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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
The test substance was incubated with trout hepatocytes. Metabolic competence of the test system was assessed using 4-nonylphenol as a positive control, and incubations containing heat-inactivated cells were used as a negative control. Incubation mixture samples were removed at selected time points and analysed for test substance. The metabolic clearance of the test substance was calculated.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
EC Number:
405-800-7
EC Name:
4,4',4''-(ethan-1,1,1-triyl)triphenol
Cas Number:
27955-94-8
Molecular formula:
C20H18O3
IUPAC Name:
4-[1,1-bis(4-hydroxyphenyl)ethyl]phenol
Details on test material:
- Purity: 99.5%
Radiolabelling:
no

Test animals

Species:
other: Rainbow trout
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Limestone Springs Fishing Preserve, Richland, PA
- Age at study initiation: not reported; 10-12 inches in length
- Weight at study initiation: 350 g
- Fasting period before study: 24 hours
- Diet: freshly hatched, live brine shrimp and AquaMax® Starter Fingerling 300 5D03; fed twice daily on weekdays and once daily on weekends and holidays

ENVIRONMENTAL CONDITIONS
- Water temperature (°C): 11.7-12.7 (mean 12.2)

Administration / exposure

Route of administration:
other: in vitro incubation of hepatocytes
Vehicle:
DMSO
Details on exposure:
PREPARATION OF STOCK SOLUTIONS:
-Test substance: final concentration of 10 mM in DMSO
-Positive control: 4-nonylphenol at a final concentration of 10 mM in DMSO
-Negative control: DMSO
Duration and frequency of treatment / exposure:
See details on dosing and sampling below.
Doses / concentrations
Remarks:
Doses / Concentrations:
The final concentration of test substance in the reaction mixture was 25 µM
No. of animals per sex per dose / concentration:
Cells were diluted to a final concentration of approximately 10 x 10E6 cells/mL in buffer
Control animals:
other: Both positive and negative control experiments were run using trout hepatocytes.
Positive control reference chemical:
4-nonylphenol
Details on study design:
Hepatocytes were isolated by three-stage collagenase perfusion. All perfusion steps were carried out at 10ºC and a flow rate of approximately 2 mL/min. Cells were diluted to a final concentration of approximately 10 x 10E6 cells/mL in buffer, and maintained on ice for approximately 30 minutes until used. Cells for control reactions were heat inactivated by microwave irradiation for 30 seconds and cooled prior to use.
The experimental design consisted of the following groups:
Viable cells + test substance (duplicate incubations)
Heat inactivated cells + test substance
Viable cells + positive control compound
Heat inactivated cells + positive control compound
Viable cells + vehicle

Incubations were carried out in closed 20 mL scintillation vials in a chilled orbital shaker at 10-12ºC. Vials containing 4 mL of cell suspension were preincubated for 5 minutes with shaking, and the reactions were initiated by addition of 10 µL of stock test substance, positive control substance, or vehicle.
Sample Processing and Analysis:
200 µL aliquots of test substance reaction mixtures were removed and added to 400 µL of ice cold acetonitrile, vortexed, and centrifuged to sediment cellular debris. The supernatant was analyzed by HPLC. 100 µL aliquots of positive control reaction mixture were removed and added to 400 µL of ethyl acetate, vortexed, and centrifuged to separate the organic and aqueous layers. The organic phase was analyzed by GC/MS.
Details on dosing and sampling:
- Time and frequency of sampling: Samples were collected and analyzed for the appropriate compound at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 24 hours after initiation of the incubation period.
Statistics:
Data were analyzed using a simple one-compartment pharmacokinetic model. Calculations were carried out using Microsoft Excel. The elimination rate constants for the test substance in hepatocyte reactions (k) were calculated as the negative slope of the semi-log plot of test substance concentration vs reaction time. Hepatocyte clearance was scaled to estimate the whole animal hepatic clearance using physiological parameters taken from the literature; see Table 1 below.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
A time dependent increase in unresolved UV-absorbing components eluting near the void volume was observed, suggesting biotransformation of the test substance to highly polar metabolites such as sulfate or glucuronide conjugates.

Any other information on results incl. tables

The liver perfusion procedure yielded 2.9 x 10E8 cells with an overall viability of approximately 100%. The metabolic competence of the cell preparation was demonstrated by rapid metabolism of the positive control compound 4-nonylphenol. The test substance disappeared rapidly in incubations containing viable trout hepatocytes, and was completely cleared within 24 hours. While there was also a time-dependent decrease in test substance concentration in incubations with heat-inactivated hepatocytes, the disappearance was slower and incomplete at the 24 hour sampling time. The lack of other UV-absorbing peaks in HPLC chromatograms suggests that the test substance was not spontaneously decomposing over the course of the reaction. Incubation of the test substance in reaction vials with buffer only, no cells, showed that the rate of disappearance was similar with and without cells. This experiment suggests that non-specific adsorption to the reaction vessel was probably responsible for the disappearance of the test substance in control reactions. The rate constant for metabolic clearance was calculated as the difference between the elimination rate constants for reactions containing viable and inactivated cells. The results of this analysis are presented in Table 2.

Table 2: Kinetic Values for Metabolism of the Test Substance

Parameter

Value

Elimination Constants

 

   Viable Cells

0.774/hr

   Heat Inactivated Cells

0.062/hr

   Metabolic Rate Constant

0.713/hr

 

 

Metabolic Clearance

0.071 mL/hr/106cells

 

 

Estimated Intrinsic Clearance

57.7 mL/hr

 

 

Estimated Hepatic Clearance

9.5 mL/hr

Analysis of the data from these experiments results in an intrinsic hepatocyte clearance of 0.0713 mL/hr/10E6 cells. Scaling this value to the whole animal yields an estimate of approximately 57.7 mL/hr. Since the expected hepatic blood flow for a 350 g rainbow trout is approximately 11.4 mL/hr, the clearance of the test substance in rainbow trout is predicted to be flow-limited. That is, the test substance would be metabolized by the liver as rapidly as it is delivered by the blood. A venous equilibration model was applied to predict hepatic clearance from intrinsic clearance. In this model, the liver is viewed as a well stirred compartment with complete mixing of portal and hepatic arterial blood in the sinuses. The further assumption is made that binding of the test substance to plasma proteins is insignificant. Under these conditions, the in vivo hepatic clearance of the test substance was predicted to be 9.5 mL/hr.

 

Applicant's summary and conclusion

Conclusions:
The test substance was rapidly metabolized in isolated trout hepatocytes, and is expected to be rapidly metabolized in whole fish. Since the hepatic intrinsic clearance of the test substance in whole animals predicted from the hepatocyte data is well in excess of the hepatic blood flow, hepatic clearance of the test substance in whole animals is likely to be flow limited.
Executive summary:

The metabolism of the test substance was examined in freshly isolated trout hepatocytes. The test substance (25 µM) was incubated with trout hepatocytes (10E6 cells/mL) in a physiological buffer at approximately 10ºC. Metabolic competence was assessed using 4-nonylphenol as a positive control, and incubations containing heat inactivated cells were used as a negative control. Samples of the incubation mixtures were removed at selected time points up to 24 hours and analyzed for the test substance by high performance liquid chromatography. Time-dependent disappearance of the test substance was observed in incubation mixtures containing viable hepatocytes, as well as in incubations containing heat-inactivated hepatocytes. A subsequent experiment conducted in reaction buffer in the absence of cellular protein suggests that disappearance of test substance in control reactions may be due to non-specific adsorption to the walls of the reaction vials. The metabolic rate constant for the test substance in trout hepatocytes was determined by difference between reactions containing viable and non-viable cells. The metabolic clearance of the test substance in trout hepatocytes was calculated to be 0.071 mL/hr/10E6 cells. This value was scaled to a whole animal intrinsic clearance of 57.7 mL/hr using literature values for physiological parameters. Since the estimated intrinsic clearance of the test substance is significantly greater than calculated hepatic blood flow for rainbow trout (11.4 mL/hr), clearance of the test substance in vivo is expected to be flow-limited. Hepatic clearance for the test substance in rainbow trout was estimated to be 9.5 mL/hr, based on a venous equilibration model.