Reaction mass of stereoisomers of 4-METHYLENE-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-TETRAHYDRO-PYRAN and; 4-METHYL-6-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN and; 4-METHYL-2-(2,2,3-TRIMETHYL-CYCLOPENT-3-ENYLMETHYL)-3,6-DIHYDRO-2H-PYRAN

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:

29 August to 15 September 2011

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 and the substance is considered to be adequately identified. However, this study is not reliable due to high concentration of solvent used. 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 in the case of moderately soluble substances such as this one. Considering the acceptably high water solubility of the substance (44.4 mg/L) and the concentrations used in this study, this method could have been avoided. 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 give preferential dissolution of 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). Acetone is suspected to have affected the definitive results. Some solvent/substance interactions may have occurred in this study with this high concentration of acetone.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 March 2011

Specific details on test material used for the study:

- Physical state: Light yellow translucent liquid
- Storage condition of test material: Stored at room temperature, protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Analytical verification of the test item was performed at the concentrations of 0.78, 1.16, 1.75, 2.52 and 3.88 mg/L

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions of the test item were prepared in acetone. 50 µL aliquot samples of the stock solutions were used for the treatment application. See Table 6.1.3/1 for further details.
- Controls:
Water control: The control tubes consisted of 10 mL of M4 mineral medium.
Solvent control: The tubes received the same volume of acetone as used for the test item treatments.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Strain was received from a commercial breeder. Organisms were regularly sub-cultured at the Phytosafe site and fed with living cells of the green algae.
- Age: Less than 24 hours at test initiation
- Feeding during test: Daphnids was not fed during the exposure period.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

20 ± 1 °C

pH:

Start of the test: 8.2-8.4
End of the test: 7.6-7.8

Dissolved oxygen:

Start of the test: 7.7 dissolved O2 mg/L
End of the test: 7.1-7.6 dissolved O2 mg/L

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0.78, 1.16, 1.75, 2.52 and 3.88 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 mL capacity glass tubes filled with 10 mL of M4 mineral medium.
- Type: The tubes were tightly capped during the exposure period.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The M4 mineral medium was freshly reconstituted from mineral stock solutions.

OTHER TEST CONDITIONS
- Photoperiod: The tubes were maintained in the dark in a climatic chamber.

EFFECT PARAMETERS MEASURED
- Immobilization: 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.
- Water quality: Dissolved oxygen and pH were measured at the start and end of the test in the controls and in the highest test substance concentration, at least (definitive test only).

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- One replicate unit for each the five test item treatments and for the control.
- Test concentrations: 0.01, 0.1, 1.0, 9.8 and 97.9 mg/L
- Results used to determine the conditions for the definitive study: No immobilization was observed in the controls and the test item treatments up to and including 0.1 mg/L. The percentage of immobilization was 20% for the 1.0 mg/L test item treatment, and 100% for both the 9.8 and the 97.9 mg/L test item treatments. Based on the results, concentrations of 0.78, 1.16, 1.75, 2.52 and 3.88 mg/L were selected for definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 0.6, 1.0 and 1.7 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence intervals: 1.33 - 1.87 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence intervals: 0.97 - 1.44 mg/L

Duration:

24 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Percentage of immobilization:
No immobilization was observed for the water controls over the test period. For the solvent control, mean value for immobilization was 5.0%.
The percentage of immobilization was 20% as a maximum for the 0.78 mg/L test item treatment. Mean final value for the group was 5.0 %.
All the daphnids were immobilized after 24 h of testing for both the 2.52 and 3.88 mg/L test item treatments.

Water quality:
Both the pH values and the oxygen concentrations for the test item treatments were similar to those measured in the controls. In every case the pH value of the mineral medium was between 7.5 and 8.5, and the oxygen concentration remained > 3 mg/L.

Results with reference substance (positive control):

EC50-24 h for Potassium dichromate was between 0.6 and 1.7 mg/L. The results fulfilled the validity criteria based on Phytosafe historical data: the EC50-24 for Potassium dichromate should be between 0.6 and 1.7 mg/L.

Reported statistics and error estimates:

EC50 calculations: After 24 hours of exposure, the regression curve was measured for the three intermediate values of the test item treatments. Both the lowest and the highest test item treatments were excluded because of the plateau-phase. After 48 hours of exposure, the two highest test item treatments were excluded.

Analytical verification of the test item treatments:

The results showed that the measured concentrations were satisfactorily maintained between 80-120 % of the nominal values throughout the test period.

 

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

 

Test item - Nominal values	Initial medium	Final medium
0.78 mg/L	105.1%	102.4%
1.16 mg/L	102.4%	98.9%
1.75 mg/L	101.4%	97.1%
2.52 mg/L	102.0%	98.0%
3.88 mg/L	100.3%	96.8%

 

 

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

 

Groups	% immobilization after 24 h				% immobilization after 48 h
	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	20	0
Test item
0.78 mg/L	0	20	0	0	0	20	0	0
1.16 mg/L	0	20	20	20	40	40	60	40
1.75 mg/L	60	60	80	60	100	100	100	80
2.52 mg/L	100	100	100	100	100	100	100	100
3.88 mg/L	100	100	100	100	100	100	100	100

 

VALIDITY OF THE TEST RESULTS

The tests were considered as valid:

Immobilization was less than 10 % in the controls,

The dissolved oxygen concentration exceeded 3 mg/L in the controls and every test item treatment.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 48-hour EC50 of the test item was calculated to be 1.18 mg/L (95% Cl: 0.97-1.44 mg/L) based on analytically confirmed nominal concentrations.
However, this study is not reliable due to high concentration of solvent used.

Executive summary:

A Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test item to Daphnia magna.

Test item was exposed to daphnids at the concentrations of 0.78, 1.16, 1.75, 2.52 and 3.88 mg/L for 48 hours. Water and solvent controls were included. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. The test temperature was 20 ± 1 °C. Before definitive test, a 48-hour range-finding test was conducted and the percent immobilization was 0, 0, 0, 0, 20, 100 and 100% at 0.01, 0.1, 1.0, 9.8 and 97.9 mg/L, respectively.

The analytical verification results showed that the measured concentrations were satisfactorily maintained between 80-120 % of the nominal values throughout the test period. Recovery of the test item at the end of test was 92-96%.

No immobilization was observed for the water controls over the test period. For the solvent control, mean value for immobilization was 5.0%. The percentage of immobilization was 20% as a maximum for the 0.78 mg/L test item treatment. Mean final value for the group was 5.0 %. All the daphnids were immobilized after 24 h of testing for both the 2.52 and 3.88 mg/L test item treatments.

EC₅₀-24 h for Potassium dichromate was between 0.6 and 1.7 mg/L. The results fulfilled the validity criteria based on the historical data.

Under the test conditions, the 48-hour EC50 of the test item was calculated to be 1.18 mg/L (95% Cl: 0.97-1.44) based on analytically confirmed nominal concentrations.

This study is not reliable due to high concentration of solvent used. 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 in the case of moderately soluble substances such as this one. Considering the acceptably high water solubility of the substance (44.4 mg/L) and the concentrations used in this study, this method could have been avoided. 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 give preferential dissolution of 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). Acetone is suspected to have affected the definitive results. Some solvent/substance interactions may have occurred in this study with 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:

July 31st, 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® toolbox – in Silico Algorithms For Environmental Risk And Toxicity version 2.4

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.8

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
isomer 1: CC1=CCC(CC2CC(CCO2)=C)C(C)1C
isomer 2: CC1=CCC(CC2C=C(C)CCO2)C(C)1C
isomer 3: CC1=CCC(CC2CC(C)=CCO2)C(C)1C

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

QSAR input value

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The purpose of this QSAR model is to accurately predict the acute toxicity to daphnids as would be expected in a laboratory experiment following OECD Guideline 202 (OECD, 2004b) and EC method C.2 (European Commission, 2008) for specific, named mechanisms of action. The model provides an in silico prediction for the 48-hour EC50 value that can effectively be used in place of an experimentally derived 48-hour EC50 value. The regression based method used to achieve this has been fully validated following the OECD recommendations (OECD, 2004a).

GLP compliance:

no

Remarks:

QSAR model

Specific details on test material used for the study:

- Water solubility: 44,4 mg/L at 25°C (KREATiS, 2019)

Analytical monitoring:

no

Remarks:

QSAR model

Details on sampling:

not applicable

Vehicle:

no

Remarks:

QSAR model

Details on test solutions:

not applicable

Test organisms (species):

other: Daphnia magna, Daphnia pulex

Details on test organisms:

No difference in terms of toxic mechanism of action 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 model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Results from a test duration of 48 hours only were used for daphnid species.

Post exposure observation period:

None

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

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 QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

Not applicable

Conductivity:

Not data

Nominal and measured concentrations:

Studies were used only where sufficient evidence was presented to determine that the substance was stable under test conditions (i.e. maintained within ± 20 % of the nominal) or, if not, the result was based on measured concentrations as geometric mean.

Details on test conditions:

Preferentially results from semi-static studies were used. However, substances tested using a static design were accepted (preferably accompanied by analytical measurements over the study period). For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 0.9 – 3.7 mg/L.

Details on results:

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.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05): 2.9 – 3.7 mg/L.
QSAR statistical parameters are given in the QMRF and the QPRF

Analysis of the Applicability Domain

Descriptor domain

The Subcooled Liquid Water Solubility value (44.4 mg/L or -3.696 in log (mol/L)) 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.

Structural fragment domain

All chemical groups within the molecular structure are taken into account by the model.

Mechanistic domain

Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the aquatic toxicity for chemicals with the following mechanisms of action of toxicity (MechoA):

•          non-polar narcosis (MechoA 1.1)

•          polar narcosis of alkyl-/alkoxy-phenols (MechoA 1.2)

•          polar narcosis of aliphatic amines (MechoA 1.2)

•          cationic narcosis of quaternary ammoniums (MechoA 1.3)

•          mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)

•          hard electrophile reactivity (MechoA 3.1)

•          RedOx cycling of primary thiols (MechoA 4.4)

•          Proton release of carboxylic acids (MechoA 5.2)

The MechoA of molecules is predicted directly from the structure. The test item as an aliphatic ether is expected to exert a MechoA 1.1 and can be taken into account by the model.

Validity criteria fulfilled:

yes

Conclusions:

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 48h-EC50 of the test item to daphnids was predicted as 3.3 mg/L.
95% confidence interval (α = 0.05): 2.9 – 3.7 mg/L.

Executive summary:

A Quantitative Structure-Activity Relationship (QSAR) model was used to calculate the acute toxicity of the test item to daphnid. This QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004a) 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" (OECD, 2004b), referenced as Method C.2 of Commission Regulation No. 440/2008 (European Commission, 2008). 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) (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 48h-EC50 of the test item to daphnids was predicted as 3.3 mg/L.

95% confidence interval (α = 0.05): 2.9 – 3.7 mg/L.

Description of key information

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

48h-EC50 = 3.3 mg/L (95% confidence interval: 2.9 – 3.7 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.3 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, 2011) 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 in the case of moderately soluble substances such as this one. Considering the acceptably high water solubility of the substance (44.4 mg/L) and the concentrations used in this study, this method could have been avoided. 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 give preferential dissolution of 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 modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the 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) (Baueret 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 48h-EC50 of the test item to daphnids was predicted as 3.3 mg/L (95% CI: 2.9 - 3.7 mg/L).