Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

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:

2013-10-28 to 2013-10-31

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 11, 2013

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses: single samples for analysis were taken from the control and all test concentrations from replicates without daphnids (except at the end of the test: replicates with daphnids).
- Frequency of sampling: at the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The standard test procedures required preparation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures. The stock and test solutions were prepared under closed conditions and gently stirred to avoid production of a dispersion.
Two stock solutions (one for the fresh medium at t=0 h and another for the fresh medium at 24 h) were prepared by slow-stirring. 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 150 mg (nominal) of the test item (actual measured amount: 151.45 mg for the fresh medium at t=0 h and 153.09 mg for the fresh medium at t=24 h) were weighed on a weighing boat that afterwards was placed above the mixing vessel and rinsed with test water. The mixing was then carefully filled with the remaining volume of test water to obtain 1 L of stock solution and thereafter was closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. The stirring speed was kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 ± 2 hours of gentle stirring, the contents of the vessel was allowed to stand undisturbed for 1 hour at room temperature. Then the first 100 mL were discarded by the drain port and 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 and filled into test tubes. Each prepared concentration was inverted several times before filling the test tube (without headspace) to ensure adequate mixing and homogeneity. After filling the vessels were sealed immediately with screwcaps after introduction of daphnids. No bubbles were observed in the test tubes. The test solution was observed to be clear and colourless and 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 old
- 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
- Acclimation: At least 48 h 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 h old were used for the test.

Test type:

semi-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)

Test temperature:

19.6-20.6 °C (average value: 20.2 °C)

pH:

At the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) in all vessels.
pH: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

At the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) in all vessels.
oxygen: ≥ 60% of the air-saturation value at the end of the test.

Salinity:

No data

Nominal and measured concentrations:

Nominal concentrations: 49, 61, 77, 96, 120 and 150 mg/L
Measured concentrations: see Table 6.1.3/1

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screwcaps.
- Aeration: No aeration of the test solutions occurred throughout the test.
- A semi-static test was performed with renewal of test solutions after 24 h, in order to maintain test concentrations within 80-120% of nominal values.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- 4 replicates with daphnids for the control and per test concentration prepared on the basis of the concentration measured in the stock solution. Moreover, 3 abiotic replicates for the control and per test concentration were prepared:
- two for sampling for analysis of test concentrations from the freshly prepared solutions (t=0 h and t=24 h fresh)
- and another for sampling from the 24 h old solutions (t=24 h old).
Chemical analyses were taken from abiotic replicates in order to avoid disturbing the daphnids at t=24 h and to prevent test item losses on opening test tubes (the range-finding test showed no significant differences between measured concentrations of old solutions with or without daphnids). The sampling for analysis of the control and test concentrations at t=48 h was performed directly from one of the 4 replicates with daphnids.
- Introduction of Daphnids: Daphnids were introduced into the test medium immediately after filling the 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
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.3
- Range finding study
- Test concentrations: 1, 5, 10, 25, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: After 48 h of exposure, immobilisations were 0% at 1, 5, 25 and 50 mg/L, 10% at 10 mg/L and 60% at 100 mg/L. Based on the results of a range-finding study, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to the test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.3): 49, 61, 77, 96, 120 and 150 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

93.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 86.64 - 101.57 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

86.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 80.03 - 93.44 mg/L

Details on results:

- After 24 h of exposure, immobilisations were 0% at 49 mg/L, 5% at 61mg/L, 20% at 77 mg/L, 55% at 96 mg/L, 80% at 120 mg/L and 100% at 150 mg/L.
- After 48 h of exposure, immobilisations were 0% at 49 mg/L, 10% at 61 mg/L, 30% at 77 mg/L, 60% at 96 mg/L, 95% at 120 mg/L and 100% at 150 mg/L.

Results with reference substance (positive control):

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

Reported statistics and error estimates:

The evaluation of the effects was based on the measured test item concentrations. The software ToxRat® Professional was used for the determination of the effective concentrations. The 24 and 48 h EC50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.

Analytical results:

The analytical results of this test showed that concentrations of test solutions prepared following the outlined procedures were reproducible. Indeed, analyses of samples taken from all test concentrations at the start and the end of the test revealed that test item levels found were stable, with losses of main constituents < 20%.For information, the results of the nominal concentrations and the summed measured concentrations of the constituents are presented in the table below. Thus, since initial and final measured concentrations were comparable to their expected test item concentration and that the concentration of main constituents (accounting for 97.2% of the test item) was satisfactorily maintained within ± 20% of the nominal concentration throughout the test, the results can be based on nominal values.

Table 6.1.3/1: Comparison between nominal concentrations and summed measured concentrations of the constituents

Concentration (mg/L)	Summed measured values of each constituent (mg/L)
	Start (t=0 h)	t=24 h Old	Relative loss to initial value (%)	t=24 h Fresh	t=48 h Old	Relative loss to initial value (%)
Control	absence	absence	N.A.	absence	absence	N.A.
49^a/47.64^b	49.58	45.11	9	44.39	45.12	-2
61^a/59.28^b	59.84	58.18	3	60.07	58.96	2
77^a/74.85^b	77.24	72.71	6	74.40	78.72	-6
96^a/93.30^b	110.61	96.51	13	96.51	91.44	5
120^a/116.64^b	130.19	112.48	14	114.85	121.16	-5
150^a/145.80^b	146.89	121.85	17	*	*	N.A.

 

N.A.: not applicable

*: no further analysis was performed for concentrations in which all daphnids were immobilized at t=24 h.

^a Nominal concentrations as test item

^b Nominal concentrations as main constituents.

Table 6.1.3/2: pH-values and Dissolved oxygen saturation (mg/L) during the final test

 

Time		Nominal concentration (mg/L)
		Control	49	61	77	96	120	150
pH-values
Start t=0 h		7.80	7.86	7.88	7.89	7.90	7.90	7.90
t=24 h	Old	7.58	7.75	7.82	7.83	7.83	7.84	7.73
	Fresh	7.94	7.95	7.96	7.96	7.97	7.97	*
End t=48 h		7.72	7.74	7.75	7.76	7.78	7.80	*
Dissolved oxygen saturation (mg/L)
Start t=0 h		8.67	8.50	8.47	8.42	8.43	8.42	8.41
t=24 h	Old	8.64	8.46	8.42	8.38	8.38	8.29	8.27
	Fresh	8.68	8.55	8.49	8.56	8.55	8.42	*
End t=48 h		8.66	8.12	8.52	8.35	8.22	8.27	*

 

*no further analysis was performed for concentrations in which all daphnids were immobilised

Table 6.1.3/3: Acute immobilisation of daphnids after 24 and 48 hours 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
49	1	5	0	0	0	0
	2	5	0		0
	3	5	0		0
	4	5	0		0
61	1	5	0	5	0	10
	2	5	0		1
	3	5	0		0
	4	5	1		1
77	1 2 3 4	5 5 5 5	1 1 0 2	20	2 1 0 3	30
96	1 2 3 4	5 5 5 5	3 3 1 4	55	3 3 1 5	60
120	1 2 3 4	5 5 5 5	3 5 4 4	80	5 5 5 4	95
150	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilised 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 fulfilled in the present study.

Validity criteria fulfilled:

yes

Conclusions:

The 48 h-EC50 for dihydroterpineol multiconstituent to Daphnia magna is 86.5 mg/L with 95% confidence limits: 80.03 - 93.44 mg/L.

Executive summary:

This study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of test item dihydroterpineol multiconstituent to Daphnia magna, under semi-static conditions.

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 49, 61, 77, 96, 120 and 150 mg/L. The immobility of the daphnids was determined in a semi-static 48h test by visual observation after 24 and 48h. The concentrations of the main constituents, representing 97.2% of test item, were determined by chemical analyses at the start (t=0 h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Concentrations measured in freshly prepared solutions and in 24h old solutions revealed that the concentration of main constituents of the test item was satisfactorily maintained within ± 20% of the initial concentration throughout the test. Thus, the evaluation of the effects on Daphnia magna was based on the nominal concentrations values of test item. After 24h of exposure, immobilisations were 0% at 49 mg/L, 5% at 61 mg/L, 20% at 77 mg/L, 55% at 96 mg/L, 80% at 120 mg/L and 100% at 150 mg/L. After 48h of exposure, immobilisations were 0% at 49 mg/L, 10% at 61 mg/L, 30% at 77 mg/L, 60% at 96 mg/L, 95% at 120 mg/L and 100% at 150 mg/L.       

   

Therefore, the 48 h-EC50 for dihydroterpineol multiconstituent to Daphnia magna is 86.5 mg/L with 95% confidence limits: 80.03 - 93.44 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

calculation (if not (Q)SAR)

Remarks:

estimated by calculation

Adequacy of study:

key study

Study period:

2014-05-26 to 2014-05-28

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

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 whic has been validated to be compliant with the OECD recommendations 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.:

59 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)
cis-p-menthan-8-ol	20.30
trans-p-menthan-8-ol	17.35
cis-p-menthan-1-ol	12.39
trans-p-menthan-1-ol	7.32

Validity criteria fulfilled:

yes

Conclusions:

48 h-EL50 for Dihydroterpineol multiconstituent is 59 mg/L.
Based on the results of this study, Dihydroterpineol multiconstituent would not be classified as acute 1 to aquatic organisms in accordance with the CLP regulation.

Executive summary:

The acute toxicity of Dihydroterpineol multiconstituent to aquatic invertebrates has been investigated using an in-house calculation method that replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). 

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study. In the calculation the second step is to remove this non-bioavailable fraction. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent were predicted using the iSafeRat QSAR model. Each value has been included in the record and QPRF as well as the QMRF has been attached in the iuclid. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Effective Loading rate of the WAF.

The 48-h EL50 of Dihydroterpineol multiconstituent was 59 mg test material/L.

Based on the results of this study, Dihydroterpineol multiconstituent would not be classified as acute 1 to aquatic organisms in accordance with the CLP regulation.

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

 

Results Synopsis

Test Type: Calculation method

EL50: 59 mg test item/L

Description of key information

A study was conducted according to OECD guideline 202. The 48 h-EC50 for dihydroterpineol multiconstituent to Daphnia magna was determined to be 86.5 mg/L with 95% confidence limits: 80.03 - 93.44 mg/L.

With a calculation method, the 48-h EL50 for dihydroterpineol multiconstituent was determined to be 59 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

86.5 mg/L

Additional information

A reliable study was available to assess the 48 h-acute toxicity of test item dihydroterpineol multiconstituent to Daphnia magna, under semi-static conditions. This study was performed according to OECD Guideline 202 with GLP statement.

Concentrations measured in freshly prepared solutions and in 24h old solutions revealed that the concentration of main constituents of the test item was satisfactorily maintained within ± 20% of the initial concentration throughout the test. Thus, the evaluation of the effects on Daphnia magna was based on the nominal concentrations values of the test item. After 24 h of exposure, immobilisations were 0% at 49 mg/L, 5% at 61 mg/L, 20% at 77 mg/L, 55% at 96 mg/L, 80% at 120 mg/L and 100% at 150 mg/L. After 48 h of exposure, immobilisations were 0% at 49 mg/L, 10% at 61 mg/L, 30% at 77 mg/L, 60% at 96 mg/L, 95% at 120 mg/L and 100% at 150 mg/L. Therefore, the 48 h-EC50 for dihydroterpineol multiconstituent to Daphnia magna is 86.5 mg/L with 95% confidence limits: 80.03 - 93.44 mg/L.

Furthermore, a calculation method prediction was available as a supportive study to assess the acute toxicity (the effective loading rate of the mixture EL50 within 48 hours) of test item dihydroterpineol 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 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 dihydroterpineol multiconstituent is 59 mg/L. This toxicity study is acceptable and can be used as supportive study for that endpoint.