2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

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:

supporting study

Study period:

From July 23, 2015 to July 31, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 202 with GLP certificate. All validity criteria were fulfilled. However, this study is considered reliable with restrictions due to the use of solvent. Indeed, 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 test solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L). In addition, the concentration/quantity of solvent used in the treatment solutions was 2 mL/L , corresponding to 1.58 g/L (with a density of 0.79), which is 20 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is 5 times lower than the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L).

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

yes

Remarks:

Solvent was used, which is not the best method existing at the time being.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection dates: 12 and 13 June 2014 / Certificate signed on 5 March 2015

Specific details on test material used for the study:

- Physical state: Colourless translucent liquid
- Storage condition: Room temperature protected from direct sun light

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the treatment solutions were prepared in acetone.
- Controls: Water control (the control tubes received 10 mL of M4 mineral medium) and 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 in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 2 mL/L. see Table 6.1.3/1 in any other information

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna
- Source: received from a commercial breeder. These organisms were regularly sub-cultured at the Phytosafe site and fed with living cells of the green algae.
- Age of parental stock (mean and range, SD): Young daphnids produced within 24 hours were used for the tests
Daphnids were not fed during the exposure period.

Test type:

static

Water media type:

other: M4 mineral medium was freshly reconstituted from mineral stock solutions

Limit test:

no

Total exposure duration:

48 h

Hardness:

no data

Test temperature:

The temperature conditions were checked to be 20.4°C kept constant at +/-1°C for both the range-finding test and the definitive test.

pH:

7.6 - 7.9

Dissolved oxygen:

7.6 - 8.4 mg/L
The final oxygen concentration remained > 3 mg/L in every case, as required.

Salinity:

Not applicable

Conductivity:

no data

Nominal and measured concentrations:

Definitive test: 0.6 - 1.2 - 2.5 - 5.1 and 10.3 mg/L based on analytically confirmed nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 mL capacity glass tubes
- Type (delete if not applicable): closed (the tubes were tightly capped during the exposure period)
- Material, size, headspace, fill volume: filled with 10 mL of M4 mineral medium
- No. of organisms per vessel: 5 daphnids per replicate unit
- No. of vessels per concentration (replicates): 4 replicate units
- No. of vessels per control (replicates): 4 replicate units
- No. of vessels per vehicle control (replicates): 4 replicate units

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.002 - 0.02 - 0.2 - 2.1 and 20.6 mg/L
- Results used to determine the conditions for the definitive study: No immobilisation was observed throughout the test period for the water controls and the solvent controls. After 24 hours of exposure, the daphnids were still mobile up to and including 2.1 mg/L, but all the daphnids were immobilized at 20.6 mg/L. After 48 hours of exposure, no effects were observed at 0.2 mg/L, 70% of the daphnids were immobilized at 2.1 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval :1.0 - 4.5 mg/L ; analytically confirmed nominal concentrations : recovery of the test item of 89.1 - 101.9 %.

Duration:

48 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: analytically confirmed nominal concentrations : recovery of the test item of 89.1 - 101.9 %.

Details on results:

No adverse effect was observed for the water controls and the solvent controls throughout the test period: the percentage of immobilization was 0% in every replicate unit.
In the 2.5 mg/L test item treatment mean percentage of immobilization was 5% on day 1, and 10% on day 2.
At 0.6 mg/L, no immobilization was observed.
10% of the daphnids (mean value) were immobilized at 1.2 mg/L, but the difference with the controls was not statistically significant at the 5% confidence level. NOEC = 1.2 mg/L.
All the daphnids were immobilized at 10.3 mg/L after 24 hours of exposure.
The 2.5 and 5.1 mg/L gave intermediate values.
Daphnids were observed at the interface air/water at 2.5, 5.1 and 10.3 mg/L. They were considered as immobilized when they failed in swimming within the whole volume after the tubes were gently rotated up and down.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ECx: 24h-EC50 for Potassium dichromate fell within 0.6 - 2.1 mg/L. Calculations gave 1.33 mg/L as the measured value.

Reported statistics and error estimates:

F values were compared to F(1,6) = 5.99 at the 5% confidence level. The NOEC was 1.2 mg/L on day 1 and day 2 of the test period.

Table 6.1.3/2 : Measured concentrations of the test substance in the test item treatments and % of the nominal values

	Nominal concentrations, mg/L
	0.6	1.2	2.5	5.1	10.3
Test initiation	0.63 0.61 99.9%	1.19 1.18 95.9%	2.15 2.18 87.5%	5.31 5.35 103.6%	9.91 9.93 96.4%
End of test	0.58 0.58 93.6%	1.22 1.21 98.6%	2.23 2.18 89.1%	5.18 5.31 101.9%	10.41 10.18 100.0%

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 substance
0.6 mg/L	0	0	0	0	0	0	0	0
1.2 mg/L	0	0	0	20	20	0	0	20
2.5 mg/L	40	20	60	80	40	20	60	80
5.1 mg/L	100	40	100	100	100	80	100	100
10.3 mg/L	100	100	100	100	100	100	100	100

Table 6.1.3/4: Definitive test - detailed calculations of EC50 values for test item

	After 24h of testing	after 48h of testing
Lowest treatment group	0.60 mg/L	0.60 mg/L
Highest treatment group	10.3 mg/L	5.1 mg/L
Regression slope	91.5	106.4
Regression constant	11.4	12.5
Coefficient of correlation	r = 0.83	r = 0.84
EC50 (mg/L)	2.6	2.2
95% confidence level	1.0 - 6.7	1.0 - 4.5

Validity criteria fulfilled:

yes

Remarks:

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 24h-EC50 for potassium dichromate was between 0.6 and 2.1 mg/L.

Conclusions:

The 48h-EC50 value was determined at 2.2 mg/L, with a 95% CI of 1.0 - 4.5 mg/L, based on analytically confirmed nominal concentrations.

Executive summary:

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

Following a preliminary range-finding study, 5 daphnids per replicate unit (4 replicate units per treatment) were exposed to the test item (prepared with acetone as solvent) at concentrations of 0.6 - 1.2 - 2.5 - 5.1 and 10.3 mg/L (analytically confirmed nominal concentrations, recovery of the test item at the end of the test of 89.1 - 101.9 %) for 48 hours under static test conditions. The number of immobilised Daphnia was recorded after 24 and 48 hours.

The 48 hour EC50 for the test item to Daphnia magna based on nominal test concentrations was 2.2 mg/L and the 95% confidence interval was 1.0 - 4.5 mg/L. The highest concentration tested without observed effect was 1.2 mg/L.

Analytic check of the test item treatments at test initiation and at the end of the test showed that the test item concentrations were satisfactorily maintained within 80 -120% of the nominal values over the test period, and so the results are based on nominal test concentrations only.

All validity criteria were fulfilled. However, this study is considered reliable with restrictions due to the use of solvent. Indeed, 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 test solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L).

In addition, the concentration/quantity of solvent used in the treatment solutions was 2 mL/L, corresponding to 1.58 g/L (with a density of 0.79), which is 20 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is 5 times lower than the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

October 16th, 2019 to October 18th, 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
CC1(C)C=C(C2(OCC(C)(C)CO2)C)CCC1
The toxicity of the test item was predicted using the iSafeRat® Ecotox module providing the Subcooled Liquid Water Solubility (SLWS) as the input. TThe SLWS has been predicted using the iSafeRat® Water Solubility module providing the experimental log KOW value as the input. Water Solubility of the test item = 15.4 mg/L (or -4.190 in log (mol/L).

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 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, 2004) 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, 2004).

GLP compliance:

no

Remarks:

QSAR model

Specific details on test material used for the study:

- Mechanism of action : MechoA 1.1: non-polar narcosis (Bauer et al., 2018)
- Log Kow: 4.37 at 20°C and pH 7.1 (Phytosafe, 2015; EEC A.8; Shake-Flask method)
- Water solubility : 15.4 mg/L (KREATiS, 2019)

Analytical monitoring:

no

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.:

1.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 1.3 – 1.7 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.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05): 1.3 – 1.7 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 (15.4 mg/L or -4.190 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 a cetal (i.e.diether) 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 1.5 mg/L.
95% confidence interval (α = 0.05): 1.3 – 1.7 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, 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" (OECD, 2004), 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 1.5 mg/L.

95% confidence interval (α = 0.05): 1.3 – 1.7 mg/L

Description of key information

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

48h-EC50 = 1.5 mg/L (95% confidence interval: 1.3 – 1.7 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 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, 2015) was considered as reliable with restrictions due to the use of solvent (acetone). 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 test solutions were prepared with a solvent (acetone), which is not an appropriate method to conduct toxicity tests for substances with a solubility at the level of this substance (15.4 mg/L).

In addition, the concentration/quantity of solvent used in the treatment solutions was 2 mL/L , corresponding to 1.58 g/L (acetone has a density of 0.79 g/cm3), which is 20 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is 5 times lower than the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L). In this Guideline study, the 48h-EC50 value for the registered substance to Daphnia magna was determined at 2.2 mg/L (95% CL: 1.0 - 4.5 mg/L) based on analytically confirmed nominal concentrations. While in this case, the use of solvent does not appear to have significantly influenced the resule, it was considered appropriate to complement the study with other data to provide a stronger case for the EC50 result. This experimental result supports the more conservative key data presented below.

The QSAR prediction (KREATiS, 2019) was considered as reliable and was used as the key data. 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 1.5 mg/L (95% CI: 1.3 - 1.7 mg/L).