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

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:

2-15 September 2011

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 and the substance is considered to be adequately identified. However, this study is considered not assignable due to non sufficient 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 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 for testing substances with a reasonable level of water solubility. 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 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)

Deviations:

yes

Remarks:

semi-static methodology used

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 0.4, 0.8, 1.5, 3.0 and 6.0 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 6.1.5/1 for further details.
- Controls:
Water control: The water controls consisted of 100 mL of mineral medium.
Solvent control: The solvent controls received 50 µL of acetone, and were further added with the same volume of acetone as used for the adjustment of the test item treatments.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Strain was provided by the Museum National d'Histoire Naturelle (Paris, France) and regularly sub-cultured in OECD medium at the Phytosafe site.
- Method of cultivation: 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 test 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

Post exposure observation period:

None

Hardness:

No data

Test temperature:

21-24 °C

pH:

Start of the test: 8.6-8.9
End of the test: 8.0-10.0

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0.4, 0.8, 1.5, 3.0 and 6.0 mg/L
Geometric means of the minimum and the maximum measured concentrations: 0.26, 0.50, 0.85, 1.50 and 2.74 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (250 mL) filled with 100 mL of culture served as test vessels.
- Type: The test vessels were capped with air-permeable stoppers.
- Renewal rate of test solution:
Range-finding test: The test item treatments were renewed each day: the test units were added with 50 µL of the stock solutions.
Full definitive test: The test item treatments were adjusted twice per day, early in the morning and late in the afternoon. The added volumes depended on the results of the analytical checks.
- 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 risk 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
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: OECD medium (OECD TG 201, according to ISO 8692) was freshly reconstituted by dilution of mineral stock solutions in pure water.

OTHER TEST CONDITIONS
- The test vessels were continuously shaken in a culture apparatus (Innova shaker 44R) so as to keep the algae in suspension and to facilitate the transfer of CO2.
- Light intensity and quality: The surface of the cultures received continuous, uniform fluorescent illumination of 60-120 µE/m^2/S. The light intensity did not deviate by more than 15 % from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED
Algal biomass: 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. The numeration was done using an electronic cell counter (Coulter Counter ZM). The results were expressed as cells per liter of solution.
pH values: The pH of the solutions was measured at the beginning and at end of test. The pH of the control medium was not to increase by more than 1.5 units during the test.
Other observations: The inoculum culture was examined microscopically to verify that it was normal and healthy in appearance and to detect any abnormal appearance of the algae (as may be caused by exposure to the test substance) at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: A range-finding test was performed using one replicate unit for each of five test item treatments at the concentrations of 0.01, 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No adverse effects were observed for the test item treatments up to and including 1.0 mg/L. The algae growth was totally inhibited at 10.0 mg/L. The definitive test was thus performed for test item treatments ranging between 0.4 and 6 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 0.2, 0.6, 0.75 and 1.0 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-confidence limits: 0.19 - 19.57 mg/L

Remarks:

Observed interval: 1.50 - 2.74 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95%-confidence limits: 0.29 - 4.10 mg/L

Remarks:

Observed interval: 0.85 - 1.50 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Definitive test:
Specific growth rate:
The specific growth rate was regular in the controls throughout the test period. The standard deviation between the three replicate units did not exceed 2.5% of the mean value at each sampling time and was less than 1 % for the average specific growth rate per day over the entire test period.
F-variance analysis at a 5% confidence level served to judge upon the difference for mean specific growth rate (section-by-section and total values) for each test item treatment compared to the water controls.
For both the 0.26 and the 0.50 mg/L test item treatments, the specific growth rate per day was considered as similar to that of the water controls for every intermediate period, and for the entire test period.
For the 0.85 mg/L test item treatment one intermediate period was significantly reduced but not the two others. For the entire test period the specific growth rate per day was considered as significantly reduced.
For both the two highest test item treatments the specific growth rate per day was considered as significantly reduced for every intermediate period and for the entire test period.
NOEC based on specific growth rate = 0.50 mg/L
The measured 95%-confidence interval was wide because the regression analysis involved only the three highest test item treatments. The two lowest test item treatments were excluded because of the plateau phase.
The observations showed that the ErC50-72 h was between 1.50 and 2.74 mg/L. This was reported as the observed interval.
ErC50-72 h for specific growth rate = 1.95 mg/L
95%-confidence limits = 0.19 - 19.57 mg/L
Observed interval = 1.50 - 2.74 mg/L

Yield:
F-variance analysis at a 5% confidence level served to judge upon the difference for yield in biomass for each test item treatment compared to the controls.
The difference with the water controls was not significant for the two lowest test item treatments. For the three highest test item treatments the yield in biomass was considered as significantly reduced.
NOEC based on yield = 0.50 mg/L
The regression analysis was performed using the four highest test item treatments. The lowest test item treatment was excluded because of the plateau-phase. Coefficient of determination for the regression analysis was 93.8%.
The observations showed that the EyC50-72 h was between 0.85 and 1.50 mg/L. This was reported as the observed interval.
EyC50-72 h for yield = 1.09 mg/L
95%-confidence level interval = 0.29 - 4.10 mg/L
Observed interval = 0.85 - 1.50 mg/L

pH values: The initial pH value was within the range 6-9 for the controls and every test item treatments. At the end of test the pH value was increased by 1.2 units for the water controls, and 1.4 units for the solvent controls. The three lowest test item treatments gave similar values, but for the two highest test item treatments the pH value was decreased.

Results with reference substance (positive control):

The EC50-72 h for Potassium dichromate was 0.67 mg/L for the specific growth rate and 0.39 mg/L for the yield. The EC50-72 h for Potassium dichromate was between 0.6 and 1.0 mg/L for the specific growth rate and between 0.2 and 0.75 mg/L for the yield. The results fulfilled the validity criteria based on Phytosafe historical data.

Reported statistics and error estimates:

Statistical determination of the NOEC: The NOEC was derived from the F-variance analysis at a 5%-confidence level of the response variable (specific growth rate or yield) for each test item treatment compared to the controls. The NOEC corresponds to the highest test item treatment that did not induce significant effects.
EC50 calculations: Consistently with the classical approach, EC50 values and the related 95% confidence intervals were derived from regression analysis of the concentration response-curves.

Range-finding test:

Daily numerations: The biomass increased by a factor of approximately 140 in the water control, and 160 in the solvent control. The test item treatments gave similar values for concentrations up to and including 10 mg/L. For higher concentrations the increase of the biomass was reduced.

Specific growth rate: The specific growth rate was considered as similar to that of the control for the test item treatments up to and including 1.0 mg/L. The specific growth rate was reduced for both the 10.0 and 100.0 mg/L test item treatments.

Yield: Yield in biomass was considered as similar to that in the control for every test item treatment up to and including 1.0 mg/L. For higher concentrations yield in biomass was almost totally inhibited.

pH values: The pH values were similar at test initiation for the controls and every test item treatments. At the end of test the pH value still remained in the controls and the test item treatments up to and including 1.0 mg/L. At both 10.0 and 100.0 mg/L the final pH value was decreased by 0.8 units.

 

Definitive test: 

Analytical verification of the test item treatments: The results showed that the test item treatments decreased down to less than 80% of the nominal values within a few hours. The test item concentrations were thus calculated as the geometric mean of the minimum and the maximum measured concentrations: 0.26, 0.50, 0.85, 1.50 and 2.74 mg/L.

 

Table 6.1.5/2: Definitive test - Percentages inhibition of specific growth rates per day (section-by-section and total)

 

Test item	0 to 24 h	24 to 48 h	48 to 72 h	Total period
0.26 mg/L	-1.7%	1.2%	-2.6%	-1.0%
0.50 mg/L	-0.6%	0.8%	-2.6%	-0.7%
0.85 mg/L	-1.9%	6.1%	2.7%	2.2%
1.50 mg/L	9.3%	40.5%	27.2%	25.3%
2.74 mg/L	36.8%	104.8%	85.4%	74.4%

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

 

Table 6.1.5/3: Definitive test - Mean measured yield inhibition

 

Test item	T0+72 h
0.26 mg/L	-6.15
0.50 mg/L	-4.55
0.85 mg/L	12.90
1.50 mg/L	79.41
2.74 mg/L	99.22

Negative values indicate that yield in biomass was increased as compared to the controls

 

VALIDITY OF THE TEST RESULTS

The test was considered as valid in the light of the following criteria:

- The biomass in the control cultures increased exponentially by a factor of at least 16 over the 72-hour test period.

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (this criterion applies to the mean value of the coefficients of variation calculated for replicate control cultures).

- The coefficient of variation for average specific growth rates over the entire test period in replicate control cultures did not exceed 7 %.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions and based on the geometric mean measured test concentrations, the ErC50 (growth rate) and EyC50 (yield) of the test item to Desmodesmus subspicatus were 1.95 mg/L (95% Cl: 0.19 - 19.57 mg/L) and 1.09 mg/L (95% Cl: 0.29 - 4.10 mg/L), respectively. The NOEC was 0.50 mg/L based on specific growth rate and yield.
However, this study is considered not assignable due to non sufficient information provided on the semi-static methodology used.

Executive summary:

In a algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Desmodesmus subspicatus was exposed to test item at the nominal concentrations of 0.4, 0.8, 1.5, 3.0 and 6.0 mg/L (geometric means of the measured concentrations: 0.26, 0.50, 0.85, 1.50 and 2.74 mg/L) (three replicates per concentration) for 72 hours, under constant illumination and shaking at a temperature of 21-24 °C. Water and solvent controls were included. The percent inhibition for growth rate and yield was determined. Before definitive test, a range-finding test was conducted (0.01, 0.10, 1.0, 10 and 100 mg/L) and no adverse effects were observed up to and including 1.0 mg/L; the algae growth was totally inhibited at 10.0 mg/L.

 

Analytical verification results showed that the test item treatments decreased down to less than 80% of the nominal values within a few hours. The test item concentrations were thus calculated as the geometric mean of the minimum and the maximum measured concentrations: 0.26, 0.50, 0.85, 1.50 and 2.74 mg/L.

 

Specific growth rate: The specific growth rate was regular in the controls throughout the test period. For both the 0.26 and the 0.50 mg/L test item treatments, the specific growth rate per day was considered as similar to that of the water controls for every intermediate period, and for the entire test period. For the 0.85 mg/L test item treatment one intermediate period was significantly reduced but not the two others. For the entire test period the specific growth rate per day was considered as significantly reduced. For both the two highest test item treatments the specific growth rate per day was considered as significantly reduced for every intermediate period and for the entire test period. The observations showed that the E_rC₅₀-72 h was between 1.50 and 2.74 mg/L, reported as the observed interval.

E_rC₅₀-72 h for specific growth rate = 1.95 mg/L

95%-confidence limits = 0.19 - 19.57 mg/L

 

Yield: The difference with the water controls was not significant for the two lowest test item treatments. For the three highest test item treatments the yield in biomass was considered as significantly reduced. The observations showed that the E_yC₅₀-72 h was between 0.85 and 1.50 mg/L, reported as the observed interval.

E_yC₅₀-72 h for yield = 1.09 mg/L

95%-confidence level interval = 0.29 - 4.10 mg/L

 

Under the test conditions and based on the geometric mean measured test concentrations, the E_rC₅₀ (growth rate) and E_yC₅₀ (yield) of the test item to Desmodesmus subspicatus were 1.95 mg/L (95% Cl: 0.19 - 19.57 mg/L) and 1.09 mg/L (95% Cl: 0.29 - 4.10 mg/L), respectively. The NOEC was 0.50 mg/L based on specific growth rate and yield.

All validity criteria were fulfilled. However, this study is considered not assignable due to non sufficient 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 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 for testing substances with a reasonable level of water solubility. 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 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:

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
The toxicity of the test item was predicted using the iSafeRat® Ecotox module providing the Subcooled Liquid Water Solubility (SLWS) as the input. The SLWS has been derived using a holistic approach: 44.4 mg/L (or -3.696 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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth 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 mechanisms of action. The model provides an in silico prediction for the 72-hour ErC50 value that can effectively be used in place of an experimentally derived 72-hour ErC50 value. The regression is method used to achieve this has been fully validated following the OECD (2004) recommendations.

GLP compliance:

no

Specific details on test material used for the study:

- Water solubility: 44.4 mg/L at 25°C (KREATiS, 2019)

Analytical monitoring:

no

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:

No difference in terms of toxic mechanism of action 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 cell size 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:

Results from a test duration of 72 hours only were used for this algorithm.

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 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:

The QSAR is based on data from reliable studies performed at acceptable oxygen concentrations.

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

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

Details on test conditions:

Following the guideline OECD 201, all studies were from a static test design. 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:

72 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 2.3 - 3.4 mg/L

Details on results:

The test item falls within the applicability domain of the model and was therefore reliably predicted for its toxicity (72h-ErC50) to algae. 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): 2.3 – 3.4 mg/L
QSAR statistical parameters are given in the QMRF and the QPRF

Analysis of the Applicability Domain of the model

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 - 4.38 to 0.49.

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 toxicity (72h-ErC50) to algae. Therefore, this endpoint value can be considered valid for use in risk assessment and classification and labelling.
The 72h-ErC50 of the test item to algae was predicted as 2.8 mg/L.
95% confidence interval (α = 0.05): 2.3 – 3.4 mg/L

Executive summary:

A Quantitative Structure-Activity Relationship (QSAR) was used to calculate the inhibition of growth to algae of the test item. 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" (OECD, 2006), referenced as Method C.3 of Commission Regulation No. 440/2008 (European Commission, 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.

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.

The 72h-ErC50 of the test item to algae was predicted as 2.8 mg/L.

95% confidence interval (α = 0.05): 2.3 – 3.4 mg/L

Description of key information

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

72h-ErC50 = 2.8 mg/L (95% confidence interval: 2.3 – 3.4 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.8 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, 2011) was considered as not assignable due to non sufficient 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 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 for testing substances with a reasonable level of water solubility. 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 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 1.95 mg/L (95% CI: 0.19 - 19.57 mg/L) and 0.50 mg/L, respectively. These results support the 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 test item to algae was predicted as 2.8 mg/L (95% CI: 2.3 – 3.4 mg/L)