Benzenepropanal, 2-methyl-4-(2-methylpropyl)-

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 April 2015 to 10 July 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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 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 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 biotransformed 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 (CYPs), 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 metabolic stability 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 - in vivo (IVIVE) 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 and the protocol was published. Recently, 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 is led by HESI is currently performed to provide information for an OECD guideline (OECD Project 3.13).

In order to determine hepatic metabolism of GR-88-0778, 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 metabolic stability of GR-88-0778 in fish liver S9 fractions. Furthermore, the reaction rate was calculated from the in vitro data and incorporated into a new in vitro – in vivo extrapolation model to generate a refined BCF estimate for a standardised fish (one that weighs 10g, has a 5% lipid content, and is living at 15°C). The model was developed by J. Nichols et al and a revised model was published recently (version: “S9spreadsheet_Public_032713.xlsx”). In addition, metabolites of GR-88-0778 in trout liver S9 fracitons were identified by GC-MS and LC-MS/MS.

Guideline:

other:

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 and the protocol was published. Recently, 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 is led by HESI is currently performed to provide information for an OECD guideline (OECD Project 3.13).

In order to determine hepatic metabolism of GR-88-0778, 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 metabolic stability of GR-88-0778 in fish liver S9 fractions. Furthermore, the reaction rate was calculated from the in vitro data and incorporated into a new in vitro – in vivo extrapolation model to generate a refined BCF estimate for a standardised fish (one that weighs 10g, has a 5% lipid content, and is living at 15°C). The model was developed by J. Nichols et al and a revised model was published recently (version: “S9spreadsheet_Public_032713.xlsx”). In addition, metabolites of GR-88-0778 in trout liver S9 fracitons were identified by GC-MS and LC-MS/MS.

GLP compliance:

no

Specific details on test material used for the study:

Batch Number : 6
Purity : 98.6%
Expiry : 29 March, 2017

Radiolabelling:

no

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-88-0778 (10 mM) was prepared freshly in methanol and diluted in water 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 (5 mg/ml; aliquots stored at -80°C) and 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 shaker (Ditabis, model: MKR23) at 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. Final concentrations of cofactors, protein and test substance are listed in the table below. The detailed methodology is described by Johanning et al.
In the range finding experiment, GR-88-0778 (1 µM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in duplicate for up to 60 minutes. As controls, the test substance (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. Reactions were stopped at 0, 5, 15, 30, and 60 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 (Eppendorf centrifuge, 12 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis.
Since decrease of GR-88-0778 (1 µM) was very rapid in the range finding experiment, a higher concentration of the test chemical was used in the main experiments. In the two independent main experiments (1st and 2nd main experiment), GR-88-0778 (10 µM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 20 minutes as described above. Reactions were stopped at time 0, 5, 7.5, 10, 15 and 20 minutes (1st main experiment) and at time 0, 1, 2, 4, 5 and 6 minutes (2nd main experiment). As control, the test substance (10 µM) was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors for 0, 10 and 20 minutes (1st main experiment) and for 0, 3, and 6 minutes in the 2nd main experiment. Furthermore, incubations in the presence of active S9 protein and in absence of any cofactors added were carried out for 20 and 6 minutes, respectively. Reactions were stopped and extracted as described above.

Vehicle:

yes

Remarks:

Stock solutions prepared in Methanol.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were prepared 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 was 452 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 Cyclohexyl Salicylate as internal fragrance reference molecule which is biotransformed by different enzyme systems (Phase I and Phase II). S9 fractions were only used for metabolism studies if significant enzymatic conversion of the reference substance was observed. Aliquots of S9 fractions were already prepared during the preparation 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.

Route of exposure:

aqueous

Remarks:

Aqueous medium (0.1M Potassium Phosphate Buffer) and R- Trout S9 liver cells

Test type:

static

Total exposure / uptake duration:

6 min

Hardness:

Not applicable

Test temperature:

12 °C (+/- 1 °C)

pH:

7.8

Dissolved oxygen:

Not applicable

TOC:

Not applicable

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Time 0 analysis in the Range-Finder study gave a recovery of 100% (99.3 % and 100.7%) and 100 % (101.2% and 98.8%) for the active S9 and inactivated S9 samples.
The average recoveries for Time 0 samples was 100% for the activated S9 and inactived S9 incubates in both the 1st main and 2nd main experiment.

Reference substance (positive control):

yes

Remarks:

Cyclohexyl Salicylate - Enzymatic turnover of Cyclohexyl Salicylate was determined in order to confirm the validity of the S9 liver fractions.

Details on estimation of bioconcentration:

An in vitro - to - in vivo extrapolation (IVIVE) model is employed to calculate the BCF. The model uses the intrinsic clearance rate value determined from the metabolism kinetics determined in the study.
Details on the application of 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. Environ. Toxicol. Chem., 2013. 32(7): p. 1611-22.

Conc. / dose:

10 other: MicroM

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

66.4 L/kg

Basis:

other:

Remarks:

In vitro intrinsic clearance rate. Asumes different binding to serum in vivo vs in vitro (fu calc)

Remarks on result:

other:

Remarks:

In vitro Intrinsic clearance rate (CLint, in vitro) = 25.81 mL/h/mg protein

Conc. / dose:

10 other: MicroM

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

59.4 L/kg

Basis:

other:

Remarks:

In vitro intrinsic clearance rate. Asumes no effect of differential binding to serum between in vivo and in vitro (fu = 1.0). binding

Remarks on result:

other:

Remarks:

In vitro Intrinsic clearance rate (CLint, in vitro) = 25.81 mL/h/mg protein

Results with reference substance (positive control):

The in vitro intrinsic clearance rate of Cyclohexyl Salicylate (18.74 ml/h/mg protein) was similar or even higher compared to previous S9 batches from other suppliers.

Details on results:

A very rapid decrease within 60 minutes of GR-88-0778 (1 µM) was observed in the range finding experiment with active S9 protein (no parent detectable after 60 min). After 5 min incubation, there was already a 85.3% decrease observed. In the absence of any cofactors added, a slightly slower decrease of GR-88-0778 was observed (75.3% decrease within 60 minutes). A slow decrease of GR-88-0778 was observed in the control samples with heat inactivated protein (15.7% decrease within 60 minutes).

Thus, incubations were carried out up to 20 minutes using a higher test chemical concentration in the first main experiment. Due to the very rapid enzymatic turnover (74.7% decrease in 5 min), the experiment was repeated using a shorter incubation time (up to 6 min; 2nd main experiment). Enzymatic turnover of GR-88-0778 (10 µM) was very rapid in presence of cofactors. 93.1% decrease was observed for GR-88-0778 in 6 minutes in the 2nd main experiments. A much slower turnover of GR-88-0778 was found with active S9 protein in the absence of cofactors added (16.6% decrease in 6 minutes). No decrease of GR-88-0778 was observed with inactive S9 protein.

A high level of replication and reproducibility between individual test replicates and experiments was observed.

We identified an alcohol as metabolite of GR-88-0778 in trout liver S9 fractions which were incubated for 60 min in presence of all cofactors or with NADPH as cofactor added by GC-MS (metabolite A). The identity of the alcohol of GR-88-0778 was confirmed by synthesized reference material (GR-88-1108-2) which had a similar retention time and m/z spectrum. As major metabolite, the “acid” of GR-88-0778 was detected as methyl ester after derivatisation with diazomethane (metabolite B) in incubations with active S9 protein and no added cofactors. This acid metabolite was not found in incubations with active S9 and all cofactors or NADPH as single cofactor added. The identity of metabolite B was confirmed by synthesized reference material (GR-88-1910-2). GR-88-1910 is not detectable by GC-MS without derivatisation.

Furthermore, we detected a putative hydroxylated GR-88-0778-acid as metabolite by LC-MS/MS (metabolite C; retention time 9.8 min; ESI-negative mode: [M-H]- = 235.1340; ESI-positive mode : [M-H]+ = 237.1485) and a putative diol of GR-88-0778 (metabolite D; retention time 9.8 min; ESI-positive mode : [M-H]+ = 223.1693).

One putative glucuronide (metabolite E) was identified by LC-MS/MS with GR-88-0778 incubated with active S9 protein and cofactors. Metabolite E (retention time 12.4 min; ESI-negative mode: [M-H]- = 381.1919; ESI-positive mode: [M-H]+ = 383.2064) was tentatively identified as glucuronide of GR-88-0778 alcohol by LC-MS/MS. We did not detect any further glucuronides like the acyl glucuronide of GR-88-0778-acid or hydroxylated glucuronides.

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 in the 2nd major experiment: 25.81 ml/h/mg protein. It was used as input into an in vitro - in vivo extrapolation model to predict the refind BCF. The predicted BCF (BCFTOT) was 59.4 L/kg wet weight for GR-88-0778 using an assumed fU = 1.0, i.e. no effect of differential binding to serum, and 66.4 L/kg wet weight assuming different binding to serum in vivo vs. in vitro (fU calc).

Very rapid enzymatic degradation by trout liver S9 fractions was observed for GR-88-0778.
The alcohol and acid of GR-88-0778 and the corresponding hydroxylated compounds were detected as Phase I metabolites. In addition, a glucuronide conjugate was found as a Phase II metabolite by trout liver S9 fractions.


BCF (BCFTOT) = 59.4 L/kg (wet wt) fu =1.0

BCF (BCFTOT) =66.4 L/kg (wet wt) fu calc

Executive summary:

Introduction

The primary objective of this study was to determine thein vitrostability of GR-88-0778 in fish liver S9 fractions. Metabolic stability was determined by monitoring the disappearance of GR-88-0778 as a function of time. The analytical method involved GC coupled with mass spectrometry (GC‑MS) to determine the GR-88-0778 concentrations and Phase I metabolites. LC coupled with mass spectrometry (LC‑MS/MS) was used to detect water soluble Phase I and Phase II metabolites. The information derived from the study provides an useful indication of the bioaccumulation potential of the test item.

 

Experimental

A GC-MS analytical method was developed for monitoring the disappearance of GR-88-0778 in fish liver S9 fractions. A range finding experiment was performed with GR-88-0778 to determine the optimal incubation time for metabolic stability analysis. Finally, GR-88-0778 (10 µM) was incubated in triplicate with trout liver S9 fraction (1 mg/ml) for 0, 5, 7.5, 10, 15 and 20 minutes in the 1^stmain experiment and for 0, 1, 2, 4, 5 and 6 minutes at 12°C in the 2^ndexperiment. Negative controls included incubation of the test substance with heat inactivated S9 protein. At the end of the incubation period, reactions were stopped by the addition of acetonitrile containing an internal standard and extracted with tert-butyl methyl ether (MTBE). Samples were submitted for GC-MS analysis. Calibration curves of GR-88-0778 were prepared in the presence of heat inactivated S9 protein in duplicate (bracketing). In addition, metabolites were analysed by GC-MS and LC-MS/MS in presence of all cofactors, with NADPH as single cofactor or without cofactors added (3^rdmain experiment). 

 

Results

Very rapid turnover ofGR-88-0778was observed over 6 minutes.GR-88-0778 demonstrated a metabolic turnover of 93.1% of the starting concentration (10 µM) within a 6 minute exposure period. In contrast, there was no decrease of GR-88-0778 with the heat inactivated S9 control within a 6 minute exposure period. A much slower disappearance of GR-88-0778 with trout S9 fractions was observed in the absence of added cofactors (16.6% decrease within a 6 minutes exposure period).

The corresponding alcohol of GR-88-0778 (metabolite A) was detected as a metabolite by GC-MS analysis and confirmed with the synthesized material as reference (GR-88-1108). The carboxylic acid of GR-88-0778 (metabolite B) was detected as methylester by GC-MS analysis and confirmed with the synthesized material as reference (GR-88-1910) after derivatisation with diazomethane. Furthermore, hydroxylated metabolites of the carboxylic acid (GR-88-1910) and of the alcohol (GR-88-1108) were detected by LC-MS (metabolite C and D, respectively). Importantly, a glucuronic acid conjugate of GR-88-1108 was detected as Phase II metabolite by LC-MS/MS analysis.

Thein vitro intrinsicclearance (CL_{int, in vitro}) wascalculated from the log-transform measured concentrations of the parent compound as a function of time in the 2^ndmajor experiment: 25.81ml/h/mg protein.It was used as input into an in vitro-in vivo extrapolation model to predict the refind BCF.The predicted BCF (BCF_TOT) was 59.4 L/kg wet weight for GR-88-0778 using an assumedf_U= 1.0, i.e. no effect of differential binding to serum, and 66.4 L/kg wet weight assuming different binding to serum in vivo vs.in vitro (f_Ucalc). 

Comparison of existing data for fragrance speciality chemicals possessing bothin vivoandin vitrofish BCF data sets, indicate that there tends to be closer similarity between the derived results when a f_U of 1.0 is employed for the calculations especially for compounds with high logK_owvalues. However, since the log K_owvalue is relatively low for GR-88-0778 (3.7; internal value, Givaudan), both of the calculated BCFs are rather similar. To verify the use of thein vitro–in vivoextrapolation (IVIVE) model, BCFs were predicted with fragrance molecules for which the BCF values were measured in fish. There is a good correlation between thein vivoBCF values and predicted BCFs using thein vitrometabolism data for the nine fragrance molecules and Pentachlorobenzene thus corroborating the degree of relevance of this model.

 

Conclusions

Very rapid enzymatic degradation by trout liver S9 fractions was observed for GR-88-0778.

The alcohol and acid of GR-88-0778 and the corresponding hydroxylated compounds were detected as Phase I metabolites. In addition, a glucuronide conjugate was found as a Phase II metabolite by trout liver S9 fractions.

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-88-0778.

The in vitro–in vivo extrapolationmodel to refine BCFs based on in vitro turnover rates of nine fragrance molecules by trout liver S9 fractions results in values which are comparable to knownin vivoBCF values especially if no effect of differential binding to serum is assumed.

Refined BCFs for GR-88-0778 incorporating in vitro metabolism indicate that the potential for in vivo bioaccumulation is likely to be significantly lower than relevant “B” threshold criteria.

Description of key information

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 in the 2nd major experiment: 25.81 ml/h/mg protein. It was used as input into an in vitro - in vivo extrapolation model to predict the refind BCF. The predicted BCF (BCFTOT) was 59.4 L/kg wet weight for GR-88-0778 using an assumed fU = 1.0, i.e. no effect of differential binding to serum, and 66.4 L/kg wet weight assuming different binding to serum in vivo vs. in vitro (fU calc).

Very rapid enzymatic degradation by trout liver S9 fractions was observed for GR-88-0778.

The alcohol and acid of GR-88-0778 and the corresponding hydroxylated compounds were detected as Phase I metabolites. In addition, a glucuronide conjugate was found as a Phase II metabolite by trout liver S9 fractions.

BCF (BCFTOT) = 59.4 L/kg (wet wt) fu =1.0

BCF (BCFTOT) = 66.4 L/kg (wet wt) fu calc

NYMPHEAL ACID (GR-88 -1910, Primary Metabolite) - NYMPHEAL transformed 100% to NYMPHEAL ACID in an OECD 301C MITI Biodegradation study. As a consequence of this, an In vivo fish bioconcentration study was performed on the NYMPHEAL ACID (GR-88 -1910) according to the OECD 305 - I test guideline. Detects of both the major- and minor-component of NYMPHEAL ACID were all below the respective Limit of Quantifications, which were respectively, < 3.4 L/kg and < 9.4 L/kg. NYMPHEAL ACID presents negligible potential to bioaccumulate in fish.

Key value for chemical safety assessment

BCF (aquatic species):

66.4 L/kg ww