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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From June 22, 2012 to July 13, 2012

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled. The test substance is adequately identified, but some data on composition is missing. This study is considered not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at the time being, a semi-static system is not accepted as an adaptation of the OECD Guideline. With this method, parent and degradation products are present simultaneously, so interactions can occured. In addition, 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 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

March 2006

Deviations:

yes

Remarks:

Semi-static methodology used.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP 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

Remarks:

Measured values represented 85-120 % of the nominal values

Details on sampling:

- Concentrations: no details
- Sampling method: The algal biomass in each flask was determined daily over the test period using small volumes removed from the test solution by pipette. These volumes were not replaced.

Vehicle:

yes

Remarks:

The treatment solutions were prepared in acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The treatment solutions were prepared in acetone. The different treatment solutions are shown in Table 6.1.5/1 how they were prepared.
- Controls: 100 mL of mineral medium.
- 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)): 0.5 mL/L. see Table 6.1.5/1

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain and source : No.72 of the Museum National d'Histoire Naturelle (Paris, France). The strain was regularly sub-cultured in OECD medium at the Phytosafe site.
- Method of cultivation: The test vessels were kept under orbital shaking so as to keep the algae in suspension and to facilitate the transfer of CO2.
The inoculum culture was prepared 2-4 days before the start of the test and incubated under the same conditions as the test cultures such to adapt the algae to test conditions and ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 °C

pH:

8.7 - 8.8

Nominal and measured concentrations:

Test item treatments were set at 120 % of the nominal values.
Definitive test concentrations : 0.8 ; 1.3 ; 2.0 ; 3.2 and 5.2 mg/L (nominal)
The initial values represented 118-120 % of the nominal values.
On day 1, the test solutions were still more than 89% of the nominal values and were not adjusted. On day 2 of the test period, the measured concentrations represented 85-89 % of the nominal values. The test item treatments were adjusted with 10 µL of the test item treatments solutions. The measured concentrations in the new media were 98-106 % of the nominal values.
At the end of the test, the test item treatments were still 92-100 % of the nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass erlenmeyer flasks (250 mL) filled with 100 mL of culture
- Type (delete if not applicable): closed : the test vessels were capped with air-permeable stoppers.

- Renewal rate of test solution (frequency/flow rate): concentrations were checked daily
- Initial cells density: the initial biomass in the test cultures was the same in all test cultures and sufficiently low to allow exponential growth throughout the incubation period without any risj of nutrient depletion. Historical data at Phytosafe site show that 2 to 5 x 10^3 cells/mL is an appropriate number.

- No. of vessels per concentration (replicates): 3 replicate units
- No. of vessels per control (replicates): 3 replicate units
- No. of vessels per vehicle control (replicates): 4 potassium dichromate treatments

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Photoperiod: continuously
- Light intensity and quality: uniform fluorescent illumination of 60 - 120 µE.m^-2.s^-1 ; light intensity did not deviate by more than 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] : algal biomass in each flask was determined daily over the test period using small volumes removed from the test solution by pipette. These volumes were not replaced. The numeration was done using an electronic cell counter. The results were expressed as cells per liter of solution.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1,6
- Range finding study : yes
- Test concentrations: 0.01 - 0.1 - 1.0 - 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: the specific growth rate was coinsidered as similar to that of the control for the test item treatments up to and including 1.0 mg/L but totally inhibited at 10 and 100 mg/L.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on analytically confirmed nominal concentrations

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on analytically confirmed nominal concentrations

Results with reference substance (positive control):

As required, the EC50-72h value was between 0.6 and 1.0 mg/L (growth rate) for Potassium dichromate. Calculation gave 0.82 mg/L as the measured value.

Reported statistics and error estimates:

F-variance analysis at a 5% confidence level was used to judge upon the difference for mean specific growth rate (section-by-sectionand total values) for each test item treatment compared to the water controls.
For the first day of testing the specific growth rate was significantly reduced for the highest test item treatment and similar to the controls for the four lowest concentrations.
For the second day of testing, the specific growth rate was significantly reduced for the two highest test item treatments.
For the third day of testing, mean specific growth rate was significantly reduced for every test item treatment.
For the entire test period, mean specific growth rate per day was considered as significantly reduced in every case except 0.8 mg/L.
NOEC based on specific growth rate = 0.8 mg/L.
Mean percentages of inhibition are presented in Table 6.1.5/3.
The regression was performed using the three highest test item treatments. The two lowest values were excluded because of the plateau phase. The coefficient of determination was 88.0 %.
ErC50-72=5.1 mg/L and 95%-confidence interval = 0.1 - 273.9 mg/L. The 95%-confidence interval was wide because the regression curve was calculated out from only three experimental points. The observed interval was preferred: the ErC50-72h was observed to be between 3.2 - 5.2 mg/L.

Table 6.1.5/2 : Mean measured concentrations of Santamanol in the test item treatments and % of the nominal values

	Nominal treatment solutions
	0,8 mg/L	1,3 mg/L	2,0 mg/L	3,2 mg/L	5,2 mg/L
Day 0	0,94 mg/L 119,0 %	1,50 mg/L 119,3 mg/L	2,41 mg/L 199,8 %	3,81 mg/L 118,4%	6,20 mg/L 120,4 %
Day 1	0,70 mg/L 88,9 %	1,22 mg/L 97,0 %	1,94 mg/L 96,4%	3,06 mg/L 94,9%	5,12 mg/L 99,4%
Day 2, old media	0,68 mg/L 86,9%	1,08 mg/L 85,9 %	1,70 mg/L 84,6 %	2,74 % 85,2 %	4,56 mg/L 88,5 %
Day 2, new media	0.77 mg/L 97,8 %	1,33 mg/L 105,5%	2,09 mg/L 104,0 %	3,33 mg/L 103,3 %	5,44 mg/L 105,7 %
End of test	0,79 mg/L 100,0 %	1,21 mg/L 96,2 %	1,86 mg/L 92,2 %	3,12 % 96,8 %	5,09 % 98,9 %

Table 6.1.5/3: F-variance analysis for mean specific growth rate per day in the test item treatments compared to the controls (Threshold value for F=7.71)

	Santamanol
	0,8 mg/L	1,3 mg/l	2,0 mg/L	3,2 mg/L	5,2 mg/L
0 to 24h	F = 0,00	0,05	0,34	1,33	26,22
24 to 48h	0,39	2,88	1,6	11,98	293,75
48 to 72h	17,13	14,34	18,17	172,94	614,93
Total period	3,55	9,3	40,82	1507,37	1151,26

Table 6.1.5/4 : Definitive test - Percentages inhibition of specific growth rates per day (section-by-section and total)

Negative values indicate that the growth rate was increased as compared to the controls

	0 to 24h	24 to 48h	48 to 72h	Total period
Santamanol (mg/L)	%
0,8	-0,2	-3,2	8	1,7
1,3	-1,1	-5,1	10,7	1,7
2	2,3	3,5	8,9	4,9
3,2	5,1	13,1	22,5	13,6
5,2	19,6	54,7	84,6	52,9

Validity criteria fulfilled:

yes

Conclusions:

The 72h-ErC50 value was determined at 5,1 mg/L. The 95%-confidence interval was wide because the regression curve was calculated out from only three experimental points. The observed interval was preferred: the ErC50-72h was observed to be between 3.2 - 5.2 mg/L. The 72h-NOEC was 0.80 mg/L. All results were based on analytically confirmed nominal concentrations.
However, this study is considered not assignable due to non sufficient information provided on the semi-static methodology used.

Executive summary:

This study was performed to assess the acute toxicity of the test item to Desmodesmus subspicatus. The method followed was the OECD Guideline No. 201.

Following a preliminary range-finding study, Desmodesmus subspicatus were exposed to the test item at concentrations of 0.8 ; 1.3 ; 2.0 ; 3.2 and 5.2 mg/L (nominal) for 72 hours. Concentrations were checked daily.

On day 1, the test solutions were still more than 89% of the nominal values and were not adjusted. On day 2 of the test period, the measured concentrations represented 85-89 % of the nominal values. The test item treatments were adjusted with 10 µL of the test item treatments solutions. The measured concentrations in the new media were 98-106 % of the nominal values. At the end of the test, the test item treatments were still 92-100 % of the nominal values.

The 72h-EC50 obtained for the test item on Desmodesmus subspicatus was 5.1 mg/L (observed interval 3.2 - 5.2 mg/L) (based on analytically confirmed nominal concentrations). The 72h-NOEC was 0.80 mg/L.

All validity criteria were fulfilled. However, this study is considered not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at the time being, a semi-static system is not accepted as an adaptation of the OECD Guideline. With this method, parent and degradation products are present simultaneously, so interactions can occured. In addition, 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 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

October 2nd, 2019

Reliability:

2 (reliable with restrictions)

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

Remarks:

Reliable with restrictions due to descriptor domain point of view. The test item falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. According to a k-NN approach, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line.

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 algae (inhibition of the exponential growth rate)

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 201 (Alga, Growth Inhibition Test)

Deviations:

not applicable

Remarks:

QSAR model

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition 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 toxicity to algae as would be expected in a laboratory experiment following the OECD Guideline 201 and EC method C.3 for specific, named modes of action to provide a value that can effectively replace a 72-hour ErC50 value from an experimental study. The regression is method used to achieve this has been fully validated following the OECD (2004) recommendations (refer to the QMRF with JRC/KREATiS QMRF identifier: Q19-46-51-448 for further details).

GLP compliance:

no

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

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

other: Pseudokirchneriella subcapitata, Desmodesmus subspicatus, Scenedesmus quadricauda

Details on test organisms:

Results from the following species were used in the regression: Pseudokirchneriella subcapitata, Desmodesmus subspicatus, Scenedesmus quadricauda.
No difference in relationship between solubility and ecotoxicity between algae (or indeed other) aquatic species is expected. Any observed differences may be attributed to lifestyle related parameters 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:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

Not applicable

Hardness:

Not applicable

Test temperature:

The temperatures varied from approximately 20 to 25 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the ErC50 values found in experimental data.

pH:

Test results were preferably taken from studies with measured pHs between 6 - 9. However it is recognized that in some cases (due to high luminosity) the pH may increase in the control and lower concentrations (which do not cause significant effect over the study period). This pH increase did not generally disqualify the study from being used in the test and validation set for non-polar chemicals.

Dissolved oxygen:

Not specified

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 ErC50 based on nominal values) were used only when sufficient justification for stability of the test item was determined.

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. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008. The criterion predicted was the ErC50 (Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.75 – 8.1 mg/L

Details on results:

The Subcooled Liquid Water Solubility value (8.07 mg/L or -4.463 in log (mol/L)) given as the input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the intermediate domain of the model between a log water solubility (in log (mol/L)) of - 9.34 to - 4.38.
The test substance is attributed to the class of non-polar narcotic compounds (MechoA 1.1).

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

95% CL: 0.75 – 8.1 mg/L.
QSAR statistical parameters are given in the QMRF and the QPRF, see the attached background material.

No additional information

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domain of the QSAR model.

Conclusions:

The 72-h ErC50 based on measured concentrations was determined to be 2.5 mg/L with 95%-Confidence interval between 0.75 and 8.1 mg/L.

Executive summary:

A QSAR prediction (KREATiS, 2019) was performed to assess the inhibition of growth to algae of the test substance. 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 201. The criterion predicted was the ErC50 (Median Effective Concentration for specific growth rate), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system within a period of 72 hours.

 

The growth inhibition of algae was determined using a validated QSAR for the Mechanism of Action (MechoA) 1.1, non-polar narcosis. This QSAR is based on validated data for a training set of 40 chemicals derived from 72 -hour ErC50 test on algae, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The Subcooled Liquid Water Solubility value (8.07 mg/L or -4.463 in log (mol/L)) given as the input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the intermediate domain of the model between a log water solubility (in log (mol/L)) of - 9.34 to - 4.38. as demonstrated in the QPRF.

The 72-h ErC50 based on measured concentrations was determined to be 2.5 mg/L with 95%-Confidence Interval between 0.75 and 8.1 mg/L.

Description of key information

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

72h-ErC50 = 2.5 mg/L (95% confidence interval: 0.75 – 8.1 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.5 mg/L

Additional information

One experimental study and one QSAR prediction are available to assess the toxicity of the registered substance to aquatic algae.

The experimental study (Phytosafe, 2012) was considered as not assignable due to insufficient information provided on the semi-static methodology used. A validation study should be provided to validate this method and, at this time, a semi-static system is not accepted as an adaptation of the OECD Guideline 201. With this method, parent and degradation products are present simultaneously, so interactions may have occured. In addition, 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 0.5 mL/L, corresponding to 395 mg/L (with a density of 0.79), which is 5 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD No. 23) but is below the NOEC of acetone (which was reported in the ECHA disseminated dossier at 530 mg/L).

Under the test conditions, the 72h-ErC50 and NOEC values of the test substance to Desmodesmus subspicatus, based on growth rate, were 5.1 mg/L (95% CI: 3.2 - 5.2 mg/L) and 0.80 mg/L, respectively. These results support the more conservative key data presented below.

The QSAR prediction (KREATiS, 2019) was considered as reliable and was used as key data. 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 201. The growth inhibition of algae was determined using 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 40 chemicals derived from 72-hour ErC50 test on algae, for which the concentrations of the test item had been determined by chemical analyses over the test period. The result below is the toxicity values anticipated during a 72-hour study on algae based on measured concentrations. Therefore, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

The 72h-ErC50 of the registered substance to algae was predicted at 2.5 mg/L (95% CI: 0.75 – 8.1 mg/L)