2,6-dimethyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)cyclohex-2-en-1-ol

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:

disregarded due to major methodological deficiencies

Study period:

From April 23, 2012 to July 20, 2012.

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was performed according to OECD Guideline 202 with GLP certificate. All validity criteria were fulfilled. The test substance is adequately identified, but some data on composition are missing. This study is not reliable due to the high concentration of solvent (acetone) used in combination with the test substance. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not necessary for aquatic toxicity testing except under exceptional circumstances. Furthermore, solvents are generally not appropriate for testing multiconstituent substances, such as this test substance (which is a mixture of isomers), as it may alter the relative composition of the consituents by changing the solubility of one or more components and thereby the toxicity. Furthermore, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L, corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) and represents almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L). Acetone is suspected to have negatively affected the definitive results. Some solvent/substance interactions may have occurred in this study due to this high concentration of acetone.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

yes

Remarks:

The two lowest of test item concentrations (i.e. 0,10 and 0,20 mg/L) were not checked because it was previously demonstrated that such low concentrations were below the limit of quantification.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate signed on March 11, 2011.

Specific details on test material used for the study:

- Physical state : Light yellow translucent liquid
- Storage : The test substance was stored at room temperature protected from direct sun light.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Test item application rate and the stability of the test item in water over the test period were checked by appropriate analytical verification of the test solutions at both test initiation and test completion.
- Recovery of the test item : Four additional tubes were prepared for each test item treatment used for the definitive test. Two replicate tubes were checked at test initiations, and two others were maintained under the same environmental conditions as the test system, ans were used for analytical verification of the test item treatments at the end of test. In both cases the measured concentrations were compared to the nominal values on a percent basis.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Treatment solutions were prepared in acetone. 15 mL capacity glass tubes were filled with 10 mL of M4 mineral medium. See table 1 for information concerning the preparation of the test item treatments.
- Water control : the control tubes received 10 mL of M4 mineral medium.
- Solvent control : another set of test tubes received 0,05 mL of acetone alone so as to ascertain that the vector had no adverse effects towards daphnids.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle: 5 mL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnids
- Source: commercial breeder Phytosafe s.a.r.l, 2 rue Marx Dormoy, 64000 Pau, France
- Age of parental stock (mean and range, SD): The organisms were regularly sub-cultured at the Phytosafe site. Adult females were transferred in a new volume of water before the test initiation. Young daphnids produced within 24 hours were used for the tests.
- Feeding during test : daphnids were not fed during the exposure period.
- Food type: They were fed with living cells of green algae

Test type:

static

Water media type:

other: reconstituted water

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 +/- 1 °C

pH:

pH between 7.6 et 8.3

Dissolved oxygen:

Between 93.8 % and 96.8 %

Nominal and measured concentrations:

The two lowest treatments were under the limit of quantification.
The three highest test item treatments were satisfactorily maintained between 80-120 % of the nominal values over the test period.
Nominal values : 0,40 mg/L; 0,80 mg/L and 1,60 mg/L
Initial medium : 100,9 % ; 100,6 % and 100,9 %
Final medium : 99,1 %; 104,0% and 96,8%

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 mL capacity glass tubes
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: filled with 10 mL

- No. of organisms per vessel: 5 daphnids per unit
- No. of vessels per concentration (replicates): 4 replicate units for each test item treatments (5 different concentrations)
- No. of vessels per control (replicates): 3 replicate units
- No. of vessels per vehicle control (replicates): 3 replicate units


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Each test tube was checked for immobilized daphnids 24 and 48 hours after the start of the test. Any abnormal behavior or appearance, if any, was reported.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.01 - 0.1 - 1.0 - 9.9 - 98.8 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation was observed throughout the test period for the water control and the solvent control. The percentage of immobilisation was 20% as a maximum for the 0.01 and 0.1 mg/L test item treatments. At 1.0 mg/L, no immobilisation was observed after 24 hours of standing but 60% were immobilised at the end of the test. At both 9.9 and 98.8 mg/L all the daphnids were immobilised after 24 hours of testing.

Reference substance (positive control):

yes

Remarks:

The 24h-EC50 for Potassium dichromate was between 0,6 and 1,7 mg/L. The results fulfilled the validity criteria recommended by the OECD guidelines.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Measured concentrations were close to nominal concentrations : Nominal values : 0,40 mg/L; 0,80 mg/L and 1,60 mg/L Initial medium : 100,9 % ; 100,6 % and 100,9 % Final medium : 99,1 %; 104,0% and 96,8%

Details on results:

Measured concentrations close to nominal concentrations:
-Nominal values : 0,40 mg/L; 0,80 mg/L and 1,60 mg/L
-Initial medium : 100,9 % ; 100,6 % and 100,9 %
-Final medium : 99,1 %; 104,0% and 96,8%
The regression curves were measured from the four highest test item treatments. The lowest test item treatment was excluded because of the plateau-phase.

- Mortality of control: 0, see Table 6.1.2/3

Results with reference substance (positive control):

- Results with reference substance valid? As recommended by the OECD guidelines, the EC50-24h value was between 0.6 and 2.1 mg/L. Calculations gave 1,20 mg/L as the measured value.

Table 6.1.3/2 : Measured concentrations of test item as % of the nominal values

Nominal values	Initial medium	Final medium
0,40 mg/L	100,9 %	99,1 %
0,80 mg/L	100,6 %	104,0 %
1,60 mg/L	100,9 %	96,8 %

Table 6.1.3/3 : Definitive test - % immobilized daphnids for the controls and the test item treatments

 

	% immobilization after 24h				% immobilization after 48h
	Rep 1	Rep 2	Rep 3	Rep 4	Rep 1	Rep 2	Rep 3	Rep 4
Water control	0	0	0	0	0	0	0	0
Solvent control	0	0	0	0	0	0	0	0
Test item
0,10 mg/L	0	0	0	0	0	0	0	0
0,20 mg/L	0	0	0	0	0	0	0	0
0,40 mg/L	0	0	20	20	0	0	40	20
0,80 mg/L	20	40	60	40	40	60	60	40
1,60 mg/L	80	80	60	100	80	80	60	100

Tableau 6.1.3/4 : Effects of the test item

	NOEC	EC50	95% confidence intervals
T0 + 24 h	0,40 mg/L	0,82 mg/L	0,39 - 1,75 mg/L
T0 + 48 h	0,40 mg/L	0,76 mg/L	0,37 - 1,54 mg/L

Validity criteria fulfilled:

yes

Remarks:

Validity criteria fulfilled : immobilization was less than 10% in the controls, the dissolved oxygen concentration exceeded 3 mg/L in the controls and every test item treatment. The EC50-24h for Potassium dichromate was between 0,6 and 1,7 mg/L.

Conclusions:

The 48h-EC50 value was determined at 0,76 mg/L, with a 95% CI of [0.37-1.54 mg/L], based on analytically confirmed nominal concentrations.
Even if all validity criteria were fulfilled, this study is not reliable due to high concentration of solvent used.

Executive summary:

This study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was the OECD Guideline No. 202.

Following a preliminary range-finding study, 20 daphnids (5 daphnids per unit, 4 replicate units for each test item treatment) were exposed to the test item at concentrations of 0,1 ; 0,2 ; 0,4 ; 0,8 and 1,6 mg/L for 48 hours, under static conditions. The number of immobilised daphnids were recorded after 24 and 48 hours.

The 48h-EC50 obtained for the test item on Daphnia magna was 0.76 mg/L (95% confidence limits of 0.37 - 1.54 mg/L) based on analytically confirmed nominal concentrations (measured concentrations between 80 -120% of the nominal). The 48h-NOEC is 0.40 mg/L.

This study is not reliable due to the high concentration of solvent (acetone) used in combination with the test substance. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not necessary for aquatic toxicity testing except under exceptional circumstances. Furthermore, solvents are generally not appropriate for testing multiconstituent substances, such as this test substance (which is a mixture of isomers), as it may alter the relative composition of the consituents by changing the solubility of one or more components and thereby the toxicity. Furthermore, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L, corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) and represents almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L). Acetone is suspected to have negatively affected the definitive results. Some solvent/substance interactions may have occurred in this study due to this high concentration of acetone.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

October 2nd, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
iSafeRat® holistic HA-QSAR v1.8

2. MODEL (incl. version number)
iSafeRat® High-Accuracy-Quantitative Structure-Activity Relationship (HA-QSAR) based on a holistic approach for predicting physicochemical and ecotoxicological endpoints: Short-term toxicity to Daphnia (immobilisation)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1CC(C=C(C1O)C)C2CC=C(C2(C)C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not applicable

Remarks:

QSAR model

Principles of method if other than guideline:

The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (4). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Remarks:

Not applicable: QSAR model

Specific details on test material used for the study:

- Mechanism of action : MechoA 1.1, non-polar narcosis
- Water solubility : 8.07 mg/L (KREATiS, 2019)

Analytical monitoring:

not required

Remarks:

QSAR

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

Results from the following species were used in the regression: Daphnia magna, Daphnia pulex.
No difference in relationship between solubility and ecotoxicity between invertebrate (or indeed other) aquatic species is expected. Any observed differences may be attributed to lifestyle related parameters (e.g. shell closing in molluscs) and relative duration of study versus bodysize rather than to a specific toxic mechanism causing species differences.

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Not applicable

Test temperature:

The temperatures varied from approximately 20 to 23 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.

pH:

Test results were taken from studies with measured pHs between 6 - 9.

Dissolved oxygen:

The dissolved oxygen concentration was more than 60% of the air-saturation value throughout the duration of the test. In exceptional cases where studies with oxygen concentrations lower than 60% were used, all aspects of the study were thoroughly evaluated in order to satisfy the evaluator that the effects found were not due to reduced oxygen concentration (i.e. the study would correctly receive a Klimisch score of 2 under the REACH Regulation (REACH, 2006).

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Studies were used only where analytical measurements were made on the control and all relevant test concentrations whenever possible. Any exceptions (initial concentrations measured only or EC50 based on nominal values) were used only when sufficient justification for stability of the test item was determined.
When nominal concentrations from training set data were used, the concentrations of the test substance had been shown to be maintained to within 80% of the nominal concentrations throughout the duration of the study either using analytical verification or by application of a weight of evidence approach that such an assumption was justified. If the deviation from the nominal concentration was >20%, the results were based on the measured concentrations.

Details on test conditions:

This QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008.
The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% C.I:: 0.78 – 1.1 mg/L.

Details on results:

Not applicable

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05): 0.78 – 1.1 mg/L. QSAR statistical parameters are precised in the QMRF, in the attached background material.

No additional information

Validity criteria fulfilled:

yes

Remarks:

The substance falls into the applicability domains of the QSAR model.

Conclusions:

The 48-h EC50 based on mobility and measured concentrations was determined to be 0.92 mg/L with 95%-Confidence Interval between 0.78 and 1.1 mg/L.

Executive summary:

A QSAR prediction (KREATiS, 2019) was performed to assess the acute toxicity of the test substance to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The criterion predicted was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The water solubility value given as the input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the descriptor domain of the model between a log water solubility (in log (mol/L)) of - 4.70 to 0.87.

As discussed in the QMRF for this model with JRC QMRF identifier: Q19-46-51-448, the toxicity of MechoA 1 compounds can be linked thermodynamically to their water solubility values. MechoA 1 is based on the principle of toxicity due to disruption of biological membrane leading to a narcosis effect.

The 48-h EC50 based on mobility and measured concentrations was determined to be 0.92 mg/L with 95%-Confidence Interval between 0.78 and 1.1 mg/L.

Description of key information

iSafeRat® High-Accuracy-Quantitative Structure-Activity Relationship, KREATIS, 2019 :

48h-EC50 = 0.92 mg/L (95% confidence interval: 0.78 – 1.1 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.92 mg/L

Additional information

One experimental study and one QSAR prediction are available to assess the short-term toxicity of the registered substance to aquatic invertebrates.

The experimental study (Phytosafe, 2012) was considered as not reliable and was disregarded due to major methodological deficiencies. Indeed, a large quantity of acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not the best method at the time being. Furthermore, solvents are generally not appropriate for multiconstituent substances, like the test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L , corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) and represent almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L). Therefore, solvent/substance interactions may have occured in this study with this high concentration of acetone and the result cannot be considered fit for use.

The QSAR prediction (KREATiS, 2019) was considered as reliable. The QSAR model has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48 -hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period. The test item falls within the applicability domain of the model and was therefore reliably predicted for its acute toxicity (48h-EC50) to daphnids. Therefore, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

The 48-h EC50 of the registered substance to daphnids was predicted at 0.92 mg/L (95% CI: 0.78 - 1.1 mg/L).