Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

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:

Experimental data of read across chemical

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 mention below

Principles of method if other than guideline:

WoE report is prepared based on toxicity to microorganisms study:1 and 2nd

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Naphthalenecarboxylic acid, 3-hydroxy-4-[(4-methyl-2-sulfophenyl)azo]-, calcium salt- Molecular formula (if other than submission substance): C18H14N2O6S.Ca- Molecular weight (if other than submission substance): 424.445 g/mole- Smiles notation (if other than submission substance): c12c(c(c(C(=O)[O-])cc1cccc2)O)\N=N\c1c(cc(C)cc1)S(=O)(=O)[O-].[Ca+2]- InChl (if other than submission substance): 1S/C18H14N2O6S.Ca/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;/h2-9,21H,1H3, (H,22,23)(H,24,25,26);/q;+2/p-2/b20-19+;- Substance type: Organic - Physical state: Solid

Analytical monitoring:

not specified

Details on sampling:

1. Sample storage conditions before analysis: Frozen samples were brought to room temperature, and centrifuged.2. Concentrations: The test chemical conc. used for the study was 1000 mg/l (0.1%) and 10000 mg/l (1%)

Vehicle:

yes

Test organisms (species):

other: 1.Vibrio fisheri and Paramaecium caudatum

Details on inoculum:

1. Details on test organisms- Laboratory culture: From eight serial dilutions, the percent concentration to decrease 20% of the luminescence of amodified strain of Vibrio fischeri(EC20) after 5 min incubation was calculated with the Microtox data analysis program- Method of cultivation: No data- Preparation of inoculum for exposure: Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH. Colour correction was done at 490 nm. 2. Method of cultivation: 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.

Test type:

static

Water media type:

freshwater

Total exposure duration:

5 min

Post exposure observation period:

2. After 20 mins, the mean survival time and the death rate was calculated.

pH:

1. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOH

Nominal and measured concentrations:

1. measured concentrations2. nominal concentrations

Details on test conditions:

1. TEST SYSTEMTest vessel: Microtox 500 AnalyzerNo. of vessels per concentration (replicates): triplicate analysis.OTHER TEST CONDITIONSAdjustment of pH: The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70 μl 1 M NaOHPhotoperiod:Light intensity: Colour correction was done at 490 nm.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The percent concentration to decrease 20% of the luminescence of amodified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program.2.Test vessel: Hollow slide glassNo. of organisms per vessel: 30 to 40 test organism for each test conc. was taken for the study.EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After 20 mins, the mean survival time and the death rate was calculated.TEST CONCENTRATIONSTest concentrations: 10000 mg/l (1%) and 1000 mg/l (0.1%)

Reference substance (positive control):

yes

Remarks:

1. A solution of 1 g/l ZnSO4.7H2O was used as the positive control

Key result

Duration:

5 min

Dose descriptor:

other: EC20

Effect conc.:

44.6 other: % dilution

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: In 1st study: EC20 is the percent dilution of the sample (v/v) to cause 20% decrease of bioluminescence in the Microtox assay

Key result

Duration:

20 min

Dose descriptor:

other: greater than EC50

Effect conc.:

10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: In 2nd study: The death rate of test organism was determine to be 77.4%.

Results with reference substance (positive control):

1. Result with reference substance (positive control)Results with reference substance valid?: YesOther: Glucose was used as negative control and it was non- toxic

1.

Table 1 : Toxicity of the components in the Microtox assay

 

Sample	EC20 (% DILUTION)
Positive control [ZnSO4.7H20]	0.72±0.1
Distilled water	>100
100mg/l test chemical	44.6±11.6
1g/l Glucose	>100

 

EC20 is the % dilution of the sample (v/v) to cause 20% decrease of bioluminescence in the Microtox assay

>100% indicates no toxic effect

 

2 study: Table 1: Effect of food dye on the mean survival time and death rate of Paramecium caudatum

 

Test chemical	Dye concentration
	1.0%		0.1%
	Mean survival time (sec)	Death rate (%)	Mean survival time (sec)	Death rate (%)
Test chemical	695	77.4	-*	3.3

-* indicates no deaths were observed for 20 minutes

Validity criteria fulfilled:

not specified

Conclusions:

1. The toxicity of test chemical was determined in terms of EC20 (% dilution) was 44.6 ± 11.6.. According to the ranking scheme for Microtox assay using EC20 values, test chemical can be categorized under moderately toxic category.2. The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum is reported as 10000 mg/lThus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.

Executive summary:

Toxicity of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was studied on the growth and other biological activity of microorganisms is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Toxicity study was performed to determine the toxic effect of the chemicals on the microorganisms. The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri. Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer from eight serial dilutions, the percent concentration to decrease 20% of the luminescence of a modified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20) was reported instead. The following rating was adapted. –

EC20: >100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of test chemical determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similar study was performed for chemical. The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. Hence a bioassay was conducted to study the toxic effect of test chemical. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Chemical was tested in 0.1% and 1% concentration. The test 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 test organisms were added, their survival times were measured microscopically. Thirty to forty test organisms 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 minutes. The mean survival time (in sec) of test organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.

 

Thus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.

Description of key information

1. The toxicity of test chemical was determined in terms of EC20 (% dilution) was 44.6 ± 11.6.. According to the ranking scheme for Microtox assay using EC20 values, test chemical can be categorized under moderately toxic category.

2. The death rate of the test organism at 10000 mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum is reported as 10000 mg/l

Thus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.

Key value for chemical safety assessment

EC50 for microorganisms:

10 000 mg/L

Additional information

Toxicity of calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was studied on the growth and other biological activity of microorganisms is predicted on the basis of it structurally and functionally similar read across chemicals. The studies are as mentioned below:

 

Toxicity study was performed to determine the toxic effect of the structurally and functionally similar read across chemicals on the microorganisms (From peer reviewed journal, 2002). The Microtox acute toxicity assay was performed by using a modified strain of Vibrio fischeri. Frozen samples were brought to room temperature, and centrifuged. The pH of the samples was adjusted where necessary to 6 by adding 0.5 ml 0.58 M KH2 PO4 and 70μl 1 M NaOH colour correction was done at 490 nm. The Microtox acute toxicity assay was performed in a Microtox 500 Analyzer on samples before and after decoloration according to the test protocols defined by the manufacturer from eight serial dilutions, the percent concentration to decrease 20% of the luminescence of a modified strain of Vibrio fischeri (EC20) after 5 min incubation was calculated with the Microtox data analysis program. A solution of 1 g/l ZnSO4·7H2O was used as the positive control and 1 g/l glucose as the negative control. Each EC20 reported is the average of triplicate analysis.

The concentration to decrease 50% of the bacterial luminescence in the Microtox acute assay (EC50) is normally reported. However, in most of these studies, the EC50 before or after decoloration was greater than 100% indicating that there was no toxicity or toxicity change. To better evaluate whether the decoloration process affected toxicity, the dilution required to decrease 20% of the bacterial luminescence relative to the control (EC20) was reported instead. The following rating was adapted. –

EC20: >100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic.

The toxicity of 100mg/l of test chemical determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similar study was performed for another structurally and functionally similar read across chemicals (from peer reviewed journal 1977). The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. Hence a bioassay was conducted to study the toxic effect of test chemical. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Chemical was tested in 0.1% and 1% concentration. The test 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 test organisms were added, their survival times were measured microscopically. Thirty to forty test organisms 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 minutes. The mean survival time (in sec) of test organism Paramecium caudatum was determined to be 695 seconds.  The death rate of the test organism at 10000mg/l was 77.4%. Therefore the Effective concentration causing more than 50% death of Paramecium caudatum was reported as 10000 mg/l.

 

Thus based on both the studies chemical calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate (CAS no. 5281-04-9) was consider as nontoxic.