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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Detailed justification for the QSAR prediction is provided in the attached QMRF and QPRF reports. A summary is provided below:

VALIDITY OF MODEL:
The model is valid according to the following five OECD principles;
1. Defined Endpoint: Bioconcentration factor (BCF) and bioaccumulation factor (BAF) in fish;
2. Unambiguous algorithm: Separate QSAR equations to predict log BAF, log BCF and log kM;
3. Applicability domain: The log BAF and BCF QSARs are applicable to non-ionized chemicals with a log Kow of <9. The log Kow and molecular weight (MW) applicability domain of the kM model are 0.31 to 8.7 and 68.08 to 959.17 respectively. The kM QSAR also includes 57 molecular substructures;
4. Statistical characteristics: For log Km model: a) internal performance; n = 421, r2 = 0.82, mean absolute error (MAE) = 0.38 log units, q2 = 0.75; b) External validation; n = 211, r2 = 0.73, MAE = 0.45 log units.;
5. Mechanistic interpretation: The Arnot-Gobas BCF and BAF model includes mechanistic processes for bioconcentration and bioaccumulation such as chemical uptake from the water at the gill surface (BCFs and BAFs) and the diet (BAFs only), and chemical elimination at the gill surface, fecal egestion, growth dilution and metabolic biotransformation.

ADEQUACY PREDICTION:
- Oxyoctaline formate is a mono-constituent substance, 2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl formate (CAS 65405-72-3, concentration range 75-85%). This constituent has a measured log Kow of 5.0 and molecular weight of 136.36. As such it falls within the applicability domain described above for the BCF QSARs.
- By manually checking the fragments used in the prediction (see attached BCFBAF v3.01 results file) with the structure of 2,4a,5,8a-tetramethyl-1,2,3,4,4a,7,8,8a-octahydronaphthalen-1-yl formate, it is concluded that the substance also falls within the structural fragment domain of the kM model, which is used to estimate biotransformation rates. The later is used as input into the BCF and BAF models.
- An in vitro study shows that the main isomer of Oxyoctaline formate undergoes enzymatic turnover by trout liver S9 fractions. Metabolites for the minor isomers were also observed, indicating that they also undergo metabolism. As such the model predictions (including biotransformation rate estimates) for the main constituent are considered to be relevant, reliable and representative for the registered substance.
- The BCF estimates (corrected to 5% lipid content, including biotransformation rate estimates) are 271 L/kg wet-wt (upper trophic), 548 L/Kg wet-wt (mid trophic) and 672 L/Kg (lower trophic). The different estimates obtained from the three general trophic levels of fish probably reflect different assumptions regarding body size. To this end, it is considered most appropriate to use the highest value of 672 L/Kg wet-wt as a relevant and reliable conservative estimate of the BCF of Oxyoctaline Formate.
Principles of method if other than guideline:
BCFBAF v3.01 Arnot-Gobas BCF & BAF method for non-ionic chemicals in 3 general trophic levels of fish (lower, middle, upper). The model is available in the BCFBAF v3.01 programme, which is part of EpiSuite v4.10 available at http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm. To estimate BAF and BCF (including biotransformation rate estimates), the whole body primary biotransformation rate constant kM (“normalized” for a 10 g fish at 15ºC) is estimated and converted to a Km value for the typical body size of an upper trophic, middle trophic and lower trophic fish.
For regulatory purposes, the predicted BCF values have been corrected to a lipid content of 5%. This is the average lipid content of small fish used in the OECD 305 and the common lipid basis used for bioaccumulation assessment under REACH (ECHA Guidance R.7.10.4.1 and R.11.1.3.2).
Specific details on test material used for the study:
SMILES O=COC1C2(C(C(=CCC2)C)(CCC1C)C)C and measured log Kow value of 5.0 (main mono-constituent isomer) were used as input for the BCFBAF v3.01 model prediction.
Type:
BCF
Value:
271 L/kg
Remarks on result:
other: Estimate for upper trophic, including biotransformation rate
Remarks:
5% lipid
Type:
BCF
Value:
548 L/kg
Remarks on result:
other: Estimate for mid-trophic, including biotransformation rate
Remarks:
5% lipid
Type:
BCF
Value:
672 L/kg
Remarks on result:
other: Estimate for lower trophic, including biotransformation rate
Remarks:
5% lipid
Details on results:
For full details see the BCFBAF v3.01 results file (attached background information) and QPRF document (attached justification).

PREDICTED VALUE (MODEL RESULT):
- Including biotransformation rate estimates:
Estimated Log BCF (upper trophic) = 2.763 (BCF = 579 L/kg wet-wt)
Estimated Log BAF (upper trophic) = 2.768 (BAF = 585.7 L/kg wet-wt)
Estimated Log BCF (mid trophic) = 2.875 (BCF = 750.2 L/kg wet-wt)
Estimated Log BAF (mid trophic) = 2.916 (BAF = 824.2 L/kg wet-wt)
Estimated Log BCF (lower trophic) = 2.905 (BCF = 804.1 L/kg wet-wt)
Estimated Log BAF (lower trophic) = 3.036 (BAF = 1085 L/kg wet-wt)
-Assuming a biotransformation rate of zero:
Estimated Log BCF (upper trophic) = 3.897 (BCF = 7882 L/kg wet-wt)
Estimated Log BAF (upper trophic) = 5.033 (BAF = 1.079E+005 L/kg wet-wt)
-Biotransformation Half-Life (HL):
Estimated Log (HL) = 0.1733 (HL = 1.49 days, normalised to 10 gram fish)
-Biotransformation Rate Constant (kM): 0.4651 /day (10 gram fish)

PREDICTED VALUE (COMMENTS):
The Arnot-Gobas model assumes default lipid contents of 10.7%, 6.85% and 5.98% for the upper, middle and lower trophic levels. For regulatory purposes, the predicted BCF values have been corrected to a lipid content of 5%. This is the average lipid content of small fish used in the OECD 305 and the common lipid basis used for bioaccumulation assessment under REACH (see sections R.7.10.4.1 and R.11.1.3.2 in the ECHA Guidance on information requirements and chemicals safety assessment, and section 5.4 of the attached QMRF).
- Corrected to 5% lipid content (Including biotransformation rate estimates):
Estimated BCF (upper trophic) = (579 / 0.107 ) * 0.05 = 271 L/kg wet-wt
Estimated BCF (mid trophic) = (750.2 / 0.0685) * 0.05 = 548 L/kg wet-wt
Estimated BCF (lower trophic) = (804.1 / 0.0598) * 0.05 = 672 L/kg wet-wt
- Corrected to 5% lipid content (Assuming a biotransformation rate of zero):
Estimated BCF (upper trophic) = (7882/ 0.107) * 0.05 = 3683 L/kg wet-wt)

ADEQUACY OF PREDICTION:
The predicted BCF of 3683 L/kg wet-wt (corrected to 5% lipid content, assuming a biotransformation rate of zero) is considered a conservative maximum estimate for Oxyoctaline formate. It is considered unrealistic given that an in vitro study shows that Oxyoctaline formate undergoes metabolism by trout liver S9 fractions. The predicted values of 271 (upper trophic), 548 (mid trophic) and 672 L/kg wet-wt (lower trophic), all corrected to 5% lipid content and including biotransformation rate estimates, are considered more relevant and reliable, particularly as Oxyoctaline formate falls within the applicability domain of the model used to estimate biotransformation rates. The different estimates obtained from the three general trophic levels of fish after correction to 5% lipid content probably reflect different assumptions regarding body size. To this end, it is considered most appropriate to use the highest conservative value of 672 L/kg wet-wt.


Conclusions:
Based on QSAR estimates of the main constituent, Oxyoctaline formate is predicted to have a BCF in the range of 271-672 L/kg wet-wt in fish (corrected to 5% lipid).
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
other: Experimental in vitro results and estimated BCF by calculation
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The in vitro data, generated from a pre-validated method, are considered reliable and have been used to produce an estimated BCF using a scientifically valid extrapolation model.
Principles of method if other than guideline:
The bioaccumulation potential is estimated from an in vitro fish liver S9 standardised assay, pre-validated by a consortium under the coordination of HESI/ILSI (Johanning et al, 2012). In vitro metabolic stability is determined by monitoring the disappearance of the test item as a function of time. The rate of substance depletion is used as input into an "in vitro - in vivo" extrapolation model (Nichol et al, 2006) to generate an estimated BCF value for a "standardised fish" (one that weighs 10g, has a 5% lipid content, and is living at 15'C).
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oxyoctaline formate
- Physical state: Liquid, colourless to pale yellow
- Analytical purity: 96.5% (sum of the four constituents)
- Structure comment: Mixture of three isomers (major isomer, constituent D, 79.7% abundance; constituent B, 6.9% abundance; constituent C, 10.9% abundance) and constituent A, 1.5% abundance)
- Batch No.: SC00010054
- Expiration date of the lot/batch: 23 Dec 2014
- Storage condition of test material: Approximately 4'C (protected from light)
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 -80°C.
The average body weight of the fish used for the preparation of S9 fractions was 452 g.
Route of exposure:
other: in vitro assay
Details on test conditions:
Initially, a range finding experiment was performed to determine the optimal incubation times to be used in the main experiments.

A stock solution of Oxyoctaline Formate (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) incubated at 12°C. 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 in the section entitled "any other information on materials and methods incl. tables".

In the range finding experiment, Oxyoctaline Formate (1 µM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in duplicate for up to 120 minutes in Hirschmann glass tubes in a Thermomixer (Eppendorf; 12°C, 700 rpm). As controls, the test substance 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, 30 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), Oxyoctaline Formate (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 60 minutes as described above. Reactions were stopped at time 0, 15, 30, 45 and 60 minutes. As control, the test substance (1 μM) was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors for 0, 30 and 60 minutes (1st main experiments) and for 15, 30, 45 minutes and 60 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 60 minutes. Reactions were stopped and extracted as described above.

Since the alcohol of Oxyoctaline Formate (GR-80-3313) was identified as the major Phase I metabolite in trout liver S9 fractions, its metabolic turnover rate by trout liver S9 was determined to assess its bioaccumulation potential. Similar to Oxyoctaline Formate, a range finding experiment was carried out first. GR-80-3313 (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, 10, 30, and 60 minutes incubation, extracted and analysed as described above.
In the main experiment (4th main experiment), GR-80-3313 (1 μM) was incubated in presence of 1 mg/ml active S9 protein and cofactors in triplicate for up to 60 minutes as described above. Reactions were stopped at time 0, 15, 30, 45 and 60 minutes. As control, the test substance (1 μM) was incubated in presence of heat inactivated S9 protein (1 mg/ml) and cofactors for 0, 30 and 60 minutes. Furthermore, incubations in the presence of active S9 protein and in absence of any cofactors added were carried out for 60 minutes. Reactions were stopped, extracted and analysed as described above.
Details on estimation of bioconcentration:
The model version used was “S9spreadsheet_Public_032713.xlsx”. The S9 in vitro substrate depletion rate is converted to a whole-fish metabolism rate constant (Kmetab) using a number of extrapolation and scaling factors. The estimated Kmetab value is then used to refine the partitioning based BCF model prediction. A binding term, fu, is used to correct for the difference in free chemical concentration between in vivo and the in vitro system. Two assumptions are possible: 1) fu can be calculated as the ratio of predicted free fractions in plasma and in the in vitro system using logKow-based algorithms, or 2) binding in vitro and in vivo can be assumed to be equal ( fu = 1.0). The refined BCF has been estimated using both approaches.
Type:
BCF
Value:
1 084 L/kg
Remarks on result:
other: Oxyoctaline Formate (major isomer), fu=calculated
Type:
BCF
Value:
191 L/kg
Remarks on result:
other: Oxyoctaline Formate (major isomer), fu=1
Type:
BCF
Value:
382 L/kg
Remarks on result:
other:
Remarks:
Major metabolite of Oxyoctaline Formate (GR-80-3313), fu=calculated
Type:
BCF
Value:
115 L/kg
Remarks on result:
other: Major metabolite of Oxyoctaline Formate, fu=1
Metabolites:
The corresponding alcohol of the major isomer of Oxyoctaline Formate (metabolite B) was detected as the major metabolite by GC-MS analysis and confirmed with the synthesized material as reference (GR-80-3313). In addition, minor peaks corresponding to the alcohols of two minor isomers were detected (metabolites A and C). Two putative glucuronides were identified by LC-MS/MS (Metabolite 1 and 5). Metabolite 5 was the glucuronide of a hydroxylated metabolite B. Apparently, hydroxylation of metabolite B is fast. Several peaks with similar m/z as the two glucuronides (metabolite 1 and 5) were found indicating the formation of these glucuronides with all isomers of Oxyoctaline Formate.
Details on results:
Moderate turnover of Oxyoctaline Formate was observed over 60 minutes. The major isomer of Oxyoctaline Formate (79.7% abundance) demonstrated a metabolic turnover of 57.8 to 57.9% of the starting concentration within a 60 minute exposure period. In contrast, there was a slow decrease of the major isomer of Oxyoctaline Formate with the heat inactivated S9 control (4.5-14.0% decrease within a 60 minute exposure period) which may be due to abiotic losses like adsorption to protein or volatility. Disappearance of Oxyoctaline Formate with trout S9 fractions was independent on the presence of added cofactors indicating the involvement of a cofactor independent esterase.The in vitro intrinsic clearance (CLint, in vitro) was calculated from the log-transformed measured concentrations of parent compound as a function of time in two independent experiments for the
major isomer: 0.84 and 0.85 ml/h/mg protein.

In addition, the in vitro intrinsic clearance was determined for the major metabolite in S9 fractions, the alcohol of Oxyoctaline Formate (GR-80-3313). Moderate enzymatic turnover was observed with 71.9% decrease of the starting concentration within a 60 minute exposure period. There was negligible decrease of GR-80-3313 with the heat inactivated S9 control (4.2% decrease). The in vitro intrinsic clearance rate of GR-88-3313 was 1.27 ml/h/mg protein.

The average in vitro substrate depletion rates were used as input into the in vitro - in vivo extrapolation model to generate estimated BCF values.
Conclusions:
Moderate enzymatic turnover of the major isomer of Oxyoctaline Formate by trout liver S9 fractions was observed. The bioaccumulation potential in vivo is likely to be low compared to the bioaccumulation potential estimated using models based solely on logKow without biotransformation estimates.

Since the enzymatic decrease of Oxyoctaline Formate was independent on the addition of cofactors, a carboxylesterase may be involved in the biotransformation. Indeed, the corresponding alcohol of the major isomer of Oxyoctaline Formate (metabolite B) was detected as the main metabolite by GC-MS analysis and confirmed with the synthesized material as reference (GR-80-3313). In addition, minor peaks corresponding to the alcohols of two minor isomers were detected (metabolites A and C). These Phase I metabolites are further transformed by hydroxylation and conjugation with glucuronic acid. The putative glucuronide of a hydroxylated metabolite B was identified as a metabolite by LC-MS/MS. In addition, an acyl glucuronide of Oxyoctaline Formate was found as Phase II metabolite. Minor peaks corresponding to the respective glucuronide metabolites of the minor isomers were also detected, indicating that probably all isomers were biotransformed. Due to the relatively low abundances of the minor isomers, only the major isomer was quantified.

Trout liver in vitro S9 metabolism is considered to be an adequate assay to assess enzymatic degradation. The most up to date in vitro – in vivo extrapolation model was applied to predict BCFs based on in vitro turnover rates of nine reference fragrance molecules by trout liver S9 fractions. These predicted BCFs are comparable to known in vivo BCFs especially if no effect of differential binding to serum is assumed (i.e. fU= 1.0). This demonstrates the applicability of using in vitro metabolism data to refine the estimation of a partitioning based BCF to assess the bioaccumulation of test chemicals.

Predicted BCFs for the major Oxyoctaline Formate isomer incorporating in vitro metabolism were 191 L/kg using the measured log Kow value (5.0) and an assumed fU of 1.0 and 1084 L/kg using a theoretically calculated fU. The predicted BCFs for GR-80-3313 were 115 L/kg using the estimated log Kow value (4.3) and an assumed fU of 1.0 and 382 L/kg using a theoretically calculated fU. Due to the more rapid biotransformation of GR-80-3313 and the lower log Kow value compared to the parent, the predicted BCFs were lower for the metabolite compared to the parent.

All BCF estimates were significantly lower than the B criterion of 2000. Overall, it can be hypothesised that the bioaccumulation potential of Oxyoctaline Formate in vivo is likely to be significantly lower than the relevant “B” threshold of 2000 L/kg wet wt.
Executive summary:

Moderate enzymatic degradation by trout liver S9 fractions was observed for the major isomer of Oxyoctaline Formate.

The corresponding alcohols of Oxyoctaline Formate were detected as Phase I metabolites. In addition, two glucuronide conjugates were found as Phase II metabolites 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 Oxyoctaline Formate.

The in vitro – in 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 Oxyoctaline Formate and its major metabolite incorporating in vitro metabolism indicate that the potential for in vivo bioaccumulation is likely to be significantly lower than the relevant “B” threshold of 2000 L/kg wet wt..

In vitro → In vivo Fish BCF Extrapolation (L/kg wet wt.)

Oxyoctaline Formate (major isomer): fU calc = 1084 L/kg; fU =1.0 = 191 L/kg

Major metabolite of Oxyoctaline Formate (GR-80-3313): fU calc = 382 L/kg; fU =1.0 = 115 L/kg

Description of key information

The BCF of Oxyoctaline formate has been estimated using an "in vitro to in vivo extrapolation" model and a valid QSAR. The former gave estimates of 191 L/kg (fu = 1) and 1084 L/kg (fu calculated); while the latter gave a predicted range of 271 -672 L/Kg. The highest worst-case value of 1084 L/kg has been selected as the key value for the CSA.

Key value for chemical safety assessment

BCF (aquatic species):
1 084 L/kg ww

Additional information

Bioaccumulation is not a specified endpoint below 100 t/y. Surrogate information, such as log Kow, may be used at a screening level for hazard assessment and PBT screening. However, the capability of fish to metabolise substances to more polar components, leads to lower bioconcentration factor (BCF) values than that predicted using log Kow alone. Given that Oxyoctaline formate is screened as potentially B based on the log Kow of 4.5, 5.0 and 5.3 (3 isomers), it was considered appropriate to further assess the bioaccumulation of the substance. Therefore, the BCF in fish has been estimated using i) in vitro metabolism data to refine the estimation of a partitioning based BCF and ii) a scientifically valid QSAR that incorporates biotransformation rate estimates.

An in vitro metabolism assay using trout S9 fractions was performed. Due to the relatively low abundance of the minor two isomers, only the major isomer (79.7% abundance in test item) was quantified. In two independent experiments, metabolic turnover of 57.8 and 57.9% of the starting concentration within a 60 minute exposure period was observed. The substrate depletion rates (0.84 and 0.85 ml/h/mg protein) were used as input into an “in vitro - in vivo” extrapolation model to give refined BCF estimates in the range of 191 L/kg (fu = 1, assuming no effect of differential binding to serum) and 1084 L/kg (fu calculated, assuming different binding to serum in vivo vs. in vitro). The Arnot-Gobas BCF QSAR model gave estimates of 271 L/kg wet-wt (upper trophic), 548 L/Kg wet-wt (mid trophic) and 672 L/Kg (lower trophic). The different estimates obtained from the three general trophic levels of fish probably reflect different assumptions regarding body size. To this end, it is considered most appropriate to use the highest conservative value of 672 L/kg wet-wt as the key value from this QSAR method.

All BCF estimates were significantly lower than 2000. Therefore for PBT/vPvB assessment purposes it can be hypothesised that the bioaccumulation potential of the major isomer of Oxyoctaline Formate in vivo is likely to be significantly lower than the relevant “B” threshold of 2000 L/kg wet wt. In contrast, for classification purposes, Oxyoctaline Formate is considered to have potential to bioconcentration since some BCF estimates are greater than the CLP threshold value of BCF ≥ 500 L/kg.

The major metabolite in the fish liver S9 assay was confirmed to be the corresponding alcohol of the major isomer of Oxyoctaline Formate. In addition, minor peaks corresponding to the alcohols of the two minor isomers were detected. Importantly, two glucuronic acid conjugates were detected as Phase II metabolites by LC-MS analysis.This is a major elimination pathway in fish.Several peaks with similar m/z as the two glucuronides were found indicating the formation of these glucuronides with all isomers of Oxyoctaline Formate.Thus metabolite dataindicates that the minor isomers in Oxyoctaline Formate are also not bioaccumulative.

Since the alcohol of Oxyoctaline Formate was identified as major metabolite in trout liver S9 fractions, its metabolic turnover rate was also determined in order to evaluate its bioaccumulation potential. The test item was a reference sample of the alcohol that had been synthesised (GR-80-3313). Enzymatic turnover was observed with 71.9% decrease of the starting concentration within a 60 minute exposure period. The in vitro intrinsic clearance rate was 1.27 ml/h/mg protein. The resulting predicted BCF was 115 L/kg (fu=1) and 382 L/kg (fu calculated). The predicted BCFs for the metabolite were lower compared to the parent due to the more rapid biotransformation of GR-80-3313 and lower log Kow value.

In summary, both Oxyoctaline Formate and the corresponding alcohol metabolite have estimated BCF values that are well below the B definitive criterion of BCF > 2000 L/kg. Based on this information, it is concluded that there is no need for further investigation of aquatic bioaccumulation with fish.