6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-11-09 to 2020-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

for analytical measurements
- Sampling intervals/frequency for test organisms: performed before daily feeding routine; twice during the uptake phase (days 12 and 14) and eight times during depuration (1, 2, 3, 5, 7, 14, 21, 28 days)
For lipid determination three specimen of each fish population were sacrificed for monitoring lipid contents at the end of uptake and at the end of depuration.
- Sample storage conditions before analysis: sample material was stored at < -18 °C until further processing
- Details on sampling and analysis of test organisms: At each sampling event, five specimen of both populations (control and test item group) were sacrificed for matrix collection (complete animal) and subsequent analytical assessment. Fish were anesthetized in MS 222, rinsed, blotted dry and shock-frozen in liquid nitrogen after recording animal parameters (length, weight). Each fish was analyzed individually for the test item to generate five replicates per sampling date. Control fish sampled at test start, end of uptake and end of depuration were analyzed first with only one replicate of the sampled specimen to monitor contaminations. Further replicates as well as samples from other time points were only considered, when test item was detected in controls.

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION OF SPIKED FISH FOOD
- Details on fish food: Commercial fish feed with a pellet size of 2 mm
- Details of spiking using an organic solvent: spiked by solvent spray application with a targeted nominal test concentration of 100 mg/kg. 200 g fish feed were placed in a 2 L pear-shaped flask connected to a rotary evaporator equipped with a stainless steel capillary. As a typical loss of about 15 % can occur during the procedure of the spray application, a surplus of 15 % of test item were added to the pursued 100 mg/kg. 4.15 mL application solution (= 23 mg WinCon-2) was thus spread among the feed particles through the spray application which were being constantly mixed by rotation to ensure a homogenous distribution of test item on pellets. During spiking, a low pressure of approximately 700 mbar was set to run the application. The test item containing vessel was rinsed with acetone, twice. This additional amount of solvent/solution was also applied to the feed for rinsing the capillary. The control feed (for uptake) was prepared the exact same way, but without the test item in the spiking solvent, here 5 mL of pure acetone were used.

- Chemical name of vehicle: acetone
- Method of mixing: a rotary evaporator equipped with a stainless steel capillary to apply the test solution via a solvent-inlet tube to the pellets under vacuum
- Method for removal of solvent, if used: vacuum of the rotary evaporator was set to 400 mbar for 15 min. Subsequently, the spiked pellets were dispersed in a tray and left in the fume hood in order to remove all potential solvent residues before use

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: Fischzucht Störk, Wagenhausen 8, 88348 Bad Saulgau, Germany
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (25 random fish of each population): 7.54 ± 0.47 cm
- Weight at study initiation (25 random fish of each population): 4.91 ± 0.91 g
- Weight at termination: 15.85 ± 095 g
- Lipid content at test initiation: 6.35 ± 0.95 %
- Health status: healthy fish, free from observable diseases and abnormalities
- Description of housing/holding area: 100 L glass aquaria filled with 75 L holding and dilution water (HDW) at a scheduled temperature of 15 ± 2 °C. The oxygen saturation in water was kept above 60 % throughout the test by aeration with a glass capillary connected to an air pump. The vessels were left open and were subject to a light/dark cycle of 16/8 hours.
- Feeding during test
- Food type: Commercially available trout feed pellets (e.g. Inicio Plus, biomar, Denmark)
- Amount: 2.0 % of the body weight (adjusted weekly)
- Frequency: daily

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test
- Type of food: Commercially available trout feed pellets (e.g. Inicio Plus, biomar, Denmark)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): No

Route of exposure:

feed

Justification for method:

dietary exposure method used because stable, measurable water concentrations cannot be maintained

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

14 d

Total depuration duration:

28 d

Test temperature:

13.5 ± 2 °C (control)
13.2 ± 2 °C (treatment)

pH:

7.13 and 7.86

Dissolved oxygen:

Oxygen saturations were between 81.6 and 112 % in both vessels

TOC:

Non-purgeable organic carbon (NPOC, as a measure of total organic carbon (TOC)) was determined between < LOQ and 0.735 mg/L for the control and < LOQ in the test vessels.

Salinity:

not applicable

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): open / closed
- Material, size, fill volume: glass, 100 L, 75 L
- Aeration: yes, with a glass capillary
- Type of flow-through: continuous flow of at least 15.6 L/h water with a metering pump system
- No. of organisms per vessel: 58 (control); 57 (treatment
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: 0.1 to 1.0 g fish (wet weight) per liter of water per day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: De-chlorinated local tap water sourced from Schmallenberg district water production plants, mostly fed by small Springs and percolation.
- Chlorine: 0.02-0.03 mg/L
- Alkalinity: 1.6-2 mmol/L
- Conductance: 271-288 µS/cm
- Holding medium different from test medium: no
- Intervals of water quality measurement: monthly (please refer to "Any other information on materials and methods incl. tables" for more details on parameters)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 (light/dark cycle)
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: at a ratio of 2 % of the body weight (adjusted weekly)
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): once
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start: 19.8-19.9 %
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure: 19.6%

Reference substance (positive control):

no

Lipid content:

9.04

Time point:

end of exposure

Lipid content:

6.35

Time point:

start of exposure

Key result

Conc. / dose:

99.9 mg/g food

Temp.:

12.7 - <= 13.6 °C

pH:

8.29

Type:

BMF

Value:

2.28 dimensionless

Basis:

normalised lipid fraction

Remarks:

, normalised growth rate

Key result

Rate constant:

growth rate constant (d-1)

Value:

0.027

Key result

Rate constant:

growth-corrected depuration rate constant (d-1)

Value:

0.009

Key result

Rate constant:

overall depuration rate constant (d-1)

Value:

0.036

Key result

Rate constant:

growth-corrected half-life (d)

Value:

73.8

Details on kinetic parameters:

- Indication of bi- or multiphasic kinetics: No

Metabolites:

n.a.

Results with reference substance (positive control):

n.a.

Details on results:

- Mortality of test organisms: No
- Behavioural abnormalities: No
- Observations on feeding behavior: there were no indications of palatability issues.
- Observations on body length and weight: the animals showed a normal body weight and length development for their age and species
- Reproduction during test period: none observed
- Other biological observations: no ther effects observed
- Organ specific bioaccumulation: not investigated
- Bound residues forming a plateau: not observed
- Mortality and/or behavioural abnormalities of control: No
- Loss of test substance during test period: no
- Non-eliminated residues (NER) at the end of elimination phase: no
- Results with vehicle control: valid

Table 2 Summary of determined parameters for BMF calculation

Assimilation efficiency
c0,d	mg/kg	concentration at start of depuration in fish	7.65
k2	d^-1	depuration rate constant	0.0361
t	d	duration of the feeding period	14
I	g feed /g fish/day	feed ingestion rate	0.02
Cfeed	mg/kg	concentration on feed	99.9
e	unitless	Euler's number	2.718
α	unitless	assimilation efficiency	0.349
Growth corrected feeding rate
Wf,0	gfish	mean fish weight at Start	4.97
Wf,end	gfish	mean fish weight at end	7.14
Ig	g feed /g fish/day	growth-corrected feeding rate	0.0139
Growth rate and depuration constant
kg	d^-1	growth rate constant	0.0267
k: ;	d^-1	growth rate corrected k2	0.00940
Substance-specific half-life
t1/2	d	substance-specific half-life	19.2
t1/2g	d	growth corrected substance-specific half-life	73.8
Lipid correction factor
Lfish	%	lipid fraction in fish	6.41
Lfeed	%	lipid fraction in feed	19.7
Lc	unitless	lipid correction factor	0.325
BMF results
BMFk	unitless	kinetic BMF	0.193
BMFkg	unitless	growth-corrected BMF	0.742
BMFkgL	unitless	growth- and lipid-corrected BMF	2.28

Validity criteria fulfilled:

yes

Conclusions:

According to this OECD 305-III compliant study, a resulting BMFkgL of 2.28 was determined in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.

Executive summary:

A study was performed to determine the biomagnification factor of the test item in the fish species Oncorhynchus mykiss.
The test item WinCon-2 was administered to a test population of fish via feed in accordance to the conditions of the OECD TG 305 for BMF studies. A test item concentration of 99.9 ± 9.68 mg/kg was achieved by applying solvent-mediated spiking on regular feed pellets. In addition, solvent-spiked feed was used for a control population of fish to monitor potential mortalities or other adverse effects over time. Both feed types were administered with a rate of 2 % mean body weight per day.
The test duration was 14 days for the uptake and another 28 days for the depuration phase. During testing, one fish from control and treatment jumped out from the vessel, but no further mortalities were recorded. Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14. The highest mean tissue concentration was 8.84 ± 0.489 mg/kg at day 14.
A growth-corrected depuration rate constant (k_2g) of 0.00940 was calculated from the elimination kinetics of WinCon-2 in fish matrix. This corresponds to a growth-corrected substance specific half-life (t_1/2g) of 73.8 days.
An assimilation efficiency (a) of 0.349 was determined with a test item concentration of C_0,d = 7.65 mg/kg in fish at the beginning of the depuration phase.
The kinetic BMF_k was determined with 0.193, which was corrected for growth-dilution effects resulting in BMFk_g = 0.742.
Finally, a lipid correction of the BMF was conducted, in order to standardize the BMF for comparative analyses. The resulting BMF_kgL of 2.28 indicates a bioaccumulative potential of the test item in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-01-01 to 2020-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Deviations:

yes

Remarks:

pre-test only

Principles of method if other than guideline:

Investigations on the bioaccumulation potential of the test substance WinCon-2 are requested by ECHA as part of the REACH regulation. Therefore, a fish flow-through test according to OECD TG 3051 is required to derive a bioconcentration factor (BCF) as the standard endpoint for bioaccumulation assessment. The performance of the fish bioconcentration study with Rainbow trout was planned to be carried out in the test facility of Fraunhofer IME, Germany. In preparation for the aqueous exposure approach the water solubility of the hydrophobic test item WinCon-2 (CAS: 89331-94-2, log K0w value > 4.7) and its stability in aqueous solutions was tested. The use of solvents and dispersants (solubilizing agents) is not generally recommended by OECD TG 305. A special focus of the preliminary investigations (non-GLP) which were carried out at Fraunhofer IME, Schmallenberg, Germany, from January 2020 to June 2020, was thus on the preparation of aqueous test solutions without using solvents.

GLP compliance:

no

Remarks:

But the main test will be performed under GLP.

Radiolabelling:

no

Details on sampling:

Water samples were taken in triplicates. During the first approach two different samplings were performed: a) 10 mL cu-red water was sampled into a 20 mL glass vial. 1 mL of this solution was transferred into a glass LC-vial, 200 pL acetonitrile (ACN) were added and the samples were measured; b) 2 mL ACN were presented in a 20 mL glass vial and 10 mL cu-red water sample was
added (stabilization). The stabilized sample was transferred into a LC-vial and analysed. During the second approach sampling was performed with routine c) a 20 mL glass vial was saturated with column outlet for 10 min, afterwards the solution was discarded and another 10 mL were taken. This sample was transferred into LC-vials spiked with 200 pL ACN and then measured. All samples have been measured immediately if possible and subsequently been stored at <-18°C. If direct measurement was not possible samples were stored at < -18 °C and have been analysed after 24 or 48 Samples of the second approach which have been taken after saturation of the glass vial (routine c) were divided into two subsamples: one of which was analysed immediately and the other after centrifugation. Centrifugation was performed in 1.5 mL Eppendorf Tubes for 5 min at 16 000 x g. 600 µL of the supernatant were taken, spiked with 600 µL acetonitrile and analysed.

Vehicle:

no

Test organisms (species):

other: No fish was used for the pretest

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: preferred route of exposure by the agency (final decision on a testing proposal)

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Key result

Remarks on result:

not measured/tested

Remarks:

This pre-test was intended to assess the technical feasibility of applying a column generated flow through system in order to accomplish aqueous exposure of fish to the test item. A BCF value was not to be determined in this pre- test.

WinCon-2 concentrations in aqueous solutions
The chosen solid phase desorption dosing system allowed to derive the highest water concentrations possible without the use of solvent. Results of the first approach with a column loading of 0.4 mg/g and a flow rate of 1.5 mL/min are shown in the Table below. The mean value of the measured samples is
2.2 µg/L, which is lower than the expected water solubility of approximately 20 µg/L according to the material safety data sheet. All triplicate measurements show relative high standard deviations (RSD) of 8.88-25.1 %. The table only represents concentrations which were sampled following routine a) (10 mL column outlet was sampled into a 20 mL glass vial and 1 mL of this solution was transferred into a glass LC-vial. After addition of 200 µL ACN the sample was measured). Samples which have been directly stabilized by the addition of ACN before collection of the column outlet showed lower test item content (routine b). Samples have been stabilized for storage purposes. During this experiment it could be observed, that a precipitate was formed during storage at a temperature < - 18 °C.

Column generated cu-red water concentration of WinCon-2

Duration of column [d]	concentration [µg/L]	RSD (n = 3) [%]
2	1.65	25.1
3	1.59	20.9
7	0.84	20.4
9	5.45	8.88
17	1.46	*n= 1
mean	2.20	83.8

 

As a higher concentration was expected, a second column with a higher loading and a higher flow rate (5 mL/min) was used. Here, 5 mg WinCon-2 were loaded per g Florisil. All samples collected from the column outlet have been analysed directly and were pretreated by centrifugation (Table below).
Additionally to the second column assembly, the analytical method was optimized. Also, the sampling routine was adjusted. As WinCon-2 tends to absorb to glass surface, the sampling vial was pre-saturated before the sample was taken. To investigate the high RSDs of the measurements of the first column a centrifugation step was included.

 

Column generated cu-red water concentration of WinCon-2 (second approach); with * = concentration above calibration, 1 = analysis after 5-fold dilution of column outlet, 2 = analysis after 2-fold dilution of column outlet

Duration of column [d]	Concentration [µg/L]	RSD (n = 3) [%]	Concentration after centrifugation [µg/L]	RSD (n = 3) [%]
3	3.71	20.7	0.833	36.6
7.1	3.46	56.6	0.355	56.5
7.2	6.26	5.67 (n = 2)	--
9	--	--	025	7.78
15	55.2*	4.23	--
20.1	6.3	23.4	--
20.2	13.0	24.6	--
21.1	6.97	53.0	--
21.2	5.76	3.16	--
mean	5.41	27.1

 

Table above shows the results of the samples taken from the column outlet. Directly measured samples showed again a high RSD, as well as a broad concentration range from 3.71-55.2 µg/L. These results indicate non-solved particles in the sample. This assumption was investigated by centrifugation of a subsample. Centrifugation of the samples enabled the separation of non-solved test item particles and the results of the centrifuged samples indeed show a significantly lower concentration than untreated samples. Nevertheless, it was tested whether the column outlet will provide stable test vessel concentrations. Typically, a 5-fold water exchange rate during a BCF study following OECD 305 requires a total water flow of 15.6 L/h (260 mL/min). This leads to a 52-fold dilution of the column outlet resulting in a theoretical concentration of 0.1 µg/L in the test vessel. As this is below the defined LOQ, the total water flow rate was reduced to 5.2 L/h (86.7 mL/min). Here a concentration of 0.31 µg/L was expected, which is still below the LOQ but within the calibration range. Unfortunately, analyses of the samples from the test vessel did not lead to quantifiable concentrations. In a last step, a total flow rate of 2.76 L/h (46 mL/min, 9.2-fold dilution) was applied. In this artificial scenario the expected concentration of 0.59 µg/L was detectable with 0.632 ± 0.088 µg/L on day 15 and 0.059 µg/L on day 20. On day 21 the concentration was not detectable anymore. Notably both values are below the LOQ of this method. All results clearly demonstrate that no constant concentration could be achieved under these conditions. To perform a bioconcentration fish test, at least 4 of the solid phase desorption dosing systems would be necessary to generate constant equal concentrations in parallel. Although the absolute concentrations of dissolved test item measured following the two different approaches differed, a similar concentration range was observed. The mean concentration of WinCon-2 in approach 1 and approach 2 was 3.08 µg/L. This provides a general indication for the water solubility of WinCon-2 in fish holding and dilution water.

 

Conclusion

Preliminary investigations were carried out to estimate the suitability of aqueous exposure of WinCon-2 to be applied in a BCF study according to OECD TG 3051. Analyzed concentrations of test item measured in the aqueous solutions after elution of a solid phase desorption dosing system showed significant lower concentrations than the expected water solubility (MSDS). The results show that no constant concentration of WinCon-2 can be maintained in aqueous solution, putting in question the aqueous exposure of the test item under flow-through conditions.
A stable concentration in water of the test item is necessary to perform a valid BCF test. The results obtained provide clear indications that due to the low solubility of the test item, a flow-through approach is not recommended or rather not possible. OECD TG 305 describes in §11 and §97 that a dietary exposure bioaccumulation fish test should be used for substances where the aqueous exposure methodology is not practicable. Reasons for constraints may be that the aqueous concentration cannot be maintained at a level that is considered to be sufficiently constant. Especially for strongly hydrophobic substances (log Kow > 5 and a solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. Due to the hydrophobicity of WinCon-2 (log Kow value of 4.7) and the low water solubility of presumably < 6 µg/L in fish holding water, as confirmed by the experiments described in this report, the dietary approach is recommended for evaluation of the bioaccumulation potential (BMF) of WinCon-2. However, it should be realized though that the endpoint from this test will be a dietary biomagnification factor (BMF) rather than a bioconcentration factor (BCF). The dietary approach should thus only be applied if the suggested test is consistent with the relevant regulatory requirements and risk assessment needs.

Conclusions:

The results show that no constant concentration of WinCon-2 can be maintained in aqueous solution, putting in question the aqueous exposure of the test item under flow-through conditions.Thus, the aqueous exposure route is considered technically not feasible with the test item.

Executive summary:

A stable concentration in water of the test item is necessary to perform a valid BCF test. The results obtained provide clear indications that due to the low solubility of the test item, a flow-through approach is not recommended or rather not possible. OECD TG 305 describes in §11 and §97 that a dietary exposure bioaccumulation fish test should be used for substances where the aqueous exposure methodology is not practicable. Reasons for constraints may be that the aqueous concentration cannot be maintained at a level that is considered to be sufficiently constant. Especially for strongly hydrophobic substances (log Kow > 5 and a solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. Due to the hydrophobicity of WinCon-2 (log Kow value of 4.7) and the low water solubility of presumably < 6 µg/L in fish holding water, as confirmed by the experiments described in this report, the dietary approach is recommended for evaluation of the bioaccumulation potential (BMF) of WinCon-2. However, it should be realized though that the endpoint from this test will be a dietary biomagnification factor (BMF) rather than a bioconcentration factor (BCF). The dietary approach should thus only be applied if the suggested test is consistent with the relevant regulatory requirements and risk assessment needs.

Reference 3

Endpoint:

bioaccumulation in aquatic species, other

Remarks:

calculation

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSAR R.6

Principles of method if other than guideline:

Estimation of BCF, BAF and biotransformation rate using BCFBAF v3.01

GLP compliance:

no

Specific details on test material used for the study:

SMILES: CCCCN(c1cc5c(cc1)C3(c2c(cccc2)C(=O)(O3))c4cc(c(cc4O5)C)Nc6ccccc6)CCCC

Test organisms (species):

other: fish

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured logPow: > 4.66
- Results from toxicokinetic study:
- Results from residue study:
- Monitoring data:

BASIS FOR CALCULATION OF BCF
- Estimation software: BCFBAF v3.01
- Result based on measured log Pow of: > 4.66

Key result

Type:

BCF

Value:

552 L/kg

Basis:

other: QSAR estimation

Remarks on result:

other: QSAR predicted value. The substance is within the applicability domain of the model.

Details on kinetic parameters:

Biotransformation half-life (days): 0.0167 (normalized to 10 g fish at 15°C)
Biotransformation rate (kM, normalized to 10 g fish): 25/day

 SMILES : CCCCN(c1cc5c(cc1)C3(c2c(cccc2)C(=O)(O3))c4cc(c(cc4O5)C)Nc6ccccc6)CCCC

 

CHEM  : 6'-(Dibutylamino)-3'-methyl-2'-(phenylamino)spiro(isobenzofuran-1(3H)

,9'(9H)xanthen)-3-one

MOL FOR: C35 H36 N2 O3

MOL WT : 532.69

 

Summary Results:

Log BCF (regression-based estimate): 2.74 (BCF = 552 L/kg wet-wt)

Biotransformation Half-Life (days) : 0.0167 (normalized to 10 g fish)

Log BAF (Arnot-Gobas upper trophic): 1.10 (BAF = 12.6 L/kg wet-wt)

 

Log Kow (experimental): not available from database

Log Kow used by BCF estimates: 4.66 (user entered)

 

Equation Used to Make BCF estimate:

Log BCF = 0.6598 log Kow - 0.333 + Correction

 

Correction(s): No Applicable Correction Factors

 

Estimated Log BCF = 2.742 (BCF = 551.7 L/kg wet-wt)

Validity criteria fulfilled:

yes

Conclusions:

Using BCFBAF v3.01 the BCF value of the test item was calculated to be 552 L/kg wet-wt. The substance is within the applicability domain of the model.

Executive summary:

The BCF value was calculated using BCFBAF v3.01 as part of EPISuite v4.11 from US Environmental Protection Agency. The BCF value of the test item was calculated to be 552 L/kg wet-wt (EPI Suite, 2014).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

 

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Reference 4

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

GLP compliance:

yes

Radiolabelling:

yes

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

Identity and concentration of auxiliary solvent for dispersal:
Tween 80 was added to the acetone stock solutions at a level of 1% w/v to aid dissolution.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Route of exposure:

aqueous

Test type:

flow-through

Test temperature:

min. 15 °C

pH:

7.4 to 7.6

Salinity:

not applicable

Details on test conditions:

Duration of test: 42 days

Nominal and measured concentrations:

0.0035 mg/L (measured from 0.005 mg/L nominal)
0.00055 mg/L (measured from 0.0005 mg/L nominal)

Key result

Conc. / dose:

ca. 0.001 - <= 0.004 mg/L

Type:

BCF

Basis:

whole body w.w.

Remarks on result:

not determinable because of methodological limitations

Remarks:

No steady state was reached after 28 days.

Key result

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

18.9 d

Details on kinetic parameters:

- Depuration rate constant k(2) per hour: 0.0012

The static bioconcentration factors (BCF) were 550-900 on day 1, 3300-3500 on day 14, and 4300-4800 on day 28 for both high and low level groups, resulting in fish concentrations of 2.5 mg/kg (0.00055 mg/L) and 15 mg/kg (0.0035 mg/L) resp. after 28 days. The uptake rate of radioactivity is described by a zero order kinetic Css (1 - e Exp (-kT)), whereas the depuration rate is best described by a mixed 1.5 order/square root kinetic (0.0035 mg/L) and a 1.5 order kinetic (0.00055 mg/L), resp. Extrapolation of the uptake kinetics indicate that 95% of the steady state concentration 29 would be reached after 48 days exposure to 14 ODB-2 at both concentration levels. Since steady state was not reached after 28 days, the static BDC values are underestimated considerably. Radioactivity after 14 days depuration amounted to 52% (0.00055 mg/L) and 58% (0.0035 mg/L), resp., of the exposure concentrations. The depuration kinetics give indications of incomplete depuration and bound residues.

Concentrations of radioactivity in whole fish after exposure in the high level tank water were generally 6 - 9 times higher than in fish after the low level exposure. Analysis of the biokinetics of uptake indicated that 95 % of the steady state would be reached after 48 days at both exposure levels. The elimination half-life was 11 days at both exposure levels. The exposure of fish to radioactivity thus appeared to be predictable from a linear relationship. Bioconcentration factors were also similar for whole fish of both high and low level groups, with values of 550 - 900 on day 1, 3300 - 3500 on day 14 and 4300 - 4800 on day 28. Analysis of the biokinetics of uptake indicate that 95 % of the steady state would be reached after 48 days exposure to 14C ODB-2 at both exposure levels.

Validity criteria fulfilled:

no

Conclusions:

The steady state was not reached after 28 days and the study has not been continued until steady state. The report was therefore marked as disregarded.

Executive summary:

The bioaccumulation of the test substance was assessed in a study according to OECD guideline 305 E (Huntingdon, ECHA RSS, 2017). Concentrations of radioactivity in whole fish after exposure in the high level tank water were generally 6 - 9 times higher than in fish after the low level exposure. Analysis of the biokinetics of uptake indicated that 95 % of the steady state would be reached after 48 days at both exposure levels. The elimination half-life was 11 days at both exposure levels. The exposure of fish to radioactivity thus appeared to be predictable from a linear relationship. Bioconcentration factors were also similar for whole fish of both high and low level groups, with values of 550 - 900 on day 1, 3300 - 3500 on day 14 and 4300 - 4800 on day 28. Analysis of the biokinetics of uptake indicates that 95 % of the steady state would be reached after 48 days exposure to 14C ODB-2 at both exposure levels.

Since steady state was not reached after 28 days, the static BDC values are underestimated considerably. Radioactivity after 14 days depuration amounted to 52% (0.00055 mg/L) and 58% (0.0035 mg/L), resp., of the exposure concentrations. The depuration kinetics give indications of incomplete depuration and bound residues.

The study was not continued until study day 48 and was therefore regarded as invalid to address the endpoint of bioaccumulation adequately.

Description of key information

Based on the results of a dietary biomagnification study in rainbow trout according to OECD 305, the test item revealed a kinetic BMF value of 0.193 (corrected for growth and lipid: BMFkgl = 2.28). Please refer to the statement on bioaccumulation attached to IUCLID Section 13 for the WoE on bioaccumulation.

Key value for chemical safety assessment

BMF in fish (dimensionless):

2.28

Additional information

pre-test for column elution

A stable concentration in water of the test item is necessary to perform a valid BCF test. The results obtained in this pre-test provide clear indications that due to the low solubility of the test item, a flow-through approach is not recommended or rather not possible. OECD TG 305 describes in §11 and §97 that a dietary exposure bioaccumulation fish test should be used for substances where the aqueous exposure methodology is not practicable. Reasons for constraints may be that the aqueous concentration cannot be maintained at a level that is considered to be sufficiently constant. Especially for strongly hydrophobic substances (log Kow > 5 and a solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. Due to the hydrophobicity of WinCon-2 (log Kow value of 4.7) and the low water solubility of presumably < 6 µg/L in fish holding water, as confirmed by the experiments described in this report, the dietary approach is recommended for evaluation of the bioaccumulation potential (BMF) of WinCon-2. However, it should be realized though that the endpoint from this test will be a dietary biomagnification factor (BMF) rather than a bioconcentration factor (BCF). The dietary approach should thus only be applied if the suggested test is consistent with the relevant regulatory requirements and risk assessment needs.

 

key study OECD 305 (dietary exposure)

A study was performed to determine the biomagnification factor of the test item in the fish species Oncorhynchus mykiss.
The test item WinCon-2 was administered to a test population of fish via feed in accordance to the conditions of the OECD TG 305 for BMF studies. A test item concentration of 99.9 ± 9.68 mg/kg was achieved by applying solvent-mediated spiking on regular feed pellets. In addition, solvent-spiked feed was used for a control population of fish to monitor potential mortalities or other adverse effects over time. Both feed types were administered with a rate of 2 % mean body weight per day.
The test duration was 14 days for the uptake and another 28 days for the depuration phase. During testing, one fish from control and treatment jumped out from the vessel, but no further mortalities were recorded. Repeated samplings were done for both fish populations and five individuals were picked at each time point for chemical analyses. During the uptake phase, fish were sampled twice, at days 12 and 14. The highest mean tissue concentration was 8.84 ± 0.489 mg/kg at day 14.
A growth-corrected depuration rate constant (k_2g) of 0.00940 was calculated from the elimination kinetics of WinCon-2 in fish matrix. This corresponds to a growth-corrected substance specific half-life (t_1/2g) of 73.8 days.
An assimilation efficiency (a) of 0.349 was determined with a test item concentration of C_0,d = 7.65 mg/kg in fish at the beginning of the depuration phase.
The kinetic BMF_k was determined with 0.193, which was corrected for growth-dilution effects resulting in BMFk_g = 0.742.
Finally, a lipid correction of the BMF was conducted, in order to standardize the BMF for comparative analyses. The resulting BMF_kgL of 2.28 indicates a bioaccumulative potential of the test item in Oncorhynchus mykiss when applied orally and uptake results from the digestive tract.

 

QSAR estimation

For the assessment of the test item (Q)SAR results were used for bioaccumulation in aquatic species.

 

In order to generate a reliable and adequate prediction to fulfil or to supplement the information requirements under REACH, a (Q)SAR result should meet the conditions listed in REACH Annex XI (1.3):

(i) (Q)SAR model should be used whose scientific validity has been established and

(ii) the substance should fall within the applicability domain of the (Q)SAR model,

(iii) the prediction should be fit for the regulatory purpose, and

(iv) the information should be well documented.

 

In order to fulfil these requirements and in accordance with ECHA guidance IR/CSA R.6 QSARs and grouping of chemicals and ECHA practical guidance 5 How to report (Q)SARs; the validity, the applicability and the relevance of the used QSAR model was assessed.

 

Assessment of the Validity of the QSAR Model

 

The validity of (Q)SAR models for regulatory purposes was characterised and documented according to the five agreed OECD principles,

1. The (Q)SAR model should be associated with a defined endpoint.

2. The (Q)SAR model should be expressed in form of an unambiguous algorithm.

3. The (Q)SAR model should be associated with a defined domain of applicability.

4. The (Q)SAR model should be associated with appropriate performance of the model.

5. The (Q)SAR model should be associated with a mechanistic interpretation for human health and ecotoxicological endpoints, if possible.

These points were addressed in the respective (Q)SAR Model Reporting Form (QMRF) attached to the endpoint study record. It was verified that the substance falls within the applicability domain of the model. Thus, the prediction was regarded as reliable.

 

Assessment of the prediction of the QSAR Model 

The following information was provided to assess the QSAR prediction:

 

1. Information on the validity of the (Q)SAR model;

2. Verification that the substance falls within the applicability domain of the (Q)SAR model;

3. Assess the adequacy of the results for the purpose of classification and labelling and/or risk assessment.

The information on these three points was compiled according to the (Q)SAR Prediction Reporting Format (QPRF) and was provided as attachment to the endpoint study record.

 

Conclusion

 The validity, the applicability and the relevance of the used QSAR model was assessed, the outcome of the assessment was documented and provided as QMRF and QPRF files attached to the study record. It was concluded that the substance lies within the applicability domain of the model, the results are adequate for the purpose of risk assessment.

Thus, the criteria listed in Annex XI of Regulation (EC) No 1907/2006 are considered to be adequately fulfilled and therefore the endpoint sufficiently covered and suitable for risk assessment.

 

Disregarded bioaccumulation study in fish

Further results of a study according to OECD guideline 305E are available (Huntingdon, ECHA RSS, 2017). Concentrations of radioactivity in whole fish after exposure in the high level tank water were generally 6 - 9 times higher than in fish after the low level exposure. Analysis of the biokinetics of uptake indicated that 95 % of the steady state would be reached after 48 days at both exposure levels. The elimination half-life was 11 days at both exposure levels. The exposure of fish to radioactivity thus appeared to be predictable from a linear relationship. Bioconcentration factors were also similar for whole fish of both high and low level groups, with values of 550 - 900 on day 1, 3300 - 3500 on day 14 and 4300 - 4800 on day 28. Analysis of the biokinetics of uptake indicates that 95 % of the steady state would be reached after 48 days exposure to 14C labeled test substance at both exposure levels.

Since steady state was not reached after 28 days, the static BDC values are underestimated considerably. Radioactivity after 14 days depuration amounted to 52 % (0.00055 mg/L) and 58 % (0.0035 mg/L), resp., of the exposure concentrations. The depuration kinetics give indications of incomplete depuration and bound residues.

The study was not continued until study day 48 and a dissolution aid has been used during stock solution preparation. It was therefore regarded as invalid to address the endpoint of bioaccumulation adequately.

 

Overall conclusion

Please refer to IUCLID section 13 for details on the WoE for PBT assessment.