Cyclopropanemethanol, 1-methyl-2-[(1,2,2-trimethylbicyclo[3.1.0]hex-3-yl)methyl]-

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:

November, 2013 through February, 2014.

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body, Ministry of Economy, Trade and Industry, Environmental Policy Bureau, Ministry of the Environment (02 April, 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

JAVANOL :
- Lot No. : VE00182094
- Purity : 94.4%
- Impurities : All impurities and unknown components < 1%
- Date of Expiry : 06 December, 2014

Radiolabelling:

no

Details on sampling:

Aqueous Phase Samples for Level 1 and Level 2 Exposure Media :
Samples of the aqueous exposure phase were sampled at the following Days :
(i) 1 day prior or just prior to Introduction of the fish;
(ii) During the Uptake-Phase at Day 7, 9, 14, 21, 24 and 28;
(iii) During the Depuration-Phase at Day 29 for Level I and Level II

Water Samples from the Control Medium were taken at Day -1, Day 28 (Uptake-Phase) and Day 29 (Depuration-Phase).


Fish Tissue Sampling for Determination of Concentrtions of Javanol :
- Control Sample : At Start (Day 0) of the Uptake-Phase and Day 28, and on Day 35 (i.e. 6.6 days in to the Depuration-Phase);
- Level 1 and Level 2 Exposure : Fish tissue samples were sampled on Day 7, 9, 14, 21, 24 and 28 of the Uptake-Phase and on Day 29, 30, 31 and 35 during the Depuration-Phase.

Fish Lipid Samples :
Samples of fish tissue were taken for lipid content analysis at the following time-points :
Control : Start of exposure, Day 28 of Uptake-Phase and Day 35 (6.6 days of Depuration)
Level 1 and Level 2 Exposure : Day 28 of Uptake-Phase and Day 35 (6.6 days of Depuration-Phase).

Vehicle:

yes

Remarks:

DMSO at 0.02 mL/L.

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

The study was performed under flow-through conditions in 100 L glass aquarium tanks.

Uptake-Phase :
Test item was fed from a stock solution at a rate of 0.04 mL/min and mixed with 2000 mL/min of test water. 2880 L/day, representing approx. 30-exchanges per day was supplied.

Depuration-Phase :
No test-item is fed in to the test system during the Depuration-Phase. 2000 mL/min of dilution water, with 2880 L/day of test water fed through the flow-through system.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Cyprinus carpio - Common Carp :
- Lot No. : TFC-130903
- Length : 8.2 - 11.0 cm
- Weight : 7.30 - 16.93 g
- Age : > 6-months and < 12-months old

Fish were acclimatised at the test facility for a period f 52 days in the same test water (from an on-site bore-hole) as employed for the definitive test at a temperature of 25 +/- 2°C.

Mortality during the acclimatisation period was < 5%.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason:

Remarks:

Aqueous exposure employed since Water Solubility of test item permitted an aqueous-phase exposure to be performed relatively comfortably, while maintaining practicality for the analytics.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

6.6 d

Hardness:

16.0 - 17.7 mg CaCO3/L

Test temperature:

23.9 to 25.0 °C

pH:

Between 7.8 and 8.0

Dissolved oxygen:

Between 7.0 and 8.1 mg O2/L

TOC:

TOC during Uptake-Phase : 7.95 and 9.33 mgC/L
TOC during Depuration-Phase : 0.874 and 1.06 mgC/L

Details on test conditions:

Aeration : Air was provided via an air-stone diffuser during the study;
Light/Dark Cycle : 14 hours light / 10 hours dark (artificial light of white fluorescent lamp);
Feeding : Feed for fry of Carp (protein content > = 43.0%, Lipid Content > = 3.0%. Two feeding sessions per day (AM and PM), each feeding session of about 1.5% body weight of fish.

Nominal and measured concentrations:

Nominal and Measured Concentrtions for the Exposure Levels :
Level 1 : Nominal exposure concentration = 9.44 ppb; Average Measured Concentrations : Peak 1 = 9.30 ppb / Peak 2 = 8.69 ppb
Level 2 : Nominal exposure concentration = 0.944 ppb; Average Measured Concentrations : Peak 1 = 0.908 ppb / Peak 2 = 0.838 ppb.

In Control : Not Detected

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The BCF has been reported based on a number of permutations :

- Based on Steady State (BCFss) or if BCF is < 100 L/kg at Day 28 of the exposure with no upward trend observed, just BCF;
- Normalised to lipid content (5% lipid content) (BCFl or BCFssl)
- Kinetic BCF (BCFk)

Lipid content:

3.64 %

Time point:

start of exposure

Remarks on result:

other: CONTROL - Day 0 of Exposure

Lipid content:

3.71 %

Time point:

end of exposure

Remarks on result:

other: CONTROL - End of Exposure

Lipid content:

3.71 %

Time point:

other: End of Depuration

Remarks on result:

other: CONTROL - End of Depuration Phase

Lipid content:

2.99 %

Time point:

end of exposure

Remarks on result:

other: Level 1 (9.44 ppb nominal exposure) - End of Uptake-Phase

Lipid content:

3.28 %

Time point:

other: End of Depuration

Remarks on result:

other: Level 1 (9.44 ppb nominal exposure) - End of Depuration-Phase

Lipid content:

3.8 %

Time point:

end of exposure

Remarks on result:

other: Level 2 (0.944 ppb nominal exposure) - End of Uptake-Phase

Lipid content:

4.07 %

Time point:

other: End of Depuration

Remarks on result:

other: Level 2 (0.944 ppb nominal exposure) - End of Depuration-Phase

Key result

Conc. / dose:

9.3 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

26 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 1 - based on Day 28 values. Tissue concentrations had attained a plateau by Day 9 of sampling

Key result

Conc. / dose:

8.69 µg/L

Temp.:

25 °C

Type:

BCF

Value:

34 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 2 - based on Day 28 values. Tissue concentrations had attained a plateau by Day 9 of sampling

Key result

Conc. / dose:

0.908 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

33 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 1 - based on Day 28 values. Tissue concentrations had attained a plateau by Day 9 of sampling

Key result

Conc. / dose:

0.838 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

43 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 2 - based on Day 28 values. Tissue concentrations had attained a plateau by Day 9 of sampling

Key result

Conc. / dose:

9.3 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

41 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 1 - Level 1 normalised to 5% lipid content, based on Day 28

Key result

Conc. / dose:

8.69 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

54 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 2 - Level 1 normalised to 5% lipid content, based on Day 28

Key result

Conc. / dose:

0.908 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

42 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 1 - Level 2 normalised to 5% lipid content, based on Day 28

Key result

Conc. / dose:

0.838 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

55 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

other: Peak 2 - Level 2 normalised to 5% lipid content, based on Day 28

Key result

Conc. / dose:

9.3 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

38 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic

Remarks:

Peak 1 - Level 1

Key result

Conc. / dose:

8.69 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

46 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic

Remarks:

Peak 2 - Level 1

Key result

Conc. / dose:

0.908 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

35 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic

Remarks:

Peak 1 - Level 2

Key result

Conc. / dose:

0.838 µg/L

Temp.:

25 °C

pH:

7.9

Type:

BCF

Value:

42 L/kg

Basis:

whole body w.w.

Time of plateau:

9 d

Calculation basis:

kinetic

Remarks:

Peak 2 - Level 2

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.215 d

Remarks on result:

other: Peak 1 - Level 1

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.166 d

Remarks on result:

other: Peak 2 - Level 1

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

0.965 d

Remarks on result:

other: Peak 1 - Level 2

Elimination:

yes

Parameter:

DT50

Depuration time (DT):

1.96 d

Remarks on result:

other: Peak 2 - Level 2.

Rate constant:

growth rate constant (d-1)

Value:

0.011

Additional analytics were performed on the Edible and Non-Edible fractions of the fish on Day 28 of the exposure in order to determine the BCF in these fractions.

The results are presented in the Final Report. However, the analyses of the edible and non-edible tissue of fish indicated that bioaccumulation in the non-edible fraction was greater than that measured in the edible portion (Non-Edible fraction : 30 - 90 L/kg wet wt., Edible fraction : 17 - 55 L/kg wet wt..

The results of this study demonstrate that Javanol presents only low- to very-low potential to bioaccumulate in fish.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions applied Javanol (Peak 1 and Peak 2) were observed to have low- to very-low potential to bioaccumulate in Common Carp with BCF values for Peak 1 at the 9.44 ppb (Level 1) and 0.944 ppb (Level 2) exposure levels on Day 28 of 26 and 33 L/kg wet wt., respectively, and, 34 and 43 L/kg wet wt. for Peak 2 at the same respective exposure levels.

Rapid depuration was observed, with an elimination half-life of 0.17 and 1.96 days. All detects in fish tissue decreased to below LOQ by Day 6.6 of the Depuration-Phase.

Separate analyses of the edible and non-edible tissue of fish indicated that bioaccumulation in the non-edible fraction was greater than that measured in the edible fraction (Non-Edible fraction : 30 - 90 L/kg wet wt.; Edible fraction : 17 - 55 L/kg wet wt.).

Javanol presents low to very-low potential to bioaccumulate in fish.

Executive summary:

BCFs at both exposure concentrations tested for both Peak 1 and Peak 2 of Javanol were well below 100 L/kg wet wt. throughout the entire exposure. Due to the low bioaccumulation potential of Javanol in fish the variation between successive sampling of fish during the Uptake-Phase resulted in deviation > 20% and thus did not attain the necessary stability to attain the steady state as per the OECD definition. That said, it appeared that a maximum bioaccumulation had been attained by Day 9 to Day 14 of the exposure. BCFss values were not calculated due to the low bioaccumulation potential and thus the +/- 20 % variation principle could not be attained. All calculated BCF values were well below 100 L/kg wet wt. However, under the Japanese TG "Testing Methods for New Chemical Substances (5. Test Method a))", it can be regarded that steady state has been reached under CSCL.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Rate Kinetics Study : 15 June to 18 September, 2018
Metabolite Screening : 15 July to 19 October, 2011
Report : 14 November, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

In vitro Fish Metabolism study performed under OECD 319B.

Qualifier:

according to guideline

Guideline:

other: OECD 319B - Determination of in vitro clearance using Rainbow Trout liver S9 sub-cellular fraction (RT-S9), OECD Test Guidelines, 2018

Version / remarks:

2018

Deviations:

no

Principles of method if other than guideline:

The approach foillowed the criteria outlined in the OECD 319B (2018) test guideline.

An earlier study was performed to screen for metabolites. These incubations also followed the principles and practices of the OECD 319B despite being performed prior to the publication of the TG.

GLP compliance:

no

Specific details on test material used for the study:

JAVANOL :
Batch No. : VE00444269;
Purity : 93.4% (sum of the two peaks)
Expiry Date : 13 December, 2018

Radiolabelling:

no

Details on sampling:

Substrate depletion assay of Javanol in trout liver S9 Fractions :

A stock solution of Javanol (10 mM) was prepared freshly in methanol and diluted in 0.1 M potassium phosphate buffer, pH 7.8 resulting in 10 µM solutions. Stock solutions of cofactors were prepared freshly in 0.1 M potassium phosphate buffer, pH 7.8. Alamethicin was dissolved in methanol (10 mg/mL; aliquots stored at -80°C) and freshly diluted in buffer (250 µg/mL).
Rainbow Trout liver S9 fractions (RT-S9) were thawed on ice. All incubations were performed in potassium phosphate buffer at pH 7.8 (0.1 M) in Hirschmann glass tubes in triplicate incubated at 12°C in a Thermomixer block with shaking capabilities (Ditabis Model MKR 23, 400 rpm). Active RT-S9 or heat inactivated S9 protein as control (1 mg/mL) was preincubated on ice in buffer with alamethicin (final concentration: 25 µg/mL). Alamethicin is a pore-forming peptide antibiotic which permeabilises microsomal membranes and activates glucuronidation by allowing free transfer of UDPGA and glucuronide product across the membrane. After addition of cofactors for Phase I (NADPH, Nicotinamide adenine dinucleotide 2′-phosphate reduced) and Phase II enzymes (UDPGA, Uridine 5′-diphosphoglucuronic acid; PAPS, Adenosine 3′-phosphate 5′-phosphosulfate; GSH, reduced L-glutathione), the reaction was initiated by addition of the test substance. The detailed methodology is described by Johanning et al [2012] and in OECD TG 319B. A final concentration of 0.5 mM GSH instead of 5 mM GSH was used. The final solvent concentration in the assay was 0.35% methanol.

In the first experiment, Javanol (1 µM) was incubated in the presence of 1 mg/mL active S9 protein and cofactors in triplicate for up to 45 minutes. As controls, the test chemical (1 µM) was incubated in presence of heat inactivated S9 protein (1 mg/mL) and cofactors for 0, 25 and 45 minutes or with active S9 protein in absence of any cofactors added for 45 minutes. Reactions were stopped at 0, 5, 15, 25, 35 and 45 minutes incubation by addition of acetonitrile (200 µL) containing methyl laurate (1 µM) as internal standard to the Hirschmann tubes. Samples were extracted with MTBE (200 µL) in the same tubes by vortexing for 3 minutes, centrifuged to allow a better phase separation and separation of protein (Heraeus Fresco 17 centrifuge, 13800g, 5 min, 4°C) and subjected to GC-MS analysis.

In the second experiment, Javanol (1 µM) was incubated in the presence of 1 mg/mL active S9 protein and cofactors in triplicate for up to 20, as described above. Reactions were stopped at time 0, 3, 6, 9, 12, 15 and 20 minutes. As control, the test chemical (1 µM) was incubated in the presence of heat inactivated S9 protein (1 mg/mL) and cofactors for 0, 9, 15 and 20 minutes. Furthermore, incubations in the presence of active S9 protein and in absence of any cofactors added were carried out for 15 and 20 minutes, respectively. Reactions were stopped and samples extracted.

Vehicle:

yes

Remarks:

A stock solution of Javanol (10 mM) was prepared freshly in methanol and diluted in 0.1 M potassium phosphate buffer, pH 7.8 resulting in 10 µM solutions.

Test organisms (species):

other: Rainbow Trout liver S9 fractions

Details on test organisms:

Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were prepared from five sexually immature fish (2 female, 3 male) at the Veterinary Institute of the University Bern, Switzerland and stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions (batch IV) was 315 g. The enzymatic activity of the S9 fractions was characterized using model substrates for CYP1A (Cytochrome P450 monooxygenase; EROD), glutathione transferase (GST), and uridine 5’-diphospho-glucuronosyltransferase (UGT) activities. Furthermore, the enzymatic activity of newly received S9 fractions was typically compared in house using testosterone, 7-hydroxycoumarin, pyrene and the fragrance chemical Cyclohexyl Salicylate as test chemicals in substrate depletion assays. S9 fractions were only used for metabolism studies if significant enzymatic conversion of the reference substance was observed. Aliquots of S9 fractions were prepared to prevent several thawing and freezing cycles to avoid inactivation of enzymes. Heat inactivated S9 fractions were prepared by heating at 100°C for 10 minutes and stored at -80 °C.

Route of exposure:

other: Rainbow Trout S9 liver fractions exposed to a solution of JAVANOL in 0.1M Potassium Phosphate Buffer (pH 7.8).

Justification for method:

other: In vitro intrinsic clearance and metabolism study - in accordance with OECD 319B

Test type:

static

Water / sediment media type:

other: Rainbow Trout S9 liver fractions exposed to a solution of JAVANOL in 0.1M Potassium Phosphate Buffer (pH 7.8).

Total exposure / uptake duration:

20 min

pH:

Rainbow Trout S9 liver fractions exposed to a solution of JAVANOL in 0.1M Potassium Phosphate Buffer : pH 7.8

Conc. / dose:

222.37 µg/L

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

307 L/kg

Basis:

other: BCF for JAVANOL Isomer 1 based on a CLint of 9.09 mL/h/mg protein and assuming that hepatic clearance is calculated taking in to account a theoretical postulated difference between in vitro and in vivo binding (fu calc)

Calculation basis:

other: BCF for JAVANOL Isomer 1 based on a CLint of 9.09 mL/h/mg protein and assuming that hepatic clearance is calculated taking in to account a theoretical postulated difference between in vitro and in vivo binding (fu calc).

Conc. / dose:

222.37 µg/L

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

502

Basis:

other: BCF for JAVANOL Isomer 2 based on a CLint of 3.35 mL/h/mg protein and assuming that hepatic clearance is calculated taking in to account a theoretical postulated difference between in vitro and in vivo binding (fu calc)

Calculation basis:

other: BCF for JAVANOL Isomer 2 based on a CLint of 3.35 mL/h/mg protein and assuming that hepatic clearance is calculated taking in to account a theoretical postulated difference between in vitro and in vivo binding (fu calc).

Conc. / dose:

222.37 µg/L

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

160 L/kg

Basis:

other: BCF for JAVANOL Isomer 1 based on a CLint of 9.09 mL/h/mg protein and assuming that hepatic clearance is calculated assuming equal in vitro and in vivo binding (fu = 1.0)

Calculation basis:

other: BCF for JAVANOL Isomer 1 based on a CLint of 9.09 mL/h/mg protein and assuming that hepatic clearance is calculated assuming equal in vitro and in vivo binding (fu = 1.0).

Conc. / dose:

222.37 µg/L

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

168 L/kg

Basis:

other: BCF for JAVANOL Isomer 2 based on a CLint of 3.35 mL/h/mg protein and assuming that hepatic clearance is calculated assuming equal in vitro and in vivo binding (fu = 1.0).

Calculation basis:

other: BCF for JAVANOL Isomer 2 based on a CLint of 3.35 mL/h/mg protein and assuming that hepatic clearance is calculated assuming equal in vitro and in vivo binding (fu = 1.0).

Metabolites:

Identification of Metabolites of Javanol in RT-S9 :

The glucuronic acid conjugate of Javanol was identified as Phase II metabolite in RT-S9 incubations with UDPGA as single cofactor in presence of alamethicin, whereas no glucuronide conjugate was detected with heat-inactivated S9 and only traces of the glucuronide with active RT-S9 and no cofactors added. The LC-MS/MS spectrum indicates the identity of a glucuronide conjugate (ESI-negative mode; m/z 397.2232 [M-H-]).

Results with reference substance (positive control):

Confirmation of Validity of S9 Liver Fractions Using Reference Chemicals :

Enzymatic activity of the trout liver S9 batch used in this study was characterised using model substrates for CYP1A (EROD assay), uridine 5’-diphospho-glucuronosyltransferase (UGT) and glutathione transferase (GST). EROD, UGT and GST activities of the RT-S9 batch IV were similar to other RT-S9 batches characterized in house.

Furthermore, substrate depletion assays were carried out with testosterone (CYP3A), 7-hydroxycoumarin (UGT and SULT), pyrene and Cyclohexyl Salicylate. The in vitro intrinsic clearance rates of these reference chemicals were similar compared to various RT-S9 batches from the same or other suppliers. The in vitro intrinsic clearance rates of pyrene and Cyclohexyl Salicylate were also comparable to the RT-S9 batches used for the international ring trial.

Details on results:

In Vitro Intrinsic Clearance of Javanol in RT-S9 :

A very rapid decrease of Javanol (86.4% within 15 minutes) was observed in the 1st experiment with active RT-S9 and cofactors for isomer 1. Concentrations of isomer 1 with active RT-S9 and cofactors were below detection level at 25, 35 and 45 min incubation. A slower, but still rapid decrease was observed for isomer 2 (45.2% within 15 minutes; 87.9% in 35 minutes). After 45 minutes incubation, the concentration of isomer 2 was below the detection level. Negligible decrease of the two Javanol isomers was observed with active RT-S9 in the absence of any cofactors added (10.7-11.8% decrease in 120 minutes) and in the control samples with heat inactivated protein (10.9%-15.0% decrease in 120 minutes).

Since concentrations of isomer 1 were below detection level for the time points >15 minutes, a preliminary rate based on only 3 sampling points (0, 5, 15 minutes) was calculated (CLIN VITRO, INT = ~7.70 mL/h/mg protein. In vitro intrinsic clearance was calculated for isomer 2 based on the concentrations measured at 0-35 minutes (5 time points): 3.66 mL/h/mg protein.

Due to the very rapid decrease in the first experiment, a second experiment with shorter incubation time (0 - 20 minutes) was carried out. Enzymatic turnover of Javanol in presence of cofactors was very rapid for isomer 1 and rapid for isomer 2. 92.5% and 67.4% decrease was observed for Javanol after 20 minutes for isomer 1 and isomer 2, respectively, in the second experiment. Negligible decrease was observed for both isomers with heat-inactivated S9 (2.4% - 4.2% decrease in 20 minutes) and with active S9 in absence of added cofactors (7.4% - 8.8% decrease in 20 minutes).

The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transformed measured concentrations of the parent compound as a function of time for the two isomers of Javanol in the second experiment: 9.09 mL/h/mg protein for isomer 1 and 3.35 mL/h/mg protein for isomer 2. These final CLint, in vitro were similar compared to the ones determined in the first experiment.

Impact of Individual Cofactors on the Biotransformation of Javanol in RT-S9 :

Cofactor dependence of Javanol biotransformation was investigated to evaluate which metabolic pathways may be involved. No decrease of the two isomers was observed with active RT-S9 in the absence of added cofactors, whereas a minor decrease (12.5% for isomer 1 and 18.9% for isomer 2) was observed in the absence of UDPGA as cofactor indicating the involvement of an UDP-glucuronosyltransferase (UGT). Decrease of Javanol was not dependent on the addition of NADPH, or PAPS.

Prediction of BCFs for Javanol :

The biotransformation rates of the two isomers of Javanol in fish liver S9 fractions were similar in the 1st experiment and in the 2nd experiments. The reaction rates determined for the two isomers in the 2nd main experiment were used for the BCF prediction: isomer 1: 9.09/h and isomer 2: 3.35/h.

BCFs were predicted for the two isomers of Javanol using the measured log Kow for Javanol of 4.5 (OECD TG 117; Givaudan Study No. 18-E140). The partitioning based BCFs assuming no metabolism for Javanol based on the measured log Kow value (log Kow = 4.5) was calculated as part of the in vitro-in vivo extrapolation model (version: “S9spreadsheet_Public_062713.xlsx”) by setting the reaction rate to zero: BCF= 1526 L/kg. Including the in vitro biotransformation rates of the two isomers of Javanol in trout S9 fractions and other parameters, the predicted BCFs were 307 L/kg for isomer 1 and 502 L/kg for isomer 2 assuming different binding to serum in vivo vs. in vitro (fU calc). Predicted BCFs setting fU = 1.0 were 160 L/kg and 168 L/kg.

These predicted BCFs based on in vitro biotransformation rates are significantly lower compared to the BCF value calculated with an assumed turnover rate of 0. Especially for chemicals with higher log Kow values, a better correlation of predicted BCFs calculated with the in vitro-in vivo extrapolation model using an assumed fU of 1.0 and measured in vivo BCFs was shown in several studies. The reason for this apparent discrepancy is not known. One possible explanation may be that the assumption of the extrapolation model that only freely dissolved chemicals are available for metabolic turnover is incorrect. Chemicals bound to proteins may desorb rapidly and thus contribute to the metabolic turnover of the chemicals. Presently, it seems to be reasonable according to OECD GD 280 to use the two different binding assumptions to estimate upper and lower limits on hepatic clearance. However, due to the rapid in vitro intrinsic clearance of the two Javanol isomers, the BCFs predicted with these two competing assumptions result in values which are all well below the B criterion.

In an in vivo Fish BCF study with common carp (Cyprinus carpio), equivalent to an OECD 305 set-up, Javanol was demonstrated to have a low potential to bioconcentrate with a whole body kinetic, lipid normalised and growth corrected BCF (BCFkGL) of 45 (low concentration) and 61 L/kg wet wt. (high concentration) for Isomer 1 and 55 (low concentration) and 75 L/kg wet wt. (high concentration) for Isomer 2.

Validity criteria fulfilled:

yes

Conclusions:

Rapid enzymatic turnover of both isomers of Javanol by trout liver S9 fractions was observed. Isomer 1 was even more rapidly transformed than isomer 1. Furthermore, a glucuronic acid conjugate was detected as Phase II metabolite in RT-S9. Usually, Phase II metabolites like conjugates with glucuronic acid or other conjugates are readily excreted.

Substrate depletion assays using RT-S9 is considered to be a reliable and adequate assay to assess biotransformation of chemicals in fish. Predicted BCFs based on in vitro biotransformation rates in RT-S9 showed a better agreement to in vivo measured BCFs for fragrance chemicals compared to the in silico predictions based on solely log Kow.

Predicted BCFs of the two isomers of Javanol based on in vitro biotransformation rates were 307 L/kg (isomer 1) and 502 L/kg (isomer 2) using the measured log Kow value (4.5) and a theoretically calculated fU. The predicted BCFs using an assumed fU of 1.0 were 160 L/kg and 168 L/kg, respectively. All predicted BCFs were below the B criterion indicating that the bioaccumulation potential of Javanol in vivo is likely to be low.

In an in vivo Fish BCF study with common carp, equivalent to an OECD 305 set-up, the lipid normalised (5%), and growth corrected kinetic BCF (BCFkGL) observed during the 28-day exposure with Javanol wasranging from 45 to 61 L/kg (wet wt.) for Isomer 1 and 55 to 75 L/kg (wet wt.) for Isomer 2.

The predicted BCFs based on the RT-S9 biotransformation rates using a theoretically calculated fU were 5.7- and 7.8-fold higher than the mean in vivo BCFs for isomer 1 and isomer 2, respectively, whereas predicted BCFs using an assumed fU of 1.0 were 3.0- and 2.6-fold higher. A similar trend of overprediction of BCFs based on in vitro biotransformation and IVIVE especially for fU calculated was also observed previously in several studies.

Description of key information

In vivo Study :

Under the test conditions applied Javanol (Peak 1 and Peak 2) were observed to have low- to very-low potential to bioaccumulate in Common Carp with BCF values for Peak 1 at the 9.44 ppb (Level 1) and 0.944 ppb (Level 2) exposure levels on Day 28 of 26 and 33 L/kg wet wt., respectively, and, 34 and 43 L/kg wet wt. for Peak 2 at the same respective exposure levels.

Rapid depuration was observed, with an elimination half-life of 0.17 and 1.96 days. All detects in fish tissue decreased to below LOQ by Day 6.6 of the Depuration-Phase.

Separate analyses of the edible and non-edible tissue of fish indicated that bioaccumulation in the non-edible fraction was greater than that measured in the edible fraction (Non-Edible fraction : 30 - 90 L/kg wet wt.; Edible fraction : 17 - 55 L/kg wet wt.).

In vitro Study :

Rapid enzymatic turnover of both isomers of Javanol by trout liver S9 fractions was observed. Isomer 1 was even more rapidly transformed than isomer 1. Furthermore, a glucuronic acid conjugate was detected as Phase II metabolite in RT-S9. Usually, Phase II metabolites like conjugates with glucuronic acid or other conjugates are readily excreted.

Substrate depletion assays using RT-S9 is considered to be a reliable and adequate assay to assess biotransformation of chemicals in fish. Predicted BCFs based on in vitro biotransformation rates in RT-S9 showed a better agreement to in vivo measured BCFs for fragrance chemicals compared to the in silico predictions based on solely log Kow.

Predicted BCFs of the two isomers of Javanol based on in vitro biotransformation rates were 307 L/kg (isomer 1) and 502 L/kg (isomer 2) using the measured log Kow value (4.5) and a theoretically calculated fU. The predicted BCFs using an assumed fU of 1.0 were 160 L/kg and 168 L/kg, respectively. All predicted BCFs were below the B criterion indicating that the bioaccumulation potential of Javanol in vivo is likely to be low.

In an in vivo Fish BCF study with common carp, equivalent to an OECD 305 set-up, the lipid normalised (5%), and growth corrected kinetic BCF (BCFkGL) observed during the 28-day exposure with Javanol wasranging from 45 to 61 L/kg (wet wt.) for Isomer 1 and 55 to 75 L/kg (wet wt.) for Isomer 2.

The predicted BCFs based on the RT-S9 biotransformation rates using a theoretically calculated fU were 5.7- and 7.8-fold higher than the mean in vivo BCFs for isomer 1 and isomer 2, respectively, whereas predicted BCFs using an assumed fU of 1.0 were 3.0- and 2.6-fold higher. A similar trend of overprediction of BCFs based on in vitro biotransformation and IVIVE especially for fU calculated was also observed previously in several studies.

Key value for chemical safety assessment

BCF (aquatic species):

43 L/kg ww

Additional information

The highest BCF determined in Carp during a 28 day In vivo expsoure was 43 L/kg (wet wt) on a whole fish basis.

JAVANOL has a very low potential to bioconcentrate in fish.