Reaction mass of 1-Propanaminium, 3-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxo-2-propen-1-yl]oxy]-N-[2-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxo-2-propen-1-yl]oxy]ethyl]-N,N-dimethyl- & chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental result of read across chemicals

Justification for type of information:

Data for the target chemical is summarized based on the structurally and functionally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

WoE report is based on short term toxicity studies on aquatic algae as - WoE-2 and WoE-3 and WoE 4.

GLP compliance:

not specified

Specific details on test material used for the study:

Name: Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chlorideMol. formula: C35H46ClN5O4Molecular Weight: 636.232 g/moleInChI:1S/C35H46N5O4.ClH/c1-7-38(8-2)32-16-12-28(13-17-32)24-30(26-36)34(41)43-22-11-20-40(5,6)21-23-44-35(42)31(27-37)25-29-14-18-33(19-15-29)39(9-3)10-4;/h12-19,24-25H,7-11,20-23H2,1-6H3;1H/q+1;/p-1/b30-24+,31-25+;Smiles:C(CCOC(\C(=C\c1ccc(cc1)N(CC)CC)C#N)=O)[N+](C)(C)CCOC(\C(=C\c1ccc(cc1)N(CC)CC)C#N)=O.[ClH-]Substance type: Organic

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

1. The stock solution (100 mg/L) was prepared by dissolving pinkish red powder in DMSO/ OECD growth medium. The stock solution was kept in ultrasonic bath for 20min Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.2.The stock solution 2 g/L was prepared by dissolving yellow powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. It was not possible to test higher substance concentrations due to solubility of the test substance.- Controls: OECD medium as specified in OECD 201, used as a control and for dilution of the stock solution.3.The test solution was prepared in aseptic condition. The test item 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 20 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 organisms (species):

other: 1, 2. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) and 3.Chlorella vulgaris

Details on test organisms:

1. TEST ORGANISM- Common name: - Strain: 86.81 SAG- Source (laboratory, culture collection): Institute of botany of the ASCR- Initial biomass concentration: 5x10(3) cells /ml - Method of cultivation: No data available3.The fresh water green alga Chlorella vulgaris, was used as the test system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Remarks on exposure duration:

±1 hour

Test temperature:

1and 2 23±2°C3.22±2°C.

pH:

1. WIthout adjustmentThe sample at concentration 40 mg/l: pH=8 changed to 7.9 during the testControl: pH= 8.1 changed to pH= 7.5 during the test2.The sample at concentration 4 mg/L : pH = 8.0 changed to pH = 7.7 during the test, Control : pH = 8.1 changed to pH = 7.8 during the testControl + acetone: pH = 8.1 changed to pH = 7.6 during the test.3.6.4 to 7.1

Nominal and measured concentrations:

1. 0, 2.5, 5, 10, 20 and 40 mg/L3. 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l and 200mg/l (All the six concentration were in geometric series spaced by a factor of 2.)

Details on test conditions:

1. TEST SYSTEM- Test vessel: 50 ml Glass vessel- Type (delete if not applicable): closed (with air permeable stopper)- Sample volume: 15 ml- Initial cells density: 5x10(3) cells/ml- No. of vessels per concentration (replicates): 3GROWTH MEDIUM- Standard medium used: yesOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: Continuous- Light intensity and quality: 6000-8000 lxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.- Other: ErC50 was calculated using non-linear regression by the software Prism 4.03.1. Study Type: Static2. Exposure Duration: 72 Hours3. Temperature: 22 °C ±2°C.4. Light Quality: White Fluorescent Light5. Light Intensity: 1500Lux6. Photoperiod: 16 Hour Light Period : 8 Hour Dark Period7. RPM Speed: 120 Revolutions per minute8. Water: Deionized Water9. Test vessel size: 100ml10. Test volume: 60ml11. Dilution wate:r Bold’s Basal Medium12. Test concentration Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l13. Replicates per concentration: Three replicates for Control, Two replicates for each test concentration

Reference substance (positive control):

yes

Remarks:

1 and 2. Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: For 1 study: 95% CL: 11.9 - 15.2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2 study

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

98.169 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 3 study

Details on results:

3.The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.

Results with reference substance (positive control):

1. Results with reference substance valid- EC50: 0.75 mg/L2.- Results with reference substance valid- ErC50: 0.69 mg/L K2Cr2O7

Validity criteria fulfilled:

not specified

Conclusions:

1. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 13.4 mg/L on the basis of effects on growth rate in a 72 hour study.2.The median effective concentration (ErC50) for the test substance, in a freshwater algae Desmodesmus subspicatus was determined to be 17.8 mg/L on the basis of effects on growth rate in a 72 hour study.3.After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 98.169 mg/l graphically and through probit analysis.Based on the above studies, it was concluded that the chemical Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride was toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

Executive summary:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of the test chemical Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride on the growth of algae. The studies are as mentioned below:

In first study Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution (100 mg/L) was prepared by dissolving pinkish red powder in DMSO/ OECD growth medium. The stock solution was kept in ultrasonic bath for 20min Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the concentrations 0, 2.5, 5, 10, 20 and 40 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 13.4 mg/L. This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as Aquatic Chronic 3 as per the CLP criteria.

Similarly in second study freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution 2 g/L was prepared by dissolving yellow powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. It was not possible to test higher substance concentrations due to solubility of the test substance and tested at the concentrations 0, 0, 0.5, 1, 2 and 4 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 17.8 mg/L. Based on the ErC50, it can be concluded that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Above studies was supported by the third study. The effect of test item 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 6.2 5 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test solution was prepared in aseptic condition. The test item 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 20 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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 98.169 mg/L. This value indicates that the substance is toxic to algae and can be classified as Aquatic chronic category 3 as per the CLP criteria.

Based on the above studies, it was concluded that the Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride was toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

Description of key information

1. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 13.4 mg/L on the basis of effects on growth rate in a 72 hour study.

2.The median effective concentration (ErC50) for the test substance, in a freshwater algae Desmodesmus subspicatus was determined to be 17.8 mg/L on the basis of effects on growth rate in a 72 hour study.

3.After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 98.169 mg/l graphically and through probit analysis.

Based on the above studies, it was concluded that the Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride was toxic and classified as aquatic chronic 3 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

13.4 mg/L

Additional information

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of the test Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride on the growth of algae. The studies are as mentioned below:

 

In first study from experimental report 2016, freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution (100 mg/L) was prepared by dissolving pinkish red powder in DMSO/ OECD growth medium. The stock solution was kept in ultrasonic bath for 20min Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the concentrations 0, 2.5, 5, 10, 20 and 40 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 13.4 mg/L. This value indicates that the substance is likely to be hazardous to aquatic algae and can be classified as Aquatic Chronic 3 as per the CLP criteria.

 

 

Similarly in second study from the experimental report freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance according to OECD Guideline 201. The stock solution 2 g/L was prepared by dissolving yellow powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. It was not possible to test higher substance concentrations due to solubility of the test substance and tested at the concentrations 0, 0, 0.5, 1, 2 and 4 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, in Desmodesmus subspicatus was determined to be 17.8 mg/L. Based on the ErC50, it can be concluded that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

 

 

Above studies was supported by the third study. The effect of test item 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 6.2 5 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test solution was prepared in aseptic condition. The test item 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 20 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 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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 98.169 mg/L. This value indicates that the substance is toxic to algae and can be classified as Aquatic chronic category 3 as per the CLP criteria.

 

 

Based on the above studies, it was concluded that the Reaction mass of 1-​Propanaminium, 3-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​-​N-​[2-​[[2-​cyano-​3-​[4-​(diethylamino)​phenyl]​-​1-​oxo-​2-​propen-​1-​yl]​oxy]​ethyl]​-​N,​N-​dimethyl-​ & chloride was toxic and classified as aquatic chronic 3 as per the CLP classification criteria.