(E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Toxicity to aquatic algae and cyanobacteria

Description of key information

Based on valid algal inhibition studies for two closely related structural analogues, the toxicity to freshwater green algae of (E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2 -one has been estimated: 72h ErC50 value of >5.1 and <= 7.47 mg/L and NOEC of 1.68 to 2.3 mg/L. The EC50 value of > 5.1mg/L represents the fact that only 33% inhibition was observed at the highest test concentration of 5.1 mgL (Methyl Ionone Alpha Iso 60 source study). Therefore the determined value of 7.47 mg/L (Raldeine A GD source study) has been chosen as the key value for chemical safety assessment. Out of the two reliable NOEC values, the lowest and more conservative NOEC of 1.68 mg/L has been selected.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.47 mg/L

EC10 or NOEC for freshwater algae:

1.68 mg/L

Additional information

A study to assess the toxicity to aquatic algae and cyanobacteria is not available for the registration substance. However, valid algal inhibition studies exist for two analogue substances; Methyl Ionone Alpha Iso 60 and Raldeine A GD. Both studies are GLP compliant, conducted to OECD 201 guidelines, covered the required exposure duration of 72 hours and included sufficient dose levels to enable the relevant determination of potency. Read-across from these two analogue substances is considered to give a reliable estimate of the toxicity of (E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one to freshwater green algae and is justified based on the hypothesis that the target substance and two source substances will have similar ecotoxicological properties as a result of structural similarity (both the target and source substance consist of isomers), the same mode of action and similar physicochemical properties. A detailed justification for the proposed read-across in-line with the ECHA RAAF guidelines is provided in the target endpoint record.

The study performed on Methyl Ionone Alpha Iso 60 (Study No AL/N51/02) employed a static design and used conventional loosely stoppered test vessels. The test material (53 -58% main isomer, tested as 100% active) is representative of the source substance. Analysis of the test solutions was performed at 0, 24, 48 and 72 hours by a gas chromatographic and mass spectrometric method. The initial measured concentrations of the test item solutions were 1.1, 1.4, 3.1, 5.3 and 12 mg/L, which gradually declined throughout the 72 hour exposure period to 0.27, 0.31, 0.44, 0.87 and 1.9 mg/L (14-25% initial). Therefore the effect data were calculated based on the geometric mean of the 0, 24, 48 and 72 hour measured concentrations i.e. 0.49, 0.61, 1.1, 2.3 and 5.1 mg/L. The study was considered reliable with no restrictions (reliability 1). The 72 hr EC50 based on growth rate was reported to be > 5.1mg/L (33% inhibition seen at highest test concentration) and the NOEC based on both growth and inhibition was determined to be 2.3 mg/L.

The study performed on Raldeine A GD (Study No 2470-FF) was conducted under static conditions using test material (94.4% sum of isomers) which is representative of the source substance. The study was considered reliable with no restrictions (reliability 1). The test was performed in sealed containers from which all air space had been removed to minimize the potential loss of test substance from the test solutions to the atmosphere. Nominal concentrations of test substance were: 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, 8.0, and 16 mg/L. Analytical confirmation of the test substance (derivatized using 2,4-dinitrophenylhydrazine) was performed at 0 and 72 hours by HPLC. Samples collected at the start of the toxicity test were collected from test solutions just prior to the addition of algae and distribution of the test solutions to sealed test vessels. Samples collected at the end of the toxicity test were centrifuged to remove algal cells from pooled replicate test vessels. Initial measured concentrations of test substance were: ND (none detected at or above the limit of quantitation; control), 0.404, 0.954, 1.68, 2.94, 6.74, and 14.0 mg/L. The final measured concentrations ranged from 51 to 62% of the initial concentrations. A stability sample, prepared with a nominal concentration of 16 mg/L and not inoculated with algae, was incubated among the test vessels during the 72 hour exposure. The final measured concentration in this stability sample was 56% of the initial measured concentration. The latter is in the same range of the 51-62% obtained for the test samples, indicating that the decrease in measured concentrations in the test samples with algae is not attributable to adsorption to the growing algal biomass. However, the test substance was stable in laboratory control samples (4mg/L standards prepared in dilution water) where 72 h measured concentrations were 83-94% initial. Thus losses seen in the test samples after 72 hours could be a result of differences in sample work up (e.g. centrifugation) and/or storage conditions. Taking this into consideration and also the fact that the test vessels were sealed to minimise any loss due to volatility, it is considered justifiable to base the biological effects on the initial measured concentrations. The 72 h EC50 based on growth rate was determined to be 7.47 and the corresponding 72h NOEC to be 1.68 mg/L.

The 72h EC50 and NOEC values for the two source substances are similar and within the same environmental classification band. Used in a weight of evidence approach, they are considered to give a reliable estimate of the toxicity to freshwater green algae of (E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one, which is a 72h ErC50 of > 5.1 and <= 7.47 mg/L and a 72h NOEC of 1.68 to 2.3 mg/L. The read-across is justified and considered adequate for the purposes of classification and risk assessment.