Benzyl butyrate

Endpoint summary

Description of key information

Short term toxicity to fish:

The acute study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. In house solubility was observed to be 281.985 mg/L. Thus the stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determined to be 12.5 mg/l. Thus on the basis of LC50 value, test chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, hence chemical can be consider to be nontoxic and not classified as per the CLP classification criteria.

 

 

Short term toxicity to aquatic invertebrates:

An acute immobilisation test was conducted for 48 hrs for assessing the short-term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used as a test organism. The stock solution 100 mg/l was prepared in reconstituted water. 0, 2.5, 5, 10, 20, 40 mg/l nominal concentrations were used in the study. 5 daphnids per concentration were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Observations including immobility, pH, Temperature, dissolved oxygen content were recorded in the interval of 24 and 48 hours. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 16.6 mg/l. EC50 value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified in aquatic chronic 3 category as per the CLP criteria. As the test chemical was readily biodegradable in water, thus on the basis of degradation criteria, test chemical considers to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to algae and cyanobacteria:

Freshwater algal growth inhibition test was carried out on green algae. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/L was prepared in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted under the static system and tested at the concentrations 0, 5, 10, 20, 40 and 80 mg/L respectively. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. Effects on the growth rate of the test organism was studied. The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study. Thus, based on the ErC50 value, chemical consider to be toxic but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

 

Toxicity to microorganisms:

The study was conducted for the determination of effect of test chemical on the various species and strains of microorganism. After the exposure period of test chemical with microorganisms for 24 hours, growth rate inhibition were calculated and the MIC was determined to be > 2000 mg/l.

Additional information

Short term toxicity to fish:

Following different studies includes experimental study for the test chemical and read-across analogues which is extracted by using mechanistic approach and functionally and structurally similar to the target chemical to observe the short-term toxicity of test chemical to fishes.

 

The acute study was designed to access the toxic effects of the test compound on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. In house solubility was observed to be 281.985 mg/L. Thus the stock solution prepared as 2g /4 liter, with the concentration of 500 mg/L and was kept for 4 hours stirring. After the completion of stirring, the sample was run through HPLC system for getting the actual water solubility of the test substance. The actual solubility obtained was 122.66 mg/L. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25mg/L, 12.5mg/L, 25mg/L, 50mg/L, 100mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra Fish Danio rerio was determined to be 12.5 mg/l. Thus on the basis of LC50 value, test chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, hence chemical can be consider to be nontoxic and not classified as per the CLP classification criteria.

 

Above study further supported by the second study from experimental source. This study was designed to access the toxic effects of the test chemical on the fishes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Zebra fish (Danio rerio) was used as a test organism. The stock solution was prepared by dissolving 1ml of the test substance in 1 liter of potable water (passed through reverse osmosis system) with 24 hrs of continuous stirring. From this stock solution, further test concentration was prepared for achieving test concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L & 100 mg/L, respectively. Aquaria containing 4 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes each. A static procedure was used for the study. Effects on the mortality rate of fishes was calculated and were observed in the interval of 24, 48, 72 and 96 hours. The median lethal concentration (LC50) value of test chemical on Danio rerio in a 96 hours study on the basis of mortality effect was determined to be >12.5 mg/L. As 100 % mortality was observed at the concentration of 25 mg/l, thus on that basis, chemical consider to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria, but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

 

Similar short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical. The study was performed following the OECD Guideline 203 (Fish, Acute Toxicity Test) under flow through conditions. Oryzias latipes (Japanese Medaka) of 28 -43 days old and 18 -71 mg weight obtained from the Environmental Research Laboratory-Duluth (ERL-D) culture unit was used as a test organism. Test fishes were nurtured in tank at 25°C and fed with live Biomaine brand brine shrimp. Before 24 hr or during the study, test organism was not fed. 5 different concentrations of test chemical along with the control were taken for the study. Stock solutions of test chemical was prepared by dissolving the test chemical in Lake Superior water, using a high speed stirrer. Stock solutions were then transferred to a glass stock bottle inside the vented diluter enclosure using Teflon tubing and air pressure. During each test, a predetermined volume (ml/min) of stock solution was continuously pumped from the stock bottle into the mixing cell of the diluter system. Test chemical concentrations were verified analytically, and analysis was carried out bya Hewlett Packard 5730A gas chromatograph (GC) equipped with a flame ionization detector (FID) linked to an HP 3350 lab automation system. All test analyses were accomplished using direct aqueous injection. GC column consists of a wall-coated open tubular silica column, 0.53 mm I.D. x 15 cm, with a 2.5 µ phase of bonded polyethlene glycol at isothermal oven temperature of 85, 120 and 110°C, respectively. Total 20 fishes/conc (10 organisms/replicate) were exposed to test chemical in a2.0 lit glass aquaria tank.2.0 l glass aquaria tank has a dimension of 18.5 X 14.0 X 13.0 cm deep. It has a 8.6 cm standpipe which resulted in a total volume of 2.0 lit. Continuous-flow mini diluter exposure system with vented enclosures was used for the study. Flow rate during the study was 25 ml/min with 90% replacement times of 2.8 hr. Lake superior water was used.It was filtered before use through sand, a 50-micron filter; a 5-micron filter; and then exposed to ultraviolet light before heating to the test temperature of 25±1°C.The test vessels were placed in a room at a temperature of 25±1°C, pH 7.88 ± 0.18 (7.31 to 8.85), dissolve oxygen (D. O) 6.8 ± 0.7 (5.0 to 8.5), hardness of water 45.8 (38.0 to 52.0) mg/l as CaCO3, alkalinity 45.9 (35.0 to 58.5 mg/l) as CaCO3 and under a 16 hr photoperiod with a light intensity of 12 to 25 lumens provided by fluorescent lamps for 96 hrs. All experiments were performed in replicate.95% confidence intervals were calculated using the binomial tests. Dissolved oxygen (D.O.) was measured by a dissolved oxygen meter. pH was determined on one set of replicate tanks atleast once and often twice during the test. On the other hand, hardness and alkalinity determinations were done at a minimum on a control, one intermediate and one at high test concentration tank; it was carried out once or twice during the study. Mortality was noted after an exposure period of 96 hrs. No mortalities were observed in the control vessel, the dissolved oxygen concentration was evaluated to be ≥ 60% (i.e, reported as 82.3%) of the air saturation value in test vessels throughout the study period and analytical monitoring of test concentrations has been carried out, thus fulfilling the validity criteria of the study. As the test concentrations were maintained within ±20% of the initial measured concentrations throughout the study, all results will be reported in nominal concentrations. On the basis of the effect on mortality of the test organism Oryzias latipes (Japanese Medaka), the 96 hr LC50 was determined to be 4.0 mg/l (95% C. I. = 3.48 to 4.60 mg/l) (nominal concentration). Thus, test chemical was considered as toxic to fish and hence, considered to be classified in ‘aquatic chronic category 2’ as per the CLP classification criteria. But as the test chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus, on the basis of above all studies and effects observation, it is concluded that the test chemical was nontoxic and not classified as per the CLP classification criteria.

 

 

Short term toxicity to aquatic invertebrates:

Following different studies includes experimental study for the test chemical and read-across analogues which is extracted by using mechanistic approach and functionally and structurally similar to the target chemical to observe the short-term toxicity of test chemical to aquatic invertebrates.

 

An acute immobilisation test was conducted for 48 hrs for assessing the short-term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used as a test organism. The stock solution 100 mg/l was prepared in reconstituted water. 0, 2.5, 5, 10, 20, 40 mg/l nominal concentrations were used in the study. 5 daphnids per concentration were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Observations including immobility, pH, Temperature, dissolved oxygen content were recorded in the interval of 24 and 48 hours. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 16.6 mg/l. EC50 value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified in aquatic chronic 3 category as per the CLP criteria. As the test chemical was readily biodegradable in water, thus on the basis of degradation criteria, test chemical considers to be nontoxic and not classified as per the CLP classification criteria.

 

First study further supported by the second study from experimental source. An short term immobilisation test was conducted for 48 hrs for assessing the short term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The stock solution 100.0 mg/l was prepared in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. 2, 4 ,8,16, 32, 64 mg/l, respectively nominal test concentrations were prepared. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 15.1 mg/L with 95% CI of 10.5-21.5 mg/l. EC50 value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified in aquatic chronic 3 category as per the CLP criteria. But as the chemical was readily biodegradable in water and degrade faster, thus chemical can be considered to be nontoxic and not classified as per the CLP classification criteria.

 

Similar study was to assess the short-term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used a test organism. The animals used for the test was Daphnia magna, which should be less than 24 h old and should not be of first brood progeny. The stock solution 100 g/L was prepared in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 0, 5, 10, 20, 40 and 80 mg/L concentrations were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. The median effective concentration (EC50) value of the test substance on Daphnia magna was determined to be 60.7 mg/L on the basis of immobilisation effects in a 48-hour study. Based on the EC50 value, substance consider likely to be hazardous to aquatic invertebrate Daphnia magna and can be classified in aquatic chronic 3 category as per the CLP classification criteria.

 

Thus, based on the criteria of readily biodegradable in water, test chemical considers to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to algae and cyanobacteria:

Following different studies includes experimental study for the test chemical and read-across analogues which is extracted by using mechanistic approach and functionally and structurally like the target chemical to observe the toxicity of test chemical to aquatic algae.

 

Freshwater algal growth inhibition test was carried out on green algae. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/L was prepared in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted under the static system and tested at the concentrations 0, 5, 10, 20, 40 and 80 mg/L respectively. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. Effects on the growth rate of the test organism was studied. The median effective concentration (ErC50) of the test substance on a freshwater algae Desmodesmus subspicatus was determined to be 90.8 mg/L on the basis of effects on growth rate in a 72 hour study. Thus, based on the ErC50 value, chemical consider to be toxic but as the chemical was readily biodegradable in water, thus chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Similarly, the principle of second weight of evidence study was to determine the effect of test substance on the growth of fresh water green algae. The study was conducted following OECD guideline 201- Alga growth inhibition test. Chlorella vulgaris was used as test organism. The test substance was prepared by adding 100 mg of test substance in 200 ml of BBM to get the final concentration of 281.985 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. The test concentrations selected for the study were 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50mg/L, 100 mg/L and 200mg/L, respectively, prepared using stock solution of the test substance using de-ionized water and the study conducted under the static system. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. The test is considered to be valid as met all the standard parameters. The green algae were exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of test substance. EC50 calculated through probit analysis was determine to be > 200 mg/l. Thus, based on the effects observed on the growth rate of algae, chemical consider to be nontoxic and not classified as per the CLP classification criteria. 

 

 

Above experimental data further supported by the third weight of evidence study. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10,16, 26, 41, 66 mg/l, respectively concentration was used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (EC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72-hour study. Based on the ErC50 value, the substance chemical was considered likely to be hazardous to aquatic algae and can be considered to be classified in aquatic chronic 3 category. But as the test chemical was readily biodegradable in water, thus on that basis, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Similar study was conducted to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 10,16, 26, 41, 66 mg/l, respectively concentration was used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (EC50) of the test substance on algae was determined to be 23.6 mg/L with 95% CI of 28.9 mg/l to 33.7 mg/l on the basis of growth rate inhibition effects in a 72 hour study. Based on the ErC50 value, the substance chemical was considered likely to be hazardous to aquatic algae and can be considered to be classified in aquatic chronic 3 category. But as the test chemical was readily biodegradable in water, thus on that basis, chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus, based on the above experimental studies from various sources, test chemical considers to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to microorganisms:

Summarized result for the determination of effect of test chemical andread-across analogues which is extracted by using mechanistic approach and functionally and structurally like the target chemicalon the growth rate inhibition of different microorganisms are as mentioned below:

 

The Minimum Inhibition (MIC) effect of test chemical was observed on 5 different species of microorganisms i.e. Corynebacterium minutissimum (CM), Arthrobacter sp. isolated from Lipo-66, Staphylococcus aureus (IAM-1011, (SA)),Staphylococcus epidermidis var. (SE) and Escherichia coli (ATCC 11775, (EC)). Microorganisms exposed with test chemical for period of 24 hrs. In culture dishes (35 × 10mm) Muller Hinton agar medium was used for the measurement of MIC. DMSO used as solvent in which various concentrations of test chemical were prepared. The bacteria which was tested were pre-propagated with sensitivity test broth of NISSUI using shaking culture. The final incubated medium was diluted by 0.75% physiological saline to the microbial concentration of 10E6 CFU/ml. In the Muller Hinton agar medium having test chemical, 0.1ml of diluted culture solution was inoculated. MIC was determined after 24 hours of exposure at 37°C. After the exposure of test chemical with different microorganism, MIC was counted. The Minimum Inhibitory Concentration of test chemical on various microorganisms Corynebacterium minutissimum (CM), Arthrobacter sp. isolated from Lipo-66,Staphylococcus aureus (IAM-1011, (SA)), Staphylococcus epidermidis var. (SE) and Escherichia coli (ATCC 11775, (EC) was determine to be >2000 mg/l (inoculum 10E6 CFU/plate) after 24 hours exposure with test chemical.

 

 

Above study further supported by the third weight of evidence report from peer reviewed journal. Toxicity to micro-organisms study was conducted using five different microorganisms. The study was performed for 24 hrs at 37°C.Test organism Staphylococcus aureus (IAM-1011, SA) was purchased from Institute of Applied Microbiology, Tokyo university. Escherichia coli(ATCC 11775, (EC)) was purchased from Institute of Medical Science, Tokyo university. Corynebacterium minutissimum (ATCC 23348, (CM)) and Staphylococcus epidermis var. (SE) were gifted from the Department of Dermatology, University of Pennsyvania. Arthrobacter sp. was isolated from Lipo-66. Culture dish of 35 X 10 mm dimension was used as a test vessel. Test chemical solutions were prepared in either ethyl alcohol or DMSO. Muller Hinton agar was used as a test medium. Test bacteria were pre-propagated with sensitivity test broth of NISSUI using shaking culture. Incubated mediums were diluted using 0.75% physiological saline to the microbial concentration of 10e6 CFU/ml. Test medium containing the test chemical was inoculated using 0.1 ml of diluted culture solution. MIC was determined after 24 hrs at 37°C. Based on the effect of test chemical on the growth inhibition of five different organisms such as Corynebacterium minutissimum, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, Arthrobacter spp. after the exposure period of 24 hours, the MIC value was determined to be > 2000 mg/l.

 

Thus, on the basis of above all studies on different microorganisms, the MIC was determined to be > 2000 mg/l.

 

Thus, on the basis of effect of test chemical on the fishes, invertebrates and algae and also considering criteria of readily biodegradability of test chemical, it was observed that the test chemical was nontoxic and not classified as per the CLP classification criteria.

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