1,1,1"((methylsilanetriyl)tris)(oxy))tris(2-methylpropan-2-amine)

Endpoint summary

Description of key information

Toxicity to Fish:

The objective of this study is to assess the Acute Toxicity of test chemical in Freshwater Fish (Danio rerio). The study was performed in compliance with OECD 203 Guideline for the testing of chemicals: Fish, Acute Toxicity Testing, (18th June 2019). Prior to performing study fishes of mean size 1.4 cm were acclimatised for at least for 7 days under similar conditions as used in main study. The acclimatisation conditions were 16:8 hours, 23.2°C, 7.7, and 7.6 mg/L of photoperiod, temperature, pH and dissolved oxygen respectively. Under acclimatisation feed was provided daily with continues aeration. Feeding was stopped prior 24 hours to initiation of main study. The test chemical was directly formulated dissolved in the RO water by weighing 1000 mg of test chemical in 1000 ml of water to obtain a final concentration of 1000 mg/L. the analytically determined solubility was 745.93mg/l , carried out using UV Visible spectrophotometer. Based on the available data, main study was conducted with limit concentration, of 100 mg/L under static conditions. Total of 7 fishes were exposed per each concentration and control. The exposure was done in 7 lit of Glass aquarium which was loosely closed to avoid cross over and to reduce chemical evaporation from vessel. The whole setup was maintained at temperature ranging form 22 -23°C and dissolved oxygen concentrations ranging form 6.0-8.3 mg/L. 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. For chemical analysis a linearity range of 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 mg/l was selected, absorbance was recorded at 199 lamda max. The analytically measured concentrations at 0hour were 88.43 mg/L and 88.10 mg/L at 96 hours, concludes that chemical is within the range of 100±20% of the exposed test concentrations. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 79.76% of the air saturation value. The evaluation study after 96 hours meets all the validity criteria as mentioned in the OECD 203 guidelines and the median lethal concentration was reported to be >100 mg/L Thus, chemical cannot be classified as per CLP classification criteria.

Toxicity to daphnia:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study was conducted using 0 (control), and 100 mg/L concentrations (limit test). 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups , there was no immobilisation was observed in both controla and exposed group. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 100 mg/L of exposure, which were found in acceptable range between 99.37 at zero hours and 98.29 at 48 hours . Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.4 -7.7), temperature (19.7 -21.9 °C), dissolve oxygen (8.8 -6.9 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 μS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to, Daphnia magna was reported to be >100mg/L. The 48-h EC50of reference item(Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Hence, as per CLP classification category the test chemical can be categorized as Not classified category.

Toxicity to algae:

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. T The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.5, 1, 2, 4 and 8 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 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.81 in all the replicates at day 0 and followed by between 8.44 on day 3. 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 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 (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Toxicity to algae:

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata

 were exposed for 72 hours to nominal test item concentration of 0.26, 0.64, 1.6, 4.0 and 10 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.18, 0.52, 1.4, 3.7 and 9.3 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.

Toxicity to microorganims:

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

Toxicity to Fish:

The short term toxicity to fresh water fish was calculated using ECOSAR of EPI suite, the outcome was 126.801 mg/l after 96 hours of exposure. Thus the chemical cannot be classified as per CLP classification criteria.

The objective of this study is to assess the Acute Toxicity of test chemical in Freshwater Fish (Danio rerio). The study was performed in compliance with OECD 203 Guideline for the testing of chemicals: Fish, Acute Toxicity Testing, (18th June 2019). Prior to performing study fishes of mean size 1.4 cm were acclimatised for at least for 7 days under similar conditions as used in main study. The acclimatisation conditions were 16:8 hours, 23.2°C, 7.7, and 7.6 mg/L of photoperiod, temperature, pH and dissolved oxygen respectively. Under acclimatisation feed was provided daily with continues aeration. Feeding was stopped prior 24 hours to initiation of main study. The test chemical was directly formulated dissolved in the RO water by weighing 1000 mg of test chemical in 1000 ml of water to obtain a final concentration of 1000 mg/L. the analytically determined solubility was 745.93mg/l , carried out using UV Visible spectrophotometer. Based on the available data, main study was conducted with limit concentration, of 100 mg/L under static conditions. Total of 7 fishes were exposed per each concentration and control. The exposure was done in 7 lit of Glass aquarium which was loosely closed to avoid cross over and to reduce chemical evaporation from vessel. The whole setup was maintained at temperature ranging form 22 -23°C and dissolved oxygen concentrations ranging form 6.0-8.3 mg/L. 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. For chemical analysis a linearity range of 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 mg/l was selected, absorbance was recorded at 199 lamda max. The analytically measured concentrations at 0hour were 88.43 mg/L and 88.10 mg/L at 96 hours, concludes that chemical is within the range of 100±20% of the exposed test concentrations. After the 96 hours of exposure, there was no mortality in the control group, and the dissolved oxygen concentration was 79.76% of the air saturation value. The evaluation study after 96 hours meets all the validity criteria as mentioned in the OECD 203 guidelines and the median lethal concentration was reported to be >100 mg/L Thus, chemical cannot be classified as per CLP classification criteria.

To evaluate the acute toxicity of the test item on Zebrafish (Danio rerio) a static 96-hour test according to OECD TG 203, was carried out in compliance with the GLP principles. Based on the results of a preliminary study 10 animals were exposed to a single limit concentration of 100 mg/L (nominal). A negative control with ISO Medium but without the test item was performed in parallel. The test item concentration was analytically verfied by using HPLC-UV and found to be 103 –104 % of the nominal at the start, and 100 % of the nominal at the end of the test. Therefore, all biological results are based on nominal concentrations. Fish were observed at approximately 2, 6, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. All validity criteria of the test guidelines were fulfilled. As a result, no toxic effect on fish was observed  at the limit concentration of 100 mg/L. Accordingly, the 96-hour LC50 value was determined to be > 100 mg/L. The 96-hour NOEC was determined to be 100 mg/L. Based on the outcomes the test chemical cannot be classified as per CLP classification criteria.

Toxicity to Daphnia:

This study was designed (as per OECD 202, adopted in 2004) to assess the acute toxicity of test chemical following exposure of daphnids up to 48h by static method. The brood daphnids were acclimatized 48 hours prior to the test item exposure. Less than 24 h old daphnids were collected from the acclimatized gravid females and exposed to the test item. After exposure on day 0, daphnids were observed for immobilization at 24 and 48 h. M7 medium was used as control, and the same was used for test item formulation . 25 mL glass beakers having a solution volume of 20 mL were used in the test. . Main study was conducted using 0 (control), and 100 mg/L concentrations (limit test). 4 replicates/concentration having 5 daphnids/replicate was used for the main study. Normal behavioural response and no immobilization (0% mortality) were observed up to 48 h followed by control groups , there was no immobilisation was observed in both controla and exposed group. UV-Visible spectrophotometer method was used for active ingredient analysis along with stability of the test item in the test medium. Test item was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during main study 0 h and 48 h avg. recovery for 100 mg/L of exposure, which were found in acceptable range between 99.37 at zero hours and 98.29 at 48 hours . Hence the results were based on nominal concentration since the deviation in the initial measured concentration didn’t exceed 20%. Environmental parameters such as pH (7.4 -7.7), temperature (19.7 -21.9 °C), dissolve oxygen (8.8 -6.9 mg/L), hardness (164 mg CaCO3/L), conductivity (0.27 μS/cm), photoperiod (16 h light- 8 h dark) was maintained in acceptable range throughout the test. Feed was not provided during the test. The 48-h EC50of test chemical to, Daphnia magna was reported to be >100mg/L. The 48-h EC50of reference item(Potassium dichromate) to daphnid, Daphnia magna(found to be in acceptable range) is 0.640 mg/L. Hence, the results of the test with reference item establish the acceptability of the test system response, test procedures followed, and results obtained with test item. Hence, as per CLP classification category the test chemical can be categorized as Not classified category.

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, 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 6.609, 12.740, 26.350, 51.560 and 103.228 mg/L after 48 hours. The test item concentration was thus 97 – 101 % of the nominal at the end of the experimental phase (at the start of the test: 102 –106 % 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 94.3 mg/L in nominal (95 % conf. limits: 77.3 – 133.3 calculated). The 48-hour EC10 was found to be 56.1 mg/L in nominal ( 95 % conf. limits: 28.1 – 70.0 mg/L). Based on the outcomes the test chemical can be classified into aquatic chronic category 3 as per CLP classification criteria.

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. T The test solution was prepared by dissolving 250 mg of test chemical in 250 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 1179.67 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.5, 1, 2, 4 and 8 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 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. the initial pH in the control vessels were 7.81 in all the replicates at day 0 and followed by between 8.44 on day 3. 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 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 (EC50) was determined to be 9.66 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 2' as per the CLP classification criteria.

Toxicity to algae

The effect of test chemical on green algae was estimated using ECOSAR model for the exposure period of 72 hours and median effective concentration was found to be 2.047 mg/l. Based on the values it can be classified into aquatic chronic category 2 as per CLP classification criteria.

To determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata a growth inhibition test according to OECD TG 201 and EU Method C.3. was carried out in compliance with GLP principles. Exponentially growing cultures of Raphidocelis subcapitata

 were exposed for 72 hours to nominal test item concentration of 0.26, 0.64, 1.6, 4.0 and 10 mg/L over several generations under defined conditions. An untreated control group (OECD Test Medium without test item) was tested parallel with the treated cultures. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. The algal cell concentration was determined in 24-hour intervals by manual cell counting using a microscope. The nominal test item concentrations were analytically verified by using a HPLC-UV method. The measured concentrations deviated more than 20 % from the nominal at the two lowest concentrations during the experiment therefore the geometric mean of the measured concentrations were calculated to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: 0.18, 0.52, 1.4, 3.7 and 9.3 mg/L. Biological results and endpoints are thus based on the measured geometric mean concentrations. All validity criteria of the guidelines were met. As a result, the 72-hour EC50 and EC10 for the growth rate was determined to be 1.92 and 0.74 mg/L, respectively. The 72-hour EC50 and EC10 based on biomass was found to be 0.95 and 0.43 mg/L, respectively. The 72-hour NOEC was 0.52 mg/L both for the growth rate and biomass.

Toxicity to microorganims:

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.

Another 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 -3.46 % inhibition, stimulation and 2.69 % inhibition were considered as being within the biological variability of the applied test system), but the inhibition was in average 53.69 % 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 and EC₅₀ calculations and for estimation of EC₈₀ value. The EC₁₀ was calculated as 472 mg/kg soil dry weight, the EC₅₀ was calculated as 962 mg/kg soil dry weight and the EC₈₀ value was estimated as 1284 mg/kg soil dry weight. The above EC_x values will be further refined in the definite test. The EC₅₀ value was determined as: EC₅₀ < 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, the NOEC < 1000 mg/L. 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 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.