4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one

Endpoint summary

Description of key information

Short term toxicity to fish:

Based on the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to fish was predicted. On the basis of this program, the LC50 value for short term toxicity to aquatic invertebrates was predicted to be 1.376 mg/l for test material in 48 hrs. Based on the above effect concentration value it can be predicted test substance is toxic to fish and can be classified as aquatic chronic 2 as per CLP 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 substance . On the basis of effects observed in a freshwater system, the NOEC value for the substance is estimated to be 0.173 mg/l for fish for 28 days of exposure duration., it can be concluded that the test chemical can be considered as oxic to fish at environmentally relevant concentrations and can be considered to be classified aquatic chronic 2 as per the CLP classification criteria. 

Short term aquatic invertebrate:

After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2.

Long-term toxicity to aquatic invertebrates:

Based on the prediction done using ECOSAR version, the long term toxicity on aquatic invertebrate was predicted for test substance. On the basis of no effects observed in a freshwater system, the NOEC value for the substance is estimated to be 0.170 mg/l for aquatic invertebrate for 21 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as toxic to aquatic invertebrate at environmentally relevant concentrations and can be classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

In the data for the lab report of the structurally similar read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

The above study was further supported by another structurally similar read across substance for the test chemical , toxicity of test material was evaluated for aquatic algae Desmodesmus subspicatus for 72 h , the EC50 was observed to be 22.2 mg/l. Based on the above effect concentration it can be considered that test material is toxic and can be classified as aquatic chronic3 as per CLP criteria.

Thus, based on the above summarized studies, test material and it’s structurally similar read across substance, it can be concluded that effect concentration value is in the range of 22.2 to 50.12mg/l. Thus, comparing this value with the criteria of CLP regulation, test material can be classified as aquatic chronic 3 for algae and cyanobacteria.

Toxicity to microorganism:

The Minimum Inhibitory Concentration (MIC) value of test chemical on the fungi  Colletotrichum musae DAR 24962 was determine to be 894.195 mg/L.

Additional information

Short term toxicity to fish:

The toxicity of test material was evaluated for fish using a predicted study and to support the prediction , data from secondary source of structurally similar read across substance was also used.

Based on the prediction done by EPI suite, ECOSAR version 1.1, on the basis of similarity of structure to chemicals for which the aquatic toxicity has been previously measured by structure-activity relationships (SARs) program, the LC 50 value for short term toxicity to fish was predicted. On the basis of this program, the LC50 value for short term toxicity to aquatic invertebrates was predicted to be 1.376 mg/l for test material in 48 hrs. Based on the above effect concentration value it can be predicted test substance is toxic to fish and can be classified as aquatic chronic 2 as per CLP criteria.

The above prediction was further supported by data from secondary source of structurally similar read across substance, experimental study for test chemical was conducted according to guideline 203. Rainbow Trout (average length, 5.8 cm), were exposed to a series of 5 test concentrations of 0, 7.8, 10.9, 15.3, 21.4, or 30 mg/L dispersed in Polysorbate 80 (10 mg/L) for 96 hours at 17.1 °C. Control fish were exposed to polysorbate 80 (10 mg/L). Fish were observed twice daily for mortality and symptoms. pH values and water temperature were monitored after substance addition at 24 hour intervals. Dissolved oxygen was measured at the beginning of the experiment and at 96 hours. The LC50 value for the given test material is 10.9 mg/L.Based on the above effect concentration it can be concluded that test material is toxic to fish and can be classified as aquatic chronic 2.

According to another study from secondary source for structurally similar read across substance , as per the guideline 203, Rainbow Trout (average length, 6-8 cm), were exposed to a series of 2 test concentrations of 0, 5, or 10 mg/L of test materialfor 96 hours at 16+/-1°C.During experiment the LC0 was determined to be 5 mg/L while LC100 as 10 mg/L. Thus LC50 value for the given test material was considered to be between 5mg/L and 10 mg/L.Based on the above effect concentration it can be concluded that test material is toxic to fish and can be classified as aquatic chronic 2.

In a data from peer reviewed handbook it was observed that,short term toxicity of test material was evaluated for Leuciscus idus fish for 96 h . The minimum leathel effect concentration (LC50) was observed to be 6.8 mg/l.

Based on the above effect concentrations it can be concluded that test material is toxic to fish and can be classified as aquatic chronic 2.

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 substance . On the basis of effects observed in a freshwater system, the NOEC value for the substance is estimated to be 0.173 mg/l for fish for 28 days of exposure duration., it can be concluded that the test chemical can be considered as oxic to fish at environmentally relevant concentrations and can be considered to be classified aquatic chronic 2 as per the CLP classification criteria. 

Short term aquatic invertebrate:

Data available for the test chemical was reviewed to determine its toxic nature upon the growth and mobility of aquatic invertebrate based on the available study of structurally similar read across substances. The studies are as mentioned below:

 

The first study was to determine the effect of test chemical on the immobilisation of test organism daphnia magna. Test conducted in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample conducted by using HPLC/UV detector. Juvenile daphnia (<24 hours old) produced from an in-house culture of adults were maintained at the contract laboratory under test conditions for 45 days. During the 48 hours prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions for 48 hours. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7days prior to the beginning of the test. The test substance was provided via an intermittent flow proportional diluter. 5 nominal concentrations 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L were used for the toxicity testing. 10 daphnia per vessel added and test conducted in duplicates. After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Similarly in the second study a Daphnia immobilization static study was conducted following Guideline “Acute Toxicity for Daphnia” (C.2), Directive 92/69/EEC by providing the exposure period of 48 hrs. Test conducted under the static system. After the exposure of 48 hrs, effect on the daphnia magna were observed and measured on the basis of immobility. The EC50 was determine to be 5.7 mg/l.

 

Above studies was supported by another third study. To study the effects of test chemical on aquatic invertebrates test was carried out according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The 48-h LC50 value is based on measured data. A synthetic fresh water was used for culture and test purposes. Properties of this medium: Total hardness: 2,20 - 3,20 mmol/l, Alkalinity up to pH 4.3: 0,80 - 1,00 mmol/l, Molar ratio Ca:Mg: about 4 : 1, pH value: 7,5 - 8,5, Conductivity: 550 - 650 μS/cm. After preparation the M4 medium was aerated for approximately 24 h until saturation. The test substance was tested in the range of concentrations between 16 and 1 mg/l. The dilution factor was 2. The number of mobile daphnid after 24 and 48 hours of exposure in the different test concentrations was measured. The EC50 (48 h) calculated using the Probit analyses The EC 50 value of test chemical was observed to be 3.7 mg/L when daphnia magna was exposed to for 48 hrs. On the basis of this value it can be concluded that the test chemical is toxic to the aquatic environment and can be considered as Aquatic chronic 2 as per the classification criteria for aquatic environment.

 

Thus based on the overall studies, chemical consider to be toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

Long-term toxicity to aquatic invertebrates:

Based on the prediction done using ECOSAR version, the long term toxicity on aquatic invertebrate was predicted for test substance. On the basis of no effects observed in a freshwater system, the NOEC value for the substance is estimated to be 0.170 mg/l for aquatic invertebrate for 21 days of exposure duration. Thus, it can be concluded that the test chemical can be considered as toxic to aquatic invertebrate at environmentally relevant concentrations and can be classified as aquatic chronic 2 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

In the data for the lab report of the structurally similar read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 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. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

The above study was further supported by another structurally similar read across substance for the test chemical , toxicity of test material was evaluated for aquatic algae Desmodesmus subspicatus for 72 h , the EC50 was observed to be 22.2 mg/l. Based on the above effect concentration it can be considered that test material is toxic and can be classified as aquatic chronic3 as per CLP criteria.

Thus, based on the above summarized studies, test material and it’s structurally similar read across substance, it can be concluded that effect concentration value is in the range of 22.2 to 50.12mg/l. Thus, comparing this value with the criteria of CLP regulation, test material can be classified as aquatic chronic 3 for algae and cyanobacteria.

Toxicity to microorganism:

Based on the various experimental data for the structurally similar read across substance of test chemical s have been reviewed to determine the toxic nature of test chemical on the growth of microorganisms. The studies are as mentioned below:  

The effects of test chemical on the growth of Colletotrichum musae on agar medium were evaluated. Test was performed on the agar medium. Agar plugs (5.5-mm diam.) were picked up from the 3-day-old cultures of decay fungi using the bottom end of a sterilized Pasteur pipet and then transferred onto the centers of new PDA media, in 9-cm plastic Petri dishes. The Petri dishes were then inverted and 7-cm Whatman No. 1 filter papers were attached onto the inner surface of their lids. Ethanol, the first tested volatile in this experiment, was impregnated into the filter paper with varying volumes from 0.1 to 1.0 mL/dish in the 4°C room. Immediately after the impregnation, the Petri dishes were sealed by wrapping them with plastic film and incubated for 10 days at 25 °C. Experiments were repeated two times with four replications for each experiment. The minimum concentration of ethanol (expressed as mmol/dish) required to give complete control or the minimum inhibitory concentration (MIC) for each microorganism was determined. The MIC of ethanol for target decay microorganism was used as the initial level to identify the MIC of other tested volatiles. If the MIC level of ethanol used for other volatiles failed to stop the growth of pathogen, the level was increased until the MIC was found. However, if the volume of 1.5 mL/dish still failed to stop the growth of pathogen, the compound was considered ineffective as a vapor to stop the growth of pathogens. When the tested compounds had the same effect as the MIC of ethanol, the concentration was decreased until the MIC of the compound for each microorganism was determined. All the unit concentrations of MIC were then expressed as mmol/dish. After the incubation of 10 days, the Minimum Inhibitory Concentration (MIC) value of test chemical on the fungi, Colletotrichum musae DAR 24962 was determine to be 894.195 mg/L.

 

First study was supported by the second experimental study. Aim of this study was to evaluate the effect of test chemical on the growth of Bacillus subtilis and other fungi. The antimicrobial activity of test compounds against various bacteria and fungi was examined by the broth dilution method. Solution of the test compound was added to 2-day-old cultures of the microorganisms. After 2-5 days of incubation, growth of the microorganisms was checked. Minimal inhibitory concentrations (MICs) were measured by two fold serial broth dilution. Based on the antimicrobial effect of test chemical on the growth of Bacillus subtilis after the exposure of chemical for 2-5 days, the MIC was observed at 100 mg/l.

 

Thus based on the above studies, MIC ranges from 100 mg/l to 894.195 mg/l after the exposure of microorganisms with the test chemical for 2-10 days.