3-methylcyclopentadecan-1-one

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:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

From 2017-02-20 to 2017-02-23

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® HA-QSAR toolbox v2.3

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.7

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1CCCCCCCCCCCCC(=O)C1
The water solubility given as input was experimentally measured: 0.43 mg/L

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not applicable

Remarks:

(QSAR model)

Principles of method if other than guideline:

The acute toxicity to aquatic invertebrates was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 48-hour EC50 tests on daphnia, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 48-h EC50 for daphnia and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

- Water solubility: 0.43 mg/L (from experimental study)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

Not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

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 QSAR 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 (generally >60%).

Salinity:

Not applicable.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

Not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

48 h

Dose descriptor:

EC50

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: > subcooled liquid water solubility value

Details on results:

The substance falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the substance falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately.
Moreover the toxicity is likely greater than the water solubility limit. Based on a k-NN approach (with k = 3), the predicted acute toxicity (48h-EC50) to daphnid is considered as greater than the water solubility limit within the exposure period of the test.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Not determinable

No additional information

Validity criteria fulfilled:

yes

Remarks:

The substance falls within the applicability domain of the model except for the descriptor domain. However, the substance falls within the intermediate descriptor domain where baseline toxicity cannot be experimentally measured accurately.

Conclusions:

The 48h-EC50 of the substance to daphnids was predicted as greater than the subcooled liquid water solubility value within the exposure period of the test. No acute toxicity is expected at levels up to the water solubility.

Executive summary:

A QSAR prediction was performed to assess the acute toxicity of the test substance to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The criterion predicted 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 immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility value of the substance given as the input was experimentally measured at 0.43 mg/L and does not fall within the descriptor domain of the model between a log water solubility (in log (mol/L)) of - 4.70 to 0.87. However it falls within the intermediate domain between a log water solubility of - 8.22 to - 4.70 where baseline toxicity cannot be experimentally measured accurately. Moreover the toxicity is likely greater than the water solubility limit. Based on a statistical k-NN approach (k = 3), the toxicity of the test substance is considered as greater than the subcooled liquid water solubility value within the exposure period of the test.

No acute toxicity to aquatic invertebrates is expected at levels up to the water solubility.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-10-16 to 2017-11-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Despite numerous attempts using this method it was not possible to attain the maximum solubility in the medium that had previously been achieved in the water solubility study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004) / using passive dosing method

Deviations:

no

Principles of method if other than guideline:

This study was carried out using a state of the art method, a passive dosing system using a silicone disk, maximally charged with the test substance in an appropriate solvent, in an attempt to determine the toxic effects (immobilisation) of this poorly soluble, volatile, test substance to Daphnia magna after 24 and 48 hours of exposure under static conditions in a passive dosing system in a limit test at maximum water solubility under test conditions.

GLP compliance:

yes

Remarks:

Date of inspection: June 07th - June 10th, 2016 & July 13th, 2016 / Date of signature: 03.01.2017

Specific details on test material used for the study:

- Relative density: 0.919 at 20 ± 0.5 °C
- Water solubility: 430 µg/L at 20 °C, pH 6 in pure water (Slow-stirring Flask Method, Noack 2016)
- Vapour pressure: 0.041 Pa at 25 °C (effusion method: Knudsen cell; provided by the sponsor)
- Log Kow: 6.056 ± 0.079 (25 ± 1°C; mean pH 5.647) (Slow-stirring method, Noack 2016)
- Storage conditions: Room temperature, protected from light, in the tightly closed original container
- Stability under test conditions: Stable at neutral pH (Firmenich stability test)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the start of the exposure (0 hours), samples of the fresh media were taken from additional replicates after completion of the equilibration phase (same incubation conditions as for the test vessels) and analyzed. After 24 hours of exposure samples of the test media were taken from additional replicates and analyzed. These replicates were prepared without daphnids and incubated under test conditions until sampling. At the end of the exposure (48 hours), samples of the old media were taken from the test vessels and analyzed.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary. Prepared samples were stored at room temperature in an autosampler until analysis.

Vehicle:

no

Details on test solutions:

- Passive dosing system: A carrier (silicone disc) was loaded with an appropriate amount of the test item. The carrier was inserted in an appropriate amount of dilution water. After an equilibration phase, a saturated test item solution with constant concentration as near as possible at the water solubility of the test item under test conditions was achieved by the continuous diffusion of the test item into the water body.
- Preparation of the limit concentration: Silicone discs were used as carrier for the test item in the passive dosing system. Each silicone disc was loaded with a 500 mg/L stock solution of the test item as specified below. The concentration of the stock solution was based on preliminary testing. Then, one silicone disc was inserted in each test vessel, which was filled with 20 mL of the dilution water. To achieve a saturated test item solution by passive dosing, the silicone discs were incubated for 24 hours under test conditions before insertion of the daphnids for equilibration of the saturated test item solution. The silicone discs also remained in the test vessels during the exposure phase. This ensured a constant concentration near the water solubility of the test item.
- Preparation of silicone discs: A stock solution of 500 mg test item/L (methanol : water = 1.5 : 1) was prepared. Discs of 25 mm diameter were prepared out of PDMS foil (thickness 1 mm). The PDMS discs were placed into glass vessels containing 160 mL of stock solution and incubated under constant shaking (150 rpm) for 96 hours. Silicone discs for use in the control were treated with methanol carrier without addition of test item and incubated under the same conditions as the test item discs.
- Control: A control silicone disc incubated in dilution water without test item under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: The daphnids were not fed during the study.

ACCLIMATION
Acclimatization is not necessary, because the composition of the dilution water is equivalent to the culture medium.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

160 to 180 mg CaCO3/L (measured at 178 mg CaCO3/L at the start of the exposure)

Test temperature:

18 - 22 °C, constant within ± 1 °C (measured at 20.3 °C at the start of the exposure).

pH:

The pH should be in the range of 6 – 9. The deviation of the final pH-values (old media) from the initial values (fresh media) should not exceed 1.5 units (measured between 7.32 to 7.62 during the test).

Dissolved oxygen:

The dissolved oxygen concentration in the old media at the end of the exposure should be ≥ 3 mg/L in all test vessels of the limit concentration and the control (measured between 6.73 to 8.13 mg/L during the test).

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal concentrations: A limit concentration at the maximum achievable water solubility of the test item under test conditions was tested. The limit concentration was prepared in a passive dosing system. The limit concentration was selected based on the results of non GLP range finding tests under static conditions = 500 mg/L
- Measured concentrations: The measured concentration of the test substance in fresh media was 85.3 µg/L at the start of the exposure (0 hours). Then, after 24 hours and 48 hours (end of the exposure), 82% and 119% of the initially measured concentration were determined, respectively. See Tables 6.1.3/1 and 6.1.3/2 in "Any other information on results incl. tables"

Details on test conditions:

Based on the results of a non-GLP preliminary range finding test, the study was performed under static conditions. The test item concentration in the passive dosing system was considered to be sufficiently stable for an exposure period of 48 hours. A constant test item concentration near the water solubility of the test item under test conditions was achieved by the continuous diffusion of the test item into the water body in the passive dosing system. No further measures had to be taken to prevent losses due to evaporation or degradation of the test item.

TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm) with a nominal capacity of 50 mL, were used and covered with watch glasses during the exposure phase to minimize volatilization of the test item. A test volume of 20 mL was provided in each test vessel. A mesh made from inert glass material (glass fiber filter) was used as separation device to prevent the daphnids from direct contact to the silicone disc.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Application: 20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. Then, one silicone disc was inserted in each test vessel and incubated for 24 hours under test conditions for equilibration. Before insertion of the daphnids, the silicone disc was covered by insertion of a glass fiber filter (MN GF-5, MACHEREY-NAGEL) to prevent the daphnids from coming into direct contact with the silicone discs. Thereafter, the daphnids were placed in the test vessel together with a small amount of dilution water by pipette.

TEST MEDIUM / WATER PARAMETERS
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Other adverse effects did not appear.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
See attached document "Preliminary Range Finding Tests (non-GLP)" in Attached background material.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 85.3 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: maximum achievable test substance concentration under test conditions (passive dosing system)

Details on results:

The percentage of immobility, determined in the limit concentration and in the control after 24 and 48 hours, is given in Table 6.1.3/3 in "Any other information on results incl. tables". The absolute numbers of immobile daphnids are presented in Table 6.1.3/4 in "Any other information on results incl. tables". Other adverse effects did not appear.
Test solutions were clear throughout the exposure. No further additional observations were noticed.

Results with reference substance (positive control):

- Results with reference substance valid? yes

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-11-02 to 2017-11-03.
EC50 value (with 95% confidents limits), based on nominal concentrations (0-24h) = 2.10 (1.92 - 2.43) mg/L.
Valid range = 0.6 to 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5

Reported statistics and error estimates:

None

Table 6.1.3/1: Measured Concentrations of the test substance during the Definitive Test

Sampling:	0 hours	24 hours		48 hours
Loading level [%]	Test substance
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Geometric mean [µg/L]
100	85.3	69.6	82	101	119	84.3
Control	< LOQ	< LOQ		< LOQ		< LOQ

Meas. conc.  = measured concentration of the test item, enrichment and dilution factor taken into account

% = percentage of the initial concentration of the test substance

LOQ  = limit of quantification of the analytical method (15 µg test item/L)

Table 6.1.3/2: Measured Concentrations of the test substance in the Stock Solution

Sampling:	0 hours		96 hours
	Test substance
Nominal loading concentration [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
501	502	100	408	81
Control	< LOQ		< LOQ

Meas. conc.  = measured concentration of the test item, enrichment and dilution factor taken into account

% = percentage of the nominal concentration of the test substance

LOQ   = limit of quantification of the analytical method (15 µg test item/L)

Table 6.1.3/3: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Initially measured concentration of the test item [µg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
85.3	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Table 6.1.3/4: Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Initially measured concentration of the test item [µg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
85.3	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Validity criteria fulfilled:

yes

Conclusions:

No immobilisation were observed during the test at the limit concentration and the control. Therefore, based on the initially measured concentration of the test substance, the 48h-EC50 for Daphnia magna was > 85.3 µg/L, the maximum achievable test substance concentration under test conditions (passive dosing system).

Executive summary:

In the acute immobilization test with Daphnia magna, the effects of the test substance were determined at the test facility according to OECD Guideline 202 with GLP compliance.

The study was performed under static conditions in a passive dosing system in a limit test at maximum water solubility. All test media were visually observed to be clear throughout the exposure period. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test substance were analytically verified via GC-MS in fresh media at the start of the exposure (0 hours), during the exposure after 24 hours and in the old test media at the end of the exposure (48 hours) in the limit concentration and the control. The measured concentration of the test substance at the start of the exposure (0 hours) was 85.3 µg/L. Then, after 24 hours and 48 hours (end of the exposure), 82% and 119% of the initial concentration were measured, respectively. The measured concentrations of the test substance were within ± 20% of the initial measured test item concentration. This indicates that the test substance concentrations were successfully maintained for the duration of the test. Therefore, all effect values given are based on the initially measured test item concentration.  

No immobilisation was observed during the test at the limit concentration and the control. Therefore, based on the initially measured concentration of the test substance, the 48h-EC50 for Daphnia magna was > 85.3 µg/L, the maximum achievable test substance concentration under these test conditions.

All validity criteria of the test guidelines were fulfilled.

Description of key information

WoE with one experimental study and one QSAR prediction:

48h-EC50 > water solubility limit

Additional information

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

The first endpoint value (NOACK, 2018) is from a valid experimental study, performed on the registered substance, according to OECD Guideline 202 with GLP compliance. This study was carried out using a state of the art method, a passive dosing system using a silicone disk, maximally charged with the test substance in an appropriate solvent, in an attempt to determine the toxic effects (immobilisation) of this poorly soluble, volatile, test substance to Daphnia magna after 24 and 48 hours of exposure under static conditions in a passive dosing system in a limit test at maximum water solubility under test conditions. Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. All test media were visually observed to be clear throughout the exposure period. The measured concentration of the test substance at the start of the exposure (0 hours) was 85.3 µg/L. Then, after 24 hours and 48 hours (end of the exposure), 82% and 119% of the initial concentration were measured, respectively. This indicates that the test substance concentrations were successfully maintained for the duration of the test (the measured concentrations were within ± 20% of the initial measured test item concentration). Therefore, all effect values given are based on the initially measured test item concentration. No immobilisation was observed during the test at the limit concentration and the control. Therefore, based on the initially measured concentration of the test substance, the 48h-EC50 for Daphnia magna was determined to be greater than 85.3 µg/L, the maximum achievable test substance concentration under these test conditions.

The second result (KREATiS, 2017) was obtained from a QSAR. This QSAR prediction (iSafeRat® holistic HA-QSAR v1.7) was performed to assess the acute toxicity of the registered substance to daphnids. The QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility value of the substance given as the input was experimentally measured at 0.43 mg/L and does not fall within the descriptor domain of the regression line which has been validated for substances with a water solubility (in mol/L) between a log water solubility (in log units) of - 4.70 to 0.87. However it does fall within a second zone with even lower water solubility which is described as the intermediate domain between a log water solubility of - 8.22 to - 4.70 where baseline toxicity cannot be experimentally measured accurately. Moreover, the solubility is so low for the registered substance that the toxicity is likely greater than the water solubility limit. Based on a statistical k-NN approach (k = 3), the toxicity of the test substance is considered as greater than the subcooled liquid water solubility value within the exposure period of the test. No acute toxicity to aquatic invertebrates is expected at levels up to the water solubility.

In conclusion, based on the experimental study and despite numerous attempts using the passive dosing method, it was not possible to attain the maximum solubility in the medium that had previously been achieved in the water solubility study. Therefore, it's not possible to prove experimentally that no effect is observed up to the water solubility limit (measured at 0.43 mg/L in an OECD 105 study) but only up to the maximum achievable concentration in the present study (0.0853 mg/L). However, an in silico prediction is available and predicts no acute effects on aquatic invertebrates up to the water solubility limit. The registered substance falls within an intermediate zone, according to an accurate in silico model, where baseline toxicity cannot be experimentally measured accurately.

Enormous effort has been made to obtain a maximum solubility concentration of the substance in test medium while avoiding the creation of an emulsion which typically occurs for this kind of low solubility liquid. It is not believed that further attempts to obtain meaningful experimental results will be successful and this is supported by the in silico model which has positioned the substance in a zone where acute toxicity is no longer expected. The substance is therefore considered not to be toxic in an acute study.