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Toxicity to microorganisms

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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:
Experimental result of 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:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mention in below principle
Principles of method if other than guideline:
WoE report is based on two toxicity study of microorganisms for the test chemical :1. and 2. To evalute growth inhibition test of test material
GLP compliance:
not specified
Specific details on test material used for the study:
IUPAC name: Reaction mass of Methylium, tris[4-(diethylamino)phenyl]- & acetate Molecular weight: 515.7375Molecular formula: C33H45N3O2Smiles: CCN(CC)c1ccc(C{+}(c2ccc(N(CC)CC)cc2)(c2ccc(N(CC)CC)cc2).O{-}C(C)=O)cc1Inchl: 1S/C31H42N3.C2H4O2/c1-7-32(8-2)28-19-13-25(14-20-28)31(26-15-21-29(22-16-26)33(9-3)10-4)27-17-23-30(24-18-27)34(11-5)12-6;1-2(3)4/h13-24H,7-12H2,1-6H3;1H3,(H,3,4)/q+1;/p-1Physical state: Solid and liquidForm: Organic
Analytical monitoring:
yes
Remarks:
In 2 study
Vehicle:
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.A. radiobacter, A. tumefaciens, Bradyrhizobium japonicum, E. coli, Erwinia atroseptica, E. herbicola, E. uredovora, P.fluorescence, P. phaseolicola, P. syringae, Rhizobium trifolii, Xanthomonas malvacearum, X. phaseoli, X. stewartii 2. P.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 available2. - 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:
not specified
Water media type:
not specified
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.
Post exposure observation period:
2. 17.28 min
Test temperature:
1. 21 ± 1 °C2. 22 deg.C
pH:
2. 7.2
Nominal and measured concentrations:
1. 0.6918 mg/l (1 µM) 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
Key result
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.692 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: For gm-negative bacteria Erwinia herbicola and Pseudomonas syringae
Remarks:
In 1st study
Key result
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
Remarks on result:
other: LOEC for remaining 12 gm-negative bacteria.
Remarks:
In 1st study
Key result
Duration:
17.28 min
Dose descriptor:
LOEC
Effect conc.:
0.1 mg/L
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.
Remarks:
2nd study
Reported statistics and error estimates:
2. The correlation coefficient between the inhibitory activity of these food dye on LAP and the death rate showed r values of 0.7952 (p < 0.05).

1.Table: Microbiological activity of synthetic dyes towards bacteria.

Test organism

Growth inhibition (in %)

Agrobacterium radiobacter

11

Agrobacterium tumefaciens

11

Bradyrhizobium japonicum

28

Escherichia coli

15

Erwinia atroseptica

8

Erwinia herbicola

0

Erwinia uredovora

13

Pseudomonas fluorescence

6

Pseudomonas phaseolicola

13

Pseudomonas syringae

0

Rhizobium trifolii

11

Xanthomonas malvacearum

6

Xanthomonas phaseoli

19

X. stewartii

9

Validity criteria fulfilled:
not specified
Conclusions:
1. 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 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.Thus based on the above studies chemical was consider toxic.
Executive summary:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity microorganisms of the test Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate.The studies are as mentioned below:

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.

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, 36.7% mortality observed of test organism i.e Paramecium caudatum after exposure of 17.28 min. Thus on that basis chemical was not consider to be very toxic.

Thus based on the above studies chemical was consider toxic.

Description of key information

1. 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 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.

Thus based on the above studies chemical was consider toxic.

Key value for chemical safety assessment

EC50 for microorganisms:
0.691 mg/L

Additional information

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity microorganisms of the test Reaction mass of Methylium, tris[4-​(diethylamino)​phenyl]​-​ & acetate.The studies are as mentioned below:

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.

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, 36.7% mortality observed of test organism i.e Paramecium caudatum after exposure of 17.28 min. Thus on that basis chemical was not consider to be very toxic.

Thus based on the above studies chemical was consider toxic.