Cyclohexanepropanol, 2,2,3,6-tetramethyl-.alpha.-propyl-

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:

22-06-2018 to 17-09-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system without headspace

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

see above

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2016 ; signature: January 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive limit-test : 0 (control), and 100% saturated solution prepared from a nominal loading of 100 mg/L renewed every 24 hours.
Equivalent concentrations were : 0 (control) and 0.85 mg/L based on geometric mean measured concentrations ; controls: Test medium without test item or other additives. Definitive limit-test concentrations were based on a preliminary (non-GLP) range-finding study : 0 (control), 1%, 10% and 100% saturated solution (where no immobilisation up to 100% saturated solution).
- Sampling method: t the start of the exposure intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: Samples were used on day of sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water one day before the start of the exposure (at day -1) and one day before the renewal of the test solutions (at day 0). Procedure: an appropriate volume of the test item (relative density: 0.902 was taken into account) was placed by pipette under the water surface and the bottle was closed with the screw cap. The headspace in the glass flask was minimised. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour for separation of undissolved test item. Thereafter, the clear water phase (saturated solution) was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. Definitive limit-test : 0 (control), and 100% saturated solution prepared from a nominal loading of 100 mg/L renewed every 24 hours.
Equivalent concentrations were : 0 (control) and 0.85 mg/L based on geometric mean measured concentrations ; controls: Test medium without test item or other additives. Definitive limit-test concentrations were based on a preliminary (non-GLP) range-finding study : 0 (control), 1%, 10% and 100% saturated solution (where no immobilisation up to 100% saturated solution)
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: details of the sensitivity check are provided in the full study report. A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item and Tyndell effect. Presence of undissolved test item during preparation and during the definitive limit-test was not observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours old daphnids from a healthy stock were used for the study
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: Juvenile ; < 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Not reported. Although the breeder and culture conditions are given in the full study report.
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): Not applicable.
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are fed 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. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: At least 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): No.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guideline.

Hardness:

Dilution water quality parameters: 0 hours: Total Hardness: 245 mg CaCO3/L ; 24 hours: Total Hardness: 270 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 19.2 °C ; 24 hours: Temperature 19.1 °C

pH:

Dilution water quality parameters: 0 hours: pH 8.08 ; 24 hours: pH 7.75

Dissolved oxygen:

Dilution water quality parameters: 0 hours: O2 dissolved: 8.84 mg/L ; 24 hours: O2 dissolved: 8.99 mg/L; mean dissolved oxygen to 48 hours old media all replicates: did not decrease to less than 7.89 mg/L.

Conductivity:

Dilution water quality parameters: 0 hours: Conductivity: 611 µS/cm ; 24 hours: Conductivity: 615 µS/cm

Nominal and measured concentrations:

Range finding tests were performed (non-GLP) prior to the definitive test: 0 (control), 1%, 10% and 100% saturated solution (where no immobilisation up to 100% saturated solution). Information is provided in table 1.
Definitive test: Definitive limit-test : 0 (control), and 100% saturated solution prepared from a nominal loading of 100 mg/L renewed every 24 hours.
Equivalent concentrations were : 0 (control) and 0.85 mg/L based on geometric mean measured concentrations
Measured concentrations were determined and are available in the full study report.

Details on test conditions:

TEST SYSTEM
- Test vessel: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL
- Type: A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) and in diffuse light was chosen to reduce contact with air and losses of the test item by evaporation.
- Material, size, headspace, fill volume: Sealed glass flasks with screw caps (made from polypropylene) ; no headspace ; fill volume 130 mL
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates)
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), according to OECD 202, Annex 3
- Culture medium different from test medium: No, Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16/8 h light/dark cycle during culture conditions and testing.
- Light intensity: Diffuse light, light intensity of max. ca. 20 µE /m2 s (or 1500 lx) during culture conditions and testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours. An organism 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 observations were completed however, no adverse effects were reported.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable (limit test)
- Justification for using less concentrations than requested by guideline: Per guideline OECD TG 202: paragraph 24: "Using the procedures described in this Guideline, a limit test may be performed at 100 mg/L of test substance or up to its limit of solubility in the test medium (whichever is the lower) in order to demonstrate that the EC50 is greater than this concentration. The limit test should be performed using 20 daphnids (preferably divided into four groups of five), with the same number in the control{s). If the percentage of immobilisation exceeds 10% at the end of the test, a full study should be conducted. Any observed abnormal behaviour should be recorded".
- Range finding study: Yes.
- Test concentrations: 0 (control), 1%, 10% and 100% saturated solution (where no immobilisation up to 100% saturated solution). Information is provided in table 1.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.185 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - mg/L

Remarks:

Effect level exceeded maximum (saturation) solubility of the test item ; no immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.185 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: - mg/L

Remarks:

Effect level exceeded maximum (saturation) solubility of the test item ; no immobilisation

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations: None reported.
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: Yes. Although this was taken account of in the study design (limit-test).

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 2.03 mg/L (C.I. 1.00 - 4.00).
- Other: The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) listed in DIN 38412 - L30 for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD Guideline TG 202

Reported statistics and error estimates:

An EC50-value was calculated for the reference item by linear dose response regression (where possible). The respective 95% confidence limits were empirically derived from the observation data as follows (where possible): The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as 95% confidence limits, since the calculated values were classified as ambiguous by the software.
All calculations were carried out from the best-fit values with the software package: GraphPad Prism.

Table 1. Immobilisation Rates in the non GLP Preliminary Range Finding Test

(n = 20, divided into 2 replicates with 10 daphnids each)

Dilution level of saturated solution (%)	Immobilisation [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1	2	MV
100	0	0	0	0	0	0
10	0	0	0	0	0	0
1	0	0	0	0	0	0
Control	0	0	0	0	0	0

 

Table 2. Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Limit-Test

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

Dilution level of saturated solution (%)		Geometric mean measured concentrations (mg/L)		Immobilisation [%]
				24 hours										48 hours
				Replicates										Replicates
				1		2		3		4		MV		1		2		3		4		MV
100		0.185		0		0		0		0		0		0		0		0		0		0
0% (Control)	0 (Control)		0		0		0		0		0		0		0		0		0		0

 

Table 3. Measured Concentrations and Percent of the Nominal and the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling	Fresh media, 0 hours Start of the exposure interval		Old media, 24 hours End of the exposure interval		Fresh media, 24 hours Start of the exposure interval		Old media, 48 hours End of the exposure interval		Geometric mean measured concentrations (mg/L)
Dilution level of saturated solution (%)	Test item concentration
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	0.163	-	0.148	91	0.246	-	0.199	81	0.185
Control	< LOQ		< LOQ		< LOQ		< LOQ		0 ( or < LOQ)

Meas. conc. = measured concentration of the test item, dilution factors taken into account

% = percent of the initial concentration of the test item

LOQ = limit of quantification of the analytical method (0.01 mg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was > 0.185 (95% C.I. – ) mg/L based on geometric mean measured concentration. No immobility was observed during the test when tested at the limit of solubility (100 % saturated solution) of the test item.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with a saturated solution of the test item in a definitive limit-test. The test item has limited water solubility. In accordance with the globally harmonized system for classification and labelling (GHS Rev. 7, 2017), availability of the substance is based on the extent to which the substance becomes soluble. Only the portion that is available will be able to lead to toxic effects, and therefore testing above the limit of solubility is not warranted. In accordance with OECD guidance document No. 23 (2000), for poorly water-soluble substances, the substance should be tested at its limit of solubility. Therefore, there is only a need to test to the limit of solubility for a substance. A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water one day before the start of the exposure and one day before the renewal of the test solutions. An appropriate volume of the test item (relative density was taken into account) was placed by pipette under the water surface and the bottle was closed with the screw cap. The headspace in the glass flask was minimised. A slow stirring procedure was applied. Gentle stirring was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour for separation of undissolved test item. Thereafter, the clear water phase saturated solution, was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam for Tyndall effect i.e. undissolved test item or formation of an emulsion. The Tyndall effect was negative. The tested concentration level was colourless and visually clear throughout the exposure period. Twenty daphnids were exposed to each concentration level and the control. In the definitive limit-test the conducted concentrations were : 0% (control), and 100% saturated solution prepared from a nominal loading of 100 mg/L renewed every 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. The test item was analytically verified via GC-MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) for the saturated solution and the control. The measured concentration of the saturated test item solution was 91% of the 0 hour measured concentration at 24 hours and 81% of the 24 hour fresh test solution measured concentration at 48 hours. Effect levels were based on geometric mean measured concentrations of the test item which were: 0 (control), and 0.185 mg/L. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. Under the conditions of this study, the 48h-EC50 was > 0.185 (95% C.I: -) mg/L based on geometric mean measured concentrations of the test item in the test system. No immobility was observed during the test when tested at the limit of solubility (100 % saturated solution) of the test item.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Software Package: iSafeRat® HA-QSAR toolbox v2.5,
Contact KREATIS
Dr. Paul Thomas
KREATiS SAS
23, rue du Creuzat
38080 L’ISLE D’ABEAU
FRANCE

2. MODEL (incl. version number)
iSafeRat® HA-QSAR toolbox v2.5 – Daphnia Sp. (48-hour EC50); including the following specific sub-models:
(i) non-polar narcosis (MechoA 1.1)
01 February 2014 (model development); 13 March 2020 (current model version)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See attached: ‘Determination of acute toxicity to Daphnids (48-hour EL50) of Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol tested as the Water Accommodated Fraction (WAF)’ version 1.0; dated August 2020.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Full details of the method are provided in the attached QMRF named 'iSafeRat® HA-QSAR based on a holistic approach for predicting physicochemical and ecotoxicological endpoints’ version 1.9; 13 March 2020.

5. APPLICABILITY DOMAIN
See ‘any other information on results incl. tables’.
See attached: ‘Determination of acute toxicity to Daphnids (48-hour EL50) of Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol tested as the Water Accommodated Fraction (WAF)’ version 1.0; dated August 2020.

6. ADEQUACY OF THE RESULT
1) QSAR model is scientifically valid. 2) The substance falls within the (general) applicability domain of the QSAR model and/or with interpretation by expert judgement. 3) The prediction is fit for regulatory purpose. The prediction is adequate for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3. Specifically when combined with further information such as physico-chemistry and environmental fate testing and modelling.

Reason / purpose for cross-reference:

reference to other study

Guideline:

other: REACH Guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

1. iSafeRat® HA-QSAR toolbox v2.5 – Daphnia Sp. 48h EC50 ; including the following specific sub-models:
(i) non-polar narcosis (MechoA 1.1)
2. Aquatic Toxicity Assessment using the Additivity Approach or based on a constituent and worst-case approach
3. Assessment of degradability and/or n-octanol/water partitioning behaviour and/or bioaccumulation for the purpose of Classification and Labelling

Specific details on test material used for the study:

Detailed information on the 'test material identity' is provided in the attached QSAR Prediction Reporting Format (QPRF) document including information on individual constituents.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Interval: 0.11 - 0.49 mg/L

1. Defined Endpoint:

QMRF 3. Ecotoxic effects

QMRF 3.1. Short-term toxicity to Daphnia (immobilisation)

Reference to type of model used and description of results:

iSafeRat® HA-QSAR toolbox v2.5 –Daphnia Sp. 48h EC50 ; including the following specific sub-models:

(i) non-polar narcosis (MechoA 1.1)

01 February 2014 (model development); 13 March 2020 (current model version)

 

2. Description of results and assessment of reliability of the prediction:

See attachment QPRF: ‘Determination of acute toxicity to Daphnids (48-hour EL50) of Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol tested as the Water Accommodated Fraction (WAF)’ version 1.0; dated August 2020.

Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol :

Principle of calculation employed for the substance: the additivity principle based on constituents acting with the same MechoA

Step 1 : determination of the water solubility of each individual constituent

Step 2 : determination of the analytically measurable aqueous phase (AMAP)

Step 3 : determination of the bioavailable toxic fraction of each constituent

Step 4: calculation of the global activity of the WAF and determination of the toxic loading rate

Results:

48-h EL50 = > water solubility limit [i.e. > 0.485 mg/L]

Water Solubility of the test item was measured to be 0.485 mg/L or -5.695 in log10 (mol/L) at 20°C (cited reference study: EU Method A.6, 2020).

i.e. the test item 48-h ErL50 is expected to be greater than the limit of solubility (according to the WAF method) of the substance

 

NOTE: Stereoisomerism is not taken into account by the calculation. By default, the reported prediction is applicable for all the stereoisomeric forms of this substance. i.e. 1-[2,2,3,6-tetramethylcyclohexyl]hexan-3-ol is considered equivalent to : all constituent possible stereoisomers including e.g. Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol; this and stereoisomer predictions are fully documented in the QSAR Prediction Reporting Format (QPRF) documentation.

 

Inputs Used for Test Item: All modelling was based on input of SMILES notation for each constituent of the test item and the process relied on modelled or measured values for Kow and water solubility to define each constituent prediction. This approach is in line with the attached QMRF named ‘iSafeRat® HA-QSAR based on a holistic approach for predicting physicochemical and ecotoxicological endpoints’ version 1.9 ; 13 March 2020, and ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R7.b for hazard assessment of substances and complex mixtures.

 

For the purpose of concluding on classification and labelling for this substance either (i) the additivity formula from Annex I, Section 4.1.3.5.2 of CLP Regulation (EC) 1272/2008 is applied or (ii) a worst case application of 48-h EC50 or EL50 effect levels is applied to known components of the substance. As applicable.

Substance rapidly degradable: No.

Log Kow: > 4.0 as surrogate to expected : BCF > 500 L/kg wet-wt.

Principle of calculation employed for the substance: the additivity principle based on constituents acting with the same MechoA

Step 1 : determination of the water solubility of each individual constituent

Step 2 : determination of the analytically measurable aqueous phase (AMAP)

Step 3 : determination of the bioavailable toxic fraction of each constituent

Step 4: calculation of the global activity of the WAF and determination of the toxic loading rate

 

Assessment of the substance within the applicability domain as documented within the corresponding QMRF named ‘iSafeRat® HA-QSAR based on a holistic approach for predicting physicochemical and ecotoxicological endpoints’ version 1.9 ; 13 March 2020 – section 5; indicates the substance:

 

(i) Falls within the Log Kow and Water Solubilities intermediate domain : (e.g. outside the descriptor regression domain log WSOL (mol/L) -4.70 to 0.87 but is within Subcooled Liquid Water Solubility of –8.33 to -4.70 in log10 (mol/L) intermediate descriptor domain, where results cannot be accurately measured). The result is therefore is considered an extrapolation. The result can be considered reliable with limitations placed on the estimate and additional assessment based on expert judgment. More information is provided below.

Comments: The Subcooled Liquid Water Solubility value given as the input to the iSafeRat® daphEC50 falls within the intermediate domain of the model between a Subcooled Liquid Water Solubility of -8.33 to -4.70 in log10 (mol/L) where baseline toxicity cannot be experimentally measured accurately. In this intermediate domain, the toxicity may to be greater than the water solubility limit. For confirmation, a statistical k-NN approach (k = 3) is performed on the data of substances found to be in the intermediate domain of the model. The toxicity of the three closest neighbours based on the solubility are considered. Based on these data, either the toxicity of the test item is expected to be greater than the limit of solubility, or the toxicity is estimated by the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits, as applicable). According to this analysis, the toxicity of the test item is estimated as greater than the water solubility limit.

(ii) Falls within the structural fragment domain

(iii) Falls within the mechanistic domain of MechoA 1.1

 

3. Uncertainty of the prediction and mechanistic domain:

An external validation set is available for the chemical class used in this prediction however is not publicly available. Interested parties are invited to contact the model author. The QMRF has been peer reviewed by Joint Research Centre, ISPRA, Italy.

Full details on the training set, internal and external validations, as applicable are included in the attached QMRF named ‘iSafeRat® HA-QSAR based on a holistic approach for predicting physicochemical and ecotoxicological endpoints’ version 1.9 ; 13 March 2020 . Interested parties are invited to contact the model author.

 

Uncertainty in the prediction relates to:

Statistical characteristics are provided (i) for the prediction: in the attached QPRF and (ii) for the model: in the attached QMRF, respectively.

Validity criteria fulfilled:

yes

Conclusions:

The results are adequate for the for the regulatory purpose.

Executive summary:

iSafeRat® HA-QSAR toolbox v2.5 – Daphnia Sp. 48h EC50 ; including the following specific sub-models:

(i) non-polar narcosis (MechoA 1.1)

Endpoint values: Daphnia Sp.

Principle of calculation employed for the substance: the additivity principle based on constituents acting with the same MechoA

Step 1 : determination of the water solubility of each individual constituent

Step 2 : determination of the analytically measurable aqueous phase (AMAP)

Step 3 : determination of the bioavailable toxic fraction of each constituent

Step 4: calculation of the global activity of the WAF and determination of the toxic loading rate

Results:

48-h EL50 = > water solubility limit [i.e. > 0.485 mg/L]

Water Solubility of the test item was measured to be 0.485 mg/L or -5.695 in log10 (mol/L) at 20°C (cited reference study: EU Method A.6, 2020).

i.e. the test item 48-h ErL50 is expected to be greater than the limit of solubility (according to the WAF method) of the substance

 

Adequacy of the QSAR:

1) QSAR model is scientifically valid. 2) The substance falls within the (general) applicability domain of the QSAR model and/or with interpretation by expert judgement. 3) The prediction is fit for regulatory purpose.

The prediction is adequate for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3. The assessment indicates that the prediction is suitable for the regulatory conclusion in accordance with the tonnage driven information requirements. Specifically when combined with further information such as physico-chemistry and environmental fate testing and modelling.

Description of key information

EC50 (invertebrates) = greater than maximum water solubility or > 0.185 mg/L based on geometric mean measured concentrations (tested at up to 100% saturation or the maximum solubility in media), 48hour-freshwater, OECD TG 202, 2018

Supporting information: QSAR: 48h-EL50 (invertebrates) = > water solubility limit [i.e. > 0.485 mg/L], iSafeRat® HA-QSAR toolbox v2.5, KREATIS SAS, 2020

 

Conclusion : The substance has apparent lower solubility in aquatic Daphnia medium Elendt M4 or OECD TG 202 annex 3 (2004), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the Daphnia exposure matrix and that of pure water. The conclusion of the experimental study was that no effects were observed (0% immobilisation) at up to 100% saturation, with maximum concentration 0.24 mg/L (after renewal at 48 hours) and geometric mean measured concentration of 0.185 mg/L. Consequently, it can be concluded the test item has comparable acute aquatic toxicity to freshwater invertebrates to that which is predicted. With no toxicity below the water solubility limit observed experimentally.

Additional information

Key data : OECD TG 202, 2018 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with a saturated solution of the test item in a definitive limit-test. The test item has limited water solubility. In accordance with the globally harmonized system for classification and labelling (GHS Rev. 7, 2017), availability of the substance is based on the extent to which the substance becomes soluble. Only the portion that is available will be able to lead to toxic effects, and therefore testing above the limit of solubility is not warranted. In accordance with OECD guidance document No. 23 (2000), for poorly water-soluble substances, the substance should be tested at its limit of solubility. Therefore, there is only a need to test to the limit of solubility for a substance. A saturated solution with a nominal loading of 100 mg/L of the test item was prepared with dilution water one day before the start of the exposure and one day before the renewal of the test solutions. An appropriate volume of the test item (relative density was taken into account) was placed by pipette under the water surface and the bottle was closed with the screw cap. The headspace in the glass flask was minimised. A slow stirring procedure was applied. Gentle stirring was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour for separation of undissolved test item. Thereafter, the clear water phase saturated solution, was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam for Tyndall effect i.e. undissolved test item or formation of an emulsion. The Tyndall effect was negative. The tested concentration level was colourless and visually clear throughout the exposure period. Twenty daphnids were exposed to each concentration level and the control. In the definitive limit-test the conducted concentrations were : 0% (control), and 100% saturated solution prepared from a nominal loading of 100 mg/L renewed every 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. The test item was analytically verified via GC-MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the test (24 and 48 hours) for the saturated solution and the control. The measured concentration of the saturated test item solution was 91% of the 0 hour measured concentration at 24 hours and 81% of the 24 hour fresh test solution measured concentration at 48 hours. Effect levels were based on geometric mean measured concentrations of the test item which were: 0 (control), and 0.185 mg/L. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. Under the conditions of this study, the 48h-EC50 was > 0.185 (95% C.I: -) mg/L based on geometric mean measured concentrations of the test item in the test system. No immobility was observed during the test when tested at the limit of solubility (100 % saturated solution) of the test item.

 

Key data : QSAR, iSafeRat® HA-QSAR toolbox v2.5, KREATIS SAS, 2020

iSafeRat® HA-QSAR toolbox v2.5 – Daphnia Sp. 48h EC50 ; including the following specific sub-models:

(i) non-polar narcosis (MechoA 1.1)

Endpoint values: Daphnia Sp.

48-h EL50 = > water solubility limit [i.e. > 0.485 mg/L]

 

Adequacy of the QSAR:

1) QSAR model is scientifically valid. 2) The substance falls within the (general) applicability domain of the QSAR model and/or with interpretation by expert judgement. 3) The prediction is fit for regulatory purpose.

The prediction is adequate for the Classification and Labelling or risk assessment of the substance as indicated in REACH Regulation (EC) 1907/2006: Annex XI Section 1.3. The assessment indicates that the prediction is suitable for the regulatory conclusion in accordance with the tonnage driven information requirements. Specifically when combined with further information such as physico-chemistry and environmental fate testing and modelling.

 

References for further information:

1. QMRF Title: ‘iSafeRat® HA-QSAR based on a holistic approach for predicting physicochemical and ecotoxicological endpoints’ version 1.9 ; 13 March 2020

2. QPRF Title: ‘Determination of acute toxicity to Daphnids (48-hour EL50) of Reaction mass of (3RS)-1-[(1RS,3SR,6SR)-2,2,3,6-tetramethylcyclohexyl]hexan-3-ol and (3RS)-1-[(1SR,3RS,6RS)-2,2,3,6-tetramethylcyclohexyl] hexan-3-ol tested as the Water Accommodated Fraction (WAF)’ version 1.0; dated August 2020.

 

Conclusion : The substance has apparent lower solubility in aquatic Daphnia medium Elendt M4 or OECD TG 202 annex 3 (2004), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the Daphnia exposure matrix and that of pure water. The conclusion of the experimental study was that no effects were observed (0% immobilisation) at up to 100% saturation, with maximum concentration 0.24 mg/L (after renewal at 48 hours) and geometric mean measured concentration of 0.185 mg/L. Consequently, it can be concluded the test item has comparable acute aquatic toxicity to freshwater invertebrates to that which is predicted. With no toxicity below the water solubility limit observed experimentally.