1,4-bis(p-tolylamino)anthraquinone

Endpoint summary

Description of key information

Short-term toxicity to fish:

Test material was evaluated for its toxicity on Cyprinus carpio as per OECD 203 and European Economic Community (EEC). Test material was added to test medium at a nominal concentration of 100 mg/I. The mixture was treated with ultrasonic waves for 5 minutes. Afterwards magnetic stirring was applied for a total period of three days in order to achieve maximum saturation of the test medium with the test substance. After stirring the 100 mg/i solution appeared to be a black turbid dispersion with a floating layer. The test solution was used as such.

Under the conditions of the present test material  induced no mortality or other visible effects in carp at a nominal concentration of 100 mg/I during the 96-hour test period. Hence, the 96h-LC50 was above 100 mg/I, the regulatory limit concentration.Based on the LC50 value it can be concluded that test material is not hazardous to fish and can not be considered to be not classified as per CLP criteria.

Short-term toxicity to aquatic invertebrates:

To evaluate toxicity of aquatic invertebrates, various experimental studies were performed.Based on the experimental data aquired from various sources following is the discription:

Test material was used as a test material to evaluate its influence on the mobility of Daphnia magna. test material was added to test medium at a nominal concentration of 100 mg/I. The mixture was treated with ultrasonic waves for 5 minutes. Afterwards magnetic stirring was applied for a total period of three days in order to achieve maximum saturation of the test medium with the test substance. After stirring the 100 mg/I solution appeared to be a black turbid dispersion with a floating layer. One part of this 100 mg/I solution was filtered in order to remove the larger undissolved test substance particles. The filtering resulted in a clear greyish coloured solution. The other part of the 100 mg/I solution was not filtered and used as such. Under the conditions of the present study test material did not induce acute immobilisation of Daphnia magna at a nominal concentration of 100 mg/l after 48 hours of exposure (NOEC).Based on the NOEC value it can be concluded that test material has no hazardous effect on aquatic invertebrate and can not be classified as per CLP criteria.

The above study was supported by another study whose aim of the study was to assess the short term toxicity of test chemical to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs.The stock solution 10.0 g/l was prepared by dissolving dark violet powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. It was not possible to test higher substance concentration due to formation of precipitate.0,0,2,3,4,6,10 mg/l concentrations were used in the study. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0.The median effective concentration (EC50) for the test substance,in Daphnia magna was determined to be 19.7 mg/L with 95% CI:13.4 -28.9 on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified as aquatic acute/ chronic 3 category as per the CLP classification criteria.

Even though varied results for short term toxicity of aquatic invertebrate were available but, based on first experimental purchased study and maximum notifiers of ECHA , we can consider that the test chemical is non toxic and cannot be classified for aquatic invertebrates.

Toxicity to aquatic algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left.The stock solution of test item  was prepared using BBM. The final concentration of this stock solution was 7.21mg/L. This stock solution was kept for stirring for 24 hours and an equilibration of another 24 hours followed by filtration through a 0.22 µm membrane filter was given to this stock solution 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 cell density of the culture was kept 1 X 104cells/ml.The nominal effect concentrations were 0.382mg/l, 0.688mg/l, 1.238mg/l, 2.228mg/l, 4.010mg/l and 7.21mg/l.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 158.892 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

For structurally similar read across substance , tetrahymena pyriformis population growth impairment assay was carried out for 48 hr to study the effects of test chemical on micro-organisms. The population growth inhibition IGC50 to 50% of Tetrahymena pyriformis when exposed to test chemical for 48 hr under static condition is 0.228 mg/L.

In a data source for another read across substance toxicity to micro-organisms test was carried out according to the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test.The 50% inhibition IC50 of micro-organsms when exposed to test chemical is >100 mg/L. The test chemical test material effect concentration value is in the range of 0.228 to >100 mg/L.

Additional information

Short-term toxicity to fish:

In an experimental study test material was evaluated for its toxicity on Cyprinus carpio as per OECD 203 and European Economic Community (EEC). Test material was added to test medium at a nominal concentration of 100 mg/I. The mixture was treated with ultrasonic waves for 5 minutes. Afterwards magnetic stirring was applied for a total period of three days in order to achieve maximum saturation of the test medium with the test substance. After stirring the 100 mg/i solution appeared to be a black turbid dispersion with a floating layer. The test solution was used as such.

Under the conditions of the present test material  induced no mortality or other visible effects in carp at a nominal concentration of 100 mg/I during the 96-hour test period. Hence, the 96h-LC50 was above 100 mg/I, the regulatory limit concentration.

In another experimental report , study was conducted to assess the effect of test chemical on the mortality of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The stock solution was prepared by dissolving 0.1gm of the test substance in 100mL of potable water (passed through reverse osmosis system) and kept it for 24 hrs stirring. By giving half an hour of settling period filtered it . The nominal concentration were 0.45 mg/L, 0.9 mg/L, 1.8mg/L, 3.6mg/L, 7.2 mg/L. Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to various nominal test concentrations, LC0 was determine to be 7.2 mg/l . No mortalities were observed in any of the tested concentration.

Based on the LC50 value it can be concluded that test material is not hazardous to fish and can not be considered to be not classified as per CLP criteria.

Short-term toxicity to aquatic invertebrates:

To evaluate toxicity of aquatic invertebrates, various experimental studies were performed.Based on the experimental data aquired from various sources following is the discription:

Test material was used as a test material to evaluate its influence on the mobility of Daphnia magna. test material was added to test medium at a nominal concentration of 100 mg/I. The mixture was treated with ultrasonic waves for 5 minutes. Afterwards magnetic stirring was applied for a total period of three days in order to achieve maximum saturation of the test medium with the test substance. After stirring the 100 mg/I solution appeared to be a black turbid dispersion with a floating layer. One part of this 100 mg/I solution was filtered in order to remove the larger undissolved test substance particles. The filtering resulted in a clear greyish coloured solution. The other part of the 100 mg/I solution was not filtered and used as such. Under the conditions of the present study test material did not induce acute immobilisation of Daphnia magna at a nominal concentration of 100 mg/l after 48 hours of exposure (NOEC).Based on the NOEC value it can be concluded that test material has no hazardous effect on aquatic invertebrate and can not be classified as per CLP criteria.

The above study was supported by another study whose aim of the study was to assess the short term toxicity of test chemical to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs.The stock solution 10.0 g/l was prepared by dissolving dark violet powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. It was not possible to test higher substance concentration due to formation of precipitate.0,0,2,3,4,6,10 mg/l concentrations were used in the study. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0.The median effective concentration (EC50) for the test substance,in Daphnia magna was determined to be 19.7 mg/L with 95% CI:13.4 -28.9 on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified as aquatic acute/ chronic 3 category as per the CLP classification criteria.

Even though varied results for short term toxicity of aquatic invertebrate were available but, based on first experimental purchased study and maximum notifiers of ECHA , we can consider that the test chemical is non toxic and cannot be classified for aquatic invertebrates.

Toxicity to aquatic algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left.The stock solution of test item  was prepared using BBM. The final concentration of this stock solution was 7.21mg/L. This stock solution was kept for stirring for 24 hours and an equilibration of another 24 hours followed by filtration through a 0.22 µm membrane filter was given to this stock solution 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 cell density of the culture was kept 1 X 104cells/ml.The nominal effect concentrations were 0.382mg/l, 0.688mg/l, 1.238mg/l, 2.228mg/l, 4.010mg/l and 7.21mg/l.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 158.892 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to microorganisms:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

For structurally similar read across substance , tetrahymena pyriformis population growth impairment assay was carried out for 48 hr to study the effects of test chemical on micro-organisms. The population growth inhibition IGC50 to 50% of Tetrahymena pyriformis when exposed to test chemical for 48 hr under static condition is 0.228 mg/L.

In a data source for another read across substance toxicity to micro-organisms test was carried out according to the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test.The 50% inhibition IC50 of micro-organsms when exposed to test chemical is >100 mg/L. The test chemical test material effect concentration value is in the range of 0.228 to >100 mg/L.