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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 3, 2003 to November 24, 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 202 (Daphnia sp. Acute Immobilisation 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:

Samples were taken from the control and each test concentration at the start and end of each renewal period i.e. 0 hour, 24 hour (before media renewal), 24 hour (after media renewal) and 48 hour.

Analytical samples (10 mL) were collected into 40 mL glass VOA vials containing 2.0 mL of 0.01 M H2SO4 (test solution samples containing the test substance were diluted with dilution water prior to collection into the VOA vials). A 2.0 mL aliquot of 0.001 M 2,4-dinitrophenylhydrazine in acetonitrile and 0.5 mL of concentrated phosphoric acid were added to each vial. The vials were shaken and set aside for approximately1 hour to derivitize. A 5.0 mL aliquot of each derivatized solution was then added to a 10 mL class A volumetric flask and the volume made to the mark with a 50/50 solution of acetonitrile and HPLC grade water. An aliquot of sample was transferred into an autosampler vial and analyzed by HPLC/UV (Hewlett Packard 1100 Series HPLC).

Vehicle:

no

Details on test solutions:

Stock solutions were prepared at 10 mg/L on June 30 and July 1, 2003, by bringing 0.0100 g of test substance to 1,000 mL with dilution water in a Class A volumetric flask. The solution was mixed on a magnetic stirrer for approximately 6 hours. A series of solutions was then prepared by bringing appropriate volumes of stock solution to 250 mL with dilution water in Class A volumetric flasks.

Test organisms (species):

Daphnia magna

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

48 h

Post exposure observation period:

The numbers of surviving organisms, the occurence of immobility and sublethal effects, and the presence of insoluble material were determined visually and recorded after 0, 24 and 48 hours.

Test temperature:

20 ± 1°C

Nominal and measured concentrations:

Nominal concentrations were 0 mg/L (control); 0.78; 1.3; 2.2; 3.6 and 6.0 mg/L.
Mean measured concentrations were: ND (none detected at or above the limit of quantitation; control), 0.621, 1.14, 1.82, 2.99, and 5.32 mg/L.

Details on test conditions:

The test was performed under static, renewal conditions in sealed vials with no head space to minimize the potential loss of the test substance from test solutions to the atmosphere (media was renewed in each test vessel after 24 hours).

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

4.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobility and sublethal effects

Remarks on result:

other: 95% confidence level = 3.40 to 6.30 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobility and sublethal effects

Remarks on result:

other: 95% confidence level = 1.82 to 5.32 mg/L

Details on results:

The 24h and 48h LC50 values based on mortality were also determined, these were respectively 5.07 mg/L and 3.11 mg/L. The 48 hour NOEC was 1.14 mg/L.

Measured concentrations at 0 hour, 24 hours (before media renewal), 24 hours (after media renewal) and at 48 hours showed that the test item was stable (see table below). The mean measured concentrations were 80-89% of the nominal. The effect and mortality results were calculated based on the mean measured concentrations, which were 0.621, 1.14, 1.82, 2.99 and 5.32 mg/L.

Measured concentrations of methyl ionone in test media during the toxicity test with the daphnid, Daphnia magna. (taken from Table 2 in report)

Nominal Concentration (mg/L)	Measured Concentration (mg/L)					Percent of nominal
	0 hour	24 hours	24 hours	48 hour	mean
0 (control)	ND	ND	ND	ND	ND	--
0.78	0.694	0.551	0.603	0.634	0.621	80
1.3	1.42	0.978	1.08	1.08	1.14	88
2.2	2.22	1.60	1.63	1.81	1.82	83
3.6	3.50	2.22	2.88	3.36	2.99	83
6.0	6.35	4.44	5.23	5.27	5.32	89

Validity criteria fulfilled:

yes

Conclusions:

Exposure of daphnids to methyl ionone resulted in a 24 hour EC50 of 4.34 mg/L (95% confidence interval = 3.48 to 6.30 mg/L) and a 48 hour EC50 of 2.65 mg/L (95% confidence interval = 1.82 to 5.32 mg/L). This exposure also resulted in a 24 hour LC50 of 5.07 mg/L (95% confidence interval = 3.88 to >5.32 mg/L) and a 48 hour LC50 of 3.11 mg/L (95% confidence interval = 1.82 to 5.32 mg/L). The 48 hour no observed effect concentration (NOEC) was 1.14 mg/L. All effect values are based on mean measured concentrations.

For the purpose of classification and labelling the 48 hr EC50 is relevant. The determined value of 2.65 mg/L for methyl ionone (test material: Raldeine A GD) is 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).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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 sup-ported 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:

48 h

Dose descriptor:

EC50

Effect conc.:

2.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

read-across / source substance; methyl ionone, trade quality Raldeine A GD

Basis for effect:

mobility

Remarks on result:

other: Read-across from Raldeine A GD

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Read-across from Methylionon 70

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.4 - < 4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

based on 100% active test item Methyl ionone alpha

Basis for effect:

mobility

Remarks on result:

other: Read-across from Methyl Ionone Alpha Iso 60

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

based on 100% active test item

Basis for effect:

mobility

Remarks on result:

other: Read-across from Methyl Ionone Alpha Iso 60

Conclusions:

Valid short-term daphnia 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 48 hours and included sufficient dose levels to enable the relevant determination of potency. The EC50 value from each study was determined to be =>2.05 and < 4.1 mg/L (source substance 1, study reliability 2), 2.65 mg/L (source substance 2, study reliability 1) and 3.7mg/L (source substance 3, study reliability 1) - 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 daphnia 48h EC50 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 EC50 results are considered to give a reliable estimate of the 48h EC50 for 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”. Furthermore, the EC50 values obtained from the three source studies are within the same environmental classification band and as such are considered adequate for regulatory purposes. The value of 2.65 mg/L has been selected as the key value for chemical safety assessment because it is the lowest value of the reliable 1 studies and the composition of source substance is closest to that of the target.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 20/06/1994 to 23/06/1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Although a GLP guideline study, analytical monitoring was based on TOC and not a chemical specific technique. Further the documentation within the report is limited.

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 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes

Specific details on test material used for the study:

Sample Code : N51
Manufacturer's Code : 260208 S/R 86352
Chemical name: 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-methyl-3-buten-2-one
Purity cited in report: 53-58% alpha-iso-isomer, mean of 55.5% used in calculation of nominal stock solution concentration as alpha-iso-isomer.
Composition cited in report: Other components are methyl ionone alpha normal, methyl ionone beta normal, methyl ionone beta iso and methyl ionone gamma

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:

Samples were taken from the control and each test concentration at Day -1, Day 0 and Day 2.

Vehicle:

no

Details on test solutions:

The stock solution in this study was prepared by dispersing 0.18g of test item in 1 litre of Elendt M7 medium to produce a 100mg/L as alpha-iso-isomer, 180 mg/L as commercial sample stock. The dispersion was achieved by mixing this stock using a high shear mixer for approximately 5 minutes at 12-14000rpm. test solutions were preared be adding known volumes of stock to 2000 ml Elendt medium. These test solutions were prepared 24 hours before the daphnia were added to allow the test substance to equilibrate.

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

249 mg/l as CaCO3

Test temperature:

20.4

pH:

7.5

Dissolved oxygen:

7.5 mg/l

Nominal and measured concentrations:

Nominal (expressed as 100% test item): 0.0 (control), 1.8, 5.8, 10, 18, 32 mg/L
Measured Mean TOC concentration attributable to test item: - (control), 0.0, 0.8, 1.8, 3.3, 5.4 mg/L (taken from Table 3, column 2 in study report)
Measured Concentration of test substance, calculated from mean TOC concentration: - (control), 0.0, 1.8, 4.0, 7.3, 12 mg/L (taken from Table 3, column 4 in studyreport, based on 81.42% calculated carbon content and corrected for 55.5% purity)
Measured Concentration of test substance - (control), 0.0, 1.0, 2.2, 4.0, 6.6 mg/L (calculated by registrant from mean TOC concentration attributable to test item, based on 81.42% calculated carbon content)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

5.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

calculated from TOC, 81.43% carbon content and corrected for 55.5% purity

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

calculated from TOC, 81.43% carbon content and corrected for 55.5% purity

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

calculated from TOC, 81.43% carbon content and corrected for 55.5% purity

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

calculated from TOC, 81.43% carbon content and corrected for 55.5% purity

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.4 - < 4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

based on 100% active test item

Basis for effect:

mobility

Remarks on result:

other: Estimated from calculated concentration of total test item and visual inspection of the data

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

based on 100% active test item

Basis for effect:

mobility

Remarks on result:

other: Reported value of 3.7mg/L multiplied by 0.555.

Details on results:

TOC analysis of the test solutions showed that the amount of test substance remained relatively stable throughout the test.

The effect results are reported to be based on calculated concentrations of the test substance (using measured TOC concentrations). The concentrations of test substance based on mean measured TOC (mg/L) reported in Table 3 of the study report (column 4) are 0.0 (control), 0.0, 1.8, 4.0 , 7.3 and 12 mg/L. The corresponding percentage immobile daphnia at these concentrations is reported in Table 4 of the study report and are:
24 hrs: 10 (control), 0, 0, 5, 100 and 100%
48 hrs: 10 (control), 0, 10, 40, 100 and 100%
Based on a visual inspection of this data, one would expect the 24h EC50 and 48h E50 to be > 4mg/L and < 7.3 mg/L. The reported 24 and 48 EC50 values calculated by the Binominal and Probit methods are respectively 5.2 and 3.7 mg/L.

From an inspection of Table 3 in the study report, the above concentrations have been obtained by converting the mean TOC concentration attributable to the test substance based on the carbon content (i.e. divided by 0.8143 ) and then correcting for the alpha-iso-isomer purity of 55.5% (i.e. divided by 0.555). Given that the other reported components present in the test item are isomers of the major constituent (i.e. purity > 92%, total isomers) and will contribute to the toxicity of the test item, the correction of test substance concentration based on the alpha-iso-isomer purity of 55.5% is not considered appropriate by the registrant. To this end, the registrant has converted the mean TOC concentrations attributable to the test substance of 0.0, 0.8, 1.8, 3.3 and 5.4 mg/L (column 2 of table 3) to test substance concentrations of 0.0, 1.0, 2.2, 4.1 and 6.6 mg/L (i.e. corrected for carbon content by dividing by 0.8143 and assuming 100% active test substance). The corresponding percentage immobile data at these concentrations after 48 hours is 0, 10, 40, 100 and 100% respectively giving an expected 48h EC50 of >2.2 and <4.1 mg/L. Alternatively the 48h EC50 for the total test item can be estimated from the reported value of 3.7mg/L (corrected for alpha-iso-isomer purity of 55.5% purity) by multiplying by 0.555, which gives a value of 2.05 mg/L.

Validity criteria fulfilled:

yes

Remarks:

The dissolved O2 concentration criteria at test end of => 3mg/L cannot be assessed as only the mean value (7.5mg/L, 0-48h) is reported. However, based on the 10% immobile daphnia in the control (criteria <= 10%) the test is considered valid.

Conclusions:

In a guideline study, conducted according to GLP, the exposure of daphnids to Methyl Ionone Alpha Iso 60 resulted in a 24 hour EC50 of 5.2 mg/L (95% confidence interval of 4.0-7.3 mg/L) and a 48 hour EC50 of 3.7 mg/L (95% confidence interval of 3.0-4.5 mg/L). These EC50 values are based on calculated concentrations of the test substance (using measured TOC concentrations) and calculated by the Binomial and Probit methods respectively. The calculated concentrations were corrected for an alpha-iso-isomer purity of 55.5%. Given that the other components present in the test item are isomers of the major constituent and will contribute to the overall toxicity of the test item, the correction for the main isomer purity is not considered appropriate by the lead registrant. Thus the EC50 based on the assumption of 100% active test item was estimated. Two approaches were used giving a value of 2.05 mg/L and a range of > 2.2 and < 4.1 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).

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation 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: Water Accomodated Fractions were prepared. The test concentrations were prepared separately according to OECD 23

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Originally obtained from the Institut National de Recherche Chemique Appliquee, France, cultured at test facility since 1978
- Age at study initiation (mean and range, SD): < 24 h

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.2 - 3.2 mmol/l

pH:

8.1 - 8.2

Dissolved oxygen:

8.5 - 8.8 mg/l

Nominal and measured concentrations:

- Nominal test concentrations (mg/l), Loading rates of 0 (control), 4.6, 10, 22, 46, 100
- Analytically determined concentration (mg/l), mean measured; not determined (control), 2.4, 4.6, 9.4, 13.6, 12.0

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers (100 ml)
- Fill volume: 50 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 0.1 animals/ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium prepared according to OECD guideline
- Ca/mg ratio:4:1
- Conductivity: 550 - 650 µE/cm

OTHER TEST CONDITIONS
- Photoperiod: no illumination

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility after 0, 24 and 48 h

TEST CONCENTRATIONS
- Range finding study: EC50 (48 h) 10-100 mg/l

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

2.42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

9.41 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

In the study report, the effect results are also report based on nominal loading rate e.g. EC50 = 7.69mg/L based on nominal.

However, since the measured concentrations deviated markedly from the loading rates, the effect concentration based on the mean measured concentrations should preferably be used for the evaluation of this substance. Thus only effect results based on mean measured concentrations are lsited in the table above.

Validity criteria fulfilled:

yes

Remarks:

≤10% immobilisation in the control; O2 concentration > 3mg/L in control and test vessels

Conclusions:

The 48 hour EC50 value for Methylionone 70 in this acute toxicity study with Daphnia magna was 3.7 mg/L based on mean measured concentrations.

This value is 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).

Executive summary:

A Daphnia test was performed according to OECD TG 202 and in compliance with GLP. Groups of daphnids (4 replicates of 5 animals) were exposed for 48 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 mean measured concentrations determined to be 2.4, 4.6, 9.4, 13.6 and 12.0 mg/L. Mobility was recorded at the start of the test and after 24 and 48 hours exposure. Based on mobility numbers, the 48- h EC50 value was determined at 6.79 mg/L (based on nominal concetrations) and 3.7 mg/L (based on measured concentrations).

Description of key information

Based on valid short-term daphnia studies for three closely related structural analogues, the daphnid 48 hour EC50 for the registered substance is estimated to be ≥ 2.05 and < 4.1 mg/L (source substance 1, study reliability 2), 2.65 mg/L (source substance 2, study reliability 1) and 3.7 mg/L (source substance 3, study reliability 1).

The value of 2.65 mg/L has been selected as the key value for chemical safety assessment because it is the lowest value from the two studies rated reliabilty 1 and because the composition of source substance 2 is closest to that of the target.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.65 mg/L

Additional information

A study to assess the short-term toxicity to aquatic invertebrates is not available for the registration substance. However, valid short-term daphnia studies exist for three analogue substances; Methyl Ionone Alpha Iso 60 (Unilever 1994), Raldeine A GD (Ward 2003) and Methyionon 70 (BASF 2010). Read-across from these three source substances is considered to give a reliable estimate of the short-term 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 1994, Study No AT/N51/02) employed a static design. It is GLP compliant, conducted to OECD guideline 202 and using test material (53 -58% main isomer) which is representative of the source substance. The study design covered the required exposure duration of 48 hours and included sufficient dose levels to enable the relevant determination of potency. Analysis of the test solutions was performed at the start of the test (0 hours) and at the end of the exposure period (48 hours). Based on TOC analysis, the amount of test item present was shown to relatively stable throughout the test period. The study was considered reliable with restrictions (reliability 2). The 48 hr EC50 was reported to be 3.7 mg/L (95% confidence interval 3.0 to 4.5 mg/L) based on calculated concentrations of test substance from mean measured TOC concentrations. These calculated concentrations were corrected for an alpha-iso-isomer purity of 55.5%. Given that the other components present in the test item are isomers of the major constituent and will contribute to the overall toxicity of the test item, this correction is not justified. Therefore the EC50 based on the assumption of 100% active test item has been estimated by the registrant. Two approaches were used giving a value of 2.05 mg/L and a range of > 2.2 and < 4.1 mg/L.

The study performed on Methyl Ionone, commercial test item = Raldeine A GD (Ward 2003, Study No 2469 -FF) was conducted under semi-static design with test medium renewal after 24 hours. It is GLP compliant, conducted according to OECD guideline 202 and using test material (94.4% sum of isomers, 78% n-isomers of methyl ionone) which is representative of the source substance. The study design covered the required exposure duration of 48 hours and included sufficient dose levels to enable the relevant determination of potency. Analytical confirmation of the test substance using GLC analysis was performed at 0 hours, 24 hours (before media renewal), 24 hours (after media renewal) and 48 hours. The measured concentrations, the means of which ranged from 80 to 89% of nominal concentrations, were steady throughout the exposure period. The study is considered reliable without restrictions (reliability 1). The 48 hr EC50 was determined to be 2.65 mg/L (95 confidence interval 1.82 to 5.32mg/L) and the 48 hr LC50 to be 3.11 mg/L (95% confidence interval of 1.82 to 5.32 mg/L) based on mean measured concentrations.

The study performed on Methylionon 70 (BASF 2010, Study No 50E0397/093103) was conducted under semi-static design with renewal after 24 hours. The test material (95.3% purity, mixture of isomers) 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 from each freshly prepared solution (0h and 24h) and at the end of each exposure period (after approx 24h and 48h). The mean measured concentrations were determined to be 2.4, 4.6, 9.4, 13.6 and 12.0 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 48 hr EC50 was determined to be 3.7 mg/L (95% confidence interval 3.17 to 4.28mg/L).

The 48h EC50 results for the three source substances are similar and within the same environmental classification band (>1 and < 10 mg/L). Further, the three source substances cover “methyl ionone” qualities with varying isomer ratios. Thus the similar EC50 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.