Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

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:

April - June, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

02 October, 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Aqueous Exposure Bioconcentration Fish Test" in "Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body (Aqueous Exposure Bioconcentration Fish Test)" Japanese CSCL (March 31, 2011, Yakushokuhatsu 0331 No. 7

Version / remarks:

01 July, 2019

Principles of method if other than guideline:

The study was performed with a single concentration because a single concentration was expected to be sufficient for the following reasons;
a) ROSYFOLIA is a non-polar organic substance and is expected to behave and bioaccumulate in a linear fashion across a broad range of environmentally relevant exposure concentrations.
b) In the test fish analysis, ROSYFOLIA can be quantified accurately at the test concentration 0.02 mg/L.
c) No concentration effects were expected because the test concentration (0.02 mg/L) is much lower than its water solubility at 25 °C (Peak 1; 112 mg/L, Peak 2; 111 mg/L, Peak 3; 107 mg/L), and acute toxic effects observed in fish toxicity studies (96-hr LC50 value; 3.2 mg/L for Oncorhynchus mykiss).
d) No concentration effects and low bioconcentration factor (BCF) values were observed in the preliminary BCF study conducted at the test concentration 0.02 mg/L (High) and 0.002 mg/L (Low).

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

Water Samples for Analysis :
Control : Prior to introduction of fish, Day 28 (end of Uptake-Phase)
Level 1 (0.02 mg/L nominal) : Prior to introduction of fish, Day 7, 14, 21, 25 and 28 (end of Uptake-Phase)

Fish Samples for Substance Specific Analyses :
Control : Prior to start of Uptake-Phase, end of Uptake-Phase (Day 28)
Level 1 (0.02 mg/L nominal) : Day 7, 14, 21, 25, 28 (end of Uptake-Phase)

Fish Samples for Lipid Analysis :
Control, only : Prior to start of Uptake-Phase, end of Uptake-Phase (Day 28).

Vehicle:

yes

Remarks:

0.005% N,N-dimethylformamide

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

Preparation of stock solutions :
Level 1
The test sample (400 mg) was dissolved in N,N-dimethylformamide to prepare a 400 mg/L stock solution (1 L).
Control
N,N-Dimethylformamide was used as the stock solution (approximately 2.5 L).

Frequency of preparation for stock solutions :
Level 1 Once / 2 weeks
Control Once during uptake phase

Test concentrations :
Test nominal concentration of the test item was set as follows. The control was set as a blank test (0.005% of N,N-dimethylformamide).
Level 1 0.02 mg/L (0.005% of N,N-Dimethylformamide)

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species Common carp (Cyprinus carpio)
Reason for selection: The previous data conducted with this species can be compared and the size of this species is adequate for handling.
Supplier CERI Kurume
Conditions for acclimatization
The external disinfection was carried out in an aqueous solution containing OTC for fisheries (oxytetracycline hydrochloride, Kyoritsu Seiyaku) and sodium chloride (The Salt Industry Center of Japan). Thereafter fish were acclimatized in the following conditions. Test fish were moved from acclimatization tank (No. 7-2) to a test tank after the acclimatization.
Period: 68 days
Temperature: 25±2ºC
Mortality during acclimatization: < 5%.
Lot No. TFC-191212
Weight The minimum fish weight at the beginning of uptake phase was more than two thirds of the maximum one.
Length 6.9–8.7 cm (at the beginning of uptake phase 7.0–7.4 cm)
Age Yearling fish
Feeding Feed: Feed for colored carp
Composition: Proteins content ≥30%
Lipid content ≥4.0%
Manufacturer: KYORIN FOOD INDUSTRIES
Feeding amount and interval:
There were two feeding sessions (AM and PM), each of approximately 1% wet body weight, and that at each feeding session the quantity was administered in two separate actions, therefore the total feeding amount was performed at approximately 2% wet body weight per day (once a day during weekends and holidays). The fish were starved for 24 hours before the sampling, that is, there was one feeding session (AM, 2% wet body weight at one time) in the day before the sampling.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: No concn. effects were expected because the test concentration (0.02 mg/L) is much lower than its water solubility at 25 °C (Peak 1; 112 mg/L, Peak 2; 111 mg/L, Peak 3; 107 mg/L), and is well below acute toxic effects (96h LC50=3.2 mg/L in O. mykiss)

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

0 d

Hardness:

Level 1 16.0 mgCaCO3/L
Control 17.0 mgCaCO3/L

Test temperature:

During uptake phase
Level 1 25.0–25.3ºC
Control 24.6–25.0ºC

pH:

During uptake phase (at the beginning and end of uptake phase)
Level 1 8.1, 8.1
Control 8.1, 8.1

Dissolved oxygen:

During uptake phase (at the beginning and end of uptake phase)
Level 1 6.9 - 7.7 mg O2/L
Control 7.1 - 7.7 mg O2/L

TOC:

During uptake phase
Level 1 23.7–24.5 mgC/L
Control 23.9–25.8 mgC/L

Details on test conditions:

Aeration The test tanks were supplied with air.
Time of irradiation with light
14 hours light /10 hours dark (artificial light of white fluorescent lamp)
Number of fish (at the beginning of uptake phase)
Levels 1 26 fish
Control 16 fish
Duration of uptake 28 days (April 16, 2020 – May 14, 2020)


Preparation of stock solutions :
a) Level 1
The test sample (400 mg) was dissolved in N,N-dimethylformamide to prepare a 400 mg/L stock solution (1 L).
b) Control
N,N-Dimethylformamide was used as the stock solution (approximately 2.5 L).

Frequency of preparation for stock solutions :
Level 1 Once / 2 weeks
Control Once during uptake phase

Test concentrations :
Test nominal concentration of the test item was set as follows. The control was set as a blank test (0.005% of N,N-dimethylformamide).
Level 1 0.02 mg/L (0.005% of N,N-Dimethylformamide)

Fish-to-water loading rate :
The fish-to-water loading rate at the beginning of exposure which was calculated with a flow rate of test water (1152 L/day), the number of fish (26 fish for Level 1 and 16 fish for Control) and the average wet weight of test fish at the beginning of exposure (4.35 g) are shown below.
Level 1 0.098 g wet weight/L per day
Control 0.060 g wet weight/L per day

Observation, measurement and cleaning :
Observation of Fish - Twice a day
Water Temp. - Once a day
Flow Rate of Test Water - Once a day
Concn. of Dissolved Oxygen - Once before uptake-phase and then once a Week;
pH - Once before uptake-phase and then twice per Week
Concn. of TOC - Twice before Uptake-Phase, then once per week;
Total water hardness - Once during Uptake-Phase
Excreta of common carp and dirt on the test tank were removed approximately once a day in the test period

Nominal and measured concentrations:

The concentrations of the test item were maintained at ≥ 88% of nominal concentrations and the variations were within ±20% of the average measured concentrations. No interfering peak was observed at the peak positions of the test item on the GC-MS chromatogram for the Control water at the end of the uptake phase.

Nominal Exposure = 0.02 mg/L

Average Exposure Concentration :
Peak 1 = 0.0193 mg/L
Peak 2 = 0.0190 mg/L
Peak 3 = 0.0187 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

Calculation of bioconcentration factor (BCF) :
BCF was calculated as follows, and it was rounded off to 2 figures.
a) Calculation of bioconcentration factor
BCF = Cf / Cw
BCF : Bioconcentration factor (L/kg)
Cf : Concentration of test item in test fish (subtracting FB) (μg/g)
Cw : Average concentration of test item in test water during uptake phase (mg/L)
FB : Average concentration of fish blank in analysis of Control (μg/g)
The results of blank test was regarded as the average concentration of fish blank.

b) Average bioconcentration factor in m-th analysis
BCFm = (BCFa + BCFb) / n
BCFm : Average BCF in m-th analysis (number of group 2 (a,b)) (L/kg)
BCFa,b : Each BCF in m-th analysis of test fish (L/kg)
n : Number of group in m-th analysis of test fish

Definition of steady-state :
The steady-state is defined to be reached when four successive analyses of concentration of the test item in test fish made on samples taken at intervals of at least two days are within ± 20% of each other, and there is no significant increase of concentration of the test item in test fish in time between the first and last successive analyses (after 14, 21, 25 and 28 days).
Criterion for the steady-state: V(m-3), V(m-2), V(m-1), V(m) ≤20 (%)
V(m-3), V(m-2), V(m-1), V(m) : Variation rate of concentration of test item in test fish from average value (%)

Calculation of bioconcentration factor at steady-state (BCFss) :
BCFSS was calculated as follows, and it was rounded off to 2 figures.
a) Calculation of average concentration of test item in test fish at steady-state
Cfs = { Cf (m-3) + Cf (m-2) + Cf (m-1) + Cf (m)}/ 4
Cfs : Average concentration of test item in test fish at steady-state (μg/g)
Cf(m) : Average concentration of test item in m-th analysis of test fish (subtract FB) (μg/g)
FB : The average concentration of fish blank in analysis of Control (μg/g)
The results of blank test was regarded as the average concentration of fish blank.

b) Calculation of BCFss
BCFss = Cfs / Cw
BCFss : Bioconcentration factor at steady-state (L/kg)
Cfs : Average concentration of test item in test fish at steady-state (μg/g)
Cw : Average concentration of test item in test water during uptake phase (mg/L)

Calculable BCF :
On the basis of the LOQ for the test item, calculable BCF are as follows (rounded off to 2 figures) :
Peak 1 27 L/kg
Peak 2 27 L/kg
Peak 3 28 L/kg

Lipid content:

5.09 %

Time point:

other: Average fish lipid content during study

Lipid content:

4.75 %

Time point:

start of exposure

Lipid content:

5.42 %

Time point:

end of exposure

Key result

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8.1

Type:

BCF

Value:

33 L/kg

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

steady state

Remarks on result:

other: Corrected to 5% Lipid content

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8.1

Type:

BCF

Value:

33 L/kg

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

steady state

Remarks on result:

other:

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8.1

Type:

BCF

Value:

<= 35 L/kg

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

other: Peak 1 - all fish tissue analyses performed during Uptake-Phase were in the range < 27 L/kg (i.e. < LOD) to 35 L/kg

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8.1

Type:

BCF

Value:

>= 28 - <= 44 L/kg

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

other: Peak 2 - all fish tissue analyses performed during Uptake-Phase were in the range of 28 - 44 L/kg

Conc. / dose:

0.02 mg/L

Temp.:

25 °C

pH:

8.1

Type:

BCF

Value:

< 28 L/kg

Basis:

whole body w.w.

Time of plateau:

7 d

Calculation basis:

other: Peak 3 - all fish tissue analyses performed during the Uptake-Phase < 28 L/kg (i.e. < LOQ for BCF)

Details on results:

Results of test fish observation :
No abnormality in behavior or appearance of the fish was noted during the uptake phase. No mortalities occurred in either Level 1 or the Control during the 28 day exposure period of the uptake phase.

Concentration of test item in test water :
The concentrations of the test item were maintained at ≥ 88% of nominal concentrations and the variations were within ±20% of the average measured concentrations. No interfering peak was observed at the peak positions of the test item on the GC-MS chromatogram for the Control water at the end of the uptake phase.

BCFs at steady-state (BCFss) :
a) Peak 1
Because the test item concentration in some test fish at four successive analyses (after 14, 21, 25 and 28 days) from the initiation of uptake phase were less than the LOQ, a BCFss value could not be calculated for Peak 1. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached during the 28 day uptake phase.
b) Peak 2
The average test item concentration in test fish made on samples taken at 14, 21, 25 and 28 days were within ± 20% of each other for all peaks. Therefore, it was confirmed that the steady-state was reached in uptake phase and the BCFss was 33 L/kg.
c) Peak 3
Because the test item concentration in all test fish at four successive analyses (after 14, 21, 25 and 28 days) from the initiation of uptake phase were less than the LOQ, a BCFss value could not be calculated. However, because all BCFs were less than 100 L/kg, it was evaluated that a steady-state was reached during the 28 day uptake phase.

Growth rate constant (kg) :
The growth rate constants (kg) of test fish are shown as follows.
Level 1 0.0140 /day
Control 0.0142 /day

Fish lipid content :
The measured fish lipid contents are shown below.
Before uptake phase 4.75%
After uptake phase 5.42%
Average fish lipid content during study 5.09%

Validity criteria fulfilled:

yes

Conclusions:

In the present study, test concentration (0.02 mg/L) of the test item (ROSYFOLIA) was set lower than the water solubility of the test item (Peak 1; 112 mg/L, Peak 2; 111 mg/L, Peak 3; 107 mg/L), and at the test concentration more than 100-times lower (actual 160-times lower) than the estimated 96-hour acute LC50 value for Oncorhynchus mykiss (3.2 mg/L).
The flow-through set-up and exposure conditions were demonstrated to be stable. Under these conditions, BCFs were estimated to be lower than 35 L/kg (Peak 1), 44 L/kg (Peak 2) and 28 L/kg (Peak 3). The BCFs at steady state was determined to be 33 L/kg (Peak 2), 33 L/kg when normalized to 5% lipid content (BCFssl). These results indicate that ROSYFOLIA presents negligible risk to bioconcentrate in fish.

BCFss = 33 L/kg (wet wt.)

BCFssl = 33 L/kg (wet wt.)

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 2013 - January 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 319B

Version / remarks:

Determination of in vitro intrinsic clearance using rainbow trout liver S9 subcellular fraction (RT-S9).

June, 2018

Deviations:

no

Principles of method if other than guideline:

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, 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 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 and the protocol was published recently. In order to determine hepatic metabolism of GR-50-1408, 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-50-1408 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”).

GLP compliance:

no

Radiolabelling:

no

Details on sampling:

Range-Finding Experiment :
Reactions were stopped at 0, 30 minutes and 120 minutes.

Two Main Experiments :
Reactions were stopped at time 0, 15, 30, 45 and 60 minutes (1st main experiment) or at 0, 15, 30, 45, 60 and 90 minutes (2nd main experiment).

Reactions were stopped via the 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.

Vehicle:

yes

Remarks:

MeOH / Water

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

A stock solution of GR-50-1408 (10 mM) was prepared freshly in methanol and diluted in water resulting in 100 µM solutions.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Trout Liver S9 Fractions :
Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were purchased from Lifetechnologies (formerly CellzDirect/Invitrogen; Invitrogen GmbH, Frankfurter Strasse 129B, 64293 Darmstadt, Germany) (“Pooled Male Hatchery Rainbow Trout Liver S9”; product code CZDTRS9PL, Lot# TR015) and stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions was 800 g. The enzymatic activity of newly received S9 fractions was typically tested using internal fragrance reference molecules which are degraded by different enzyme systems (Phase I or Phase II). Furthermore, enzymatic activity was tested with Testosterone and Umbelliferone (7-Hydroxycoumarin) as controls to determine the activity of CYP3A (Cytochrome P450 monooxygenase), UDP-Glucuronosyltransferase and Sulfotransferase. S9 fractions were only used for metabolism studies if significant enzymatic conversion of the reference substances 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.

Route of exposure:

other: In vitro exposure of isolated Rainbow Trout liver S9 fractions

Justification for method:

other: Exposure of Rainbow trout S9 liver cells to determine In vitro metabolic transformation rate of test item as an indication of bioconcentration potential.

Test type:

static

Total exposure / uptake duration:

>= 15 - <= 120 min

Details on test conditions:

Metabolic Turnover Rate of GR-50-1408 in Fish Liver S9 Fractions :

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-50-1408 (10 mM) was prepared freshly in methanol and diluted in water resulting in 100 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 Thermomixer (Eppendorf) at 700 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-50-1408 (10 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 substance (10 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, 30 minutes 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 (Eppendorf centrifuge, 12 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis.
In the two independent main experiments (1st and 2nd main experiment), GR-50-1408 (10 µM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 60 minutes (1st main experiment) or 90 minutes (2nd main experiment) as described above. Reactions were stopped at time 0, 15, 30, 45 and 60 minutes (1st main experiment) or at 0, 15, 30, 45, 60 and 90 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 and 60 minutes (1st main experiment) or 0 and 90 minutes (2nd main experiment) and in the presence of active S9 protein in absence of any cofactors for 60 (1st main experiment) or 90 minutes (2nd main experiment). Reactions were stopped and extracted as described above.

Addition of: Final concentration
90 µl K-Phosphate buffer, pH 7.8 (0.1 M) 0.1 M K-Phosphate buffer, pH 7.8
10 µl S9 protein (20 mg/ml) 1 mg/ml protein
Preincubation for 5 min at 12.5 ± 2.5 °C (700 rpm)
20 µl Alamethicin (250 µg/ml) 25 µg/ml Alamethicin
Incubation on ice for 15 min; pre-incubation at 12°C for 10 min
Addition of cofactors:
20 µl NADPH (10 mM) 1 mM NADPH
20 µl UDPGA (20 mM) 2 mM UDPGA
20 µl GSH (5 mM) 0.5 mM GSH
20 µl PAPS (1 mM) 0.1 mM PAPS
20 µl test substance in water (100 µM) 10 µM test substance
Final volume in Hirschmann tubes: 200 µl
Incubation at 12.5 ± 2.5 °C (700 rpm) for different time points

Nominal and measured concentrations:

The Range-Finding and two main experiments were performed with Nominal starting concentrations of 10 microM of test item.

Isomer 1 = 51.6% abundance in the test item

Isomer 2 = 31.2% abundance in the test item

Average time 0 concentrations of both Isomers 1 and 2 in the Range-Finder and two Main experiments are all 100.0%, and are used as the Time 0 baseline reference from which metabolic turnover will be derived.

Range-Finding Experiment :

Isomer Time 0 60 Minutes (% remaining)
Isomer 1 100.0% 42.6%
Isomer 2 100.0% 50.5%


1st Main Experiment :

Isomer Time 0 60 Minutes (% remaining)
Isomer 1 100.0% 70.7%
Isomer 2 100.0% 77.7%


2nd Main Experiment :

Isomer Time 0 90 Minutes (% remaining)
Isomer 1 100.0% 52.7%
Isomer 2 100.0% 61.7%

Details on estimation of bioconcentration:

Determination of in Vitro Intrinsic Clearance Rate of GR-50-1408 in Fish Liver S9 Fractions :

The remaining measured concentrations of GR-50-1408 (1st and 2nd main experiments) were log-transformed and plotted as a function of time (h). The first order reaction rate (1/h) is calculated by multiplying the fitted slope term from the regression equation by -2.3 (conversion from log (base 10) to ln). Then the in vitro intrinsic clearance rate (Clint, in vitro; ml/h/mg S9 protein) is calculated by multiplying the reaction rate by the volume of the incubation system (i.e. 0.2 ml) and dividing by the amount of protein (0.2 mg).


Calculation of Refined BCF Estimates :

The final in vitro-in vivo extrapolation model by J. Nichols et al. (version: “S9spreadsheet_Public_032713.xlsx”) which was published recently is used to calculate refined BCF estimates.
The model provides BCF predictions for a "standard fish", defined as a 10 g rainbow trout held at 15°C which contains 5% whole-body lipid. In addition to specific inputs to the metabolism extrapolation model, e.g. modelled body weight (10 g), liver weight as a fraction of body
weight (0.015), other parameters are taken from Arnot & Gobas. The liver S9 protein content (163 mg/g liver) was determined based on CYP content and glucose-6-phosphatase activity as markers. The in vitro S9 turnover rate was used to estimate an in vivo intrinsic clearance (CLIN VIVO, INT; l/d/kg fish) which then was scaled for 10 g fish and divided by the compound’s estimated volume of distribution (VD; l) to calculate a whole-body biotransformation rate (kMET; 1/d). To calculate the hepatic clearance (CLH; l/d/kg fish), the well-stirred liver model is applied. Two possibilities are explored in regard of plasma binding applying the binding correction term fU: (a) hepatic clearance is calculated taking into account a theoretically postulated difference between in vitro and in vivo binding (“fU calc”); (b) hepatic clearance is calculated assuming equal in vitro and in vivo binding, i.e. same chemical concentration available to metabolizing enzymes in vivo and in vitro, by setting fU = 1.0.
Finally, kMETAB (1/d) is combined with estimates of k1 (gill uptake rate constant), k2 (gill elimination rate constant), and kE (fecal egestion rate constant) to simulate the concentration in fish (CFISH) and the BCF is predicted (log Kow = 3.5 for both isomers, 10 g fish, 5% lipid content, 15°C).

Conc. / dose:

10 other: microM

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

58 L/kg

Basis:

other: Result for Isomer 1 - Based on fu = 1 - assumes no differential effect of binding to serum In vivo vs In vitro

Calculation basis:

other: Extrapolated from CLint using IVIVE Model (Nichols, J.W., et al., 2013 (version: “S9spreadsheet_Public_032713.xlsx"

Remarks on result:

other: 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 clear-ance. Environ Toxicol Chem, 2013. 32(7): p. 1611-22

Conc. / dose:

10 other: microM

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

61 L/kg

Basis:

other: Result for Isomer 2 - Based on fu = 1 - assumes no differential effect of binding to serum In vivo vs In vitro

Calculation basis:

other: Extrapolated from CLint using IVIVE Model (Nichols, J.W., et al., 2013 (version: “S9spreadsheet_Public_032713.xlsx"

Remarks on result:

other: 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 clear-ance. Environ Toxicol Chem, 2013. 32(7): p. 1611 - 22

Conc. / dose:

10 other: microM

Temp.:

12 °C

pH:

7.8

Type:

BCF

Value:

127 L/kg

Basis:

other: Result for Isomer 1 - Based on fu calc - assumes different binding to serum In vivo vs In vitro

Calculation basis:

other: Extrapolated from CLint using IVIVE Model (Nichols, J.W., et al., 2013 (version: “S9spreadsheet_Public_032713.xlsx"

Remarks on result:

other: 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:

133 L/kg

Basis:

other: Result for Isomer 2 - Based on fu calc - assumes different binding to serum In vivo vs In vitro

Calculation basis:

other: Extrapolated from CLint using IVIVE Model (Nichols, J.W., et al., 2013 (version: “S9spreadsheet_Public_032713.xlsx")

Remarks on result:

other: 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.

Details on results:

Moderate turnover of GR-50-1408 was observed over 90 minutes. Isomer 1 of GR-50-1408 (51.6% abundance at time 0 with the GC-method used) demonstrated a metabolic turnover of 47.3% of the starting concentration within a 90 minute exposure period. Isomer 2 of GR-50-1408 (31.2% abundance at time 0 with the GC-method used) was slightly slower metabolized with a metabolic turnover of 38.3% of the starting concentration within a 90 minute exposure period. In contrast, there was a neglibile decrease of both GR-50-1408 isomers with the heat inactivated S9 control (3.2 – 5.5% decrease within a 90 minute exposure period).

The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transformed measured concentrations of the two isomers of the parent compound as a function of time in two independent experiments: 0.36 and 0.43 ml/h/mg protein for isomer 1; 0.26 and 0.33 ml/h/mg protein for isomer 2. It was used as input into an in vitro - in vivo extrapolation model to generate a refined BCF estimate.

The refined BCF estimate (BCFTOT) was 58 l/kg wet wt for isomer 1 of GR-50-1408 and 61 l/kg wet wt for isomer 2 of GR-50-1408 using an assumed fU = 1.0, i.e. no effect of differential binding to serum and 127 l/kg wet wt for isomer 1 and 133 l/kg wet wt for isomer 2 assuming different binding to serum in vivo vs. in vitro (fU calc).

 

		In vitro - in vivo fish BCF extrapolation ^1 (L/kg wet wt.)
		fU calc	fU =1.0
GR-50-1408 ^2	Isomer 1	127	58
	Isomer 2	133	61

 

¹ J. Nichols 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 (version: “S9spreadsheet_Public_032713.xlsx”)

² The two major isomers of GR-50-1408 were quantified separately: isomer 1 ((1S,2R)-1-methyl-2-((S)-5-methylhex-4-en-2-yl)cyclopropyl)methanol, rel-; 51.6% abundance with the GC-method used) and isomer 2 ((1S,2R)-1-methyl-2-((R)-5-methylhex-4-en-2-yl)cyclopropyl)methanol, rel-; 31.2% abundance with the GC-method used).

 

Validity criteria fulfilled:

yes

Conclusions:

The two monitored Isomers of GR-50-1408 demonstrated a moderate rate of metabolic turnover under the experimental conditions :

in vitro intrinsic clearance (CLint, in vitro)
0.36 and 0.43 ml/h/mg protein for isomer 1;
0.26 and 0.33 ml/h/mg protein for isomer 2

The refined BCF estimate (BCFTOT) was 58 l/kg wet wt for isomer 1 of GR-50-1408 and 61 l/kg wet wt for isomer 2 of GR-50-1408 using an assumed fU = 1.0, i.e. no effect of differential binding to serum and 127 l/kg wet wt for isomer 1 and 133 l/kg wet wt for isomer 2 assuming different binding to serum in vivo vs. in vitro (fU calc).

Executive summary:

Moderate turnover of GR-50-1408 was observed over 90 minutes. Isomer 1 of GR-50-1408 (51.6% abundance at time 0 with the GC-method used) demonstrated a metabolic turnover of 47.3% of the starting concentration within a 90 minute exposure period. Isomer 2 of GR-50-1408 (31.2% abundance at time 0 with the GC-method used) was slightly slower metabolized with a metabolic turnover of 38.3% of the starting concentration within a 90 minute exposure period. In contrast, there was a neglibile decrease of both GR-50-1408 isomers with the heat inactivated S9 control (3.2 – 5.5% decrease within a 90 minute exposure period).

The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transformed measured concentrations of the two isomers of the parent compound as a function of time in two independent experiments: 0.36 and 0.43 ml/h/mg protein for isomer 1; 0.26 and 0.33 ml/h/mg protein for isomer 2. It was used as input into an in vitro - in vivo extrapolation model to generate a refined BCF estimate.

The refined BCF estimate (BCFTOT) was 58 l/kg wet wt for isomer 1 of GR-50-1408 and 61 l/kg wet wt for isomer 2 of GR-50-1408 using an assumed fU = 1.0, i.e. no effect of differential binding to serum and 127 l/kg wet wt for isomer 1 and 133 l/kg wet wt for isomer 2 assuming different binding to serum in vivo vs. in vitro (fU calc).

Description of key information

An In vivo Fish bioconcentration study (Key Study), required for notification within another region, exists for the substance as well as an In vitro metabolic stability assay using fish liver S9 fractions (Supporting Study).

 

Key Study In vivo (CERI Study No. 46336) :

The bioconcentration of Rosyfolia was studied in a flow-through system in Common Carp (C. carpio) according to Japanese CSCL test guideline, and comparable to the OECD 305 test guideline, also.  Three peaks were followed to determine the aqueous exposure concentration and concentrations in fish tissue.  A 28-day Uptake-Phase was performed.  The determined BCF values were low (<< 100 L/kg (wet wt.)) and thus a Depuration-Phase was not necessary.   

The flow-through set-up and exposure conditions were demonstrated to be stable.  Under these conditions, BCFs were estimated to be lower than 35 L/kg (Peak 1), 44 L/kg (Peak 2) and 28 L/kg (Peak 3). 

The BCF at steady state was determined to be 33 L/kg (Peak 2), 33 L/kg when normalized to 5% lipid content (BCFssl). 

These results indicate that ROSYFOLIA presents negligible risk to bioconcentrate in fish.

 

Supporting Study In vitro (Givaudan Report No. 153'380) :

An In vitro study on the metabolic stability of the registration substance when exposed to Rainbow Trout (O. mykiss) liver S9 cells was performed, and serves as supporting information.

The in vitro intrinsic clearance (CL_{int, in vitro}) was calculated from the log-transformed measured concentrations of the two isomers of the parent compound as a function of time in two independent experiments: 0.36 and 0.43 ml/h/mg protein for isomer 1; 0.26 and 0.33 ml/h/mg protein for isomer 2. It was used as input into an in vitro - in vivo extrapolation model to generate a refined BCF estimate.

The refined BCF estimate (BCF_TOT) was 58 l/kg wet wt for isomer 1 of GR-50-1408 and 61 l/kg wet wt for isomer 2 of GR-50-1408 using an assumed f_U = 1.0, i.e. no effect of differential binding to serum and 127 l/kg wet wt for isomer 1 and 133 l/kg wet wt for isomer 2 assuming different binding to serum in vivo vs. in vitro (f_U calc).

 

Both the In vivo and In vitro studies demonstrate strong similarity in the BCF values derived, with the In vitro liver S9 approach illustrating a more conservative output.

Key value for chemical safety assessment

BCF (aquatic species):

33 L/kg ww