Reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 3, 2003 to November 26, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Percent Active Ingredient Cited in report: 94.4% (sum of isomers); 78.0% (normal isomers of methylionone)
Supplier component data for Product Code 8620103 (in-house information, not cited in report): typically n-alpha 60%, n-beta 15%, iso-alpha 10%

Analytical monitoring:

yes

Details on sampling:

Analytical samples (approximately 10 mL) were collected into 40 mL glass VOA vials that contained 2 mL of 0.01M H2SO4 and 0.5 mL methanol in HPLC water. Samples were derivatized by adding 2 mL of 0.001M 2,4-dinitrophenylhydrazine in acetonitrile and 0.5 mL of concentrated phosphoric acid to each vial. The vials were capped, shaken, and allowed to derivatize for approximately 1 hour at approximately 50°C. A 5 mL aliquot of each derivatized sample was then brought to a total volume of 10 mL with 50/50 acetonitrile/HPLC water. 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.

Analytical confirmation of the test substance was performed at 0 and 72 hours in the following:
- the control and 5 test item concentrations
- stability sample, prepared with a nominal concentration of 16 mg/L which was not inoculated with algae, was incubated among the test vessels during the 72 hour exposure
- Laboratory control samples, 4mg/L standards prepared in dilution water

Vehicle:

no

Details on test solutions:

A 16 mg/L stock solution was prepared by weighing 0.0320 g of test substance into a 2,000 mL Class A glass volumetric flask and adjusting the volume of dilution water to the line. This stock solution was mixed and a series of solutions was then prepared on by bringing 31, 63, 126, 250, 500, and 1,000 mL of the 16 mg/L stock solution to 1,000 mL with dilution water. Nominal concentrations of methyl ionone were 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, 8.0, and 16 mg/L. A portion of each solution was transferred into a 1.0 L glass beaker and a 1.0 L portion of dilution water was also transferred to a glass beaker to serve as a control. A stability sample, prepared with a nominal concentration of 16 mg/L which was not inoculated with algae, was incubated among the test vessels during the 72 hour exposure.

Test organisms (species):

Selenastrum sp.

Details on test organisms:

Selenastrum capricornutum, UTEX 1648

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

A 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (2.94, 6.74, and 14.0 mg/L methyl ionone concentrations) was combined in a 250-mL flask with 100 mL of fresh media to determine whether toxic effects were algicidal or algistatic. These cultures were incubated under test conditions for 72 to 120 hours.

Test temperature:

24 ± 2°C

pH:

7.5 +/- 0.1

Nominal and measured concentrations:

Nominal concentrations of methyl ionone were 0 mg/L (control), 0.50, 1.0, 2.0, 4.0, 8.0, and 16 mg/L.
Initial measured concentrations of methyl ionone 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.

Details on test conditions:

Solutions were subdivided into 11 clear glass 40 mL vials for each treatment (the control was subdivided into 20 replicates) and the vials, which were filled to capacity to eliminate any head space, were sealed with Teflon-lined caps. Test vessels were randomly arranged on a rotary shaker adjusted to approximately 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel) and vessels were repositioned daily. A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 400 to 420 footcandles (approximately 54 μEin/m2sec).

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer. At 24, 48, and 72 hours, three treatment vessels and six control vessels were randomly selected and sacrificed (opened to the atmosphere) to allow daily determination of the number of algal cells/mL. The remaining two vessels at each concentration were used for the determination of methyl ionone concentration at the end of the test.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.68 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on the number of cells and area under the growth curve were also reported (see attached full study report and conclusions). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Initial measured concentrations were used for all calculations, which 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 which was 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, which is in the same range of the 51-62% obtained for the test samples indicating that the decrease in measured concentrations seen 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.

At the conclusion of the definitive toxicity test, a 0.5 mL aliquot of test media from each test vessel where growth was maximally inhibited (the 2.94, 6.74, and 14.0 mg/L methyl ionone concentrations) was combined with fresh media. These cultures were incubated under test conditions for 72 to 120 hours. During this period the number of algal cells increased from an initial concentration of approximately 3,200 cells/mL to approximately 322,000 cells/mL at 2.94 mg/L, from 1,450 cells/mL to approximately 152,000 cells/mL at 6.74 mg/L, and from 270 cells/mL to 202,000 cells/mL at 14.0 mg/L, indicating that the toxic effects were algistatic rather than algicidal.

Validity criteria fulfilled:

yes

Conclusions:

In a guideline study, conducted according to GLP, exposure of Selenastrum capricornutum to methyl ionone (test item: Raldeine A GD) for 72 hours resulted in an EC50 of 7.47 mg/L when calculated using the average specific growth rate, 3.23 mg/L when calculated using the number of cells/mL, and 2.89 mg/L when calculated using the area under the growth curve. The 72 hour NOEC is 1.68 mg/L methyl ionone when determined using the number of cells/mL or the average specific growth rate, and 0.404 when calculated using the area under the growth curve.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v2.0, OECD 201 Guideline). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus the 72-h EC50 and NOEC based on growth rate have been selected as the key values for classification and risk assessment purposes, which were determined in this study to be 7.47 mg/L and 1.68 mg/L respectively.

These values are being used in to read-across to the registration substance, “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one” (see target endpoint record).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

A detailed read-across justification in-line with the ECHA RAAF guidelines is provided as an attached document in this IUCLID endpoint record. In summary, the read-across is based on the hypothesis that the target and three source substances have similar ecotoxicological properties as a result of structural similarity, the same expected mode of action and similar physicochemical properties. The target and three source substances consist of methyl ionone isomers (C14H22O, MW 206.33). The substances differ in the ratio of the isomers present and whether individual isomers are considered as constituents (≥10%) or impurities (<10%) under the REACH naming convention. The registered substance (target) is a multi-constituent substance consisting of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one (concentration range 10-50%) and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one (30-77%). These two constituents are also present in the three source substances. Constituent 2 (trivial name = alpha-n-methylionone), is a major constituent in all three source substances present at 22-26% (Source substance 1), 60% (source substance 2) and >10 to < 30% (source substance 3). Constituent 1 (common name = beta-n-methylionone) is considered an impurity in source substance 1 (4.5 to 6.5%) and 3 (8.2%) and a major constituent in source substance 2 (typically 15%). Source substances 2 and 3 also have the alpha-iso-methylionone isomer as a main constituent (approximately 60%). All of the aforementioned isomers have the same functional group chemistry (vinyl ketones). The only structural difference between them is the position of the double bond associated with the cyclohexyl ring and/or the position of the methyl group in the side chain (see generic structure, attached in illustration section of this IUCLID endpoint record). These slight differences will not affect the mode of action. This is supported by the OECD QSAR toolbox aquatic toxicity profiles which are identical for all these isomers. Given also the similar lipophilicity of the isomers (as modelled by log Kow), it is expected that all individual isomers will exhibit a similar strength of aquatic toxicity and that any variation in the ratio of isomers between substances will not significantly influence the aquatic toxicity properties. Any minor components present in either the target or 3 source substances are also methyl ionone structural isomers and thus will not impact the prediction.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.47 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Raldeine A GD

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.68 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Raldeine A GD

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Methyl Ionone Alpha Iso 60

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Methyl Ionone Alpha Iso 60

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Methylionon 70

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: read-across from Methylionon 70

Details on results:

The EC50 values of > 5.1mg/L and > 9.42 mg/L represent the fact that only 33% inhibition was observed for Methyl ionone Alpha Iso 60 and no significant inhibition based on growth rate for Methylionon 70 at the highest test concentrations of 5.1 and 9.42mg/L respectively. Therefore the determined value of 7.47 mg/L has been chosen as the key value for chemical safety assessment. Out of the NOEC values, the lowest and more conservative NOEC of 1.68 mg/L has been selected. Furthermore, these key values are both derived from the Raldeine A GD study, which out of the three source substances has the closest isomeric composition to that of the target.

Conclusions:

Valid algal inhibition studies exist for three analogue substances; Methyl Ionone Alpha Iso 60, Raldeine A GD and Methylionon 70. All studies are GLP compliant, conducted to OECD 202 guidelines, covered the required exposure duration of 72 hours and included sufficient dose levels to enable the relevant determination of potency. The 72 ErC50 value from the respective studies was determined to be > 5.1, 7.47 mg/L and > 9.42mg/L and the corresponding NOEC values are 2.3, 1.68 and ≥ 9.42 mg/L - see source study records for details.

The detailed information provided in the attached read-across justification document indicates that the aquatic ecotoxicity of “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one (target substance)” and the three source sub-stances are expected to be similar as a result of structural similarity. Both the target and source sub-stances are made up of various methyl ionone isomers, which are expected to exhibit a similar strength of aquatic toxicity based on the same expected mode of action and similar lipophilicity. This is supported by the above algae ErC50 and NOEC results for the three source substances, which show that this family of isomeric substances exhibit similar aquatic toxicity irrespective of the isomer ratios present.

Therefore, the above effect values are considered to give a reliable estimate of the toxicity to aquatic algae of the registered material, “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one”. The determined 72h ErC50 value of 7.47 mg/L and the lowest NOEC of 1.68mg/L have been selected as the key values for chemical safety assessment. Furthermore, these key values are both derived from the Raldeine A GD study, which out of the three source substances has the closest isomeric composition to that of the target.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 24/6/96 to 27/6/96

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Specific details on test material used for the study:

Commerial name: Methyl Ionone Alpha ISO 60
Manufacturer's Code : 260208 SR 86352
Chemical name: 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-methyl-3-buten-2-one
Composition (cited in report): Major component = Methyl Ionone alpha iso isomer; minor components = methyl ionone alpha normal isomer, methyl ionone beta normal isomer, methyl ionone beta iso isomer and methyl ionone gamma isomer
Purity (cited in report): 53-58% alpha-iso-isomer
Sample tested as 100% active.

Supplier historical QC data (not included in report):
GLC total isomers by rpa = 93-97% (specification > 92%)
alpha iso methyl ionone = 55-62%
alpha n methyl ionone = 22-26%
beta n methyl ionone = 4.5-6.5%
gamma n methyl ionone < 4%

Analytical monitoring:

yes

Details on sampling:

Concentrations of the test item were determined at 0, 24, 48 and 72 hours. The control and the five test concentrations were analysed.

Vehicle:

no

Details on test solutions:

A nominal 500 mg/L stock was prepared by dispersing 0.250 g of Methyl Ionone Alpha ISO 60 in 500ml of distilled water by warming to 50'C and then sonicating at maximum energy for 10 minutes prior to being mixed with a high shear mixer at 12,000 rpm for 15 minutes. A range of test substance concentrations (nominal 0, 5.6, 10, 18, 32 and 56 mg/L) was prepared by diluting the stock with ISO medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The mean measured 0 hours algal density was 1.25E +04 cells/ml.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

23-25°C

pH:

Test media pHs at 0 hours : 7-0-7.6
Test media pHs at end of test : 7.6-9.7

Nominal and measured concentrations:

Nominal: 5.6, 10, 18, 32, 56 mg/L
0 hours: 1.1, 1.4, 3.1, 5.3, 12 mg/L
24 hours: 0.47, 0.63, 1.5, 3.5, 7.2 mg/L
48 hours: 0.42, 0.51, 0.80, 1.7, 4.0 mg/L
72 hours: 0.27, 0.31, 0.44, 0.87, 1.9 mg/L
Geometric mean: 0.49, 0.61, 1.1, 2.3, 5.1 mg/L

Details on test conditions:

Test vessels were loosely stoppered and placed according to a randomised distribution in an orbital illuminated incubator. The algal concentrations in the test media were determined at 0, 24, 48 and 72 hours using a Coulter Counter.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5.1 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Remarks on result:

other: 33% inhibition of growth at highest test concentration of 5.1mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Details on results:

Analysis of the test solutions was performed at 0, 24, 48 and 72 hours by GC-MS. 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.

Effects based on biomass and growth rate were determined (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Validity criteria fulfilled:

yes

Remarks:

65-fold mean increase in cell density of control cultures over 72h (OECD201 criteria is at least 16-fold growth)

Conclusions:

In a guideline study, conducted according to GLP, the toxicity of Methyl Ionone Alpha Iso 60 to the freshwater green alga Scenedesmus subspicatus was determined. The effect concentrations were based on geometric means of the measured concentrations (0, 24, 48 and 72 hour). After 72 hours, the percentage inhibition of growth rate at the highest test concentration of 5.1 mg/L was 33%. Thus the 72h EC50 is considered to be > 5.1mg/L. The corresponding NOEC was 2.3 mg/L.

These values are being used to read-across to the registration substance, “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one” (see target endpoint record).

Executive summary:

In a guideline study, conducted according to GLP, the toxicity of Methyl Ionone Alpha Iso 60 to the freshwater green alga Scenedesmus subspicatus was determined. The effect concentrations were based on geometric means of the measured concentrations (0, 24, 48 and 72 hour). After 72 hours, the percentage inhibition of growth rate at the highest test concentration of 5.1 mg/L was 33%. Thus the 72h EC50 is considered to be > 5.1mg/L. The corresponding NOEC was 2.3 mg/L.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to reaction mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Methylionon 70
- Lot/batch No.: ch. 1436 7956 P0
- Analytical purity: 95.3 area %

Analytical monitoring:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation according to OECD guideline 23 (water accomodated fraction). Each test concentration was prepared separately.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Collection of algal cultures, University of Goettingen, Germany

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9 - 23.2 °C

Nominal and measured concentrations:

- Nominal test concentrations (mg/l): 0, 4.6, 10, 22, 46 and 100
- Measured concentrations (mg/l): 2.49, 4.45, 7.67, 9.68, and 9.42

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (250ml) closed with gas permeable silicone sponge caps.
- Fill volume: 100 ml
- Initial cells density: 5000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates):

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD guideline 201

OTHER TEST CONDITIONS
- Photoperiod: permanent illumination
- Light intensity and quality: actificial white, universal white, 5240 lux, wave length 400-700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: pulsed excitation with light flashes having a wavelength of 432 nm

TEST CONCENTRATIONS
- Range finding study: EC50 fluorescence: >100 mg/l, ErC50 > 100 mg/l

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Since the measured concentrations deviated markedly from the loading rates, the effect concentration based on the mean measured concentrations have been selected as the key values for the evaluation of this substance.

The study report also contain effects based on yield (see attached full study report). However, the preferred observational endpoint in the algal inhibition study is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v 2.0, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. Thus only the effects based on growth rate are presented in the above "effects concentration" table.

Validity criteria fulfilled:

not specified

Conclusions:

The 72-h ErC50 and 72-h NOErC values are >9.4 mg/L, and ≥9.4 mg/L, respectively, in freshwater green algae (D. subspicatus).

These values are being used to read-across to the registration substance, “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one” (see target endpoint record).

Executive summary:

An algae toxicity test was performed according to OECD TG 201 and in compliance with GLP. Cultures of freshwater green algae (D. subspicatus; initial cell density of ca. 5E+03 cells/mL), were exposed for 72 hours to test solutions of Ionone, methyl- prepared as water accommodated fractions of loading rates of 4.6, 10, 22, 46 and 100 mg/L. Test substance analysis was performed and measured concentrations determined to be 2.49, 4.45, 7.67, 9.68 and 9.42 mg/L. Chlorophyll measurements were performed throughout the test and growth rates at the different test concentrations determined.  At the end of the test, no effects were seen up to the solubility limit in test medium. The 72-h ErC50 and 72-h NOErC were determined at >9.4 mg/L and ≥9.4 mg/L, respectively (based on measured concentrations).

Description of key information

Based on valid algal inhibition studies for three 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 based on 72h ErC50 values of > 5.1 mg/L ((Methyl Ionone Alpha Iso 60, source study), 7.47 mg/L (Raldeine A GD, source study) and > 9.42mg/L (Methylionon 70, source study). The corresponding NOEC values are 2.3, 1.68 and ≥ 9.42 mg/L.

The EC50 values of > 5.1mg/L and > 9.42 mg/L represent the fact that only 33% inhibition and no significant inhibition based on growth rate were observed at the highest test concentrations in the respective studies. Therefore the determined value of 7.47 mg/L has been chosen as the key value for chemical safety assessment. Out of the NOEC values, the lowest and more conservative NOEC of 1.68 mg/L has been selected. Furthermore, these key values are both derived from the Raldeine A GD study, which out of the three source substances has the closest isomeric composition to that of the target.

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 three analogue substances; Methyl Ionone Alpha Iso 60 (Unilever 1997), Raldeine A GD (Ward 2003) and Methylionon 70 (BASF 2010). All 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 three analogue substances is considered to give a reliable estimate of the toxicity of “Reaction Mass of 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)pent-1-en-3-one and 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)pent-1-en-3-one (target substance)” to aquatic invertebrates and is justified based on the hypothesis that the target substance and source substances will have similar ecotoxicological properties as a result of structural similarity. Both the target and source substances are made up of various methyl ionone isomers, which have the same expected mode of action for aquatic toxicity and similar physicochemical properties relevant for the read-across ecotoxicological endpoint. A detailed justification for the proposed read-across in-line with the ECHA RAAF guidelines is provided in the target endpoint record. A summary of the three source studies is provided below.

The study performed on Methyl Ionone Alpha Iso 60 (Unilever 1997, 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 (Ward 2003, 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 study performed on Methylionon 70 (BASF 2010, Study No 60E0397/093104) was conducted under static conditions using test material (95.3% purity, mixture of isomers) that was representative of the source substance. Test solutions were prepared as water accommodated fractions (WAFs) using loading rates of 4.6, 10, 22, 46 and 100mg/L. Samples for concentration analysis were taken at the start (0h) and end of exposure (72h). The mean measured concentrations were determined to be 2.49, 4.45, 7.67, 9.68 and 9.42 mg/L. The study is considered reliable without restrictions (reliability 1). Since the measured concentrations deviated markedly from the loading rates, the effect concentration based on the mean measured concentrations was used for the evaluation of this substance. The 72 hr EC50 was determined to be > 9.42mg/L.

The 72h EC50 and NOEC values for the three source substances are similar. Further, the three source substances cover “methyl ionone” qualities with varying isomer ratios. Thus the similar effect values support the hypothesis that the various methyl ionone isomers exhibited a similar strength of aquatic toxicity based on the same expected mode of action for aquatic toxicity and similar lipophilicity; and that any variation in the ratio of isomers between substances will not significantly influence the aquatic toxicity properties. Therefore the read-across to the target substance is justified and considered adequate for the purposes of classification and risk assessment.