Reaction mass of Diethyl[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]ammonium and acetate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

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:

of read across substance

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

WoE report is based on toxicity study of microorganism for the test chemical :
1. Determination of toxicity of methyl violet on the growth of 14 different gram – negative microorganisms. Inhibitory activity of test chemical was determined by the traditional agar gel method.

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.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate
- Molecular formula: C29H36N2O2
- Molecular weight: 444.615 g/mole
- Substance type: Organic
- Physical state: maroon lustrous liquid

Analytical monitoring:

not specified

Details on test solutions:

2) PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Food dye of various concentrations were put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):phosphate buffer
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):0.1 M

Test organisms (species):

other: 1) gm-negative bacteria Erwinia herbicola and Pseudomonas syringae , 2) Paramaecium caudatum

Details on inoculum:

1) - Laboratory culture: Test organisms were taken from the collection of the Plant Protection Institute, Hungarian Academy of Sciences (Budapest, Hungary).
- Name and location of sewage treatment plant where inoculum was collected: No data available
- Method of cultivation: 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.
- Preparation of inoculum for exposure: 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.
- Pretreatment: No data available
- Initial biomass concentration: No data available

2) - Laboratory culture: PC was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes.
- Preparation of inoculum for exposure:One week after inoculation, PC culture was filtered through two thicknesses of muslin. This was centrifuged at 8000 g for 10 min and the packed cells were then washed in a solution of 0.12 M NaCl and resedimented by centrifugation at 8000 g for 10 min. The pellet of cells was then sonicated in 0.1 M sodium phosphate buffer, pH 7.2, for 20 sec. The resulting sonicate was centrifuged at 8000 g for 10 min, and the supernatant fraction was used as the crude enzyme extract. The centrifugation and the subsequent procedures were carried out at 0-4°C.

Test type:

static

Water media type:

freshwater

Total exposure duration:

24 h

Remarks on exposure duration:

1) After 24 hrs of incubation, colony diameters were determined and growth inhibition was calculated in percentage of dye free colony growth.

Test temperature:

21 deg C

Nominal and measured concentrations:

1) 0.6918 mg/l
2) 0.1 and 1.0 % test concentration

Details on test conditions:

1)TEST SYSTEM
- Test vessel: Petri dishes
- Material, size, headspace, fill volume: The diameter of Petri dishes was 90 mm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After 24 hrs of incubation, colony diameters were determined and growth inhibition was calculated in percentage of dye free colony growth.
- Test concentrations: 0.6918 mg/l (1 µM)

2)test material of 0.1 and 1.0 % concentrations were 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 PC were added, their survival times were measured microscopically. Thirty to forty PC for each concentration were tested by the same method, and the mean survival time and the death rate were calculated. 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.

Reference substance (positive control):

not specified

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

0.692 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

17.28 min

Dose descriptor:

LOEC

Effect conc.:

0.1 other: %

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

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

Validity criteria fulfilled:

not specified

Conclusions:

The test chemical Reaction mass of Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & acetate is likely to be toxic to microorganism atleast in the concentration 0.6918 mg/l

Executive summary:

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 Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & 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.

Description of key information

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 Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & 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.

Key value for chemical safety assessment

EC50 for microorganisms:

0.692 mg/L

Additional information

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 Ethanaminium, N-​[4-​[[4-​(diethylamino)​phenyl]​phenylmethylene]​-​2,​5-​cyclohexadien-​1-​ylidene]​-​N-​ethyl-​ & 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.