Registration Dossier

Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 May 2017 to 16 June 2017
Reliability:
other: Currently non-guideline study. However, methodology and draft guideline has been submitted to OECD test guideline committee for review and evaluation
Justification for type of information:
Bioaccumulation refers to an increase of chemical concentration in an organism through all environmental sources like water, food and sediment. Thus, bioaccumulation can be considered as the net result of absorption, distribution, metabolism and excretion (ADME). The bioaccumulation potential of chemicals is scrutinised on a global basis by regulatory agencies in their risk assessment of chemicals. The Bioconcentration factor (BCF) is routinely used to evaluate the bioaccumulation potential of chemicals (i.e. the ratio of concentration of a substance in an organism like fish to the concentration of the water in a steady state). BCF values of chemicals are usually predicted with computation models that are based mainly on the hydrophobicity of the molecule which is either estimated or measured as n-octanol-water partition coefficient (Kow) of the chemical. The usefulness of the computer models is limited for the estimation of BCFs due to the broad variety of chemical classes and structures. Xenobiotic metabolism in fish is not well understood. Even models which consider e.g. metabolism using a screening-level quantitative structure-activity relationship (QSAR) model for estimation of biotransformation rate constants (kMet) can produce inaccurate estimates of bioaccumulation potential. Definitive determination of the BCF value involves the valid implementation of an OECD 305 fish bioconcentration test.

After absorption or ingestion of a substance by the fish, the chemical may be distributed to various tissues where it may be metabolized by enzymes. Metabolism is considered to be the dominant mechanism of elimination of hydrophobic substances which can significantly reduce their bioaccumulation potential. The first phase of biotransformation (Phase I) is usually the introduction of a polar group e.g. catalysed by Cytochrome P450 monooxygenases, which increases water solubility and renders it a suitable substrate for Phase II reactions. In Phase II reactions, xenobiotics are conjugated to endogenous substrates such as carbohydrates, amino acids, glutathione, or inorganic sulfate. The general trend of these metabolic transformation processes is the enzymatic conversion of lipophilic compounds to more polar hydrophilic metabolites which are usually less toxic and are normally readily excreted. The primary site of xenobiotic metabolism is typically the liver in most fish species and in mammalian systems. Therefore, determination of the in vitro metabolism of chemicals using liver cellular/subcellular fractions can provide an indication of their bioaccumulation potential. Furthermore, in vitro metabolism data can be used as an indication of the in vivo hepatic intrinsic clearance and may be utilized in in vitro to in vivo BCF extrapolation models.

The most commonly used in vitro methods to assess metabolism involve either liver S9 fractions or primary hepatocytes. A pre-validation study with trout liver S9 fractions has been done by a consortium under the coordination of HESI/ILSI (Health and Environmental Sciences Institute) and the protocol was published. The intra- and interlaboratory reliability of a cryopreserved trout hepatocyte assay was compared by three laboratories using six chemicals. A ring trial to compare intra- and interlaboratory reliability of trout liver S9 fractions and hepatocytes by several laboratories from academia, governmental institutions and industry which was led by HESI was recently performed to provide information for OECD guidelines (OECD Project 3.13). The study showed good intra- and interlaboratory reliability and resulted into the preparation of two draft OECD guidelines on the in vitro substrate depletion assays using trout liver hepatocytes or S9 fractions and a guidance document.

In order to determine hepatic metabolism of GR-50-3010, fish liver S9 fractions (Rainbow trout, Oncorhynchus mykiss) have been chosen as a model system. Fish liver S9 fractions contain both Phase I and Phase II enzyme systems. The primary objective of this study was to determine the in vitro intrinsic clearance (CLINT, IN VITRO) of GR-50-3010 in trout liver S9 fractions. Furthermore, in vitro metabolic rates were incorporated into an in vitro – in vivo extrapolation model to predict the BCF for a standardised fish (one that weighs 10g, has a 5% lipid content, and is living at 15 °C).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Principles of method if other than guideline:
The most commonly used in vitro methods to assess metabolism involve either liver S9 fractions or primary hepatocytes. A pre-validation study with trout liver S9 fractions has been done by a consortium under the coordination of HESI/ILSI (Health and Environmental Sciences Institute) and the protocol was published. The intra- and interlaboratory reliability of a cryopreserved trout hepatocyte assay was compared by three laboratories using six chemicals. A ring trial to compare intra- and interlaboratory reliability of trout liver S9 fractions and hepatocytes by several laboratories from academia, governmental institutions and industry which was led by HESI was recently performed to provide information for OECD guidelines (OECD Project 3.13). The study showed good intra- and interlaboratory reliability and resulted into the preparation of two draft OECD guidelines on the in vitro substrate depletion assays using trout liver hepatocytes or S9 fractions and a guidance document.

In order to determine hepatic metabolism of GR-50-3010, fish liver S9 fractions (Rainbow trout, Oncorhynchus mykiss) have been chosen as a model system. Fish liver S9 fractions contain both Phase I and Phase II enzyme systems. The primary objective of this study was to determine the in vitro intrinsic clearance (CLINT, IN VITRO) of GR-50-3010 in trout liver S9 fractions. Furthermore, in vitro metabolic rates were incorporated into an in vitro – in vivo extrapolation model to predict the BCF for a standardised fish (one that weighs 10g, has a 5% lipid content, and is living at 15 °C).

GLP compliance:
no

Test material

Specific details on test material used for the study:
Batch No. : VE00466805
Purity : 93.6% (sum of 2 diastereoisomers)
Expiry : 23 January 2019
Radiolabelling:
no

Sampling and analysis

Details on sampling:
Initially, a range finding experiment was performed to determine the optimal incubation times to be used in the main experiments.
A stock solution of GR-50-3010 (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 were thawed on ice. All incubations were performed in potassium phosphate buffer at pH 7.8 (0.1 M) in Hirschmann glass tubes in duplicate or triplicate incubated at 12°C in a Thermomixer block with shaking capabilities (Ditabis Model MKR 23, 400 rpm). Active S9 fractions protein or heat inactivated protein as control (1 mg/ml) was preincubated on ice 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. 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 range finding experiment, GR-50-3010 (1 µM) was incubated in presence of 1 mg/mL active S9 protein and cofactors in duplicate for up to 120 minutes. As controls, the test chemical
(1 µM) was incubated in presence of heat inactivated S9 protein (1 mg/mL) and cofactors or with active S9 protein in absence of any cofactors added. Reactions were stopped at 0, 30, 60, 90 and 120 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 30 seconds, centrifuged to allow a better phase separation and separation of protein (Heraeus Fresco 17 Centrifuge, 12 000 rpm, 5 min, 4°C) and subjected to GC-MS analysis.
In the main experiment, GR-50-3010 (1 µM) was incubated in presence of 1 mg/mL active S9 protein and cofactors in triplicate for up to 15 minutes as described above. Reactions were stopped at time 0, 3, 6, 9, 12 and 15 minutes. As control, the test chemical (1 µM) was incubated in presence of heat inactivated S9 protein (1 mg/mL) and cofactors for 0, 9 and 15 minutes. Furthermore, incubations in the presence of active S9 protein and in absence of any cofactors added were carried out for 15 minutes. Reactions were stopped and extracted as describe above.

Test solutions

Vehicle:
yes
Remarks:
Stock solutions of GR-50-3010 prepared in Methanol.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were prepared from five fish (mixed gender) 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 to determine the activity of CYP1A (Cytochrome P450 monooxygenase; EROD), and glutathione transferase (GST). The enzymatic activity of newly received S9 fractions was typically compared in house using Testosterone, 7-Hyroxycoumarin, and Pyrene as test chemicals. In addition, Cyclohexyl Salicylate as internal fragrance reference molecule which is biotransformed by different enzyme systems (Phase I and Phase II) was used. 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 100 µl aliquots at 100°C using a Biometra Thermocycler and stored at -80°C.

Study design

Route of exposure:
aqueous
Remarks:
Aqueous medium (0.1 M Potassium Phosphate Buffer) and R. Trout S9 liver cells
Test type:
static
Total exposure / uptake duration:
15 min

Test conditions

Hardness:
Not applicable
Test temperature:
12 °C (+/- 1 °C)
pH:
pH 7.8
Dissolved oxygen:
Not applicable
TOC:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Time 0 test concentrations in the Range-Finder incubates were between 98.3 and 101.7% and 96.4% to 111.4% in the Time 0 incubates for the definitive study.
Reference substance (positive control):
yes
Remarks:
7-Hydroxycoumarine and Cyclohexyl Salicylate were used as Reference Substances.
Details on estimation of bioconcentration:
An in vintro to in vivo extrapolation (IVIVE) model is employed to calculate the BCF. This model uses the intrinsic clearance rate value determined from the metabolism kinetics determined in the study. Deatils on the IVIVE model can be found at :
Nichols, J.W., et al., Towards improved models for predicting bioconcentration of well-metabolized compounds by rainbow trout using measured rates of in vitro intrinsic clearance. Environmental Toxicology and Chemistry, 2013. 32(7): p. 1611-22.

Results and discussion

Bioaccumulation factoropen allclose all
Conc. / dose:
1 other: microM
Temp.:
12 °C
pH:
7.8
Type:
BCF
Value:
76 L/kg
Basis:
other: Isomer 1
Remarks:
In vitro intrinsic clearance rate. Assumes different binding to serum in vivo vs in vitro (fu calc)
Conc. / dose:
1 other: micorM
Temp.:
12 °C
pH:
7.8
Type:
BCF
Value:
82 L/kg
Basis:
other: Isomer 2
Remarks:
Invitro intrinsic clearance rate. Assumes different binding to serum in vivo vs in vitro (fu calc)
Conc. / dose:
1 other: microM
Temp.:
12 °C
pH:
7.8
Type:
BCF
Value:
52 L/kg
Basis:
other: Isomer 1
Remarks:
Invitro intrinsic clearance rate. Assuming no effect of differential binding to serum between in vivo and in vitro (fu = 1.0)
Conc. / dose:
1 other: MicroM
Temp.:
12 °C
pH:
7.8
Type:
BCF
Value:
53 L/kg
Basis:
other: Isomer 2
Remarks:
Invitro intrinsic clearance rate. Assuming no effect of differential binding to serum between in vivo and in vitro (fu = 1.0)
Rate constantsopen allclose all
Rate constant:
other: In vitro intrinsic clearance rate (CLint, in vitro)
Remarks:
mL/h/mg protein
Value:
5.49
Remarks on result:
other: Isomer 1 (45.3% content)
Remarks:
Represents rapid metabolism rate
Rate constant:
other: In vitro intrinsic clearance rate (CLint, in vitro)
Remarks:
mL/h/mg protein
Value:
3.91
Remarks on result:
other: Isomer 2 (49.4% content)
Remarks:
Represents rapid metabolism rate
Results with reference substance (positive control):
The in vitro intrinsic clearance rates of the reference chemicals were similar compared to previous S9 batches from the same or other suppliers.

Any other information on results incl. tables

A rapid decrease of the two isomers of GR-50-3010 (ca. 90% within 30 minutes) was observed in the range finding experiment with active S9 protein. In the absence of any cofactors added, a very slow decrease of GR-50-3010 was observed (11.6% and 18.6% decrease for isomer 1 and isomer 2, respectively, within 120 minutes). A negligible decrease of GR-50-3010 was observed in the control samples with heat inactivated protein (< =5% decrease within 120 minutes). Preliminary rates were estimated based on the concentrations at t0 and 30 min:~4.5 and ~4.2mL/h/mg protein for isomer 1 and isomer 2, respectively.

Final incubations were carried out up to 15 minutes in the main experiment. Enzymatic turnover of GR-50-3010 was rapid in presence of cofactors. 77.0% and 63.0% decrease was observed for isomer 1 and isomer 2 of GR-50-3010 within 15 minutes. A very slow turnover of the two isomers of GR-50-3010 was found with active S9 protein in the absence of cofactors added (17.6% and 12.4% decrease, respectively, in 15 minutes), whereas negligible decrease was observed with inactive S9 protein for the two isomers (0.2% and 4.2%, respectively, decrease in 15 minutes).

Thein vitrointrinsicclearance(CLint, in vitro)wascalculated from the log-transformed measured concentrations of parent compound as a function of time for the two isomers of GR-50-3010: 5.49 and 3.91mL/h/mg protein. These final rates were similar to the preliminary rates estimated in the range-finding experiment.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transform measured concentrations of the parent compound as a function of time: 5.49 and 3.91 mL/h/mg protein for isomer 1 and isomer 2, respectively. They were used as inputs into an in vitro - in vivo extrapolation model to predict the BCF using the measured log Kow value of 3.4. The predicted BCF (BCFTOT) was 52 L/kg wet weight and 53 for isomer 1 and isomer 2 of GR-50-3010, respectively, using an assumed fU = 1.0, i.e. no effect of differential binding to serum. The predicted BCFs were 76 L/kg wet weight and 82 L/kg wet weight for isomer 1 and isomer 2, respectively, assuming different binding to serum in vivo vs. in vitro (fU calc).

Executive summary:

Rapid and similar enzymatic biotransformation by trout liver S9 fractions was observed for both isomers of GR-50-3010.

Trout liver in vitro S9 metabolism is considered to be an adequate assay to assess enzymatic degradation and thus may be utilised as an important tool to determine the bioaccumulation potential of GR-50-3010.

The in vitroin vivo extrapolation model to predict the BCFs based on in vitro turnover rates of nine fragrance molecules by trout liver S9 fractions results in values which are comparable to known in vivo BCF values especially if no effect of different binding to serum is assumed.

The predicted BCFs for the two isomers of GR-50-3010 incorporating in vitro metabolism indicate that the potential for in vivo bioaccumulation is likely to be low.