Reaction mass of α-3,3-trimethyl-(αS,1R)-cyclohexanemethanol and α-3,3-trimethyl-(αR,1S)-cyclohexanemethanol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 20 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 202. All validity criteria were fulfilled

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2013

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses: Single samples for analysis were taken from the control and all test concentrations at the start of the test (t=0h) and from the control, the highest test concentration with no effect and the lowest test concentration causing 100% immobilisation at the end of the test (t=48h).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
-Method: The standard test procedures required preparation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures. A stock solution was prepared with a nominal concentration of 100 mg.L-1 for the definitive test. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and test water (2.2.) was added. Then 100 mg (nominal) of the test item (actual weight: 104.5 mg) were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing vessel was then carefully filled with the remaining volume of test water to obtain 1L of stock solution and thereafter was closed immediately. After 24 ± 2 hours of gentle stirring, the content of the vessel was allowed to stand undisturbed for at least 1 hour at room temperature. The first 100 mL were discarded by the drain port and samples were taken from the resulting stock solution and chemically analysed (measured concentration: 83.13 mg.L-1). Then the stock solution was diluted with test water as necessary into 200-mL volumetric flasks (filled up to the meniscus) to obtain the required test concentrations based on the measured concentration of the stock solution. Each prepared test solution was inverted several times before filling the test tubes (without headspace) to ensure adequate mixing and homogeneity. After filling, the vessels were sealed with screwcaps immediately after the introduction of daphnids. No bubbles were observed in the test tubes. The test solution was observed to be clear and colourless at all concentrations.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass beakers containing purified drinking water (Composition (mg/L): Calcium 11.5 – Magnesium 8.0 – Sodium 11.6 – Potassium 6.2 – Silica 31.7 – Bicarbonates 71.0 – Sulfates 8.1 – Chlorides 13.5 – Nitrates 6.3; pH = 7). Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. During the week the stock daphnids were fed daily with a suspension of freshwater algae (mix of 3 algae strains: Chlorella vulgaris = 2.5x10^6 cells/mL/day/daphnid, Desmodesmus subspicatus = 2.5x10^6 cells/mL/day/daphnid and Pseudokirchneriella subcapitata = 5x10^6 cells/mL/day/daphnid). The water was changed once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

ACCLIMATION
- At least 48 hours prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 hours old were used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Total water hardness was approximately 250 mg/L (as CaCO3). It can be noted that the water hardness of the batch of test water used for acclimation and preparation of the fresh medium at t=0h was 251 mg.L-1 (as CaCO3).

Test temperature:

Between 19.9 and 20.7°C throughout the test (average value: 20.4°C)

pH:

7.71 - 7.90
These test conditions remained within the limits prescribed (pH: 6.0-9.0, not varying by more than 1.5 units).

Dissolved oxygen:

8.66 - 9.05 mg O2/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal: 20, 26, 35, 46, 61 and 80 mg test item.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screwcaps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Test type: static test was performed because the substance was soluble and stable in test water.
- No. of daphnids: 20 per control and test concentration, divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Introduction of Daphnids: Daphnids were introduced into the test medium immediately after filling of test tubes with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202
- Conductivity: < 10 μS/cm

OTHER TEST CONDITIONS
- Light regime: 16 h light : 8 h darkness

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels.
- pH and dissolved O2: At the beginning and at the end of the test, for the highest test concentration and the control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.3
- Range finding study: Daphnids were exposed to a series of nominal test concentrations of 0, 1, 10, 100 and 250 mg.L-1 and to a concentration close to the solubility limit of test item in test water (maximum solubility).
- Results used to determine the conditions for the definitive study: The percent immobility was 0, 0, 0, 100, 100 and 100 (24 h and 48h) at 0, 1, 10, 100, 250 mg.L-1 and at the maximum solubility respectively. Based on the results of a range-finding test, the following nominal concentrations were prepared for the definitive test (spaced by a factor of approximately 1.3): 20, 26, 35, 46, 61 and 80 mg test item.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

45.583 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 42.093 - 49.364 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

45.021 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 41.586 - 48.740 mg/L

Details on results:

- After 24 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 35% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1.
- After 48 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 40% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1.

Results with reference substance (positive control):

On July 24, 2013 (most recent test), the 24h-EC50 was 0.98 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal test item concentrations. The software ToxRat® Professional (7) was used for the determination of the effective concentrations.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

 

Nominal concentration* (mg/L)	Replicate	Number of daphnids exposed	Response at 24 h		Response at 48 h
			Number	Total %	Number	Total %
Control	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
20.0	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
26.0	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
35.0	1	5	1	15	1	15
	2	5	1		1
	3	5	1		1
	4	5	0		0
46.0	1	5	3	35	3	40
	2	5	3		3
	3	5	0		1
	4	5	1		1
61.0	1	5	5	100	5	100
	2	5	5		5
	3	5	5		5
	4	5	5		5
80.0	1	5	5	100	5	100
	2	5	5		5
	3	5	5		5
	4	5	5		5

Water quality parameter values throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L-1) remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: 60% of the air-saturation value at the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

However, it can be noted that the water hardness (251 mg.L-1 as CaCO3) of the batch of test water used for acclimation and preparation of the fresh medium at t=0h was slightly above the 250 mg.L-1 (as CaCO3) given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

Furthermore, the temperature of the test medium was situated between 19.9 and 20.7 °C throughout the test (average value: 20.4 °C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water nor showed signs of stress.

Dissolved [O2]: The dissolved oxygen concentration at the end of the test was >= 60% of the air-saturation value in controls and test vessels.

Thus the validity criteria have been respected in the present study.

Validity criteria fulfilled:

yes

Conclusions:

The test substance was found to be stable over the study period. After 48 hours of experimentation, the EC50 value determined was of 45 mg test item.L-1 based on nominal concentrations.

Executive summary:

A study was performed to assess the acute toxicity of test item α-3,3-trimethylcyclohexanemethanol multiconstituent to Daphnia magna. The toxic effect of the test item was investigated in a static test, according to OECD Guideline No. 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and it was performed with GLP compliance. The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item at the required nominal test concentrations of 20, 26, 35, 46, 61 and 80 mg test item.L-1. The immobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by chemical analyses at the start and at the 48-hour incubation period.

 

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on daphnids was based on the nominal concentrations. After 24 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 35% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1. After 48 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 40% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1. Thus, the highest concentration resulting in no immobilisation at 48 hours was 26 mg.L-1 and the lowest concentration resulting in 100% immobilisation at 48 hours was 61 mg.L-1. The EC50 at each of the observation times was as follows:

- 24 h EC50: 45.583 mg/L (95% Confidence limits 42.093 - 49.364 mg/L)

- 48 h EC50: 45.021 mg/L (95% Confidence limits 41.586 - 48.740 mg/L)

 

The test substance was found to be stable over the study period. Therefore, after 48 hours of experimentation, the EC50 value determined was 45 mg test item.L-1 based on nominal concentrations. The substance is classified toxic to aquatic life (chronic category 3) according to CLP regulation (EC) No 1272/2008.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2014-05-26 to 2014-05-28

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:

Calculation method is used ; calculation method applicable for the endpoint

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Calculation method

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

Calculation method

Principles of method if other than guideline:

The acute toxicity to the daphnids was determined using a calculation method for the Mode of Action in question. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004). 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 EL50 tests on daphnid, for which the concentrations of the test item had been determined by chemical analyses over the test period.

GLP compliance:

no

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Daphnia magna

Details on test organisms:

Not applicable

Test type:

other: calculation method

Water media type:

not specified

Total exposure duration:

48 h

Remarks on exposure duration:

48h-EL50 (effective loading rate of WAF)

Post exposure observation period:

Not applicable

Hardness:

Hardness is not a necessary component of the WAF calculation

Test temperature:

The Temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine EL50s for daphnia between 12 and 28 °C.

pH:

The pH is not a necessary component of the WAF calculation

Dissolved oxygen:

The oxygen concentration is not a necessary component of the WAF calculation

Salinity:

Salinity is not a necessary component of the WAF calculation. However as the fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater organisms

Conductivity:

No data

Nominal and measured concentrations:

The calculation determines measured concentrations

Details on test conditions:

Calculation method

Reference substance (positive control):

not required

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

44 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Not applicable

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not applicable

Table 6.1.3/1: Expected concentrations at this 48 h-EL50

Constituents	concentration in the WAF (mg/L)
(αS,1R)-α-3,3-trimethyl-cyclohexanemethanol and (αR,1S)-α-3,3-trimethyl-cyclohexanemethanol	31.37
(1R-trans)-2,6,6-trimethyl-cycloheptanol and (1S-trans)-2,6,6-trimethyl-cycloheptanol	5.24
(R*,R*)-α-3,3-trimethyl-cyclohexanemethanol and (S*,S*)-α-3,3-trimethyl-cyclohexanemethanol	3.74
(1R-cis)-2,6,6-trimethyl-cycloheptanol and (1S-cis)-2,6,6-trimethyl-cycloheptanol	0.84

Validity criteria fulfilled:

yes

Conclusions:

48 h-EL50 for α-3,3-trimethylcyclohexanemethanol multiconstituent is 44 mg/L.

Executive summary:

A calculation method prediction was performed to assess the acute toxicity of test item α-3,3-trimethylcyclohexanemethanol multiconstituent, a multiconstituent substance, to Daphnia magna using the WAF method. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008 adapted for testing as a mixture using the WAF method. The criterion measured was the 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 the daphnids was determined using a calculation method for the Mode of Action in question. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004) (1). 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 EL50 tests on daphnid, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The iSafeRat® mixture module first uses Phase Equilibrium Thermodynamics to determine the analytically measurable concentrations in aqueous solutions expected from known WAF loading rates of characterised mixtures for fish, daphnids or algae. These loading rates are then divided into two parts: the bioavailable and non-bioavailable fractions per constituent. Existing experimental ecotoxicity endpoint values or predictions of these (from the single constituent ecotoxicity modules) are then used to determine the mixture ecotoxicity based on the sum of the chemical activities of the remaining bioavailable fractions of the individual constituents. The resulting value is converted back to the equivalent loading rate providing a EL50 value.

 

The EC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF. Using this approach, the 48-h EL50 for α-3,3-trimethylcyclohexanemethanol multiconstitent is 44 mg/L. The substance is therefore classified toxic to aquatic life (chronic category 3) according to CLP regulation (EC) N° 1272/2008.

 

This toxicity study is acceptable and can be used for that endpoint.

 

Description of key information

An experimental study is available on the registered substance: the 48h-EC50 of α-3,3-trimethylcyclohexanemethanol multiconstituent to Daphnia magna was determined to be 45 mg test item.L-1 based on nominal concentrations (analytically verified).

This result is confirmed by a reliable QSAR data that is available. The predicted 48 h-EL50 for the registered substance is 44 mg/L.

The substance is classified toxic to aquatic life (chronic category 3) according to CLP regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

44 mg/L

Additional information

Two valid acute studies were available to assess the acute toxicity of test item α-3,3-trimethylcyclohexanemethanol multiconstituent to Daphnia magna.

In one of the studies, the toxic effect of the test item was investigated in a static test, according to OECD Guideline No. 202, referenced as Method C.2 of Commission Regulation No. 440/2008, and it was performed with GLP compliance. The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

The test item levels were found to be relatively stable throughout the test and the evaluation of the effects on daphnids was based on the nominal concentrations. After 24 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 35% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1. After 48 hours of exposure, immobilisations were 0% at 0, 20 and 26 mg.L-1, 15% at 35 mg.L-1, 40% at 46 mg.L-1 and 100% at 61 and 80 mg test item.L-1. Thus, the highest concentration resulting in no immobilisation at 48 hours was 26 mg.L-1 and the lowest concentration resulting in 100% immobilisation at 48 hours was 61 mg.L-1. After 48 hours of experimentation, the EC50 value determined was of 45 mg test item.L-1 based on nominal concentrations.

Furthermore, a QSAR prediction was available as a supportive study and has been performed to assess the acute toxicity of test item α-3,3-trimethylcyclohexanemethanol multiconstitent, a multiconstituent substance, to Daphnia magna using the WAF method. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008 adapted for testing as a mixture using the WAF method. The criterion measured was the 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 EC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF. Using this approach, the 48-h EL50 for α-3,3-trimethylcyclohexanemethanol multiconstituent is 44 mg/L.