1-(2-methyl-4-(2-methylphenylazo)phenylazo)-2-naphthol

Endpoint summary

Description of key information

Hydrolysis

In accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable.

Biodegradation in water

Biodegradation study of test chemical was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days (Haiyan Xu et. al., 2010).The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various test chemical conc.. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Test chemical stock solutions were added to the medium at final concentrations of 1.5µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometry(LC/ESI-MS). Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains,B. vulgatus,B.ovatus,B. uniformis, B. distasonis,B. fragilis ,B. thetaiotaomicron,B. caccae,B. infantis, C. perfringens, C. indolis, C. clostridioforme,E. aerofaciens, E. limosum , E. faecalis, E. faecium,F. russi, F. nucleatum,L. paracasei,L. reuteri, L. rhamnosus,R. obeum and R. gnavuswere able to completely degrade (100%) the test chemical.The bacteria which are unable to degrade the test chemical areBifidobacterium catenulatum,Clostridium ramosum, Eubacterium tenue, Escherichia coliandPeptostreptococcus magnus ,respectively. The metabolite produced from test chemical by E. faecalis was identified aso-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH⁺+acetonitrile].2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced byE. coliwas detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

Bioaccumulation: aquatic / sediment

Bioaccumulation study was conducted on test organism Cyprinus carpiofor 6 weeks for evaluating the bioconcentration factor (BCF value) of test chemical (from authoritative database and secondary source).The study was performed according to other guideline"Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances" under flow through conditions at a temperature of25°C and pH range 6.0-8.5, respectively.Cyprinus carpio (length - 8 dimensionless) was used as a test organism for the study. Test chemical was prepared inHCO-40.Test chemical nominal conc. used for the study was 0.35mg/land 0.035 mg/l, respectively. Analytical method involve therecovery ratio:Test water: 98.3 %, Fish : 96.7 %, - Limit of quantitation : Test water :1st concentration area : 100 %, 2nd concentration area : 98.6 %, Fish : 90.8 %, - Limit of detection : Fish : 0.103 ppm.Range finding study involve theTLm(48h) ≥ 100 ppm (w/v) on Rice fish (Oryzias latipes).The bioconcentration factor (BCF value) of test chemical on Cyprinus carpio was determined to be in the range of 0.29-2.9 L/Kg at a conc. of 0.35 mg/l and 2.9-11 L/Kg at a conc. of 0.035 mg/l, respectively, 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

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the Reverse Phase High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals (Experimental study report, 2018). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 4 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.9. Each of the reference substance and test substance were analysed by HPLC at 210 nm. After equilibration of the HPLC system, Urea was injected first, the reference substances were injected in duplicate, followed by the test chemical solution in duplicate. Reference substances were injected again after test sample, no change in retention time of reference substances was observed. Retention time tR were measured, averaged and the decimal logarithms of the capacity factors k were calculated. The graph was plotted between log Koc versus log k(Annex - 2).The linear regression parameter of the relationship log Koc vs log k were also calculated from the data obtained with calibration samples and therewith, log Koc of the test substance was determined from its measured capacity factor. The reference substances were chosen according to functionally similarity with the test substance and calibration graph prepared. The reference substances were 4-methylaniline(p-Tolouidine), Aniline, Xylene, Ethylbenzene, Toluene, Naphthalene having Koc value ranging from 1.9 to 2.75. The Log Koc value of test chemical was determined to be 1.523± 0.006 at 25°C. This log Koc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.

Additional information

Hydrolysis

In accordance with column 2 of Annex VIII of the REACH regulation, testing for this endpoint is scientifically not necessary and does not need to be conducted since the test chemical is readily biodegradable.

Biodegradation in water

Various experimental studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from peer reviewed journal (Haiyan Xu et. al., 2010),biodegradation experiment of test chemical was performed. The test is carried at a temperature of 37ᵒCwith a duration period of 2 days.The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various test chemical conc.. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Test chemical stock solutions were added to the medium at final concentrations of 1.5µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometry(LC/ESI-MS). Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B. vulgatus, B.ovatus, B. uniformis, B. distasonis, B. fragilis , B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. indolis, C. clostridioforme, E. aerofaciens, E. limosum , E. faecalis, E. faecium, F. russi, F. nucleatum, L. paracasei, L. reuteri, L. rhamnosus, R. obeum and R. gnavus were able to completely degrade (100%) the test chemical.The bacteria which are unable to degrade the test chemical are Bifidobacterium catenulatum, Clostridium ramosum, Eubacterium tenue, Escherichia coli and Peptostreptococcus magnus , respectively. The metabolite produced from test chemical by E. faecalis was identified aso-toluidine, based on an identical retention time of 7.42 min and ions atm/z108 [MH+] and 149 [MH++acetonitrile].2,5-diaminotoluene from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

For the test chemical, biodegradation study was performed. The test is carried at a temperature of 37ᵒC with a duration period of 2 days (peer reviewed journal, 2010). The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC).All strains were preserved at-80ᵒC in 10 to 15% glycerol stocks and revived as needed.The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10 mg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test chemical were isolated and identified by HPLC and Liquid chromatography/ electrospray ionization mass spectrometric(LC/ESI-MS).Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B. infantis, C. indolis, E. faecalis, L. rhamnosus and R. obeum were able to completely degrade (100%) the test chemical whereas partial degradation of the test chemical occurred by the bacterial strains such as B. ovatus, B. distasonis, B. thetaiotaomicron, B.caccae, C. clostridioforme, E. faecium, F. russi and L. paracasei. Thus, the test chemical is readily biodegradable in water. The bacteria which are unable to degrade the test chemical areB. vulgatus, B. uniformis, B. fragilis, B. longum, B. adolesecentis, B. catenulatum, B. angulatum, C. perfringens, C. butyricum, C. ramosum, C. difficle, C. leptum, E. aerofaciens, E. limosum, E. tenue, E. coli, F. nucleatum, L. bifidus, L. reuteri, L. ruminis, P. magnus and R. gnavus. The metabolite produced from test chemical by E. faecalis was identified as 2,4-dimethylaniline, based on an identical retention time of 16.55 min and ions at m/z 122 [MH+] and 163 [MH+ + acetonitrile]. 1-Amino-2-naphthol from test chemical could not be detected in the extracted samples. No metabolites of test chemical produced by E. coli was detected by LC/ESI-MS, indicating that the test chemical were not degraded by the bacterium. Thus, based on percentage degradation, the test chemical is considered to be readily biodegradable in water.

 

In a supporting study from peer reviewed journal (D. Brown et. al., 1987),biodegradation experiment was carried out for determining the biodegradability rate of the test chemical. Activated sludge was used as an inoculum and the study was performed under anaerobic conditions at a temperature of 35°C for a period of 56 days. Samples of the aqueous phase were analyzed either qualitatively or quantitatively by an appropriate chromatographic method for the presence of certain of the expected aromatic amine metabolites. The percentage degradation of test chemical was determined to be 100% degradation by appropriate chromatography method in 7 days. The metabolites identified by the appropriate chromatographic method were 4,4 '-diamino-3,3'-dimethyl biphenyl and 4-methyl benzenesulphonic acid- (4'-aminophenyl) ester, respectively. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

On the basis of above results for test chemical, it can be concluded that the test chemical can be considered to be readily biodegradable in nature.

Bioaccumulation: aquatic / sediment

Experimental studies and predicted data of the test chemical were reviewed for the bioaccumulation end point which are summarized as below:

 

In an experimental key study from authoritative database and secondary source (2018),bioaccumulation experiment was conducted on test organism Cyprinus carpio for 6 weeks for evaluating the bioconcentration factor (BCF value) of test chemical. The study was performed according to other guideline "Bioaccumulation test of a chemical substance in fish or shellfish" provided in "the Notice on the Test Method Concerning New Chemical Substances" under flow through conditions at a temperature of25°C and pH range 6.0-8.5, respectively. Cyprinus carpio (length - 8 dimensionless) was used as a test organism for the study. Test chemical was prepared inHCO-40.Test chemical nominal conc. used for the study was 0.35mg/land 0.035 mg/l, respectively. Analytical method involve the recovery ratio: Test water: 98.3 %, Fish : 96.7 %, - Limit of quantitation : Test water :1st concentration area : 100 %, 2nd concentration area : 98.6 %, Fish : 90.8 %, - Limit of detection : Fish : 0.103 ppm. Range finding study involve the TLm(48h) ≥ 100 ppm (w/v) on Rice fish (Oryzias latipes).The bioconcentration factor (BCF value) of test chemical on Cyprinus carpio was determined to be in the range of 0.29-2.9 L/Kg at a conc. of 0.35 mg/l and 2.9-11 L/Kg at a conc. of 0.035 mg/l, respectively.

 

In a prediction done using the BCFBAF Program 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 10 L/kg whole body w.w (at 25 deg C).

 

For the test chemical,bioaccumulation study was conducted for estimating the BCF (bioaccumulation factor) value of test chemical (HSDB, 2017). The bioaccumulation factor (BCF) value was calculated using a logKow of -0.14 and a regression-derived equation. The estimated BCF (bioaccumulation factor) value of test chemical was determined to be 0.5 dimensionless.

 

In a supporting study from authoritative database (2017),the bioaccumulation study in fish was conducted for estimating the BCF (bioaccumulation factor) value of test chemical. The bioaccumulation factor (BCF) value was calculated using a logKow of 3.81 and a regression-derived equation. The estimated BCF (bioaccumulation factor) valueof test chemical was determined to be 3 dimensionless.

 

On the basis of above results for test chemical, it can be concluded that the BCF value of test chemical was evaluated to beranges from0.29 to 10, respectively,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

The adsorption coefficient Koc in soil and in sewage sludge of test chemical was determined by the Reverse Phase High Performance Liquid Chromatographic method according to OECD Guideline No. 121 for testing of Chemicals (Experimental study report, 2018). The solutions of the test substance and reference substances were prepared in appropriate solvents. A test item solution was prepared by accurately weighing 4 mg of test item and diluted with Acetonitrile up to 10 ml. Thus, the test solution concentration was 400 mg/l. The pH of test substance was 6.9. Each of the reference substance and test substance were analysed by HPLC at 210 nm. After equilibration of the HPLC system, Urea was injected first, the reference substances were injected in duplicate, followed by the test chemical solution in duplicate. Reference substances were injected again after test sample, no change in retention time of reference substances was observed. Retention time tR were measured, averaged and the decimal logarithms of the capacity factors k were calculated. The graph was plotted between log Koc versus log k(Annex - 2).The linear regression parameter of the relationship log Koc vs log k were also calculated from the data obtained with calibration samples and therewith, log Koc of the test substance was determined from its measured capacity factor. The reference substances were chosen according to functionally similarity with the test substance and calibration graph prepared. The reference substances were 4-methylaniline(p-Tolouidine), Aniline, Xylene, Ethylbenzene, Toluene, Naphthalene having Koc value ranging from 1.9 to 2.75. The Log Koc value of test chemical was determined to be 1.523± 0.006 at 25°C. This log Koc value indicates that the test chemical has a low sorption to soil and sediment and therefore have moderate migration potential to ground water.