N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

Endpoint summary

Description of key information

Hydrolysis

Hydrolysis study of test chemical was carried out at pH 4.0, 7.0 and 9.0 for determining the half-life value of test chemical. Study was performed in accordance with the OECD Guideline 111 (Hydrolysis as a Function of pH).Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. High Performance Liquid Chromatograph (HPLC) was equipped with DAD and PC based data system. Column used was column: Inertsil ODS-3V, [250 mm x 4.6 mmi.d. x 5 μm particle size] or equivalent. Mobile phase used in the study was Milli-Q water and Acetonitrile, respectively. It has a detector wavelength of 210 nm, flow rate of 1.0 ml/min, injection volume of 100 μl. All the parameters were maintained constant throughout the analysis. The analyte peak in the sample was identified by comparing its retention time with that of analyte peak in reference standard (absence of such a peak in control was also checked). Calibration curve was prepared for the analyte by plotting peak area versus concentration (μg /mL), corrected for purity, for each standard. Best-line fit equation (Y = a + bX) was calculated using the method of least squares. In this equation, ‘Y’ is peak area, ‘X’ is concentration (μg/mL), ‘a’ is Y-intercept and ‘b’ is the slope of the line. For LOD, the minimum quantity of the analyte, which was detected by the HPLC with one-third of LOQ concentration, was determined. An aliquot of 0.2 mL of the DLC-7 solution was diluted to 10 mL with acetonitrile and the same solution was injected as DLC-1 to check the LOD of the method. The LOQ of the equipment for the analyte was determined by analyzing lowest concentration of the reference standard solution (1.5 mg/L) with an acceptable accuracy and precision. For the linearity range, a stock solution of 2033.22 mg/L was prepared by dissolving accurately weighed quantity of 0.0103 g of test chemical reference standard (98.7%) in 5 mL volumetric flask. The contents were dissolved by adding about 2.0 mL of acetonitrile by sonication for 5 minutes. After equilibrating to room temperature, the volume was made up with acetonitrile.Later, the solutions for detector linearity were prepared by diluting known aliquots of the stock solution to a known volume with acetonitrile. Each of the standard solutions from DLC 1 to DLC 7 was injected in triplicate and the detector response (peak area) for each injection was recorded. A graph of the peak area (Y-axis) versus concentration (X-axis) was plotted and the intercept (a), slope (b) and linear regression coefficient (r) were calculated. A test chemical solution was prepared by dissolving 0.0103 g of test chemical in 2 ml of acetonitrile into a 5 ml of volumetric flask. The contents were sonicated for 2 minutes and finally the volume was made up to the mark with acetonitrile. The concentration of the stock solution was 2033.22 mg/L (calculated considering the purity of the test item as 98.7%). An aliquot of 0.075 mL of the same was fortified in pH 4, 7 and 9 buffer solutions at the concentration of 15.25 mg/L (with <1% co-solvent) in duplicate and incubated for 5 days at 50 + 0.5°C along with untreated buffer solutions. The test samples were prepared in laminar flow chamber under aseptic comditions. Sterility of the samples were checked at both start and end of the hydrolysis experiment for each of the test system. 0.2 M acetic acid solution, 0.2 M of monobasic sodium phosphate solution and 0.1 M of boric acid solution were used as a buffer solution during the study. For preparing the buffer Solution of pH 4; an aliquot of 205 mL of 0.2M acetic acid solution and 45 mL of 0.2M anhydrous sodium acetate solution was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 4.01.for preparing the buffer solution of pH 7.0; an aliquot of 97.5 mL of 0.2M monobasic sodium phosphate solution and 152.5 mL of 0.2 M dibasic sodium phosphate solution were transferred into a 1000 mL volumetric flask. The contents were diluted to volume with Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 7.01 and for preparing the buffer solution of pH 9.0; an aliquot of 500 mL of 0.1M boric acid solution and 500 mL of Milli-Q® water was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 9.01. All buffer solutions were sterilized by passing through 0.2 μm sterilized filters. 20 ml amber-coloured glass containers was used as a test vessel. Prepared test chemical solutions were taken in amber-colored volumetric flasks and kept in a dark incubatoer in order to avoid photolytic effects. The buffer solutions were bubbled with nitrogen to avoid oxygen.Test system was kept closed with PTFE caps and glass stoppers. Empty vessels and stoppers were sterilized by autoclaving at 121ºC for 20 mins prior to use. Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. All experiments were performed in triplicates. The mean analyte concentration in the sterile pH 4.0, 7.0 and 9.0 buffer samples were 14.86, 15.06, 15.18 pg/mL and 14.32, 14.94 and 14.52 g/mL at 0 and 5 days of post treatment, respectively. Percentage degradation of test chemical was determined to be 3.63%, 0.80% and 4.35% at pH 4.0, 7.0 and 9.0 at a temperature of 50°C, respectively. The half-life value of test chemical was determined to be > 1 year. Since the test chemical is not degraded more than 10% in the test conditions (Tier-1), test chemical was considered to be stable in water.

Biodegradation in water

Estimation Programs Interface Suite was run to predict the biodegradation potential of the test chemical in the presence of mixed populations of environmental microorganisms. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that test chemical is expected to be not readily biodegradable.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 78.1% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be 120 days (2880 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

Bioaccumulation: aquatic / sediment

BCFBAF model (v3.01) of Estimation Programs Interface was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 176.1 L/kg whole body w.w (at 25 deg C) which does not exceed the bio concentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

Adsorption / desorption

KOCWIN model (v2.00) of Estimation Programs Interface was used to predict the soil adsorption coefficient i.e Koc value of test chemical.The soil adsorption coefficient i.e Koc value of test chemical was estimated to be 30460 L/kg (log Koc=4.4837) by means of MCI method (at 25 deg C). This Koc value indicates that the test chemical has a very strong sorption to soil and sediment and therefore have negligible migration potential to ground water.

Henry's law constant

Henry's Law constant of test chemical was estimated to be 0.00000000000000942 (9.42E-015) Pa-m3/mole at 25 deg.C

Additional information

Hydrolysis

Hydrolysis study of test chemical was carried out at pH 4.0, 7.0 and 9.0 for determining the half-life value of test chemical. Study was performed in accordance with the OECD Guideline 111 (Hydrolysis as a Function of pH).Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. High Performance Liquid Chromatograph (HPLC) was equipped with DAD and PC based data system. Column used was column: Inertsil ODS-3V, [250 mm x 4.6 mmi.d. x 5 μm particle size] or equivalent. Mobile phase used in the study was Milli-Q water and Acetonitrile, respectively. It has a detector wavelength of 210 nm, flow rate of 1.0 ml/min, injection volume of 100 μl. All the parameters were maintained constant throughout the analysis. The analyte peak in the sample was identified by comparing its retention time with that of analyte peak in reference standard (absence of such a peak in control was also checked). Calibration curve was prepared for the analyte by plotting peak area versus concentration (μg /mL), corrected for purity, for each standard. Best-line fit equation (Y = a + bX) was calculated using the method of least squares. In this equation, ‘Y’ is peak area, ‘X’ is concentration (μg/mL), ‘a’ is Y-intercept and ‘b’ is the slope of the line. For LOD, the minimum quantity of the analyte, which was detected by the HPLC with one-third of LOQ concentration, was determined. An aliquot of 0.2 mL of the DLC-7 solution was diluted to 10 mL with acetonitrile and the same solution was injected as DLC-1 to check the LOD of the method. The LOQ of the equipment for the analyte was determined by analyzing lowest concentration of the reference standard solution (1.5 mg/L) with an acceptable accuracy and precision. For the linearity range, a stock solution of 2033.22 mg/L was prepared by dissolving accurately weighed quantity of 0.0103 g of test chemical reference standard (98.7%) in 5 mL volumetric flask. The contents were dissolved by adding about 2.0 mL of acetonitrile by sonication for 5 minutes. After equilibrating to room temperature, the volume was made up with acetonitrile.Later, the solutions for detector linearity were prepared by diluting known aliquots of the stock solution to a known volume with acetonitrile. Each of the standard solutions from DLC 1 to DLC 7 was injected in triplicate and the detector response (peak area) for each injection was recorded. A graph of the peak area (Y-axis) versus concentration (X-axis) was plotted and the intercept (a), slope (b) and linear regression coefficient (r) were calculated. A test chemical solution was prepared by dissolving 0.0103 g of test chemical in 2 ml of acetonitrile into a 5 ml of volumetric flask. The contents were sonicated for 2 minutes and finally the volume was made up to the mark with acetonitrile. The concentration of the stock solution was 2033.22 mg/L (calculated considering the purity of the test item as 98.7%). An aliquot of 0.075 mL of the same was fortified in pH 4, 7 and 9 buffer solutions at the concentration of 15.25 mg/L (with <1% co-solvent) in duplicate and incubated for 5 days at 50 + 0.5°C along with untreated buffer solutions. The test samples were prepared in laminar flow chamber under aseptic comditions. Sterility of the samples were checked at both start and end of the hydrolysis experiment for each of the test system. 0.2 M acetic acid solution, 0.2 M of monobasic sodium phosphate solution and 0.1 M of boric acid solution were used as a buffer solution during the study. For preparing the buffer Solution of pH 4; an aliquot of 205 mL of 0.2M acetic acid solution and 45 mL of 0.2M anhydrous sodium acetate solution was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 4.01.for preparing the buffer solution of pH 7.0; an aliquot of 97.5 mL of 0.2M monobasic sodium phosphate solution and 152.5 mL of 0.2 M dibasic sodium phosphate solution were transferred into a 1000 mL volumetric flask. The contents were diluted to volume with Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 7.01 and for preparing the buffer solution of pH 9.0; an aliquot of 500 mL of 0.1M boric acid solution and 500 mL of Milli-Q® water was transferred into a 1000 mL volumetric flask and the volume was made up to the mark using Milli-Q® Water. The pH of the resulting buffer solution was measured using a pre-calibrated pH meter. The pH of the resulting buffer solution was found to be 9.01. All buffer solutions were sterilized by passing through 0.2 μm sterilized filters. 20 ml amber-coloured glass containers was used as a test vessel. Prepared test chemical solutions were taken in amber-colored volumetric flasks and kept in a dark incubatoer in order to avoid photolytic effects. The buffer solutions were bubbled with nitrogen to avoid oxygen.Test system was kept closed with PTFE caps and glass stoppers. Empty vessels and stoppers were sterilized by autoclaving at 121ºC for 20 mins prior to use. Hydrolysis reactions were monitored by analyzing the analyte concentrations after 5 days incubated at 50 ±0.5°C using a validated HPLC method. All experiments were performed in triplicates. The mean analyte concentration in the sterile pH 4.0, 7.0 and 9.0 buffer samples were 14.86, 15.06, 15.18 pg/mL and 14.32, 14.94 and 14.52 g/mL at 0 and 5 days of post treatment, respectively. Percentage degradation of test chemical was determined to be 3.63%, 0.80% and 4.35% at pH 4.0, 7.0 and 9.0 at a temperature of 50°C, respectively. The half-life value of test chemical was determined to be > 1 year. Since the test chemical is not degraded more than 10% in the test conditions (Tier-1), test chemical was considered to be stable in water.

Biodegradation in water

Predicted data for the test chemical and various supporting weight of evidence studies for its structurally and functionally similar read across substance were reviewed for the biodegradation end point which are summarized as below:

 

In a prediction using the Estimation Programs Interface Suite (2017), the biodegradation potential of the test chemical in the presence of mixed populations of environmental microorganisms was estimated. The biodegradability of the substance was calculated using seven different models such as Linear Model, Non-Linear Model, Ultimate Biodegradation Timeframe, Primary Biodegradation Timeframe, MITI Linear Model, MITI Non-Linear Model and Anaerobic Model (called as Biowin 1-7, respectively) of the BIOWIN v4.10 software. The results indicate that test chemical is expected to be not readily biodegradable.

 

In a supporting weight of evidence study from peer reviewed journal (U. Pagga, et. al., 1986) for the test chemical,the aerobic biodegradation experiment was performed for test chemical using activated sludge at concentration 0.5 g/L dry material as inoculums and initial concentration of chemical taken was 100mg/L for 42 days. By considering DOC removal parameter test chemical showed 29 % degradation in 42 days. The test chemical belongs to B category according to table 1 and 2 as its limit value falls in that range. On the basis percentage value it is concluded that test chemical is not readily biodegradable.

 

Another biodegradation study was conducted for 5 days for evaluating the percentage biodegradability of test chemical using 5 day BOD test under aerobic conditions (authoritative database HSDB, 2017). Sewage sludge was used as a test inoculum. Initial test substance conc. used in the study was 2.5 mg/l. The percentage degradation of test substance was determined to be 0% by BOD parameter in 5 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

For the test chemical,biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test substance (authoritative database J-CHECK and EnviChem, 2017). The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I). Activated sludge was used as a test inoculums for the study. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test substance was determined to be 1 and 3% by BOD and HPLC parameter in 28 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

In an additional study from authoritative databases (2017), biodegradation experiment was conducted for 14 days for evaluating the percentage biodegradability of test substance. The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I)) under aerobic conditions. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test substance was determined to be 0.2 and 0.3% by BOD and GC parameter in 14 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

On the basis of above results of test chemical, it can be concluded that the test chemical can be expected to be not readily biodegradable in nature.

Biodegradation in water: simulation testing on ultimate degradation in surface water

Aerobic mineralisation of test chemical in water was studies as per the principles of the OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test) (Adopted 13th April 2004) under aerobic conditions. The surface water was collected from Kaveri River, Sangama, Ramnagar District, Karnataka State, India in a thoroughly cleansed container. The sampling site for collection of the surface water was selected ensuring that no known history of its contamination with the test item or its structural analogues within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The pH and temperature of the water was measured at the site of collection and the depth of sampling and the appearance of the water sample. (e.g. color and turbidity) was also noted. Oxygen concentration of the surface layer was measured in order to demonstrate aerobic conditions. Depth of sampling was 1 feet and surface water was clear with no turbidity. The test water was stored at 4°C with continuous aeration prior use for a period not more than 4 weeks. Temperature (°C) at time of collection was 21.1°C, pH of temperature was 6.73, Oxygen concentration (mg/l) of 5.1 mg/l,  Dissolved organic carbon (%) of 2.4 mg/kg dm, colony count consists of 4000 CFU/ml, Total organic carbon (TOC) of 2.6 mg/l, Nitrate (NO3- ) of 3 mg/l, Nitrite (NO2- ) of <0.005 mg/l, P of 0.3 mg/l, Orthophosphates (PO43-) of 0.22 mg/l, Total ammonia tot (NH4+ ) of <0.3 mg/l and BOD of <2.0 mg/l, respectively. Prior to use of surface water, the coarse particles were removed by filtration through a 100 μm mesh sieve. Test chemical conc. used in the study was 10 μg/L as low dose and 100 μg/L as high dose, respectively. The surface water was also treated at 500 µg/L (0.5 µg/mL) which was used for identification of degradation products. Study was performed in duplicates in a 250 ml conical flasks which was covered with cotton plugs under continuous darkness. Test conditions involve a temperature of 12±2°C, pH of  6.73. Test vessel was kept in an incubator shaker at 12 ± 2°C in dark. Aerobic condition was maintained in the test system by continuous shaking. Agitation was provided to facilitate oxygen transfer from the headspace to the liquid so that aerobic conditions were adequately maintained. Additional to test vessels, 1 blank test vessel containing only the test water for all sampling intervals was included, 1 blank test vessel containing only the sterile test water was also treated at 10 µg/L (0.01 µg/mL) and 100 µg/L (0.1 µg/mL) conc., 1 blank test vessel containing only test chemical with co-solvent and duplicate test vessels with reference (aniline) (conc. 10 μg/l i.e. 0.01 mg/l) was also kept in the study. All experiments were performed in duplicates. The concentration of test chemical residues in samples collected at different pre-determined interval zero-time (immediately after treatment day 0), day 1, day 3 day 7, day 14, day 28, day 45 and day 60 were diluted suitably with acetonitrile and at each sampling occasion, triplicate aliquots from each test concentration were subjected to total radioactivity analysis by LSC and the components were quantified by reverse phase radio-HPLC with on-line radiochemical detection. Additionally, an aliquot of each sample was subjected for 14CO2 determination by indirect method followed by LSC analysis and trapped 14CO2 in KOH and ethylene glycol by LSC analysis. Each sample was analyzed by HPLC-UV detection with on-line radiochemical detection. High performance liquid chromatograph (Exion HPLC) equipped with a mass spectrometer (TQ 5500) was used with a column of Column: Shimpack C18(2), 250 mm × 4.6 mm i.d., 5 µm, column oven temperature of 30°C, mobile phase consists of Solvent A : 5 mM ammonium formate in Milli-Q® water and Solvent B : Acetonitrile in a ratio of 30 : 70, v/v, flow rate of 0.5 mL/min with splitter, respectively. Detection method involve the use of MS. Using the method of Currie L. A. (1968), the LOD and LOQ of the LSC analyses were 28 and 111 dpm, respectively. During method validation, acceptable recoveries were generated for the samples fortified at LOQ and 10 LOQ level. The % RSD (precision) was ≤20% at each fortification level. Recovery data from these samples demonstrated that test chemical was unstable during analysis. The identification and quantification of the degradation product was carried out using mass spectrometry. Analysis of the Day 0 samples at 10 μg/L and 100 μg/L test concentrations demonstrated quantitative recovery of test chemical. The average amount of test chemical present was 98.8% and 0% & 101.7% and 46.7% at Day 0 and Day 60, respectively following application of test chemical to test water at 10 μg/L (low dose) and 100μg/L (high dose). The average amount of test chemical present was 100.9% and 60.6% at Day 0 and Day 60, respectively following application of test chemical to sterile test water at 100μg/L (high dose). The DT50 value was determined to be 7.2 d and 45.3 d at test chemical conc. of 10 μg/l and 100 μg/l at 12°C, respectively. 90% degradation of test chemical in natural surface water was determined after 23.9 d and 150 d at test chemical conc. of 10 μg/l and 100 μg/l, respectively. Test chemical was unstable in natural water and test chemical was completely converted into degradation product (1-hydroxy-4-(phenylamino)anthracene-9,10-dione) by end of incubation period of 60 days. Based on the these results, test chemical was considered to be not persistent in water.

Biodegradation in water: sediment simulation testing

In accordance with Annex IX column 2 of REACH regulation, test for this endpoint is scientifically not necessary and does not need to be conducted, since the substance is readily biodegradable i.e. not persistent based on the experimental result of surface water simulation biodegradation study.

Biodegradation in soil

The half-life period of test chemical in soil was estimated using Level III Fugacity Model by EPI Suite version 4.1 estimation database (2018). If released into the environment, 78.1% of the chemical will partition into soil according to the Mackay fugacity model level III. The half-life period of test chemical in soil is estimated to be 120 days (2880 hrs). Based on this half-life value of test chemical, it is concluded that the chemical is not persistent in the soil environment and the exposure risk to soil dwelling animals is moderate to low.

 

Bioaccumulation: aquatic / sediment

Various predicted data of the test chemical and supporting weight of evidence studies for its structurally and functionally similar read across substance were reviewed for the bioaccumulation end point which are summarized as below:

 

In aprediction done using theBCFBAF Program(v3.01) of Estimation Programs Interface was used to predict the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 176.1 L/kg whole body w.w (at 25 deg C).

 

In an another prediction done by using Bio-concentration Factor (v12.1.0.50374) module Bio-concentration Factor over the entire pH scale of the test chemical was estimated to be 4.57, 31.3, 186, 387, 434, 438, 430, 360, 137, 19.4, 2.30 and 1.0 at pH 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 & 11-14, respectively.

 

Bioconcentration Factor (BCF) of test chemical was estimated using Chemspider database. The bioconcentration factor of test chemical was estimated to be 1068.6 at pH 5.5 and 693.19 at pH 7.4, respectively.

 

Another predicted data was estimated usingSciFinder database (American Chemical Society (ACS), 2017) was used for predicting the bioconcentration factor (BCF) of test chemical. The bioconcentration factor (BCF) of test chemical was estimated to be 44.6, 266, 552, 618, 625, 613, 514, 196, 27.6 and 3.27 at pH range 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively (at 25 deg C).

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical), the bioaccumulation i.e BCF for test chemical was estimated to be 19 dimensionless . The predicted BCF result based on the 5 OECD principles.

 

In supporting weight of evidence study from authoritative database (2017) for the test item,the BCF value of test chemical estimated was 500 dimensionless by using log Kow of 8.69 and regression derived equation and it is less than 2000 criteria therefore it is concluded that test chemical is non-bioaccumulative in aquatic organisms.

 

For the test chemical (authoritative database, 2017),the BCF value of test chemical estimated was 260 dimensionless by using log Kow of 4.0 and regression derived equation and it is less than 2000 criteria therefore it is concluded that test chemical is non bioaccumulative.

 

On the basis of above results of the test chemical, it can be concluded that the BCF value of test chemical ranges from 1 –1068.6 which does not exceed the bioconcentration threshold of 2000, indicating that the test chemical is not expected to bioaccumulate in the food chain.

 

Adsorption / desorption

Various predicted data of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across substance were reviewed for the adsorption end point which are summarized as below:

 

In aprediction done using theKOCWIN Program(v2.00) of Estimation Programs Interface was used to predict the soil adsorption coefficient i.e Koc value of test chemical. The soil adsorption coefficient i.e Koc value of test chemical was estimated to be 30460 L/kg (log Koc=4.4837) by means of MCI method (at 25 deg C). This Koc value indicates that the test chemical has a very strong sorption to soil and sediment and therefore have negligible migration potential to ground water.

 

In an another prediction done by using ChemSpider Database (2017), theSoil Adsorption Coefficient i.e Koc value of test chemical was estimated. The adsorption coefficient (Koc) value of test chemical was estimated to be 5113 (Log Koc = 3.7086) at pH 5.5 and 3317 (Log Koc = 3.52) at pH 7.4, respectively. This Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

Additional soil adsorption coefficient i.e Koc value of test chemical was estimated using the SciFinder database (2017).The soil adsorption coefficient i.e Koc value of test chemical was estimated to be 249, 1480, 3080, 3450, 3490, 3420, 2870, 1090, 154 and 18.3 at pH range 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively (at 25 deg C) (logKoc ranges from 1.26 to 3.5428). The logKoc value (3.4578 at pH 7.0) indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

From CompTox Chemistry Dashboard using OPERA (OPEn (quantitative) structure-activity Relationship Application)  V1.02 model in which calculation based on PaDEL descriptors (calculate molecular descriptors and fingerprints of chemical), the adsorption coefficient i.e KOC for test chemical was estimated to be 5170 L/kg (log Koc = 3.713).The predicted KOC result based on the 5 OECD principles. This Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

In a supporting weight of evidence study from authoritative database (2017) for the test item,adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical. The adsorption coefficient (Koc) value was calculated using a structure estimation method based on molecular connectivity indices. The adsorption coefficient (Koc) value of test substance was estimated to be 4800000 (Log Koc = 6.681). This Koc value indicates that the test substance has a very strong sorption to soil and sediment and therefore have negligible migration potential to ground water.

 

For the test chemical, adsorption study was conducted for estimating the adsorption coefficient (Koc) value of test chemical (HSDB and PubChem, 2017). The adsorption coefficient (Koc) value was calculated using a structure estimation method based on molecular connectivity indices. The adsorption coefficient (Koc) value of test chemical was estimated to be 4000 (Log Koc = 3.602). This Koc value indicates that the test chemical has a strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

 

On the basis of above overall results of the test chemical, it can be concluded that the logKoc value of test chemical was estimated to be ranges from 3.4578 to 4.4837 (at pH 7.0) indicating that the test chemicalhas a strong to very strong sorption to soil and sediment and therefore have negligible to slow migration potential to ground water.

Henry's law constant

Henry's Law constant of test chemical was estimated to be 0.00000000000000942 (9.42E-015) Pa-m3/mole at 25 deg.C