Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Short term toxicity to fish:

Different food dyes were subjected to median tolerance limit (TLm) test by use of Himedaka (Oryzias Latipes) for the comparison of the toxicity. Himedaka(Oryzias Latipes) was used.The same age fish (about 2 cm long, weight ca 0.2 g) were chosen and acclimated for 10 days in the tap water before experiment. One liter of deionized water was placed in a 2 liter tank.The food dyes were obtained from National Institute of Hygienic Sciences, Japan. Survival rate test: In one liter of pH 7.0 containing 3000mg/l of dye solution, 10 fishes were kept in the tank without direct sunlight for 48 hours. Water temperature was 20C. Aeration was performed with bubbler.  TLm test: TLm was determined according to the procedure in JIS K0102 (Japanese Industrial Standards Committee, 1971). Amaranth was tested at 4 – 6 steps of concentration utilizing ten fish per one group and the number of survivals were counted after 24 and 48 hours. The values were mean of 3 trials. 100% survival rate was determined after 48 hours exposure of fishes to Amaranth. Based on these observations, the test chemical can be considered non-toxic to fishes.

 

Short term toxicity to aq. invertebrates:

Determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test wasperformed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

Toxicity to aq. algae and cyanobacteria:

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance 2,7-Naphthalenedi- sulfonic acid, 3-hydroxy-4-[(4-sulfo-1- naphthalenyl), sodium salt (Amaranth dye) according to OECD Guideline 201. The stock solution (200 mg/L) was prepared by dissolving brown powder in  OECD growth medium. 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, 12.5, 25, 50, 100, 200 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1- -naphthalenyl), sodium salt, in Desmodesmus subspicatus was determined to be 356.2 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and can not be classified as toxic as per the CLP criteria.

Toxicity to 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 amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. 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 from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic. The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

Additional information

Short term toxicity to fish:

From the available experimental data for the target chemical, the information is summarized as below:

In the first study from The Journal of Toxicological Sciences Different food dyes were subjected to median tolerance limit (TLm) test by use of Himedaka (Oryzias Latipes) for the comparison of the toxicity. Himedaka(Oryzias Latipes) was used.The same age fish (about 2 cm long, weight ca 0.2 g) were chosen and acclimated for 10 days in the tap water before experiment. One liter of deionized water was placed in a 2 liter tank.The food dyes were obtained from National Institute of Hygienic Sciences, Japan.

Survival rate test:

In one liter of pH 7.0 containing 3000mg/l of dye solution, 10 fishes were kept in the tank without direct sunlight for 48 hours. Water temperature was 20C. Aeration was performed with bubbler.  TLm test: TLm was determined according to the procedure in JIS K0102 (Japanese Industrial Standards Committee, 1971). Amaranth was tested at 4 – 6 steps of concentration utilizing ten fish per one group and the number of survivals were counted after 24 and 48 hours. The values were mean of 3 trials. 100% survival rate was determined after 48 hours exposure of fishes to Amaranth. Based on these observations, the test chemical can be considered non-toxic to fishes.

Similarly in the second estimated data from ecotox, The acute toxicity of Amaranth dye to fishes was estimated using ECOSAR v 1.11. The estimated LC50 values for fish after 96 hours of exposure with chemical 2,7-Naphthalenedisulfonic acid, 3- hydroxy-4-[2-(4-sulfo-1-naphthalenyl)diazenyl]-, sodium salt (1 :...; Amaranth dye; trisodium 3-hydroxy-4- [(4-sulfonato-1-naphthyl) diazenyl]naphthalene-2,7-disulfonate was 950.627 mg/l. Based on the estimated data, 2,7-Naphthalenedisulfonic acid, 3- hydroxy-4-[2-(4-sulfo-1-naphthalenyl)diazenyl]-, sodium salt (1 :...; Amaranth dye; trisodium 3-hydroxy-4-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate was consider as nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Short term toxicity to aq. invertebrates:

From the available experimental data for the target chemical, the information is summarized as below:

In the first experimental data for the chemical chemical from ABITEC report, determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test wasperformed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

 

Similarly the toxic effects of Amaranth were studied (a screening method for the toxicity of food dyes using artemia salina larvae, 1977) on Artemia salina larvae. Artemia salina (A. salina eggs) a crustacean, commonly known as brine shrimp eggs, are commercially available, and are easily cultured in the laboratory because they are resistant to environmental stresses. Active larvae can be obtained within 1 to 2 days and no live culture is required for a few days thereafter. A salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -200°C. Eggs used in experiments were washed and stored at room temperature in a desiccators over anhydrous granular CaCl2. Larvae were obtained by incubating eggs in petri dishes containing muslin-filtered sea water at 30°C for 24 hours. The larvae were separated from shells, dead larvae and unhatched eggs by their phototactic movements towards a light source. Amaranth at concentrations of 6044.7mg/l and 604.47 mg/l were placed in a petri dish, and sea water containing 20 to 30 larvae was added. After this was incubated at 30°C for 24 hours and 48 hours, larvae surviving were measured by direct count. The same method was tested from 5 to 6 times for each concentration, and the death rate was calculated. Death was assumed to have occurred when there was no movement. The death rate was defined as the average of the percentage of deaths observed for 24 hours and 48 hours. 100% death rate was noted after 48 hours when 6044.7 mg/l of Amaranth was exposed to the test organism and 0% death rate after 24 hours in case of exposure to 604.47 mg/l of test chemical.

 

Structure-toxicity relationships (The Hydractinia echinata Test-System. III: Structure-Toxicity Relationship Study of Some Azo-, Azo-Anilide, and Diazonium Salt Derivatives, 2014) for Acid Red 27 were developed usingHydractinia echinata(H. echinata) as model species.Test chemical was purchased from leading chemical suppliers from their catalogues. Colonies of H. echinata (Biologische Anstalt, Helgoland, Germany) were used to obtain eggs and larvae. The culture medium was artificial seawater (980 mosmol, pH 8.2, 18 °C). In laboratory an artificial metamorphosis can be synchronically started by the introduction of Cs+ ions or by using seawater without Mg2+ ions; it then lasts only 24 h. Under the action of external stimulus of Cs+ ions or Cs+ ions together with the tested compounds, one part of larvae further lives as such and another one is metamorphosized to the polyp form. The evaluation of the influence of the tested substance is very clear this way, the proposed method being based on this aspect.H. echinatalarvae were exposed to seawater containing Cs+ and simultaneously one of the test substances for 3 h. The percentage of animals that underwent metamorphosis (development into polyps) was determined after 24 h. During the following days the frequency of inductions did not further increase. A concentration of inducers was chosen which caused about three half to three quarters of the larvae to metamorphose in order to have conditions which are highly sensitive against an inhibitory influence. The concentration of the test substances (expressed in mol/L) was varied in such a way that we were able to determine the concentration at which the frequency of induction was reduced by 50% with respect to a control. This concentration was termed MRC50 (forMetamorphosisReductionConcentration) and is similar to the effective EC50 concentration that gives half maximal effective response.The logarithm of the reciprocal value (log1/MRC50) values, the average (C) concentrations of these values was calculated for the test chemical. The MRC50value calculations result from the graphical representation of the metamorphosis variation, M (%), (Y axis) function of the xenobiotic’s concentration (mol/L) (X axis), where the metamorphosis decreases with the rise of the xenobiotic’s concentration. Thus, the MRC50value represents the xenobiotic’s concentration (mol/L) necessary for a 50% decrease of metamorphosis, with respect to control.The MRC 50 value for Hydractinia echinata after 3 hours of exposure to test chemical was determined to be 2827.360 mg/l. The test chemical can be considered non- toxic to Hydractinia echinata under the test conditions.

 

On the basis of available information for the target substance, the test substance cannot be considered nonhazardous to the aquatic organisms at environmentally relevant concentrations and cannot be classified as per the CLP classification.

Toxicity to aq. algae and cyanobacteria:

Summarized result for the toxicity of chemical 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye (915 -67 -3) on the growth of aquatic invertebrates are as follows:

In the key study from ABITEC report for the target chemical (Amaranth dye) freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance 2,7-Naphthalenedi- sulfonic acid, 3-hydroxy-4-[(4-sulfo-1- naphthalenyl), sodium salt (Amaranth dye) according to OECD Guideline 201. The stock solution (200 mg/L) was prepared by dissolving brown powder in  OECD growth medium. 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, 12.5, 25, 50, 100, 200 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (ErC50) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1- -naphthalenyl), sodium salt, in Desmodesmus subspicatus was determined to be 356.2 mg/L. This value indicates that the substance is likely to be non-hazardous to aquatic algae and can not be classified as toxic as per the CLP criteria.

Toxicity to microorganisms:

Based on the various experimental data for the target chemical study have been reviewed to determine the toxic nature of target chemical aluminium, 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex (915 -67 -3). The studies are as mentioned below:

In the first key study for target chemical (915-67-3) Biotechnology Letters, 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 amodified strain ofVibrio fischeri(EC20)after 5 min incubation was calculated with the Microtox data analysis program [Microtox Omni Software(1999) Azur Environmental, Newark, Del.]. 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 from Coleman & Qureshi (1985) –

EC20:>100%=nontoxic;

>75–100%=slightly non-toxic;

 >50–75%=toxic;

>25–50%=moderately toxic;

<25% very toxic. The toxicity of 100mg/l of Amaranth determined in terms of EC20 (% dilution) was 44.6 ± 11.6.

 

Similarly in the second supporting study from (Indian J Microbiol 2011). This investigation was aimed at identifying the effects of the Amaranth dye and its degradation products on microbial growth. Amaranth dye was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India. Aspergillus ochraceus NCIM 1146 was obtained from National Chemical Laboratory, Pune, India. E. coli MTCC 452, B. subtilis MTCC 6910 and Penicillium ochrochloron MTCC 517 were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. It was regularly maintained and preserved at 4°C on nutrient agar slants contained in (g/l); bacteriological peptone 10.0, beef extract 10.0 and NaCl 5.0 Microbial toxicity of control dye amaranth and metabolites obtained after its decolorization (final concentration 1,000 ppm) was carried out in relation to E. coli, Bacillus substilis, Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 and zone of inhibition (diameter in mm) was recorded. The diameter of the discs used was 10mm. Amaranth and its degradation products were not toxic to Aspergillus ochraceus and Penicillium ochrochloron MTCC 517 at 1,000 ppm concentration. Amaranth inhibited growth of E. coli and Bacillus substilis.

 

 

The third study for the chemical (915-67-3) was used (from, TOXICOLOGY AND APPLIED PHARMACOLOGY, 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 Amaranth. Paramecium Caudatum was maintained at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter aerogenes. Amaranth 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 the above toxicity data for the target chemical from various database for fish, aquatic invertebrates and algae, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.