3-methyl-5-propylcyclohex-2-enone

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June-August 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid GLP compliant study is available for the analogue substance, Azarbre, which was conducted in accordance to internationally accepted test guidelines with no or minor deviations and is considered reliable without restriction. As the study is being used in a read-across approach, it is rated reliability 2 (the maximum for read-across). The read-across is an analogue approach based on the hypothesis that the target substance (Celery Ketone) and source substance (Azarbre) will have similar ecotoxicological properties as a result of structural similarity, the same expected mode of action and similar physicochemical properties. Based on the relative hydrophobicity of the two substances, the read-across is conservative and worst-case.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Preliminary solubility work indicated that it was not possible to obtain a testable 100 mg/L stock solution using traditional methods of preparation e.g. ultrasonication and high shear mixing, and so the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).

A media preparation trial determined that a prolonged stir method could produce a 100 mg/L solution.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diethyldimethylcyclohex-2-en-1-one

EC Number:

272-462-3

EC Name:

Diethyldimethylcyclohex-2-en-1-one

Cas Number:

68845-36-3

IUPAC Name:

2,3-diethyl-4,4-dimethylcyclohex-2-en-1-one

Constituent 2

Reference substance name:

Azarbre

IUPAC Name:

Azarbre

Test material form:

other: liquid

Details on test material:

- Molecular formula (if other than submission substance): C12H20O
- Molecular weight (if other than submission substance): 180
- Smiles notation (if other than submission substance): O=C1[C@H](C)[C@](C)(CC)CC(CC)=C1 and C1(=O)C(C)=C(CC)CC(C)(CC)C1
- Structural formula attached as image file (if other than submission substance): see Fig 1
- Name of test material (as cited in study report): Diethyldimethylcyclohex-2-en-1-one
- Substance type: Pure active substance
- Physical state: Extremely pale yellow liquid
- Analytical purity: 99.78% purity (sum of isomers)
- Impurities (identity and concentrations): Not applicable
- Composition of test material, percentage of components: Diethyldimethylcyclohex-2-en-1-one, 99.78%
- Isomers composition: Not applicable
- Purity test date: Not available
- Lot/batch No.: SC00009973
- Expiration date of the lot/batch: 15 September 2014
- Stability under test conditions: Not available
- Storage condition of test material: Room temperature, protected from light

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 4.0Pa at 25'C
- Water solubility: 0.496 mg/L
- log Pow: 3.1-3.3


OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: not readily biodegradable
- Results for algal growth inhibition test: 72h ErC50 = 12mg/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Not applicable

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (1100 mg) was dissolved in 11 litres of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give the 100 mg/L test concentration.
- Eluate: Deionised reverse osmosis water
- Differential loading: No
- Controls: Test water only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): undissolved test item was filtered off

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: no data
- Source: In-house laboratory cultures
- Age at study initiation (mean and range, SD): neonates <24 hours old (1st instar)
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Method of breeding: parthenogenesis
- Feeding during test: No
- Food type: no data
- Amount: no data
- Frequency: no data

ACCLIMATION
- Acclimation period: Not available
- Acclimation conditions: same as test
- Type and amount of food: mixture of Algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no data

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

250 mg/L as calcium carbonate

Test temperature:

20-21 deg C

pH:

7.7 - 8.0

Dissolved oxygen:

8.8 - 9.2 mg(O2)/L

Salinity:

Not applicable

Nominal and measured concentrations:

0.10, 1.0, 10 and 100 mg/L nominal test item concentrations (range finding test). Measured concentrations were in the range of 82 % to 90 % of the nominal concentrations.

10, 18, 32, 56 and 100 mg/L nominal test item concentrations (definitive test) . Measured concentrations were in the range of 81 to 88 % of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not available
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium): CaCl2.2H2O (294 mg/L); MgSO4.7H2O (123 mg/L); NaHCO3 (64.75 mg/L); KCl (5.75 mg/L)
- Culture medium different from test medium: Yes, Elendt M7 was used as culture medium
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: If necessary with NaOH or HCl
- Photoperiod: 16 hours light, 8 hours darkness (20 minute dawn and dusk intervals)
- Light intensity: 502-521 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility at 24 and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.8
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10, 100 mg/L in range finding study
- Results used to determine the conditions for the definitive study: Noimmobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However immobilisation was observed at 100 mg/L. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were used for the definitive test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Remarks on result:

other: 30-42 mg/L (95% CL)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Details on results:

No detailed results from the tested concentrations are presented in the report.
Other biological observations: No data

READ

Results with reference substance (positive control):

- Results with reference substance valid? Yes, within normal range (0.6-2.1 mg/L; EC50-24 hour)
- Mortality: None stated
- EC50: 0.87 mg/L (24 hour); 0.71 mg/L (48 hour)

Any other information on results incl. tables

Cumulative immobilisation data in the definitive test

Nominal concentration (mg/L)	Cumulative immobilised Daphnia (Initiall population: 5 per replicate)
	24 hours						48 hours
	1	2	3	4	Total	%	1	2	3	4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0	0	0	0	0
32	3	2	2	1	8	40	5	3	2	1	11	55
56	3	1	3	5	12	60	4	3	4	5	16	80
100	4	5	5	4	18	90	5	5	5	5	20	100

ANALOGUE APPROACH JUSTIFICATION:

- See Attached " Daphnia Toxicity Read-Across Justification for Celery Ketone" document for full details.

 

In summary, the read-across is an analogue approach based on the hypothesis that the target substance (Celery Ketone) and source substance (Azarbre) will have similar ecotoxicological properties as a result of structural similarity, the same expected mode of action and similar physicochemical properties. Important considerations for the use of the read-across are:

- There are structural similarities between the two chemicals. Both substances are alkyl substituted cyclohex-2-enones. The difference in alkyl groups and substitution pattern are not expected to affect the aquatic toxicity behaviour of the two substances.

- Both substances are expected to act via the same mode of action for aquatic toxicity

- There are no impurities that are expected to affect the ecotoxicological properties of the source and target substance.

- Both substances have similar physico-chemical properties relevant for fate and behaviour of the substance under aquatic toxicity test conditions (e.g. vapour pressure, water solubility and log Kow). Both substances are not readily biodegradable.

- Hydrophobicity (as modelled by log Kow) is a known determinant of the toxicity in aquatic organisms. The measured log Kow values for Celery Ketone and Azarbre, determined under identical conditions, are 2.5 and 3.1-3.3 respectively. As the source substance (Azarbre) has the higher log Kow it is expected to exhibit the highest aquatic toxicity of the two substances and thus read-across of aquatic toxicity data from this source substance to Celery Ketone will be conservative and worst-case.

- Both chemicals have been tested for growth inhibition to algae. The 72h ErC50 for Azarbre of 12 mg/L is slightly lower than the value of 23 mg/L for Celery ketone confirming that read-cross from Azarbre to Celery ketone for other aquatic toxicity endpoints is expected to be conservative.

- The algae 72h ErC50 results for both substances and the daphnia 48h EC50 for the source substance are in the same classification band (10-100mg/L).

Based on the above, the read-across is considered adequate for the purpose of classification and labelling and/or risk assessment

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a guideline study, conducted according to GLP, the closely-related substance Azarbre was found to have an EC50 (48 hr) of 35 mg/L.

The detailed information provided in the attached read-across justification document indicates that the aquatic ecotoxicity of Celery Ketone (target substance) and Azarbre (source substance) are expected to be similar as a result of structural similarity, similar mechanistic profiles and similar physicochemical properties. In fact, based on the relative hydrophobicity (as modelled by log Kow), the strength of effect(s) in the target substance is expected to be slightly lower than that observed for the source substance. Hence the prediction constitutes a worst case.

Therefore, read-across from Azarbre is considered to give a reliable conservative estimate of the daphnia 48-hr LC50 for Celery Ketone, which is a value of 12 mg/L

The read-across is justified and considered adequate for the purpose of classification and labelling and/or risk assessment.

Executive summary:

In an OECD guideline study (TG 202), to GLP, Azarbre was observed to have a 48-hour Daphnid EC50 of 35 mg/L (95% CL = 30 - 42 mg/L). The results were based on nominal concentrations as measured concentrations were > 80% nominal. The test was considered to be valid given that none of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was greater than or equal to 3 mg/L in the control and test vessels.