Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene

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:

key study

Study period:

08 April 2020 to 27 July 2020 (experimental phase)

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) -III: Dietary Exposure Bioaccumulation Fish Test

Version / remarks:

OECD Guidelines for Testing of Chemicals 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: On Day 0, at initiation of the test, five individual fish were randomly sampled from the main stock tank for analysis. On Day 14, at the end of the uptake phase, ten individual fish were randomly sampled from the control and ten individual fish were sampled from the treated group. During the depuration phase on Days 1, 4, 7, 14, 28, 35 and 42 five fish were taken from the control and treatment tanks for analysis. In addition, on Day 42 all remaining fish were taken from the control and treated tanks and stored frozen for further analysis if necessary.
Control fish taken during the depuration phase were taken for lipid determinations
- Sampling intervals/frequency for test medium samples: As the study was conducted using dietary exposure, feed and water samples were taken for analysis.
Feed sampling: Samples of the treated feed were taken for analysis on the day of preparation (duplicate samples 2 days prior to initiation of the uptake phase), on Day 0 (four samples taken, three sent for analysis and one retained frozen for further analysis if required) and Day 14 (four samples taken for analysis).
Triplicate samples of the untreated feed were also taken for analysis on the day of preparation of the treated feed.
Water sampling: Water samples were taken on days 0, 7 and 14 of the uptake phase and on days 1, 4, 7, 14, 21, 28, 35 and 42 of the depuration phase.
- Sample storage conditions before analysis: The test samples were analyzed on the day of sampling, or stored and allowed to equilibrate to room temperature prior to analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Test organism samples: A fish (circa 5 g) was liquidized to obtain a homogenous paste. Approximately 5 g of this fish paste was accurately weighed into a 250 ml beaker and 150 ml of hexane added. The mixture was macerated with a high speed macerator for at least 1 minute, or until no recognisable fish tissue was visible. The solids were allowed to settle then the hexane extract decanted into a 1 litre separating funnel. The fish solids and macerator were washed with a further 150 ml of hexane, allowed to settle, then the washings added to the separating funnel.
The hexane extract was washed with 3 x 100 ml of 1 M NaOH solution, (approx. 1 min per wash), discarding the aqueous extract after each wash. The hexane extract was then washed with 3 x 250 ml of RO water (approx. 1 min per wash with gentle agitation to avoid emulsion forming), discarding the aqueous extract after each wash.
The hexane extract was dried by filtering through anhydrous sodium sulphate into a 500 ml round bottomed flask, evaporated to dryness, then the residue re-dissolved in 10 mL acetonitrile with sonication and shaking. The samples were transferred to a glass centrifuge tube, centrifuged at 4800 rpm for 5 minutes, then 1 mL of supernatant was mixed with 1 mL acetonitrile and analysed.
Fish food samples: A mass (5 g) of fish food was weighed and placed in a volumetric flask, 150 mL acetonitrile added, then sonicated for 15 minutes. The samples were shaken and then sonicated for a further 10 minutes, and then made to a final volume of 250 mL with acetonitrile. Where necessary, the samples were diluted into the calibration range with acetonitrile.
An aliquot of the acetonitrile was placed into a freezer for 30 minutes and then filtered through a cold 0.45 µm PTFE filter, discarding the first 3 mL of filtrate, then vialled for analysis.
Test media samples: An aliquot (200 mL) was passed through a previously primed Strata C8 (500 mg/3 mL) solid phase extraction cartridge. The cartridge was then dried and the analyte eluted with 10 mL of acetonitrile. The solutions were further diluted with acetonitrile where necessary.
All samples were analyzed by High Performance Liquid Chromatography with Tandem Mass Spectrometric Detection (HPLC-MS-MS).

Vehicle:

yes

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

PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): Inicio 917 1.1 mm trout pellets (50% protein and 16% fat content)
- Details of spiking (e.g. i) liquid test material (neat); ii) with a vehicle (corn or fish oil); or iii) using an organic solvent:
The treated diet was prepared by dissolving 150 mg of Type A and 150 mg of Type B in ethanol and making up to a final volume of 5.0 mL in ethanol to give a solvent stock solution at a combined concentration of 60 mg/mL. The solvent stock solution was inverted several times to ensure adequate mixing and homogeneity. Once fully dissolved, 2.5 mL of the 60 mg/mL solvent stock solution was added to 4.0 mL corn oil, and the ethanol removed by evaporation. The resultant corn oil stock solution had a concentration of 37.5 mg/mL. Once all the ethanol had been removed the corn oil containing the test item was added to 100 g of trout pellets in a glass jar and mixed thoroughly by shaking by hand for 2 minutes and on a rotary mixer for one hour to ensure thorough incorporation and distribution. The treated feed was therefore prepared at a concentration of 1500 µg combined test item/g.
- Quantity of corn or fish oil vehicle, if used, per unit mass of fish food: 4.0 mL corn oil / 100 g of trout diet
- Chemical name of vehicle (organic solvent), if used: ethanol
- Method for removal of solvent, if used: evaporation

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK
- Age at study initiation (mean and range, SD): juveniles
- Length at study initiation (length definition, mean, range and SD): The mean length of a subsample of 10 fish, selected at random from the batch to be used in the test prior to initiation of exposure (Day -1) was calculated as 7.9 cm.
- Weight at study initiation (mean and range, SD): The mean weight of these fish was calculated as 4.92 g. Prior to initiation of the test a selection of fish were weighed to ensure that the control and test tank had fish that fell within a weight range of 4.15 g to 6.22 g, ensuring that the smallest fish were not smaller than two-thirds of the weight of the largest fish.
- Weight at termination (mean and range, SD):
- Method of breeding: not specified
- Lipid content at test initiation (mean and range, SD): .6% to 6.3% body weight
- Feeding during test
- Food type: Fish were fed with Inicio 917 1.1 mm trout pellets (50% protein and 16% fat content) either treated or untreated.
- Amount: The fish in both the treatment and control vessels were fed at a rate of 2% of body weight during the exposure and depuration phases. The feeding rate during the depuration phase was adjusted on sample days and were based on the weight of the fish removed for analysis.
- Frequency: daily

ACCLIMATION
- Acclimation period: at least 2 weeks
- Acclimation conditions (same as test or not): The fish were acclimatized in holding tanks.
- Type and amount of food: commercial trout pellets
- Feeding frequency: not specified
- Health during acclimation (any mortality observed): No mortality was observed during the acclimatization period.

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:

42 d

Hardness:

176 - 180 mg CaCO3 (Day -1)

Test temperature:

14 - 15 °C (variation less than 2 °C)

pH:

control: 7.9 - 8.3
1500 µg/g tretament group: 7.9 - 8.4

Dissolved oxygen:

≥ 8.2 mg O2/L (equivalent to 80% ASV)

TOC:

control: 3.0 mg C/L
1500 µg/g tretament group: 1.3 mg C/L

Conductivity:

513 μS/cm at 20 °C specified as typical test water characteristic

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: glass vessels with approx. 140 liters in volume, filled with a volume of approx. 100 liters
- Aeration: yes, aerated via narrow bore glass tubes
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous flow through of approximately 1152 liters per day
- Renewal rate of test solution (frequency/flow rate): nominal flow rate 800 mL/minute
- No. of organisms per vessel: 50
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination unit) and a proportion of the incoming water softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main supply to give a water hardness of approximately 140 mg/L as CaCO3 (within the range 100 to 200 mg/L as CaCO3) and pH 6.0 to 8.5. After dechlorination and softening the water was passed through a series of computer controlled heat exchangers to achieve the required temperature.
- Holding medium different from test medium: no
- Intervals of water quality measurement: not specified
- Intervals of test medium replacement: flow through system with a flow rate of 800 mL/minute

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 14 days for the uptake phase
- Light intensity: 770 - 968 lux
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2% of body weight during the uptake and depuration phases
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): The fish in both the treatment and control vessels were fed once per day.
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used:
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: 16% lipid food concentration

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: nominal concentrations of 10, 100 and 1000 µg/g
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary palatability test 1000 µg/g was selected for the main bioaccumulation test. The exposure level was selected on the basis that observations on feeding behaviour showed the fish had no adverse reaction to the feed provided. Furthermore no toxicity was observed during the preliminary palatability test.
An error which occurred at the time of the feed preparation resulted in a higher test item concentration being prepared than what was proposed. The actual test concentration prepared for the main test was 1500 µg/g.

Nominal and measured concentrations:

nominal: 1500 µg/g (750 µg/g Type A (n = 1) and 750 µg/g Type B (n = 1))
mean measured:
Day 0: 828 µg/g Type A (n = 1) and 673 µg/g Type B (n = 1)
Day 14: 1036 µg/g Type A (n = 1) and 705 µg/g Type B (n = 1)
average concentration across the uptake phase: 932 µg/g Type A (n = 1) and 689 µg/g Type B (n = 1)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logKow: 7.93 (measured) for Type A (n = 1) and Type B (n = 1); 8.63 (calculated) for Type A (n = 1) and 8.06 (calculated) for Type B (n = 1)

BASIS FOR CALCULATION OF BCF
- Estimation software: spreadsheet provided and used in conjunction with the Guidance Document to OECD TG 305. Accordingly BCF estimates are calculated for the 3 methods presented in the Guidance Document.

Lipid content:

>= 3.6 - <= 6.3 %

Time point:

other: throughout the depuration phase

Key result

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

other: BMFkgl (lipid normalised and growth corrected BMF)

Value:

0.295 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Remarks:

Type A

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

other: BMFkgl (lipid normalised and growth corrected BMF)

Value:

0.954 dimensionless

Basis:

whole body w.w.

Calculation basis:

kinetic, corrected for growth

Key result

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

4 801.9 dimensionless

Basis:

other: growth and lipid corrected BMF

Remarks:

Type A

Calculation basis:

kinetic

Remarks on result:

other: estimation method 3 according to Inoue et al. (2012)

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

12 675.5 dimensionless

Basis:

other: growth and lipid corrected BMF

Remarks:

Type B

Calculation basis:

kinetic

Remarks on result:

other: estimation method 3 according to Inoue et al. (2012)

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

9 247.5 dimensionless

Basis:

other: growth and lipid correctd depuration rate constant

Remarks:

Type A

Calculation basis:

kinetic

Remarks on result:

other: estimation method 2 according to Brookes and Crooke (2012)

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

6 454 dimensionless

Basis:

other: growth and lipid corrected depuration rate constant

Remarks:

Type B

Calculation basis:

kinetic

Remarks on result:

other: estimation method 2 according to Brookes and Crooke (2012)

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

>= 337.2 - <= 27 866.7 dimensionless

Basis:

other: weight and/or log Kow

Remarks:

Type A

Calculation basis:

kinetic

Remarks on result:

other: estimation method 1 according to several calculation references

Conc. / dose:

1 500 µg/g food

Temp.:

>= 14 - <= 15 °C

Type:

BCF

Value:

>= 251.2 - <= 20 761.8 dimensionless

Basis:

other: weight and / or log Kow

Remarks:

Type B

Calculation basis:

kinetic

Remarks on result:

other: estimation method 1 according to several calculation references

Elimination:

no

Parameter:

DT50

Depuration time (DT):

12 d

Remarks on result:

other: Type A

Elimination:

no

Parameter:

DT50

Depuration time (DT):

9 d

Remarks on result:

other: Type B

Rate constant:

growth rate constant (d-1)

Remarks:

Type A (n = 1) and Type B (n = 1)

Value:

0.012

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

Type B (n = 1)

Value:

0.076

Rate constant:

growth-corrected depuration rate constant (d-1)

Remarks:

Type A (n = 1)

Value:

0.057

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

Type A (n = 1)

Value:

0.005

Rate constant:

overall uptake rate constant (L kg-1 d-1)

Remarks:

Type B (n = 1)

Value:

0.022

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Type A (n = 1)

Value:

0.069

Rate constant:

overall depuration rate constant (d-1)

Remarks:

Type B (n = 1)

Value:

0.088

Rate constant:

growth-corrected half-life (d)

Remarks:

Type A (n = 1)

Value:

12.073

Rate constant:

growth-corrected half-life (d)

Remarks:

Type B (n = 1)

Value:

9.061

Details on results:

- Mortality of test organisms: Mortality in the control and 1500 µg/g treated groups over the full duration of the test was 2% and 4%, respectively.
- Behavioural abnormalities: On Day 4 of the uptake phase one fish in the control group was observed with darkened pigmentation, lethargic and with exophthalmos (pop-eye). On Day 6 of the uptake phase one fish in the 1500 µg/g treated group was observed as lethargic and with exophthalmos (pop-eye). On Day 9 of the uptake phase one fish in the 1500 µg/g treated group was observed with darkened pigmentation, lethargic and with exophthalmos (pop-eye). With consideration to animal welfare implications (Animals (Scientific Procedures) Act 1986) these fish were humanely killed and were classed as mortalities for this time point. No other sub-lethal effects of exposure were observed throughout the test.

Reported statistics:

Test item concentration-time data in whole fish were analyzed using the software R version 3.6.3 (2020-02-29) (The R Foundation for Statistical Computing) and OECD-TG305 R-Package bcmfR (v0.4-18) consistent with OECD 305 Guidance (2017).
Model fitting and simulations were performed using lipid food contents of 16%.
Three optimization models were tested against the data for Type A and Type B test items. Linear regression, a Box-Cox transformation and a Natural Log transformation. Model diagnosis (see Figure 1) suggests that the model residuals are two sided distributed for both test articles; however, the best fit for Type A test item was achieved with a Box-Cox fit and the best fit for Type B test item was achieved with a non-transformed fit.

Concentration of Test Item in Treated Feed

The mean concentration of the test item on the feed during exposure was found to be 828 µg/g for Type A (n = 1) and 673 µg/g for Type B (n = 1) measured on the day of preparation and at the beginning of the uptake phase and 1036 µg/g for Type A (n = 1) and 705 µg/g for Type B (n = 1) indicating there was no notable reduction recorded from the start to end of the 14-Day uptake period. The average exposure concentration across the uptake phase was therefore determined to be 932 µg/g for Type A (n = 1) and 689 µg/g for Type B (n = 1).

 

Concentration of Test Item in Fish

Concentrations of test item in fish are reported as µg/g (µg equivalents /g fresh weight). 

Mean measured concentrations of the test item in the fish at the end of the uptake phase were determined to be 5.4 µg/g for Type A (n = 1) and 13.4 µg/g for Type B (n = 1).

Elimination of the test item during the depuration phase was relatively slow, declining to mean values of 0.42 µg/g for Type A (n = 1) and 0.95 µg/g for Type B (n = 1) by Day 28 of the depuration phase and to 0.43 µg/g for Type A (n = 1) and 0.50 µg/g for Type B (n = 1) by the end of the 42-Day depuration phase.

 

Concentration of Test Item in Tank Water

Analysis of the tank water showed no test item to be present on Day 0, prior to addition of any treated feed; however, measured concentrations on Days 7 and 14 of the uptake phase were 0.0022 and 0.0014 mg/L for Type A (n = 1) and 0.00066 and 0.00027 mg/L for Type B (n = 1), respectively.

Analysis of the tank water on Day 1 of the depuration phase showed measured concentrations of 0.0012 mg/L for Type A (n = 1) and 0.00025 mg/L for Type B (n = 1). Measurements on all other occasions during the depuration phase showed measured concentrations were below the limit of quantification, determined to be 0.00010 mg/L for both Type A (n = 1) and Type B (n = 1).

Validity Criteria

The following validity criteria were achieved during the main test (validity criteria were not applied during the preliminary test):

 

	Required	Achieved
Temperature variation	Less than 2 °C	Test temperatures recorded between 14 and 15 °C therefore confirming the variation was less than 2 °C.
Dissolved oxygen concentration	Does not fall below 60% Air Saturation Value (ASV)	All dissolved oxygen concentrations recorded as equal to or greater than 8.2 mg O2/L (equivalent to 80% ASV).
Concentration of test item in the feed	Maintained within ± 20% at the start and end of the uptake phase	The mean measured concentration was 1549 µg/g on Day 0 and 1741 µg/g on Day 14 giving a deviation of 11% across the duration of the uptake phase
Mortality in control and treated fish	Less than 10%	Mortality in the control and the 1500 µg/g treated groups were 2% and 4% respectively.
Total organic carbon (TOC) in the test water	Should be as low as possible	The calculated TOC for the control and 1500 µg/g treated groups were 3.0 and 1.3 mg C/L respectively.

Conclusion

The potential for two main constituents of the UVCBPhenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene (CAS number 68610-51-5: EC number 271-867-2) to bioaccumulate in rainbow trout (Oncorhynchus mykiss) via the dietary exposurewas studied.

All the validation criteria of the test were met.

The mean concentration of the test item on the feed during the uptake phase was found to be 828 µg/g for Type A and 673 µg/g for Type B measured on the day of preparation and at the beginning of the uptake phase and 1036 µg/g for Type A and 705 µg/g for Type B indicating there was no notable reduction recorded from the start to end of the 14-Day uptake phase. The average exposure concentration across the uptake phase was therefore determined to be 932 µg/g for Type A and 689 µg/g for Type B.

Mean measured concentrations ofthe test item in the fish at the end of the uptake phase were determined to be 5.4 µg/g for Type A and 13.4 µg/g for Type B.

Elimination of the test item during the depuration phase was relatively slow, declining to mean values of 0.42 µg/g for Type A and 0.95 µg/g for Type B by Day 28 of the depuration phase and to 0.43 µg/g for Type A and 0.50 µg/g for Type B by the end of the 42-Day depuration phase.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD 305-III (Dietary exposure Bioaccumulation Fish Test). As the test material is an UVCB, ECHA requested in the Final Decision of 11 April 2018 to test an equal mixture of the representative constituents. These constituents have been identified to be Type A (n=1) and B (n=1) and thus, have been used for the test, rather than test the whole substance. These constituents are the only two identified to be present >= 0,1 % (w/w) by the Sponsor and which potentially contribute to any bioaccumulation potential as determined by ECHA’s Final Decision of 11 April 2018. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the bioaccumulative potential of the test substance towards fish.
According to the results of the non-GLP Pharmacokinetics the examination of the chemical analysis data indicated that levels less than 0.4 µg/g of Type A (n = 1) test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.29533 with a half-life of approximately 12 days. The growth corrected depuration rate (k2g) was determined to be 0.0574.
Examination of the chemical analysis data of Type B (n = 1) indicated that levels less than 0.5 µg/g test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.95373 with a half-life of approximately 9 days. The growth corrected depuration rate (k2g) was determined to be 0.076482.
As defined in the Guidance document to OECD TG 305 a BMFkgl > 1 allows the clear categorisation of a test chemical as highly bioaccumulative (i.e. the BCF would be > 5000 L/kg) and a BMFkgl < 0.1 the clear categorisation as not bioaccumulative (i.e. the BCF would be < 2000 L/kg) according to many regulatory schemes in OECD member countries that allow the use of surrogate bioaccumulation data other than the BCF. Since the BMFkgl of both constituents was determined to be > 0.1 and < 1 a clear categorisation is not possible, wherefore the results for Type A (n = 1) and Type B (n = 1) were considered to have a bioaccumulative potential fulfilling the B criterion.
For the purposes of classification and risk assessment the obtained results were also used to generate a BCF estimate using the spreadsheet that accompanies the guidance document to OECD 305 (2nd Draft), which automates the BCF estimation using three different estimation methods as recommended by the TG and the respective guidance documents.
According to estimation method 1 (uptake rate constant estimation method) a BCF of 337.2 – 27866.7 was determined for Type A (n = 1) and a BCF in the range of 251.2 – 20761.8 was determined for Type B (n = 1). The results obtained by estimation method 2 (relating depuration rate constant directly to BCF) indicate a BCF of 9247.5 for Type A (n = 1) and 6454.0 for Type B (n = 1). Based on the estimates according to method 3 (correlating dietary BMF with BCF) the BCF for Type A (n = 1) is 4801.9 and the BCF for Type B (n = 1) is 12675.5. From these results it can be assumed that the constituents have a bioaccumulative (B) to highly bioaccumulative potential (vB).
Considering the fact that the two main constituents are molecularly large with a molecular weight > 1100, have a high log Kow (> 8) and low study assimilation efficiencies (α < approx. 0.1), the first and second BCF estimation methods may be less reliable as outlined in the guidance document. For these kinds of substances the estimation method 3 should be preferred even when the substance properties and the assimilation efficiencies may be outside the ranges of these parameters for method 3’s training set chemicals.
However, due to the uncertainty in the applicability domains of the three methods predictions and due to the fact that the results on BMF and BCF for the major constituents Type A (n = 1) and Type B (n = 1) do not allow a clear categorisation, the results were regarded for the whole substance using a weight of evidence approach involving other information relevant for bioaccumulation as recommended in the ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.11: PBT/vPvB Assessment(Version 3.0, June 2017).
Based on ECHA’s Final Decision of 11 April 2018, registrants had to identify the relevant constituents in their registered substance. Due to the UVCB status of the registered substance, different samples have been analysed and a summary of the analysis has been provided. Taking into account this analytical information for a full characterisation of the UVCB test material (Intertek, 2020), the constituents Type A (n = 1) and Type B (n = 1) are the only two identified to be present >= 0.1 % (w/w) and which potentially contribute to any bioaccumulation potential. Constituent Types C, E and F with n=0 were determined to be present in the registered substance at a concentration <= 0.1 % (w/w) and therefore not relevant for the PBT assessment.
According to the analytical information the content of the major constituents Type A (n = 1) and Type B (n = 1) was determined to be in a range of 50.6 – 58.3 % and 1.2 – 10.9 %, respectively. Thus, the content of constituent Type A (n = 1) is significantly higher than the content of Type B (n = 1). All the other constituents identified to be present at a content of >= 0.1 % (w/w) (Type B n = 0, Type A n = 2 and Types C-F with n ≥ 1) sum up to a total in the range of approx. 30 – 48 %.
As described in the in ECHA`s Guidance on Information Requirements and Chemical Safety Assessment Chapter R.11: PBT/vPvB Assessment and as summarised by ECHA within the Final Decision of 11 April 2018 these constituents are considered to have a low potential for bioaccumulation, due to their molecular size and thus decreasing bioavailability and based on logKow values below 4.5 or above 10.
Using the Software EpiWin v4.1 implemented in OECD QSAR Toolbox v4.4, a QSAR estimation was performed for the major constituents Type A (n = 1) and Type B (n = 1), which is presented as supporting information. The used BCFBAF v3.01 model predicts a BCF of 733 L/kg for Type A (n = 1) and a BCF of 2420 L/kg for Type B (n = 1) following a regression based estimation. In addition, the BAF (Arnot-Gobas upper trophic) was calculated to be 1220 L/kg for Type A (n = 1) and 1330 L/kg for Type B (n = 1) and the growth corrected half-life was estimated to be 8.26 and 3.67, respectively.
Based on these supporting QSAR results the BCF is predicted to be far below 5000 L/kg (criterion for non-vB) for the major constituent Type B (n = 1), and even below 2000 L/kg (criterion for non-B) for the major constituent Type A (n = 1).
 
Summarising the explanations and data above, the experimentally determined BMF of the two main constituents (Type A (n = 1) and Type B (n = 1)) does not allow a clear classification and indicates a bioaccumulative rather than a very bioaccumulative potential, if at all. The estimated BCF values using the three different recommended estimation methods are very inconsistent and indicate a bioaccumulative or even very bioaccumulative potential for both constituents. At last, by the effort of a more precise analytical characterization of the registered substance, it could be proven that the Type A constituents (including the main constituent with n = 1) represent the most typical structures of the registered substance with a total content of more than about 75%. Therefore, it is concluded that the results obtained for the main constituent Type A (n = 1) can be regarded as most representative for the whole test material. Thus, it is considered meaningful and scientifically reasonable to use the respective BMF of 0.29533 and the estimated BCF of 4801.9 according to estimation method 3 (Inoue et al., 2012) for further risk assessment, whereby the bioaccumulation potential of the registered substance finally fulfils the "B" criterion for PBT/vPvB set in Annex XIII of REACH.
This classification is confirmed by the results of the supporting QSAR estimation, predicting at most a bioaccumulative but not a highly bioaccumulative potential for the representative worst-case constituents Type A (n = 1) and Type B (n = 1).

Executive summary:

The potential for the UVCB substance Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene (CAS number 68610-51-5: EC number 271-867-2) to bioaccumulate via the diet in rainbow trout (Oncorhynchus mykiss )was studied according to the OECD Guidelines for Testing of Chemicals (2012) No. 305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" in a continuous flow-through system.

Rather than test the whole substance, an equal mixture of the representative constituents of Types A (n=1) and B (n=1) have been used as the test material. These constituents are the only two identified to be present >= 0,1 % (w/w) by the Sponsor and which potentially contribute to any bioaccumulation potential as determined by ECHA’s Final Decision of 11 April 2018.

Fish were exposed to these constituents of the UVCB Phenol, 4-methyl-, reaction products with dicyclopentadiene and isobutylene (CAS number 68610-51-5: EC number 271-867-2) dosed on to commercial diet at a nominal concentration of 1500 µg/g, over a 14-Day uptake period. Feed dosed using the major constituents Type A and Type B test items at nominal concentration of 750 µg/g. Depuration of the test item was determined over a further 42‑Day period. A dietary uptake level of 1000 µg/g was selected based on results generated during a preliminary 7-day palatability study at concentrations of up to 1000 µg/g; however, a technical error resulted in the main test being conducted at a nominal concentration of 1500 µg/g.

All the validation criteria of the test were met.

The mean concentration of the test item on the feed during uptake was found to be 828 µg/g for Type A and 673 µg/g for Type B measured on the day of preparation and at the beginning of the uptake phase and 1036 µg/g for Type A and 705 µg/g for Type B indicating there was no notable reduction recorded from the start to end of the 14-Day uptake period. The average exposure concentration across the uptake phase was therefore determined to be 932 µg/g for Type A and 689 µg/g for Type B.

Mean measured concentrations of the test item in the fish at the end of the uptake period were determined to be 5.4 µg/g for Type A and 13.4 µg/g for Type B.

Elimination of the test item during the depuration phase was relatively slow, declining to mean values of 0.42 µg/g for Type A and 0.95 µg/g for Type B by Day 28 of the depuration phase and to 0.43 µg/g for Type A and 0.50 µg/g for Type B by the end of the 42-Day depuration phase.

The lipid content of the fish remained constant throughout the depuration phase and was within the range of 3.6% to 6.3% body weight.

 

According to the results of the non-GLP Pharmacokinetics the examination of the chemical analysis data indicated that levels less than 0.4 µg/g of Type A test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.29533 with a half-life of approximately 12 days.

Examination of the chemical analysis data of Type B indicated that levels less than 0.5 µg/g test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.95373 with a half-life of approximately 9 days.

By the use of the spreadsheet provided in conjunction with the Guidance Document to OECD TG 305, a BCF was estimated for Type A and Type B. Due to the fact that the two major constituents have a high log Kow and low study assimilation efficiencies (α< approx.. 0.1), the first and second BCF estimation methods are considered to be less reliable according to the guidance document. Accordingly, the results obtained by estimation method 3 based on the growth and lipid corrected BMF (Inoue et al., 2012) are considered most meaningful with a BCF of 4801.9 for Type A and 12675.5 for Type B.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

7 JAN 2021

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:

1. SOFTWARE
EpiWin v4.1 implemented in OECD QSAR Toolbox v4.4

2. MODEL (incl. version number)
BCFBAF v3.01

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Type A: Cc1cc(C5CC3C4CC(C3C5)C(C4)c2cc(C)cc(c20)C(C)(C)C)c(O)c(c1)C(C)(C)C
Type B: Cc4ccc(O)c(c4)C5CC2C3CC(C2C5)C(C3)c1cc(C)cc(c10)C(C)(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please refer to attached QMRF

5. APPLICABILITY DOMAIN
Please refer to attached QPRF

Guideline:

other: ECHA Guidance R.6

Principles of method if other than guideline:

- Software tool(s) used including version: EpiWin v4.1 implemented in OECD QSAR Toolbox v4.4
- Model(s) used: BCFBAF v3.01
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

Key result

Type:

BCF

Value:

733 L/kg

Basis:

whole body w.w.

Calculation basis:

other: regression-based estimate

Remarks on result:

other: type A

Key result

Type:

BCF

Value:

2 420 L/kg

Basis:

whole body w.w.

Calculation basis:

other: regression-based estimate

Remarks on result:

other: type B

Type:

BAF

Value:

1 220 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Arnot-Gobas upper trophic

Remarks on result:

other: type B

Type:

BAF

Value:

1 330 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Arnot-Gobas upper trophic

Remarks on result:

other: type A

Key result

Rate constant:

growth-corrected half-life (d)

Remarks:

normalized to 10 g fish

Value:

8.26

Remarks on result:

other: type A

Key result

Rate constant:

growth-corrected half-life (d)

Remarks:

normalized to 10 g fish

Value:

3.67

Remarks on result:

other: type B

Details on results:

The predictions are within the applicability domain of the model

Validity criteria fulfilled:

yes

Conclusions:

The BCF, BAF and Biotransformation Half-Life of type A and type B, the main constituents of the registered substance were predicted as follows:

Type A
Log BCF (regression-based estimate): 2.86 (BCF = 733 L/kg wet-wt)
Biotransformation Half-Life (days) : 8.26 (normalized to 10 g fish)
Log BAF (Arnot-Gobas upper trophic): 3.12 (BAF = 1330 L/kg wet-wt)

Type B
Log BCF (regression-based estimate): 3.38 (BCF = 2420 L/kg wet-wt)
Biotransformation Half-Life (days) : 3.67 (normalized to 10 g fish)
Log BAF (Arnot-Gobas upper trophic): 3.09 (BAF = 1220 L/kg wet-wt)

Executive summary:

The BCF, BAF and Biotransformation Half-Life of type A and type B, the main constituents of the registered substance was calculated by the QSAR model BCFBAF v3.01 implemented in EPIWIN v4.1. The following results were obtained:

Type A

Log BCF (regression-based estimate): 2.86 (BCF = 733 L/kg wet-wt)

Biotransformation Half-Life (days) : 8.26 (normalized to 10 g fish)

Log BAF (Arnot-Gobas upper trophic): 3.12 (BAF = 1330 L/kg wet-wt)

Type B

Log BCF (regression-based estimate): 3.38 (BCF = 2420 L/kg wet-wt)

Biotransformation Half-Life (days) : 3.67 (normalized to 10 g fish)

Log BAF (Arnot-Gobas upper trophic): 3.09 (BAF = 1220 L/kg wet-wt)

The predictions are within the applicability domain of the model.

Description of key information

1) Covance conducted a guideline study (OECD 305 -III) by exposing rainbow trout (Oncorhynchus mykiss) via diet over a 14-Day uptake period followed by a 42 -Day depuration period in a flow-through limit test with the nominal concentration of 1500 µg/g. A BMF of 0.29533 and an estimated BCF of 4801.9 resulted as key values.

2) As supporting information the BCF, BAF and Biotransformation Half-Life of Type A (n = 1) and Type B (n = 1), representing the main constituents of the registered substance, were calculated by the QSAR model BCFBAF v3.01 implemented in EPIWIN v4.1. A BCF of 733 L/kg and a BCF of 2420 L/kg resulted as main values for the major constituents Type A (n = 1) and Type B (n = 1). The predictions are within the applicability domain of the model.

 

Key value for chemical safety assessment

BCF (aquatic species):

4 801.9 dimensionless

BMF in fish (dimensionless):

0.295

Additional information

Experimental Biomagnification study

The study was conducted under GLP according to OECD 305-III (Dietary exposure Bioaccumulation Fish Test). As the test material is an UVCB, ECHA requested in the Final Decision of 11 April 2018 to test an equal mixture of the representative constituents. These constituents have been identified to be Type A (n=1) and B (n=1) and thus, have been used for the test, rather than test the whole substance. These constituents are the only two identified to be present >= 0,1 % (w/w) by the Sponsor and which potentially contribute to any bioaccumulation potential as determined by ECHA’s Final Decision of 11 April 2018. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the bioaccumulative potential of the test substance towards fish.

According to the results of the non-GLP Pharmacokinetics the examination of the chemical analysis data indicated that levels less than 0.4 µg/g of Type A (n = 1) test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.29533 with a half-life of approximately 12 days. The growth corrected depuration rate (k2g) was determined to be 0.0574.

Examination of the chemical analysis data of Type B (n = 1) indicated that levels less than 0.5 µg/g test item remained at the end of the depuration phase. The lipid normalised and growth corrected biomagnification factor (BMFKgL) was calculated to be 0.95373 with a half-life of approximately 9 days. The growth corrected depuration rate (k2g) was determined to be 0.076482.

As defined in the Guidance document to OECD TG 305 a BMFkgl > 1 allows the clear categorisation of a test chemical as highly bioaccumulative (i.e. the BCF would be > 5000 L/kg) and a BMFkgl < 0.1 the clear categorisation as not bioaccumulative (i.e. the BCF would be < 2000 L/kg) according to many regulatory schemes in OECD member countries that allow the use of surrogate bioaccumulation data other than the BCF. Since the BMFkgl of both constituents was determined to be > 0.1 and < 1 a clear categorisation is not possible, wherefore the results for Type A (n = 1) and Type B (n = 1) were considered to have a bioaccumulative potential fulfilling the B criterion.

For the purposes of classification and risk assessment the obtained results were also used to generate a BCF estimate using the spreadsheet that accompanies the guidance document to OECD 305 (2nd Draft), which automates the BCF estimation using three different estimation methods as recommended by the TG and the respective guidance documents.

According to estimation method 1 (uptake rate constant estimation method) a BCF of 337.2 – 27866.7 was determined for Type A (n = 1) and a BCF in the range of 251.2 – 20761.8 was determined for Type B (n = 1). The results obtained by estimation method 2 (relating depuration rate constant directly to BCF) indicate a BCF of 9247.5 for Type A (n = 1) and 6454.0 for Type B (n = 1). Based on the estimates according to method 3 (correlating dietary BMF with BCF) the BCF for Type A (n = 1) is 4801.9 and the BCF for Type B (n = 1) is 12675.5. From these results it can be assumed that the constituents have a bioaccumulative (B) to highly bioaccumulative potential (vB).

Considering the fact that the two main constituents are molecularly large with a molecular weight > 1100, have a high log Kow (> 8) and low study assimilation efficiencies (α < approx. 0.1), the first and second BCF estimation methods may be less reliable as outlined in the guidance document. For these kinds of substances the estimation method 3 should be preferred even when the substance properties and the assimilation efficiencies may be outside the ranges of these parameters for method 3’s training set chemicals.

However, due to the uncertainty in the applicability domains of the three methods predictions and due to the fact that the results on BMF and BCF for the major constituents Type A (n = 1) and Type B (n = 1) do not allow a clear categorisation, the results were regarded for the whole substance using a weight of evidence approach involving other information relevant for bioaccumulation as recommended in the ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.11: PBT/vPvB Assessment(Version 3.0, June 2017).

Based on ECHA’s Final Decision of 11 April 2018, registrants had to identify the relevant constituents in their registered substance. Due to the UVCB status of the registered substance, different samples have been analysed and a summary of the analysis has been provided. Taking into account this analytical information for a full characterisation of the UVCB test material (Intertek, 2020), the constituents Type A (n = 1) and Type B (n = 1) are the only two identified to be present >= 0.1 % (w/w) and which potentially contribute to any bioaccumulation potential. Constituent Types C, E and F with n=0 were determined to be present in the registered substance at a concentration <= 0.1 % (w/w) and therefore not relevant for the PBT assessment.

According to the analytical information the content of the major constituents Type A (n = 1) and Type B (n = 1) was determined to be in a range of 50.6 – 58.3 % and 1.2 – 10.9 %, respectively. Thus, the content of constituent Type A (n = 1) is significantly higher than the content of Type B (n = 1). All the other constituents identified to be present at a content of >= 0.1 % (w/w) (Type B n = 0, Type A n = 2 and Types C-F with n ≥ 1) sum up to a total in the range of approx. 30 – 48 %.

As described in the in ECHA`s Guidance on Information Requirements and Chemical Safety Assessment Chapter R.11: PBT/vPvB Assessment and as summarised by ECHA within the Final Decision of 11 April 2018 these constituents are considered to have a low potential for bioaccumulation, due to their molecular size and thus decreasing bioavailability and based on logKow values below 4.5 or above 10.

Using the Software EpiWin v4.1 implemented in OECD QSAR Toolbox v4.4, a QSAR estimation was performed for the major constituents Type A (n = 1) and Type B (n = 1), which is presented as supporting information. The used BCFBAF v3.01 model predicts a BCF of 733 L/kg for Type A (n = 1) and a BCF of 2420 L/kg for Type B (n = 1) following a regression based estimation. In addition, the BAF (Arnot-Gobas upper trophic) was calculated to be 1220 L/kg for Type A (n = 1) and 1330 L/kg for Type B (n = 1) and the growth corrected half-life was estimated to be 8.26 and 3.67, respectively.

Based on these supporting QSAR results the BCF is predicted to be far below 5000 L/kg (criterion for non-vB) for the major constituent Type B (n = 1), and even below 2000 L/kg (criterion for non-B) for the major constituent Type A (n = 1).

 

Summarising the explanations and data above, the experimentally determined BMF of the two main constituents (Type A (n = 1) and Type B (n = 1)) does not allow a clear classification and indicates a bioaccumulative rather than a very bioaccumulative potential, if at all. The estimated BCF values using the three different recommended estimation methods are very inconsistent and indicate a bioaccumulative or even very bioaccumulative potential for both constituents. At last, by the effort of a more precise analytical characterization of the registered substance, it could be proven that the Type A constituents (including the main constituent with n = 1) represent the most typical structures of the registered substance with a total content of more than about 75%. Therefore, it is concluded that the results obtained for the main constituent Type A (n = 1) can be regarded as most representative for the whole test material. Thus, it is considered meaningful and scientifically reasonable to use the respective BMF of 0.29533 and the estimated BCF of 4801.9 according to estimation method 3 (Inoue et al., 2012) for further risk assessment, whereby the bioaccumulation potential of the registered substance finally fulfils the "B" criterion for PBT/vPvB set in Annex XIII of REACH.

This classification is confirmed by the results of the supporting QSAR estimation, predicting at most a bioaccumulative but not a highly bioaccumulative potential for the representative worst-case constituents Type A (n = 1) and Type B (n = 1).

Supporting QSAR calculations

The BCF, BAF and Biotransformation Half-Life of Type A (n = 1) and Type B (n = 1), representing the main constituents of the registered substance, was calculated by the QSAR model BCFBAF v3.01 implemented in EPIWIN v4.1. The following results were obtained:

Type A

Log BCF (regression-based estimate): 2.86 (BCF = 733 L/kg wet-wt)

Biotransformation Half-Life (days) : 8.26 (normalized to 10 g fish)

Log BAF (Arnot-Gobas upper trophic): 3.12 (BAF = 1330 L/kg wet-wt)

Type B

Log BCF (regression-based estimate): 3.38 (BCF = 2420 L/kg wet-wt)

Biotransformation Half-Life (days) : 3.67 (normalized to 10 g fish)

Log BAF (Arnot-Gobas upper trophic): 3.09 (BAF = 1220 L/kg wet-wt)

The predictions are within the applicability domain of the model.