 ppm. Observations of each test were measured approx. six times, at various intervals, during the 24 hr test period. At each observation, the condition of every test specimen was determined and recorded. Chronological histories were thus obtained of any symptoms of illness and the occurrence of death.No effects were observed when the test organisms sea lamprey, bluegill and rainbow trout were exposed to the test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride for 24 hrs. Thus, the EC0 value was determined to be 5 mg/l.

 

In a supportingstudy from authoritative database (ECOTOX database, 2018) of the read across chemical 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride (CAS no. 3648-36-0),short term toxicity to Leuciscus idus (Ide, Silver or Golden Orfe) study was carried out for 48 hrs. The study was based on the effects of the read across compound 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride (CAS no. 3648-36-0) on Leuciscus idus in a static freshwater system at a temperature of 10 to 13°C and pH 7.2 to 7.4, respectively. Hardness of water is 120 to 140 mg/l CaCO3 and dissolved oxygen is ˃4 mg/l, respectively. Leuciscus idus (Ide, Silver or Golden Orfe) was used as a test organism. Based on effect on mortality of the test organism Leuciscus idus, the 48 hr LC50 value was determined to be 40 mg/l, respectively. Thus, based on the LC50 value, it can be concluded that the substance 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Another short term toxicity to Leuciscus idus (Ide, Silver or Golden Orfe) study was carried out for 48 hrs (ECOTOX database, 2018). The study was based on the effects of the read across compound 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride (CAS no. 3648-36-0) on Leuciscus idus in a static freshwater system at a temperature of 13°C and pH 7.0 to 8.0, respectively. Hardness of water is 270 mg/l CaCO3 and dissolved oxygen is ˃5 mg/l, respectively. Leuciscus idus (Ide, Silver or Golden Orfe) was used as a test organism. Based on effect on mortality of the test organism Leuciscus idus, the 48 hr LC50 value was determined to be in the range 38 to 75 mg/l, respectively.Thus, based on the LC50 value, it can be concluded that the substance 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

For the same read across chemical compound 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride (CAS no. 3648-36-0), short term toxicity to Oncorhynchus mykiss (Rainbow Trout) study was carried out for 48 hrs (ECOTOX database, 2018). The study was based on the effects of the read across compound 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride on Oncorhynchus mykiss in a static freshwater system at a temperature of 13°C and pH 7.0 to 7.4, respectively. Hardness of water is 180 mg/l CaCO3 and dissolved oxygen is ˃7 mg/l, respectively. Oncorhynchus mykiss (Rainbow Trout) was used as a test organism. Based on effect on mortality of the test organism Oncorhynchus mykiss, the 48 hr LC50 value was determined to be 80 mg/l, respectively. Thus, based on the LC50 value, it can be concluded that the substance 2-(2-{4-[(2-chloroethyl)(methyl)amino]phenyl}vinyl)-1,3,3-trimethyl-3H-indolium chloride can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (from peer reviewed journal) and for its read across substance (from authoritative database ECOTOX),it can be concluded that the EC0 value of the test substance 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride was determined to be 5 mg/l.

Short term toxicity to aquatic invertebrates

Predicted data for the target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS No. 6441-82-3) and various supporting weight of evidence studies for its read across substanceswere reviewed to summarize the following information:

 

Short term toxicity on aquatic invertebrate of target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS No. 6441-82-3) is predicted using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances (2018). On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 10.5 mg/l for Daphnia magna for 48 hr duration.

 

In a supportingweight of evidence study from experimental study report (Sustainability Support Services (Europe) AB, Report no. 307/16, 2016) of the read across chemical Diethylaniline (CAS no. 91-66-7),experimental study for inhibition of the mobility of daphnids was carried out with the Diethylaniline (CAS no. 931 -66 -7) according to OECD Guideline 202. The animals used for the test shall be less than 24 h old and should not be of first brood progeny. The stock solution (100 mg/L) was prepared by dissolving clear yellow oily liquid in DMSO. The test solutions of required concentration as were prepared by mixing the stock solution of the test sample in reconstituted test water. The test substance was tested at the concentration of 0, 4, 10, 17, 30, 55 and 100 mg/L, respectively. The test was performed under static conditions in fresh water system at 20±1°C temperature. Effects on immobilisation were observed for 48 hours. EC50 was calculated using non-linear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance Diethylaniline (CAS no. 91 -66 -7) in Daphnia magna was determined to be 35.2 mg/L (95% CL: 24.8 -50.0 mg/l) for immobilisation effects.

 

Another short term toxicity to Daphnia magna (Water flea) study was carried out for 48 hrs (J-CHECK, 2017). The study was performed according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The study was based on the effects of the read across compound Diethylaniline (CAS no. 91-66-7) on Daphnia magna in a static fresh water system. Daphnia magna (Water flea) was used as a test organism. Based on effect on mobility of the test organism Daphnia magna (Water flea), the 48 hr EC50 value was determined to be 11 mg/l, respectively.

 

Thus, based on the overall reported results for target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (OECD QSAR toolbox version 3.4, 2017) and for its read across substance (from experimental study report andauthoritative database J-CHECK),it can be concluded that the test substance 2 -[2 -[4 -[(2 -chloroethyl)ethyl amino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride can be considered as toxic to aquatic invertebrates and can be considered to beclassified in aquatic chronic category 3 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Predicted data for the target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS No. 6441-82-3) and supporting weight of evidence study for its closest read across substances with logKow as the primary descriptorwere reviewed for toxicity to aquatic algae and cyanobacteria endpoint to summarize the following information:

 

Short term toxicity on aquatic algae and cyanobacteria of target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (CAS No. 6441-82-3) is predicted using the OECD QSAR toolbox version 3.4 with log kow as the primary descriptor and considering the five closest read across substances (2018).On the basis of effects observed in a static freshwater system, the effect concentration EC50 value for the substance is estimated to be 11.6 mg/l for Desmodesmus subspicatus for 72 duration.

 

In a supporting weight of evidence study from authoritative database (J-CHECK, 2017) of the read across chemical disodium 4,4'-bis[(4 -anilino-6 -morpholino-1,3,5 -triazin-2 -yl)amino]stilbene-2,2'-disulphonate (CAS no. 16090-02-1),short term toxicity to green algae study was carried out for 72 hrs. The study was based on the effects of the read across compound disodium 4,4'-bis[(4 -anilino-6 -morpholino-1,3,5 -triazin-2 -yl)amino]stilbene-2,2'-disulphonate (CAS no. 16090 -02 -1) 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 > 65 and 6.3 mg/l, respectively and on the basis of AUG, the 72 hr EC50 and NOEC value was determined to be 20 and 3.3 mg/l, respectively.

 

Thus, based on the overall reported results for target chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride (OECD QSAR toolbox version 3.4, 2017) and for its read across substance (from authoritative database J-CHECK),it can be concluded that the test substance 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride can be considered as toxic to aquatic algae and cyanobacteria and can be considered to be classifiedin aquatic chronic category 3 as per the CLP classification criteria.

Toxicity to microorganisms

Experimental key and supporting study for the target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride(CAS No. 6441-82-3)were reviewed for toxicity to microorganism endpoint to summarize the following information:

 

In an experiment key study from peer reviewed journal (F. Malpei et. al; 1998),Toxicity to micro-organisms study was conducted using anaerobic granular sludge or methanogens for 72 hrs. Anaerobic granular sludge or methanogens were used as a test organism during the study. Anaerobic granular sludge sampled from a UASB reactor fed with soft-drink wastewater was used as active biomass.Test chemical concentration used for the study were 0, 300, 600, 1200 and 2400 mg/l, respectively. Serum vials (120 ml total volume) with butyl rubber stoppers were used as a test vessel during the study. The basal medium M9 was used with laboratory grade yeast extract (1 g/l), trypticase (1 g/l), sodium acetate, a microelements solution. All manipulations were carried out in an anaerobic chamber. The autoclaved medium was aliquoted in the vials (50 ml/each), to which was added: approximately 0.5 ml of whole granules of the sludge; concentrated dye solutions to obtain final test dye concentrations of 0, 300, 600, 1200 and 2400 mg/l, respectively. For test chemical, four replicate vials were prepared.Methane production were monitored in each vial every 24 h for 3 or 4 days. The final biomass concentration in each vial was determined at the end of the test.Test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (Red Astrazon 6B) completely inhibited the methanogenic activity at every test concentration. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the biomass of test organism anaerobic granular sludge or methanogens, the inhibitory conc. (IC100) value was determined to be 300 mg/l, respectively.

 

Another supporting toxicity to micro-organisms study was conducted on Bacillus subtilis, strain IFO 3022 (Toshihiko Ogawa et. al; 1988). Bacillus subtilis (IFO 3022), a bacterial population in activated sludge, was obtained from Institute for Fermentation, Osaka.Spizien medium was used for the asynchronous culture and consisted of 0.2% (NH4)2SO4, 1.4% K2HP04, 0.6% KH2PO4, 0.1% sodium citrate-2H20, 0.02% MgSO4.7H2O, 0.0002% FeCI3.6H2O, 0.0002% MnCI2.4H20 and 0.5% glucose. The medium composition of the synchronous culture was the same as that described above but contained 0.0072% glucose as well. The cell population was synchronized by the stationary phase method. These precultivate broths, 1 ml each, were inoculated in 1000 ml mediums, and the cultures were shaken at 37°C. The cell concentrations of the asynchronous cultures were determined by measurement of transmittance at 660 nm, and those of the synchronous cultures were read under a microscope using Thoma's hemacytometer, After harvesting by centrifugation at 8,000 rpm for 15 min from the cultivate broth, RNA and DNA were each fractionated by the method of Schmidt, Thannhauser and Schneider. Their concentrations were determined respectively by measurement of absorbance at 260 and 270 nm. Based on the inhibitory effect of the chemical 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride on the growth of test organism Bacillus subtilis, the inhibitory conc. (IC) value was determined to be 0.0229 mg/l, respectively.

 

Thus, based on the overall reported results for target chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride (from peer reviewed journals),it can be concluded that the IC and IC100 value for the test chemical 2-[2-[4-[(2-chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3- trimethyl-3H-indolium chloride was determined to be 0.0229 and 300 mg/l, respectively.

On the basis of above mentioned available information for short term toxicity to fish, aquatic invertebrates and toxicity to aquatic algae and cyanobacteria, test substance 2 -[2 -[4 -[(2 -chloroethyl)ethylamino]-o-tolyl]vinyl]-1,3,3 - trimethyl-3H-indolium chloride can be considered as toxic to aquatic organisms and can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.