3-ETHYL-5,5-DIMETHYL-GAMMA BUTYROLACTONE

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:

(Q)SAR

Adequacy of study:

key study

Study period:

03 August 2020

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® – in Silico Algorithms For Environmental Risk And Toxicity

2. MODEL (incl. version number)
iSafeRat® algErC50 v1.9

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Isomer 3R: CC[C@@H]1CC(C)(C)OC1=O
Isomer 3S: CC[C@H]1CC(C)(C)OC1=O

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 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not applicable

Remarks:

QSAR model

Principles of method if other than guideline:

The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) was determined using iSafeRat® algEC50 and iSafeRat® algNOEC, two validated QSAR models for the Mechanism of Action (MechoA) in question (MechoA 2.1, i.e. mono-/poly-esters whose hydrolysis products are narcotics) (Bauer et al., 2018). The QSAR models provide in silico predictions for the 72-hour ErC50 and NOECr values that can effectively be used in place of an experimentally derived results. The QSAR models are based on validated data for a training set of 44 chemicals derived from 72-hour ErC50 for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR model for 72-hour NOECr was not applicable for the test item due to the limitation of the mechanistic domain.

GLP compliance:

no

Remarks:

QSAR model

Specific details on test material used for the study:

- Water Solubility: 5610 mg/L (Phytosafe, 2009)
- Mechanism of action: MechoA 2.1, mono-/poly-esters whose hydrolysis products are narcotics (Bauer et al., 2018)

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: 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 model

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:

not applicable

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 models. This small difference is not expected to significantly contribute to the variability of the 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 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.

Salinity:

not applicable

Conductivity:

not applicable

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):

not required

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

not determinable because of methodological limitations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

130 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval (α = 0.05): 110 – 160 mg/L.

Details on results:

The test item falls within the applicability domain of the model iSafeRat® algErC50 and was therefore reliably predicted for its TOXICITY TO ALGAE (72-HOUR ErC50).
The QSAR model for 72-hour NOECr was not applicable for the test item due to the limitation of the mechanistic domain.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05) for 72h-ErC50: 110 – 160 mg/L.

Applicability Domain

Descriptor domain
The Subcooled Liquid Water Solubility value (5610 mg/L or -1.404 in log10(mol/L)) given as the input to the iSafeRat® algErC50 model falls within the descriptor domain of the model between a Subcooled Liquid Water Solubility of -5.57 to 0.93 in log10(mol/L).

Structural fragment domain
All chemical groups within the molecular structure are taken into account by the models.

Mechanistic domain
Currently, the iSafeRat® algErC50 model 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 iSafeRat® algNOECr model can only reliably predict the aquatic toxicity for chemicals with the mechanism of action of non-polar narcosis (MechoA 1.1).

The MechoA of molecules is predicted directly from the structure. The test item as an ester is expected to exert a MechoA 2.1 (Bauer et al., 2018) and can be taken into account by the model iSafeRat® algErC50 but not by the model iSafeRat® algNOECr.

Validity criteria fulfilled:

yes

Conclusions:

The QSAR models used to achieve the study have been fully validated following the OECD recommandations (OECD, 2004). The test item falls within the applicability domain of the model for 72-HOUR ErC50 and was therefore reliably predicted for its TOXICITY TO ALGAE (72-HOUR ErC50). Therefore, this endpoint value can be considered valid for use in risk assessment and classification and labelling.

The test item falls out of the mechanistic domain of the model for TOXICITY TO ALGAE (72-HOUR NOECr).

The TOXICITY TO ALGAE (72-HOUR ErC50) of the test item was predicted as 130 mg/L.
95% confidence interval (α = 0.05) for 72h-ErC50: 110 – 160 mg/L.

Executive summary:

Two Quantitative Structure-Activity Relationship (QSAR) models were used to calculate the TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) of the test item. These QSAR models have been validated to be compliant with the OECD  recommendations for QSAR modeling (OECD, 2004) and predict the endpoint values 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 criterions predicted were the Median Effective Concentration (ErC50), a statistically derived concentration which is expected to cause 50% inhibition of intrinsic rate of growth of the test system and the No Observed Effect Concentration (NOECr), a tested concentration which is expected to cause no effect on intrinsic rate of growth of the test system. Both criterions were determined for a period exposure of 72 hours.

 

The TOXICITY TO ALGAE (72-HOUR ErC50 and NOECr) was determined using iSafeRat® algEC50 and iSafeRat® algNOEC, two validated QSAR models for the Mechanism of Action (MechoA) in question (MechoA 2.1, i.e. mono-/poly-esters whose hydrolysis products are narcotics) (Bauer et al., 2018). The QSAR models provide in silico predictions for the 72-hour ErC50 and NOECr values that can effectively be used in place of an experimentally derived results. The QSAR models are based on validated data for a training set of 44 chemicals derived from 72-hour ErC50 for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR model for 72-hour NOECr was not applicable for the test item due to the limitation of the mechanistic domain.

 

The QSAR models used to achieve the study have been fully validated following the OECD recommandations (OECD, 2004). The test item falls within the applicability domain of the model for72-HOUR ErC50 and was therefore reliably predicted for its TOXICITY TO ALGAE (72-HOUR ErC50).Therefore, this endpoint value can be considered valid for use in risk assessment and classification and labelling. The test item falls out of the mechanistic domain of the model for TOXICITY TO ALGAE (72-HOUR NOECr).

 

The TOXICITY TO ALGAE (72-HOUR ErC50) of the test item was predicted as 130 mg/L.
The TOXICITY TO ALGAE (72-HOUR NOECr) of the test item was not predicted.
95% confidence interval (α = 0.05) for 72h-ErC50: 110 – 160 mg/L.
95% confidence interval (α = 0.05) for 72h-NOECr: not determined.

 

 

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-07-24 to 2009-09-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

This study was performed according to OECD Guideline 201 with GLP certificate. All validity criteria were fulfilled and the substance is adequately identified. 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 use of solvent is not the best method at the time being. Considering the sufficiently high water solubility of the substance (5610 mg/L) and the concentration used in this study (limit test at 100 mg/L), this method could have been avoided. 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 (Alga, Growth Inhibition Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection on July 15-16 2008, implementation on July 16 2008.

Specific details on test material used for the study:

- Storage condition of test material: Room temperature protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The test substance was quantified in the treatment solutions used for the limit test at both test initiation and test completion in one replicate unit taken at random.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The treatment solutions were prepared in acetone. The concentrations of the treatment solutions were set in such a manner that treatment concentrations were achieved when delivering 50 µL of the treatment solutions per test vessel.
- Method: To prepare the limit test concentration (99.8 mg/L), 2.0006 g of test item were mixed with acetone qsp 10 mL. 0.05 mL were taken and completed to 10 mL.
- Controls: Sovent control units served to ascertain that acetone had no detrimental effect on the culture growth.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone was used as solvent.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.5 mL/L

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): The strain was provided by in December 2007 by the Muséum National d'Histoire Naturelle (Paris, France) and regularly sub-cultured in the OECD medium at Phytosafe site.
- Age of inoculum (at test initiation): 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 th etest solutions.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test.

Test type:

static

Water media type:

other: reconstituted water

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

The temperature was set in the range of 21 to 24 °C and kept constant to within +/- 2°C.

pH:

From 8.25 to 8.64

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

A range finding test was performed, testing the following nominal concentrations: 0.01 - 0.1 - 1.0 - 10.0 and 99.8 mg/L.
A limit test was performed at a concentration of 99.8 mg/L.
The measured concentrations were reported in the table 6.1.5/2 in "Any other information on results incl. tables". The measured concentrations represented 104.2% and 96.6% the nominal value at test initiation and at the end of test, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (250mL) filled with 100 mL of culture served as test vessels. They were capped with air-permeable stoppers.
- Initial cells density: 2 to 5 x 10 E-3 cells/mL
- No. of vessels per concentration (replicates): 6 replicate units for the 100 mg/L treated groups, 3 replicate units for each of four concentrations of Potassium dichromate
- No. of vessels per control (replicates): 6 replicate units for the controls (water control, solvent control)

GROWTH MEDIUM
- Standard medium used: yes, OECD medium from OECD TG 201 according to ISO 8692

OTHER TEST CONDITIONS
- Light intensity and quality: Cultures received continuous uniform fluorescent illumination within 4440 - 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic cell counter (Coulter Counter ZM)

TEST CONCENTRATIONS
- Range finding study : yes, testing the following cocnentrations : 0.01 - 0.1 - 1.0 - 9.9 and 98.7 mg/L.
- Test concentrations: one limit concentration tested of 98.8 mg/L.
- Results used to determine the conditions for the definitive study: In the range-finding test, neither the specific growth rate nor the yield was affected in the 100 mg/L test item treated unit. The definitive test was thus performed as a limit test for the 100 mg/L treatment concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as reference substance.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The control cultures showed exponential growth throughout the test period. The multiplication rate was approximately 105 (mean value). Similar values were observed for the solvent controls.
The multiplication rate was slightly decreased in the test substance treated units (100 mg/L) as compared with the control even through the growth rate appeared to remain exponential throughout the test period: the final cell density represented only approximately 93 times the initial value (mean value).

Results with reference substance (positive control):

- Results with reference substance valid? yes
EC50 for specific growth rate between 0.60 and 1.03 mg/L
EC50 for yield between 0.20 and 0.75 mg/L

Reported statistics and error estimates:

NOEC was derived from the F-variance analysis at a 5%-confidence level of the response variable (specific growth rate) in each treated group as compared to the control group. NOEC corresponds to the highest tested concentration that failed to induce significant effects.

Table 6.1.5/2 : Analysis of the test solutions

The test material was quantified in the treatment solutions used for the limit test at both test initiation and test completion, in one replicate unit taken at random.

Nominal (mg/L)	Measured concentration (mg/L)	Percent recovery (%)
Test initiation	104.7	105.0
	103.3	103.5
Mean	-	104.2
Test completion	97.0	97.2
	95.9	96.1
Mean	-	96.6

Table 6.1.5/3 : Measured % inhibition of specific growth rate per day (section-by section and total), for the range-finding test and the limit test

Time \ Treatment (mg/L)	0,01  (RF test)	0,1  (RF test)	1  (RF test)	10,0  (RF test)	99,8  (RF test)	99,8 (limit test)
0 to 24 h	-6,1	-23,80	-21,30	8,10	5,7	-0.5
24 to 48 h	-27,00	-9,9	24,60	-19,10	11,3	-0.5
48 to 72 h	-50,10	-41,80	-87,10	35,50	-31,50	7.8
total period	-25,50	-23,60	-22,20	5,60	-2,30	2.7

Table 6.1.5/4 : Measured specific growth rates per day (section-by-section and total), for the range-finding test (RF test) and the limit test

Time \ Treatment (mg/L)	solvent control (limit test)	water control (limit test)	solvent control (RF test)	water control  (RF test)	0,01  (RF test)	0,1  (RF test)	1  (RF test)	10,0  (RF test)	99,8  (RF test)	99,8 (limit test)
0 à 24	1,48	1,47	1,23	1,19	1,26	1,48	1,45	1,09	1,12	1,48
24 à 48	1,46	1,40	1,27	1,18	1,50	1,30	0,89	1,41	1,05	1,41
48 à 72	1,71	1,78	1,05	0,87	1,31	1,24	1,63	0,56	1,15	1,64
total period	1,55	1,55	1,18	1,08	1,36	1,34	1,32	1,02	1,11	1,51

The specific growth rate was slightly affected for the overall 72h testing period (2.7% inhibition) due to a significant decrease for the last day of testing (7.8% inhibition).

NOEC obtained for growth rate is < 100 mg/L.

The ErC50 based on specific growth rate is > 100 mg/L.

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks".

Conclusions:

The specific growth rate was slightly affected for the over-all 72 hour testing period (2.7% inhibition) due to a significant decrease for the last day of testing (7.8% inhibition). Therefore, the NOEC for growth rate was < 100 mg/L and the ErC50 based on specific growth rate was > 100 mg/L.
Even if all validity criteria were fulfilled, this study is considered reliable with restrictions.

Executive summary:

The influence of the test substance on algal growth inhibition (Desmodesmus subspicatus) was investigated according to OECD Guideline 201. Algae were exposed to the test substance under static conditions during 72 hours.

Based on the results of a range-finding test, a limit test was performed at a concentration of 99.8 mg/L. Six replicate units were performed for the solvent control, the water control and the 99.8 mg/L treated group (limit test). The test item concentration was quantified in the treatment solutions used for the limit test at both test initiation and completion, in one replicate unit taken at random. The resulting solution was assessed by GC/FID, using external calibration.

The specific growth rate was slightly affected for the over-all 72 hour testing period (2.7% inhibition) due to a significant decrease for the last day of testing (7.8% inhibition). Therefore, the NOEC for growth rate was < 100 mg/L and the ErC50 based on specific growth rate was > 100 mg/L.

Even if all validity criteria were fulfilled, this study is considered reliable with restrictions for the following reasons:

- 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. The use of solvent is not necessary or even useful to test soluble test substances.

- Considering the sufficiently high water solubility of the substance (5610 mg/L) and the concentration used in this study (limit test at ca. 100 mg/L), this method could have been avoided.

- 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).

Description of key information

iSafeRat® High-Accuracy-Quantitative Structure-Activity Relationship, KREATIS, 2020 :

72h-ErC50 Algae = 130 mg/L (95% confidence interval: 110 – 160 mg/L)

72h-NOECr Algae = not determined

Key value for chemical safety assessment

EC50 for freshwater algae:

130 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, 2009) was performed according to OECD Guideline 201 with GLP statement but was 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 use of solvent is not necessary for water soluble substances. Considering the high water solubility of the substance (5,61 g/L) and the test concentrations used in this study, this method could have been avoided.  Furthermore, 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 specific growth rate was slightly affected for the over-all 72 hour testing period (2.7% inhibition) due to a significant decrease for the last day of testing (7.8% inhibition), therefore the 72h-NOECr reported in the study report, based on growth rate of the algae Desmodesmus subspicatus, was lower than 100 mg/L, the limit test concentration. However, the 72h-ErC50 value based on growth rate was greater than 100 mg/L. This result supports the (more quantitative) key data presented below.

 

The QSAR prediction (KREATiS, 2020b) was considered as reliable and was used as key data. The QSAR model (iSafeRat® algEC50) 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 2.1, i.e. mono-/poly-esters whose hydrolysis products are narcotics) (Bauer et al., 2018). The QSAR model is based on validated data for a training set of 44 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 130 mg/L (95% CI: 110– 160 mg/L)

This quantitative value was considered as the key value for chemical safety assessment.

Another QSAR model (iSafeRat® algNOEC) was assessed to determine the 72-hour NOECr but was not applicable for the test item due to the limitation of the mechanistic domain.