L-Glutamic acid, N-coco acyl derivs., monosodium salts

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 1.5 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 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. 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, respectively. Total 7 fishes were exposed to test chemical in a 7 lit of glass aquarium containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 22.6°C, pH of 7.9 and DO of 78.1 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. At regular intervals of 24 hours vessels were observed for any behavioural changes and mortality along with that the DO, pH and temperature was measured. Test chemical was sampled from test vessel for analytical determinations at 0 hour and 96 hours. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were maintained in the range of 80 -120%. Therefore, the analysis of the results was based on nominal concentration. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% (i.e. above 60%) of the air saturation value throughout the exposure period. Thus, the study fulfilling the validity criterion as mentioned in the OECD TG 203. 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 determined to be >120 mg/L (nomnal conc.). Thus, based on LC50 value, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' category as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test chemical. On the basis of no effects observed in a freshwater fish, the 28 d NOEC value for the test chemical was estimated to be 23.58 mg/l. Based on this value, test chemical was considered as non-toxic to fish at environmentally relevant concentrations and hence, considered to be not-classified as per the CLP classification criteria. 

Short term toxicity to aquatic invertebrates

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 48 hr EC50/LC50 value can be expected to be > 100 mg/l.

Long term toxicity to aquatic invertebrates

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on aquatic invertebrates was predicted for test chemical. On the basis of no effects observed on a freshwater aquatic invertebrates, the 21 d NOEC value for the test chemical was estimated to be 13.477 mg/l for daphnia. Based on this value, test chemical was considered as non-toxic to daphnia at environmentally relevant concentrations 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 test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final 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 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°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. All control and test vessel conc. were performed in six replicates. 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, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.79%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 8.24%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, 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

On the basis of effect on growth inhibition of microorganism (Tetrahymena pyriformis), the 48 hr IGC50 value was estimated to be 2433.12 mg/L.

Additional information

Short term toxicity to fish

Experimental study of the test chemical and supporting weight of evidence studies for its read across substance were reviewed for the short term toxicity to fish endpoint which are as summarized below:

In an experimental 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 1.5 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 1250 mg of test chemical in 5000 ml of RO water. After stirring the stock solution was filtered and analytically detected. The final solubility value obtained after analytical detection was 226.81 mg/l. The remaining test solutions were prepared by dilution from the above stock solution. 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, respectively. Total 7 fishes were exposed to test chemical in a 7 lit of glass aquarium containing 4000 ml of potable water. The test vessels were placed in a room at a temperature of 22.6°C, pH of 7.9 and DO of 78.1 mg/l and under a photoperiod of 16:8 hr light: dark conditions, respectively. At regular intervals of 24 hours vessels were observed for any behavioural changes and mortality along with that the DO, pH and temperature was measured. Test chemical was sampled from test vessel for analytical determinations at 0 hour and 96 hours. All the test concentrations were analytical determined and at 96 hours of the exposure durations which were maintained in the range of 80 -120%. Therefore, the analysis of the results was based on nominal concentration. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 87.14% (i.e. above 60%) of the air saturation value throughout the exposure period. Thus, the study fulfilling the validity criterion as mentioned in the OECD TG 203. 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 determined to be >120 mg/L (nomnal conc.). Thus, based on LC50 value, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be 'not classified' category as per the CLP classification criteria.

In a supporting weight of evidence study from secondary source, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The test was performed following the principles of the OECD Guideline 203 (Fish, Acute Toxicity Test).Oryzias latipes (Japanese rice fish) of length 2.38 cm (S.D. = 0.11 cm) and 0.12 g (S.D. = 0.02g) weight obtained from National Institute of Environmental Research (Environmental Research Complex, Kyungseo-dong, Seo-gu, Incheon, Korea) was used as a test organism. Upon receipt, suitable male and female fish, at a ratio of 3 to 2 respectively, without any visible abnormalities and of similar length were selected and placed in a 50 L glass breeding chamber containing maintenance water. Approximately 50 fish (±10%) were bred. Eggs were harvested from the breeding chambers and placed in hatching chambers. After hatching, the fry were placed in a breeding chamber at 23±2°C and bred. Approximately 30% of the holding water is exchanged once a week. The holding room was artificially illuminated; 16 hours light, 8hours dark. Fish were fed Brine Shrimp (Ocean Star International, Inc., U.S.A.) in the morning and Top Meal (Jaeilfeed Co., Korea) in the afternoon, approximately 3% of their body weight daily, except on Sunday when they were fasted. Prior to initiation, fish without any visible abnormalities and of similar length were selected and acclimated in maintenance water for 10 days. During the acclimation period, the water temperature and dissolved oxygen concentration in maintenance water were maintained at 22.1–23.8°C and 86.4–96.1%, respectively. The room was artificially illuminated with 16 hours of light and 8 hours of dark. The fish were fed Brine Shrimp eggs (Ocean Star International, Inc., U.S.A.) in the morning and Top meal in the afternoon, at an amount of approximately 3% of their body weight, once daily except on Sunday when they were fasted. Mortalities were observed from 48 hours after initiation of acclimation. During the seven days prior to exposure, mortalities of a batch of fish were less than 5% of populations, so a batch of fish was used. No food during the 24 hours period immediately prior to exposure. Public tap water was filtered and irradiated by ultraviolet light and used as maintenance and dilution water. The request amounts (active ingredient: 0.998 g) of the test substance were measured using an electronic balance. For preparation of stock and test solutions, the required amounts of the test chemical were added to test chamber filled with dilution water. For the range finding study, a stock solution of 100 mg/L of the test substance was prepared using dilution water. This stock solution was diluted to concentrations of 10 and 1 mg/L of the test substance with dilution water. In definitive study, 100 mg/L test solution of the test substance was prepared. Each of 3 L test solution per test chamber was prepared, and the dilution water was used for the control group. Thus, limit test was performed using 100 mg/l of test chemical concentration. Test chemical concentrations were determined analytically. The concentrations of test chemical were analyzed using HPLC in all test solutions at the beginning (0 hour) and at the end (96 hours) of the study. The samples were taken from mid-layer of 100 mg/L test solutions and centrifuged at 3000 rpm for 5 minutes. Duplicate samples from the top layer of each batch of test solutions diluted within the concentration range of calibration samples and analyzed. Also, duplicate samples from mid-layer of control group were centrifuged at 3000 rpm for 5 minutes and analyzed. Total 10 fishes/conc. were exposed to the test chemical in a test chamber for 96 hrs. Test conditions during the study include hardness of 61 mg/l as CaCO3, temperature of 23.1 to 23.8°C, pH 7.33 to 7.53 and dissolved oxygen of 7.09 to 7.64 mg/l under a photoperiod of 16 hour light, 8 hour dark cycle in a fluorescent lighting. Temperature and oxygen levels were maintained. All experiments including the control were performed in replicate. Mortality of the test organisms were noted. Copper (II) Sulfate was used as a positive control. The 96 hr LC50 value of the reference substance Copper (II) Sulfate was determined to be 0.31 mg/l and it was within the historical control range (mean±2 SD: 0.24–0.48 mg/L). At the end of the test, total length and weight of the control fish were 2.38±0.11 cm and 0.12±0.02 g (mean ± SD), respectively. During the exposure period, the water temperature, dissolved oxygen concentration, and pH of test solution were 23.1–23.8°C, 7.09–7.64 mg/L (converted at air saturation value: 84.5–91.1%) and 7.33–7.53, respectively. These were within the range permitted for the study. The test solutions in the control group were observed for transparency prior to exposure initiation and during the exposure period. The test solutions in the 100 mg/L dosed group were observed for transparency prior to exposure initiation and 24, 48 hours after exposure, while it was shown turbidity at 72 and 96 hours after exposure. All the fish in the control group and the dosed group at 100 mg/L nominal concentration were normal without deaths or abnormal signs. Thus, after an exposure period of 96 hrs, the mortality of the test organism was less than 50% (actual measurement: 0%) in the highest concentration dosed group, so the LC50 at 24, 48, 72, and 96 hours was determined as >100 mg/L, respectively. The concentration of the test substance during the exposure period was within (+/-) 20% of the nominal concentrations. Therefore, all test results were determined as the nominal concentrations. In the control vessel, the mortality of the test organism was not exceed 10% at the end of the exposure, dissolved oxygen concentration was ≥60% of the air saturation value in all test vessels throughout the exposure and analytical measurement of test concentrations were carried out. Thus, validity criteria of the study was fulfilled. On the basis of effect on mortality of the test organism, the 96 hr LC50 value was determined to be > 100 mg/l (nominal conc.) and > 99.47 mg/l (measured conc.), respectively. Thus, based on this value, test chemical was considered as non-toxic to fish and hence, considered to be ‘not classified’ as per the CLP classification criteria.  

 

For the test chemical, another short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. Study was performed in accordance with the OECD Guideline 203 (Fish, Acute Toxicity Test) under static conditions. On the basis of the effect of test chemical on mortality of the test fishes, the 96 hrs median lethal concentration (LC50) value was determined to be >21 mg/l. Thus, based on the LC50 value, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations.

On the basis of the above result, test chemical was considered as non-toxic to aquatic fishes and hence, considered to be non toxic as per the CLP classification criteria.

Long term toxicity to fish

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on fish was predicted for test chemical. On the basis of no effects observed in a freshwater fish, the 28 d NOEC value for the test chemical was estimated to be 23.58 mg/l. Based on this value, test chemical was considered as non-toxic to fish at environmentally relevant concentrations and hence, considered to be not-classified as per the CLP classification criteria. 

Short term toxicity to aquatic invertebrates

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

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical.The test was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test).Daphnia magna (Water flea) (7 days old) obtained from National Institute of Agricultural Science & Tech was used as a test organism. Twenty five daphnids were kept in a glass beaker with 800 mL of dilution water. Young daphnids, which are bone from not less than 14 days old daphnids that were above third brood progeny and less than 24 hours old, were used for this study. Test chemical concentration used for the study was control (0), 100 mg/l (nominal concentration) and 83.14 mg/l (measured concentration). Thus, in a definitive test, a limit test was performed. Test chemical concentrations were determined analytically.The concentrations of test chemical were analyzed using HPLC in all test solutions at the beginning (0 hour) and at the end (48 hours) of the study. The samples from each one dish of the control and treatment groups were collected twice, and then each sample was analyzed.The concentrations at initial exposure (0 hours) and at the end of exposure (after 48 hours) were determined as 100 and 69.12 mg/L (measured concentration), respectively. Test daphnids (total 20 daphnids) were exposed to the test chemical conc. in a glass beaker under static conditions for an exposure period of 48 hrs. Test conditions during the study were hardness of 250 mg/l as CaCO3, temperature of 20.0 to 20.6°C, pH of 7.24 to 7.87 and dissolved oxygen of 6.88 to 7.41 mg/l under a photoperiod of 16 hours of continuous artificial light and 8 hours of continuous darkness with 30 minutes dawn and dusk transition period between light and darkness respectively. No aeration was provided in the test vessel during the study. Test organisms were not fed during the 48 hr exposure period. After the exposure period of 48 hrs, the numbers of immobile daphnia were noted. Potassium dichromate was used as a reference substance during the study. The results of this study were confirmed within the range permitted for the test (mean ± 2SD) by historical control data in this laboratory of Biotoxtech Co., Ltd. Exposure conditions for the positive control study were in the same conditions as the definitive test. The concentration at the end of exposure exceeded ±20% of the measured initial values (0 hours). Therefore, all test results were determined as the geometric means.During the exposure period, the percentage of immobilisation in the control group was below 10%, and no abnormal signs were foundsuchas trapping at watersurface.Inthe definitive test, the percentage of immobilisation was less than 50% in the highest concentration treatment group during the exposure period.The dissolved oxygen concentration at the end of the test should be 3 mg/l in control and test vessels. Thus, the validity criteria has been fulfilled.On the basis of the effect of the test chemical on mobility of the test organism Daphnia magna,the 48 hr NOEC value was determined to be 100 mg/l (nominal conc.) and 83.14 mg/l (measured conc.). The median effect concentration (EC50) value was determined to be>100 mg/l (nominal conc.) and>83.14 mg/l (measured conc.), respectively.

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical.The test was performed following the principles of the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) and EC Council Regulation No 440/2008 C.2 Acute Toxicity for Daphnia, respectively.Daphnia magna (Water flea) was used as a test organism.Test chemical concentration used for the study was 100% v/v saturated solution (nominal concentrations).Test chemical concentrations were determined analytically.The concentrations of test chemical were analyzed using High performance liquid chromatography with mass spectrometry (HPLC-MS) at 0 and 48 hours. The study was performed under static conditions using total 20 daphnids under test conditions with hardness of 250 mg/l CaCO3 for 48 hrs. The effect measured was immobilization of the daphnids. After the exposure period of 48 hrs, the numbers of immobile daphnia were noted. Analysis of the test solutions at 0 and 48 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method. The concentration of the test chemical could not be determined in the test media (at the highest attainable test concentration of 1.7 mg/l, no immobilisation or adverse reactions to exposure were observed). Therefore the results are based on concentration as % v/v saturated solution. On the basis of the toxic effect of the test chemical on mobility of the test organism Daphnia magna, the 48 hr NOEC value was determined to be 100% v/v (saturated solution) and the median lethal concentration (LC50) value was determined to be > 100% v/v (saturated solution), respectively. Thus, 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.

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 48 hr EC50/LC50 value can be expected to be > 100 mg/l. Thus, 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.

Long term toxicity to aquatic invertebrates

Based on the prediction done using ECOSAR version 1.1, the long term toxicity on aquatic invertebrates was predicted for test chemical. On the basis of no effects observed on a freshwater aquatic invertebrates, the 21 d NOEC value for the test chemical was estimated to be 13.477 mg/l for daphnia. Based on this value, test chemical was considered as non-toxic to daphnia at environmentally relevant concentrations 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 test solution was prepared by dissolving 200 mg of test chemical in 200 ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final 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 and 100 mg/l was from the stock test concentrations. Thus, limit test was performed. Test vessel were placed in orbital shaking incubator for 72 hrs at a temperature of 21 to 24 ± 2°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. All control and test vessel conc. were performed in six replicates. 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, the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (i.e., reported to be 0.79%) and the mean coefficient of variation of specific growth rate was not exceeded 35% (i.e., reported to be 8.24%), respectively thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (ErC50) value was determined to be >100 mg/l (nominal conc.).Thus, 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

On the basis of effect on growth inhibition of microorganism (Tetrahymena pyriformis), the 48 hr IGC50 value was estimated to be 2433.12 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 to aquatic organisms at environmental relevant concentrations and hence, considered to be classified in ‘not classified’ as per the CLP classification criteria.