Reaction mass of (3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aR*,5aS*,9aS*,9bS*)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

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:

mixture rules calculation

Adequacy of study:

key study

Study period:

From 02 november 2021 to 22 november 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

(tested according to the Water Accommodated Fraction (WAF) approach, i.e. OECD technical guideline 23)

Principles of method if other than guideline:

The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). 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. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction, this approach is based on validated data derived from 48-hour tests on daphnid, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Remarks:

calculation model

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

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: calculation method based on QSAR model predictions

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Only results from a test duration of 48 hours were included in the training set of the model.

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 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:

The test resultats is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (>60% of the air-saturation value throughout the duration of the test). In exceptional cases where studies with oxygen concentrations lower than 60% were used, all aspects of the study were thoroughly evaluated in order to satisfy the evaluator that the effects found were not due to reduced oxygen concentration (i.e. the study would correctly receive a Klimisch score of 2 under the REACH Regulation [REACH, 2006]).

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, 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):

not required

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.73 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate of Water Accomodated Fraction (WAF)

Basis for effect:

mobility

Remarks on result:

other: > solubility limit

Results with reference substance (positive control):

Not applicable

The calculation method used in this study is based on toxic additivity principle. That means the toxic parts of each constituent are added up. Therefore the constituents considered within the mixture should act with a similar MechoA.
The MechoA of the consituents are determined using the methodology described by Bauer et al. (2018) and reported

MechoA of the constituents.
constituents             MechoA             Description
Isomer1                     1.1                   non-polar narcotic
Isomer2                     1.1                   non-polar narcotic
Isomer3 (impurity)      1.1                   non-polar narcotic
Since the constituents of the test item act with the same general MechoA, the calculation method is directly applicable.

Validity criteria fulfilled:

yes

Conclusions:

The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item has been determined using the iSafeRat® calculation method for mixtures tested according to the Water Accomodated Fraction (WAF) approach. Each constituent of the test item does not completely fall within the applicability domain of the QSAR model used to determine their individual ACUTE TOXICITY TO DAPHNIDS (48-HOUR EC50). Their toxicity values were extrapolated. However, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as an extrapolation (reliable with restrictions). The result remains valid for use in risk assessment and classification and labelling.
The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item tested according to the WAF method was predicted as greater than the solubility limit.

Executive summary:

A calculation method was used to assess the ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item,  tested according to the Water Accommodated Fraction (WAF) approach. This calculation method 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, 2004a), referenced as Method C.2 of Commission Regulation No. 440/2008 (European Commission, 2008) adapted for testing of a mixture using the WAF method. The criterion predicted was the median effective loading rate of the mixture EL₅₀ (Median Effect Loading), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). 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. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction, this approach is based on validated data derived from 48-hour tests on daphnid, for which the concentrations of the test item had been determined by chemical analyses over the test period.

Each constituent of the test item does not completely fall within the applicability domain of the QSAR model used to determine their individual ACUTE TOXICITY TO DAPHNIDS (48-HOUR EC50). Their toxicity values were extrapolated. However, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as an extrapolation (reliable with restrictions). The result remains valid for use in risk assessment and classification and labelling.
The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item tested according to the WAF method was predicted as greater than the solubility limit.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2014-09-02 to 2014-09-04 and from 2014-11-05 to 2014-11-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non-GLP range finding test performed on the registered substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Specific details on test material used for the study:

- Relative density: 1.04
- Water solubility: 7.18 mg/L (20 °C, pH 7, in Daphnia medium Elendt M4) (Isomer 1: 3.45 mg/L, Isomer 2; 2.52 mg/L, Isomer 3: 1.20 mg/L)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h)
- Sampling method: At the start of the exposure intervals (0 and 24 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure intervals (24 and 48 h) samples were taken from additional replicates prepared with test media, but without daphnids, because volumes of the test replicates were insufficient for the analytical determination of the test concentrations. These additionally prepared replicates were incubated under test conditions until sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of the Test Solutions in the 1st Test
- Method: The stock solution (1 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h).
- The stock solution was stirred with approximately 1100 rpm for 1 h at room temperature, then treated with ultrasound for 30 minutes and stirred for further 1 h with approximately 1100 rpm at room temperature until the stock solution was visually clear.
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Preparation of the Test Solutions in the 2nd Test
- Method: The stock solution (7 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of both exposure intervals (at 0 and 24 h).
- The stock solution was treated with ultrasound for 15 minutes at room temperature and stirred thereafter with a magnetic stirrer at approximately 1100 rpm for 24 h at room temperature.
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut fur Wasser-, Boden- und Lufthygiene (WaBoLu)
- Culture: In glass vessels (2-3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination; light intensity of max. 20 µE/m2/s
- Culture feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL
- Age at study initiation: 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding: 2 to 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels latest 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test after an acclimatisation phase of 2 h in the dilution water. No first brood progeny was used for the test.
- Feeding during test: Daphnids were not fed during the study

ACCLIMATION
- Acclimation period: At least 2 h in dilution water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

160 to 180 mg CaCO3/L

Test temperature:

18-22 °C

pH:

1 mg/L: 7.77 (0 h); 7.46 (24 h)
7 mg/L: 7.79 (0 h); 7.70 (24 h)

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

1st Test: 0.01, 0.1 and 1 mg/L
2nd Test: 0.07, 0.7 and 7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm) 50 mL capacity, loosely covered with watch glasses
- Type (delete if not applicable): Closed
- Test volume: 20 mL
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Application: Per replicate, 20 g test solution was weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h/8 h light/dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE/m2/s

EFFECT PARAMETERS MEASURED:
Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other relevant observations were not made.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
1st test: 3 test item concentrations in a geometric series with a separation factor of 10, prepared by diluting the stock solution of 1 mg/L with dilution water were tested as follows: 0.01, 0.1 and 1 mg/L.
2nd test: 3 test item concentrations in a geometric series with a separation factor of 10, prepared by diluting the stock solution of 7 mg/L with dilution water were tested as follows: 0.07, 0.7 and 7 mg/L.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Biological effects of the 1st and the 2nd range finding test are consistent at 0.7 mg/L (5 % immobilization) and 1 mg/L (10 % immobilization).

Details on results:

The biological effects of the 1st and the 2nd range finding test are consistent at 0.7 mg/L (5 % immobilization) and 1 mg/L (10 % immobilization). 100 % immobilisation at 7 mg/L. The EC50 is expected to be > 1 mg/L based on analytically confirmed nominal concentrations.

Results with reference substance (positive control):

None

Reported statistics and error estimates:

None

Table 1: Immobilisation Rates

Nominal test item concentration [mg/L]	IMMOBILISATION [%]
	24 h			48 h
	Replicates			Replicates
1^stRange Finding Test (non GLP)
	1	2	MV	1	2	MV
1	0	0	0	0	20	10
0.1	0	0	0	0	0	0
0.01	0	0	0	0	0	0
Control	0	0	0	0	0	0
2^ndRange Finding Test (non GLP)
7	100	100	100	100	100	100
0.7	0	0	0	10	0	5
0.07	0	0	0	0	0	0
Control	0	0	0	0	0	0

 

MV = mean value

The isomer distribution in the 1st exposure interval of the 1st range finding test is equal to the isomer distribution of the standards. In the 2nd exposure interval of the 1st range finding test, a slight shift in the isomer distribution was observed and should be taken with caution, because when preparing the standards there is no shift. However, the determined recoveries in the 1st range finding test are close to the nominal values (72 to 103 %).

Recoveries during the 2nd definitive were very low (in the range of 28 - 55 % of the nominal values). This is likely to be due to the fact that a too high concentration of 7 mg/L has been dosed. Although visually clear test item solutions were observed, the isomer distribution in the test solutions differs from the distribution in the standards. The isomers were not in solution. The stock solution of 7 mg/L was further diluted to lower concentrations and such the error was propagated. Those analytical results are not reliable, they should not be used.

Validity criteria fulfilled:

yes

Conclusions:

The biological effects of the 1st and the 2nd range finding test are consistent at 0.7 mg/L (5 % immobilization) and 1 mg/L (10 % immobilization). The EC50 is expected to be > 1 mg/L based on analytically confirmed nominal concentrations.

Executive summary:

Two non GLP acute immobilisation tests with Daphnia magna (STRAUS) were performed as range finding tests to determine the effects of the test substance according to OECD 202 and EU Method C.2 guidelines.

 

The range finding tests were conducted under semi-static conditions over a period of 48 h with 3 concentrations of the test substance prepared with dilution water in a geometric series with a separation factor of 10. The test concentrations in the 1st range finding test were 0.01, 0.1 and 1 mg/L. In the 2nd range finding test, the test concentrations were 0.07, 0.7 and 7 mg/L. There were two replicates per treatment with ten daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 18-22 °C.

 

The biological effects of the 1st and the 2nd range finding test are consistent at 0.7 mg/L (5 % immobilization) and 1 mg/L (10 % immobilization). 100 % immobilisation at 7 mg/L. The EC50 is expected to be > 1 mg/L based on analytically confirmed nominal concentrations. As the substance was difficult to test the definitive study was not performed and alternative methods were preferred.

Description of key information

KREATis 2021, calculation method, 

48h-EL50 greater than solubility limit (2.7 mg/L)  

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 2.7 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, two data are available.

 

The first data (QSAR-KREATiS, 2020) is assessed as the key study and is a calculation method. The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) was determined using iSafeRat® calculation
method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This method has been published for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2021). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004b, 2007). 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. Further to this the effective loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction, this approach is based on validated data derived from 48-hour tests on daphnid, for which the concentrations of the test item had been determined by chemical analyses over the test period.The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item has been determined using the iSafeRat® calculation method for mixtures tested according to the Water Accomodated Fraction (WAF) approach. Each constituent of the test item does not completely fall within the applicability domain of the QSAR model used to determine their individual ACUTE TOXICITY TO DAPHNIDS (48-HOUR EC50). Their toxicity values were extrapolated. However, each constituent of the test item acts with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling. Therefore, the final result for the test item is considered as an extrapolation (reliable with restrictions). The result remains valid for use in risk assessment and classification and labelling.
The ACUTE TOXICITY TO DAPHNIDS (48-HOUR EL50) of the test item tested according to the WAF method was predicted as greater than the solubility limit (2.7 mg/l)

The second data (NOACK, 2015) is assessed as a supporting data and is an experimental study. In this study, two non GLP range-finding tests were performed on the registered substance according to OECD Guideline 202 and EU Method C.2. These range finding tests were conducted under semi-static conditions over a period of 48 hours with three concentrations of the test substance prepared with dilution water in a geometric series with a separation factor of 10. The test concentrations in the first range finding test were 0.01, 0.1 and 1 mg/L. In the second range finding test, the test concentrations were 0.07, 0.7 and 7 mg/L. There were two replicates per treatment with ten daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. The biological effects of the first and the second range-finding tests are consistent at 0.7 mg/L (5% immobilisation) and 1 mg/L (10% immobilisation). Therefore, the 48h-EC50 value is expected to be greater than 1 mg/L based on analytically confirmed nominal concentrations. As this value is in accordance with the iSafeRat® prediction and the substance falls under the definition of "difficult to test" according to OECD guidance No. 23, the definitive study was not considered relevant.

 

In conclusion, the experimental range-finding tests performed on the registered substance supports the key prediction data. Therefore, the 48h-EC50 value was considered to be 2.7 mg/L.