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EC number: 243-797-2 | CAS number: 20407-84-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_environmental-fate-and-pathways.png)
Bioaccumulation: aquatic / sediment
Administrative data
Link to relevant study record(s)
- Endpoint:
- bioaccumulation in aquatic species: fish
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: In vitro data, generated from a pre-validated method, which is considered reliable and provides evidence for a reduced BCF due to metabolism for linear aliphatic saturated and unsaturated aldehydes.
- 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
- Test organisms (species):
- Oncorhynchus mykiss (previous name: Salmo gairdneri)
- Details on test organisms:
- TEST ORGANISM
Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were purchased from a recognised supplier: “Pooled Male Hatchery Rainbow Trout Liver S9”; product code CZDTRS9PL, Lot# TR015. The liver fractions were stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions was 800 g. 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.
Enzymatic activity was tested and internally verified. S9 fractions were only used for metabolism studies if significant enzymatic conversion
of the reference substances was observed.
ACCLIMATION
Not applicable. - Route of exposure:
- other: in vitro assay
- Details on test conditions:
- The in vitro assay utilised the following Test System:
A range finding experiment was performed to determine the optimal incubation times to be used in the main experiments:
1. A stock solution of test item (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).
2. 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 incubated at 12°C in a Thermomixer 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 in the section entitled "any other information on materials and methods incl. tables".
3. In the range finding experiment: test item (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 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, 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 (centrifuge, 12 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis.
(see 'Details on Analytical Methods' section).
In the two independent main experiments:
1. Test item (1 µM) was incubated in presence of 0.25 mg/ml active S9 protein and cofactors in triplicate for up to 10 minutes (1st main experiment) or 2 minutes (2nd main experiment) as described above. Reactions were stopped at time 0, 2.5, 5,7.5 and 10 minutes (1st main experiment) or at time 0, 0.5, 1, 1.5 and 2 minutes (2nd main experiment). As control, the test substance was incubated in presence of heat inactivated S9 protein (0.25 mg/ml) and cofactors for 0 minutes and 10 or 2 minute, and in presence of active S9 protein in absence of any cofactors for 10 or 2 minutes, respectively (1st and 2nd main experiment).
2. Reactions were stopped and extracted as previously described. - Details on estimation of bioconcentration:
- The model version used is indicated in the full study report and corresponds with that used in from Nichol [2006] primary reference. 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 Kmetabvalue 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. The partition based BCF (assuming no metabolism) and the refined BCF (incorporating biotransformation rate estimates) for the test item have been estimated in the full study report; and are presented by the applicant. - Type:
- BCF
- Value:
- 3 972 L/kg
- Remarks on result:
- other: Partitioning based BCF; assumes no metabolism
- Type:
- BCF
- Value:
- 194 L/kg
- Remarks on result:
- other: Refined BCF; includes biotransformation rate estimates (fu calculated)
- Type:
- BCF
- Value:
- 159 L/kg
- Remarks on result:
- other: Refined BCF; includes biotransformation rate estimates (fu =1)
- Details on results:
- 1. Metabolic Turnover Rate of test item in Fish Liver S9 Fractions
An almost complete, very rapid decrease of test item aldehyde was observed within 30 minutes incubation (91.7% decrease; 92.0% decrease in 120 minutes) in the range finding experiment with active S9 protein (1 mg/ml). In the absence of any cofactors added, a decrease of test item was also observed (89.5% decrease within 120 minutes). A slight decrease of test item was observed in the control samples with heat inactivated protein (1 mg/mL) (6.3% decrease within 120 minutes).
Incubations were carried in the presence of lower concentrations of S9 protein (0.25 mg/mL) and for shorter incubation periods (up to 10 minutes in the 1st main experiment). However, enzymatic turnover was still very rapid and a 64.6% decrease of test item was observed already in 2.5 minutes. Thus, the experiment was repeated using an even shorter incubation period (2nd main experiment). Enzymatic turnover of test item was very rapid in presence of cofactors: 79.7% decrease was observed in 2 minutes.
A slower turnover of test item was found with active S9 protein in the absence of cofactors (36.5% decrease in 2 minutes). A similar, slow decrease of the parent was observed with inactive S9 protein (27.7% decrease in 2 minutes).
The in vitro intrinsic clearance was calculated from the log-transformed measured concentrations of parent compound as a function of time for test item: 135.68 ml/h/mg protein.
Since there was a significant, but slower decrease of the parent in presence of inactive S9 protein a corrected in vitro intrinsic clearance was calculated by subtraction of the putative abiotic decrease: 85.98 ml/h/mg protein. This putative abiotic decrease followed first order kinetic and was slower in the range finding experiment with a higher concentration of inactive protein. We do not know the reason for this difference. Causes for the putative abiotic decrease could be due to solubility issues, abiotic reaction with proteins, or most likely due to abiotic oxidation to corresponding fatty acid. This effect was not further studied, since the difference in the two enzymatic turnover rates did have only a minor impact on the refined BCF estimate.
2. Calculation of Refined BCF Estimates for test item aldehyde
Further details are provided in sections “Details on estimation of the bioconcentration” and "Any other information on results incl. tables").
Metabolic turnover rates of the test item in fish liver S9 fractions were very rapid and similar in both major, independent experiments. The following average reaction rates were used for the calculation of the refined BCF estimates: 33.92/h and 21.49/h (corrected rate).
The partitioning based BCF assuming no metabolism (BCFp) for the test item based on the measured Log Kow values of 4.9 was calculated as part of the in vitro – in vivo extrapolation model of Nichols [2006] and is presented by the applicant.
Including the in vitro metabolism rates of test item in trout S9 fractions and other parameters (see full study report), the refined BCF estimates (BCFTOT) were 159 L/kg for both enzymatic turnover rates using an assumed fu of 1.0. Since both reaction rates, 33.92/h and 21.49/h (corrected rate) are very high, the impact of the decrease of the rate by correction due to abiotic losses on the refined BCF estimate using a fu= 1.0 is negligible. Nichols et al. showed that increasing a hypothetical hepatic biotransformation rate from 1.0 to 10/h did not have an impact on the BCFTOT, whereas an increase of lower rates by factor 10, e.g. from 0.01 to 0.1/h did result in a decrease of the BCFTOT when fu is set to 1.0
The refined BCF estimates were 181 L/kg and 194 L/kg (corrected rate) using different binding to serum in vivo vs. in vitro. (See table 1.). - Validity criteria fulfilled:
- yes
- Conclusions:
- Metabolic turnover by trout liver S9 fractions was observed for the test substance, indicating that the test substance is expected to be metabolised in vivo. Refined BCF estimates incorporating in vitro metabolism were 194 L/kg using a theoretically calculated fu and 159 L/kg assuming no effect of differential binding to serum (fu=1.0).
- Executive summary:
A study was performed to assess the in vitro stability of test item aldehyde in fish liver S9 fractions. The method followed was a standardised assay, prevalidated by a consortium under the coordination of HESI/ILSI and under corresponding peer reviewed methodology. Full details of the in vitro assay methodology are provided in the full study report and are presented by the applicant. Very rapid enzymatic turnover of the test item aldehyde 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 on Log Kow without biotransformation estimates. 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 refine BCF estimates based on in vitro turnover rates of six reference fragrance molecules by trout liver S9 fractions. These refined BCF estimates are comparable to known in vivo BCF values (as detailed in the full study report). 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 substances. Refined BCF estimates for test item incorporating in vitro metabolism was 159 L/kg using an assumed fu of 1.0 and 181-194 L/kg using a theoretically calculated fu.
- Endpoint:
- bioaccumulation in aquatic species: fish
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: In vitro data, generated from a pre-validated method, for analogue substance which is considered reliable and provides evidence for a reduced BCF due to metabolism for linear aliphatic saturated and unsaturated aldehydes.
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have common structural features in the same relative positions. The source and target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action. Further information is included in attachment to IUCLID section 13.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Further information is included in attachment to IUCLID section 13
3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a chemically similar substance with common metabolism and common or similar degradants of the target substance. Further information is included in attachment to IUCLID section 13
4. DATA MATRIX
Further information is included in attachment to IUCLID section 13. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- 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
- Test organisms (species):
- Oncorhynchus mykiss (previous name: Salmo gairdneri)
- Details on test organisms:
- TEST ORGANISM
Rainbow Trout (Oncorhynchus mykiss) liver S9 fractions were purchased from a recognised supplier: “Pooled Male Hatchery Rainbow Trout Liver S9”; product code CZDTRS9PL, Lot# TR015. The liver fractions were stored at -80°C. The average body weight of the fish used for the preparation of S9 fractions was 800 g. 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.
Enzymatic activity was tested and internally verified. S9 fractions were only used for metabolism studies if significant enzymatic conversion
of the reference substances was observed.
ACCLIMATION
Not applicable. - Route of exposure:
- other: in vitro assay
- Details on test conditions:
- The in vitro assay utilised the following Test System:
A range finding experiment was performed to determine the optimal incubation times to be used in the main experiments:
1. A stock solution of test item (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).
2. 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 incubated at 12°C in a Thermomixer 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 in the section entitled "any other information on materials and methods incl. tables".
3. In the range finding experiment: test item (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 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, 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 (centrifuge, 12 000 rpm, 5 min, room temperature) and subjected to GC-MS analysis.
(see 'Details on Analytical Methods' section).
In the two independent main experiments:
1. Test item (1 µM) was incubated in presence of 0.25 mg/ml active S9 protein and cofactors in triplicate for up to 10 minutes (1st main experiment) or 2 minutes (2nd main experiment) as described above. Reactions were stopped at time 0, 2.5, 5,7.5 and 10 minutes (1st main experiment) or at time 0, 0.5, 1, 1.5 and 2 minutes (2nd main experiment). As control, the test substance was incubated in presence of heat inactivated S9 protein (0.25 mg/ml) and cofactors for 0 minutes and 10 or 2 minute, and in presence of active S9 protein in absence of any cofactors for 10 or 2 minutes, respectively (1st and 2nd main experiment).
2. Reactions were stopped and extracted as previously described. - Details on estimation of bioconcentration:
- The model version used is indicated in the full study report and corresponds with that used in from Nichol [2006] primary reference. 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 Kmetabvalue 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. The partition based BCF (assuming no metabolism) and the refined BCF (incorporating biotransformation rate estimates) for the test item have been estimated in the full study report; and are presented by the applicant. - Type:
- BCF
- Value:
- 3 972 L/kg
- Remarks on result:
- other: Partitioning based BCF; assumes no metabolism
- Type:
- BCF
- Value:
- 194 L/kg
- Remarks on result:
- other: Refined BCF; includes biotransformation rate estimates (fu calculated)
- Type:
- BCF
- Value:
- 159 L/kg
- Remarks on result:
- other: Refined BCF; includes biotransformation rate estimates (fu =1)
- Details on results:
- 1. Metabolic Turnover Rate of test item in Fish Liver S9 Fractions
An almost complete, very rapid decrease of test item aldehyde was observed within 30 minutes incubation (91.7% decrease; 92.0% decrease in 120 minutes) in the range finding experiment with active S9 protein (1 mg/ml). In the absence of any cofactors added, a decrease of test item was also observed (89.5% decrease within 120 minutes). A slight decrease of test item was observed in the control samples with heat inactivated protein (1 mg/mL) (6.3% decrease within 120 minutes).
Incubations were carried in the presence of lower concentrations of S9 protein (0.25 mg/mL) and for shorter incubation periods (up to 10 minutes in the 1st main experiment). However, enzymatic turnover was still very rapid and a 64.6% decrease of test item was observed already in 2.5 minutes. Thus, the experiment was repeated using an even shorter incubation period (2nd main experiment). Enzymatic turnover of test item was very rapid in presence of cofactors: 79.7% decrease was observed in 2 minutes.
A slower turnover of test item was found with active S9 protein in the absence of cofactors (36.5% decrease in 2 minutes). A similar, slow decrease of the parent was observed with inactive S9 protein (27.7% decrease in 2 minutes).
The in vitro intrinsic clearance was calculated from the log-transformed measured concentrations of parent compound as a function of time for test item: 135.68 ml/h/mg protein.
Since there was a significant, but slower decrease of the parent in presence of inactive S9 protein a corrected in vitro intrinsic clearance was calculated by subtraction of the putative abiotic decrease: 85.98 ml/h/mg protein. This putative abiotic decrease followed first order kinetic and was slower in the range finding experiment with a higher concentration of inactive protein. We do not know the reason for this difference. Causes for the putative abiotic decrease could be due to solubility issues, abiotic reaction with proteins, or most likely due to abiotic oxidation to corresponding fatty acid. This effect was not further studied, since the difference in the two enzymatic turnover rates did have only a minor impact on the refined BCF estimate.
2. Calculation of Refined BCF Estimates for test item aldehyde
Further details are provided in sections “Details on estimation of the bioconcentration” and "Any other information on results incl. tables").
Metabolic turnover rates of the test item in fish liver S9 fractions were very rapid and similar in both major, independent experiments. The following average reaction rates were used for the calculation of the refined BCF estimates: 33.92/h and 21.49/h (corrected rate).
The partitioning based BCF assuming no metabolism (BCFp) for the test item based on the measured Log Kow values of 4.9 was calculated as part of the in vitro – in vivo extrapolation model of Nichols [2006] and is presented by the applicant.
Including the in vitro metabolism rates of test item in trout S9 fractions and other parameters (see full study report), the refined BCF estimates (BCFTOT) were 159 L/kg for both enzymatic turnover rates using an assumed fu of 1.0. Since both reaction rates, 33.92/h and 21.49/h (corrected rate) are very high, the impact of the decrease of the rate by correction due to abiotic losses on the refined BCF estimate using a fu= 1.0 is negligible. Nichols et al. showed that increasing a hypothetical hepatic biotransformation rate from 1.0 to 10/h did not have an impact on the BCFTOT, whereas an increase of lower rates by factor 10, e.g. from 0.01 to 0.1/h did result in a decrease of the BCFTOT when fu is set to 1.0
The refined BCF estimates were 181 L/kg and 194 L/kg (corrected rate) using different binding to serum in vivo vs. in vitro. (See table 1.). - Validity criteria fulfilled:
- yes
- Conclusions:
- Metabolic turnover by trout liver S9 fractions was observed for the source substance, indicating that the source and target substance are expected to be metabolised in vivo. Refined BCF estimates incorporating in vitro metabolism were 194 L/kg using a theoretically calculated fu and 159 L/kg assuming no effect of differential binding to serum (fu=1.0).
- Executive summary:
A study was performed on a source substance to assess the in vitro stability of test item aldehyde in fish liver S9 fractions. The method followed was a standardised assay, prevalidated by a consortium under the coordination of HESI/ILSI and under corresponding peer reviewed methodology. Full details of the in vitro assay methodology are provided in the full study report and are presented by the applicant. Very rapid enzymatic turnover of the test item aldehyde 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 on Log Kow without biotransformation estimates. 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 refine BCF estimates based on in vitro turnover rates of six reference fragrance molecules by trout liver S9 fractions. These refined BCF estimates are comparable to known in vivo BCF values (as detailed in the full study report). 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 substances. Refined BCF estimates for test item incorporating in vitro metabolism was 159 L/kg using an assumed fu of 1.0 and 181-194 L/kg using a theoretically calculated fu.
Referenceopen allclose all
Table 1. Selected parameters, refined BCF estimates calculated with the in vitro – in vivo extrapolation model for test item aldehyde #1
Parameter |
Test item (initial rate)
|
Test item (corrected rate) #2 |
||
|
fu calc #3 |
fu=1.0 #4 |
fu calc #3 |
fu=1.0 #4 |
Input parameter: in vitro data |
|
|
|
|
S9 concentration (CS9) (mg/mL) |
0.25 |
0.25 |
0.25 |
0.25 |
Reaction rate (Rate) (1/h) |
33.92 |
33.92 |
21.49 |
21.49 |
Input Parameter |
|
|
|
|
Log Kow |
4.9 |
4.9 |
4.9 |
4.9 |
|
|
|
|
|
Calculated Parameters |
|
|
|
|
Partitioning based BCF, assuming no metabolism (BCFp) |
3972 |
3972 |
3972 |
3972 |
In vitro intrinsic clearance (CLIN VITRO,INT) (ml/h/mg protein) |
167.0 |
167.0 |
105.8 |
105.8 |
In vivo intrinsic clearance (CLIN VIVO,INT) (L/d/kg fish) |
3007 |
3007 |
1905 |
1905 |
Scaled clearance for 10 g fish (CLIN VIVO,INT,10) (L/d/kg fish) |
8992 |
8992 |
5697 |
5697 |
|
|
|
|
|
Corrected clearance for 10 g fish (CLIN VIVO,INT,10,CORR) (l/d/kg fish) |
22481 |
22481 |
14243 |
14243 |
Hepatic clearance (CLH) (L/d/kg fish) |
21.4 |
24.5 |
19.9 |
24.5 |
Whole-body metabolism rate (kMETAB) (1/d) |
3.3 |
3.7 |
3.0 |
3.7 |
BCF, on a total conc. basis, w/out lipid norm.(BCF TOT ) (L/kg) |
181 |
159 |
194 |
159 |
|
|
|
|
|
#1; J. Nichols, personal communication, based on a previous publication from Nichols et al. [2006] (primary reference and listed in full study report)
#2: corrected rate: Enzymatic turnover rate of test item was corrected by subtracting the putative abiotic decrease of the parent in presence of inactive S9 protein
#3: fu, plasma binding correction term; “fu calc”, hepatic clearance is calculated taking into account a theoretically calculated difference between in vitro and in vivo binding
#4: fu, plasma binding correction term; “fu = 1.0”, hepatic clearance is calculated assuming equal in vitro and in vivo binding by setting fu = 1.0
Table 1. Selected parameters, refined BCF estimates calculated with the in vitro – in vivo extrapolation model for test item aldehyde #1
Parameter |
Test item (initial rate)
|
Test item (corrected rate) #2 |
||
|
fu calc #3 |
fu=1.0 #4 |
fu calc #3 |
fu=1.0 #4 |
Input parameter: in vitro data |
|
|
|
|
S9 concentration (CS9) (mg/mL) |
0.25 |
0.25 |
0.25 |
0.25 |
Reaction rate (Rate) (1/h) |
33.92 |
33.92 |
21.49 |
21.49 |
Input Parameter |
|
|
|
|
Log Kow |
4.9 |
4.9 |
4.9 |
4.9 |
|
|
|
|
|
Calculated Parameters |
|
|
|
|
Partitioning based BCF, assuming no metabolism (BCFp) |
3972 |
3972 |
3972 |
3972 |
In vitro intrinsic clearance (CLIN VITRO,INT) (ml/h/mg protein) |
167.0 |
167.0 |
105.8 |
105.8 |
In vivo intrinsic clearance (CLIN VIVO,INT) (L/d/kg fish) |
3007 |
3007 |
1905 |
1905 |
Scaled clearance for 10 g fish (CLIN VIVO,INT,10) (L/d/kg fish) |
8992 |
8992 |
5697 |
5697 |
|
|
|
|
|
Corrected clearance for 10 g fish (CLIN VIVO,INT,10,CORR) (l/d/kg fish) |
22481 |
22481 |
14243 |
14243 |
Hepatic clearance (CLH) (L/d/kg fish) |
21.4 |
24.5 |
19.9 |
24.5 |
Whole-body metabolism rate (kMETAB) (1/d) |
3.3 |
3.7 |
3.0 |
3.7 |
BCF, on a total conc. basis, w/out lipid norm.(BCF TOT ) (L/kg) |
181 |
159 |
194 |
159 |
|
|
|
|
|
#1; J. Nichols, personal communication, based on a previous publication from Nichols et al. [2006] (primary reference and listed in full study report)
#2: corrected rate: Enzymatic turnover rate of test item was corrected by subtracting the putative abiotic decrease of the parent in presence of inactive S9 protein
#3: fu, plasma binding correction term; “fu calc”, hepatic clearance is calculated taking into account a theoretically calculated difference between in vitro and in vivo binding
#4: fu, plasma binding correction term; “fu = 1.0”, hepatic clearance is calculated assuming equal in vitro and in vivo binding by setting fu = 1.0
Description of key information
BCF (in vitro): 194 L/kg ww, Rainbow Trout (Oncorhynchus mykiss) in vitro BCF including biotransformation using Liver S9 Fraction, 2013
Key value for chemical safety assessment
- BCF (aquatic species):
- 194 L/kg ww
Additional information
Key data : BCF in vitro estimate, 2013: Read-Across - SOURCE (dodecanal): A study was performed to assess the in vitro stability of test item aldehyde in fish liver S9 fractions. The method followed was a standardised assay, prevalidated by a consortium and under corresponding peer reviewed methodology. Full details of the in vitro assay methodology are provided in the full study report and are presented by the applicant. Very rapid enzymatic turnover of the test item aldehyde 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 on Log Kow without biotransformation estimates. 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 refine BCF estimates based on in vitro turnover rates of six reference fragrance molecules by trout liver S9 fractions. These refined BCF estimates are comparable to known in vivo BCF values (as detailed in the full study report). 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 substances. Refined BCF estimates for test item incorporating in vitro metabolism was 159 L/kg using an assumed fu of 1.0 and 181-194 L/kg using a theoretically calculated fu.
The applicant has provided in vitro BCF of a corresponding structural analogue and provided a detailed BCF assessment utilising (Q)SAR models supporting the read-across conclusion.
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