Recyclamine R 501

Endpoint summary

Description of key information

Short term toxicity to fish

An acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.9 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test chemical was prepared by dissolving 2000 mg of test chemical in 2000 ml of RO water. After stirring the stock solution was filtered and analytically detected and the concentration found to be 1000 mg/L.  The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 100 mg/l.Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical conc. used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l. Total 7 fishes were exposed to test chemical in a 7 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -25°C, pH of 7.9 (average) and DO of 7.2 mg/l (average) and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were not maintained in the range of 80 -120%. Therefore, the analysis of the results was based on measured concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be >80.11 mg/L (measured conc.) and >120 mg/l (nomnal conc.), respectively. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

The short-term toxicity of the test chemical to aquatic invertebrates was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 531.896 mg/l. Thus, based on the LC50 value, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 7, 14, 28, 56 and 112 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be >112 mg/l (nominal conc.). Thus, test chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 3 hr NOEC and EC50 value can be expected to be 1000 mg/l and < 1000 mg/l.

Additional information

Short term toxicity to fish

Experimental and predicted data of the target chemical were reviewed for short term toxicity to fish endpoint which are summarized as below:

 

In an experimental study from study report, an acute toxicity test was conducted for 96 hrs for assessing the effect of test chemical on Zebra fish (Danio rerio). The test was performed in accordance to OECD guideline No. 203 “Fish Acute Toxicity Test”. Zebra fish (Danio rerio) of average length of 1.9 cm was used as a test organism for the study. Test fishes were kept in a static tank in tap water passed through reverse osmosis system, under natural conditions along with proper feed and aeration. During the housing period, test fishes were fed once daily with standard brand fed. Aeration in test vessels was provided till 1 day before the start of the experiment. The test chemical was prepared by dissolving 2000 mg of test chemical in 2000 ml of RO water. After stirring the stock solution was filtered and analytically detected and the concentration found to be 1000 mg/L.  The remaining test solutions were prepared by dilution from the above stock solution. Test chemical concentrations used for the study were 100 mg/l.Test chemical concentrations were analytically determined by UV-VIS spectrophotometer. Test chemical conc. used for the study were 0, 7.5, 15, 30, 60 and 120 mg/l. Total 7 fishes were exposed to test chemical in a 7 lit Polypropylene (PP) fish tank containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 21 -25°C, pH of 7.9 (average) and DO of 7.2 mg/l (average) and under a photoperiod of 16:8 hr light: dark conditions, respectively. Mortality in the control was 0%. The dissolved oxygen concentration remained above 60% of the air saturation value throughout the exposure period. Thus, fulfilling the validity criterion. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were not maintained in the range of 80 -120%. Therefore, the analysis of the results was based on measured concentration. On the basis of effect of test chemical on mortality of the test organism, the 96 hr median lethal concentration [LC50 (96 h)] for test chemical on Danio rerio (Zebra Fish) was reported to be >80.11 mg/L (measured conc.) and >120 mg/l (nomnal conc.), respectively. Since, the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' as per the CLP classification criteria.

In a prediction done using EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to aquatic fish was predicted. On the basis of effect of test chemical observed in a static system on the mortality of the test organism during the 96 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 1011.995 mg/l.

 

On the basis of the above results, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

Predicted data of the target chemical and various supporting weight of evidence studies for its read across analogue were reviewed for short term toxicity to aquatic invertebrates endpoint which are summarized as below:

 

The short-term toxicity of the test chemical to aquatic invertebrates was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 531.896 mg/l.

 

In a supporting weight of evidence study from study report,to evaluate the influence of the test item on the mobility and survival of Daphnia magna an Acute Immobilisation Test according to OECD TG 202, EU Method C.2. and US EPA OCSPP 850.1010 was carried out under static conditions and in compliance with the GLP principles. Five animals in four replicates were exposed for 48 hours to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. In addition, a blank control including the test medium but without the test item was performed. The test item concentrations were analytically verified by using HPLC-UV to be 5.715, 12.43, 25.93, 50.37 and 102.66 mg/L after 48 hours. The test item concentration was thus 91 – 104 % of the nominal at the end of the experimental phase (at the start of the test: 95 –100 of the nominal). Therefore, all biological results are based on nominal concentrations. The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate (24h EC50 = 1.12 mg/L). All validity criteria of the test guidelines were fulfilled. The mobility of the test animals was recorded after 24 and 48 hours of exposure. As a result, the 48-hour EC50 of the test item was determined to be >100 mg/L in nominal (95 % conf. limits: 91.4 – 331.8 calculated). The 48-hour EC10 was found to be 66.1 mg/L in nominal (95 % conf. limits: 18.7 – 82.5 mg/L).

 

For the test chemical, an acute Immobilisation Test according to OECD TG 202, EU Method C.2. and US EPA OCSPP 850.1010 was carried out under static conditions and in compliance with the GLP principles. Five animals in four replicates were exposed for 48 hours to nominal test item concentrations of 6.25, 12.5, 25, 50 and 100 mg/L. In addition, a blank control including the test medium but without the test item was performed. The test item concentrations were analytically verified by using HPLC-UV to be 5.715, 12.43, 25.93, 50.37 and 102.66 mg/L after 48 hours. The test item concentration was thus 91 – 104 % of the nominal at the end of the experimental phase (at the start of the test: 95 –100 of the nominal). Therefore, all biological results are based on nominal concentrations. The suitability of the test system was confirmed by a positive control with the reference substance potassium dichromate (24h EC50 = 1.12 mg/L). All validity criteria of the test guidelines were fulfilled. The mobility of the test animals was recorded after 24 and 48 hours of exposure. As a result, the 48-hour EC50 of the test item was determined to be >100 mg/L in nominal (95 % conf. limits: 91.4 – 331.8 calculated). The 48-hour EC10 was found to be 66.1 mg/L in nominal (95 % conf. limits: 18.7 – 82.5 mg/L).

 

On the basis of the above results, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Experimental and predicted data of the target chemical were reviewed for toxicity to aquatic algae and cyanobacteria endpoint which are summarized as below:

In an experimental study from study report, afreshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 1000 mg/L, verified analytically by UV-Vis Spectrophotometer. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/mL. Further, exposure concentrations of 0, 7, 14, 28, 56 and 112 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 23 °C and with a continuous uniform illumination of 6000 -10000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has not been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on measured concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be >112 mg/l (nominal conc.). Thus, test chemical was considered as non-toxic to aquatic algae and hence, considered to be 'not classified' as per the CLP classification criteria.

 

Toxicity of the test chemical to green algae was predicted using EPI Suite ECOSAR version 1.11 (2019). On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 287.873 mg/l.

On the basis of the above results, test chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

Data available of the read across chemicals has been reviewed to determine the effect of the test chemical on toxicity to microorganisms. The studies are as mentioned below:

In a 3-hour pre-test test according to OECD 209 the influence of the test item on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This pre-test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect was noticed at 10 and 100 mg/L (the observed slight, 0.18 and 3.70 % inhibitions were considered as being within the biological variability of the applied test system), but the inhibition was in average 86.16 % at 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate. The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test item had unequivocal influence on the pH at the concentrations of 100 and 1000 mg/L. Before inoculum addition the pH of the test item containing test mixtures at these concentration levels was above the acceptable pH 7 - 8 range. Based on measured oxygen consumption values and calculated specific respiration rates, unequivocal test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined. During the performance of the pre-experiment unequivocal, significant test item effect on the pH was realized especially at the concentrations of 100 and 1000 mg/L. In conclusion, these pre-test results demonstrate the significant inhibition of oxygen consumption by the test substance in the examined concentration range; therefore, a definite test with parallel test series (with and without pH adjustment) was considered as necessary. The definite test will be performed under a new study code; the concentration levels to be examined in the definite test will be based on the results of the present pre-experiment.

In a another study, a 3-hour pre-test according to OECD 209 the influence of the test item on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This pre-test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption. The test item was investigated in this study at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect was noticed at 10 and 100 mg/L (the observed 5.26 and 3.31 % inhibitions were considered as being within the biological variability of the applied test system), but the inhibition was in average 85.90 % at 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate. The test was performed including abiotic controls. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The test item had influence on the pH in all examined concentration levels. Before inoculum addition the pH of the test item containing test mixtures was above the acceptable pH 7 - 8 range. The examined concentrations covered the appropriate inhibition range for EC10, EC50 and EC80 calculations. The EC10 was calculated as 241 mg/kg soil dry weight, the EC50 was calculated as 654 mg/kg soil dry weight and the EC80 value was calculated as 926 mg/kg soil dry weight. The above ECx values will be further refined in the definite test. The EC50 value was determined as: EC50 < 1000 mg/L. Based on measured oxygen consumption values and calculated specific respiration rates, strong test item effect was established at the highest examined concentration of 1000 mg/L. For demonstration of a clear dose related inhibitory effect, the concentration spacing of this pre-test was not appropriate; however the 3-hour EC50 values of the test item is < 1000 mg/L. Due to the statistically significant differences (2-Sample T-test (α=0.05)) in the comparison with the blank control and 1000 mg/L concentration values exact NOEC value could not be determined. During the performance of the pre-experiment significant test item effect on the pH was realized in whole examined concentration range of 10 - 1000 mg/L. In conclusion, these pre-test results demonstrate the significant inhibition of oxygen consumption by the test substance in the examined concentration range; therefore, a definite test with parallel test series (with and without pH adjustment) was considered as necessary. The definite test will be performed under a new study code; the concentration levels to be examined in the definite test will be based on the results of the present pre-experiment.

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 3 hr NOEC and EC50 value can be expected to be 1000 mg/l and < 1000 mg/l.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.