Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

basic toxicokinetics in vitro / ex vivo
Adequacy of study:
other information
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable publication which meets basic scientific principles.

Data source

Reference Type:
Comparative metabolism of geranyl nitrile and citronellyl nitrile in mouse, rat, and human hepatocytes
Kemper R.A., Nabb D.L., Gannon S.A., Snow T.A., Api A.M.
Bibliographic source:
Drug Metabolism and Diposition, Vol 34, No. 6, 1019-1029

Materials and methods

Objective of study:
Test guideline
no guideline followed
Principles of method if other than guideline:
The metabolic clearance and biotransformation of citronellyl nitrile was determined in primary hepatocytes from mice, rats, and humans. For determination of intrinsic clearance, citronellyl nitrile was incubated with hepatocytes in sealed vials and the headspace was sampled periodically by solid-phase microextraction and analyzed by gas chromatography/mass spectrometry. For metabolite identification, citronellyl nitrile was incubated with hepatocytes from each species for 60 min, and reaction mixtures were extracted and analyzed by mass spectroscopy.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Citronellyl nitrile (3,7-dimethyl-6-octenenitrile)

Administration / exposure

Details on study design:
- Hepatocyte Isolation. Male Sprague-Dawley rats and CD mice (6-8 weeks old) were obtained from Charles River Laboratones (Raleigh, NC). Rat and mouse hepatocytes were prepared by two-stage collagenase perfusion by a modification of the method of Seglen (1976) using commerciaily available reagents. Isolated rat and mouse hepatocytes were purified by centrifugation through 40% Percoll at 4°C. Upon receipt, human hepatocytes were washed twice with L-15 medium, diluted to the desired concentration, and maintained on ice until used. Cell viability and yield in all species were determined by trypan blue exclusion. In all cases, hepatocyte viability was >85%.
Details on dosing and sampling:
- Clearance Kinetics. Hepatocytes were suspended in L-15 medium at a concentration of 2.5E6 cells/mL. Reactions were carried out in 10mL headspace vials fitted with Teflon septa in a final volume of 1 mL/vial. Kinetic experiments were conducted using an automated assay system composed of a Gerstel MPS-2 autosampler (Gerstel Inc., Baltimore, MD) with a temperature controlled shaking incubator coupled to a GC/MS system as described below. Hepatocyte suspensions were preincubated at 37°C for 5 min, after which the test compound was introduced into the liquid phase of the reaction mixture through the septum, resulting in an initial concentration of 25 µM. The concentration of acetonitrile in the incubation mixtures was 0.5% (v/v). At selected time points, the headspace above the reaction mixtures was sampled for 10 seconds by solid-phase microextraction (SPME) using a 1 cm x 100-µm nonbonded polydimethylsiloxane SPME fiber (Sigma-Aldrich, St. Louis, MO). Headspace samples were analyzed by GC/MS using selected ion monitoring as described below. Calibration standards were prepared by incubating varying concentrations of test compounds with heat-inactivated hepatocytes at 37°C in septum-cap vials. Quantitative analysis was accomplished by GC/MS with electron impact (EI) ionization using selected ion monitoring. Ion m/z 69 was used as the target ion for quantification. Ion m/z 136 was used as qualifier ion. Analyses were conducted using an HP6890 GC coupled to 5970 GC/MSD (Agilent Technologies, Wilmington, DE) equipped with a 30m x 0.25mm x 1-µm DB-5MS column (J&W; Agilent Technologies). The following chromatographic parameters were used for quantitative analysis: injection port temperature: 280°C, desorption time: 30 seconds, split ratio: 10:1, carrier gas: helium, flow rate: 0.4 mL/min, oven temperature: 280°C; MS transfer line temperature: 280°C. Under these conditions, citronellyl nitrile eluted at 1.28 min.

- Biotransformation Experiments. For biotransformation experiments, hepatocytes were suspended at a concentration of 5E6 cells/mL in L-15 medium. Incubations were carried out in 24-mL reaction vials fitted with Teflon-lined septa in a final volume of 5 mL. Cell suspensions were preincubated for 5 min at 37°C before the introduction of the test compound to provide an initial concentration of 250 µM. Cell suspensions were incubated with shaking for 60 min, after which reactions were terminated by rapid cooling in an ice bath. Hepatocytes were lysed using an ultrasonic probe, saturated with NaCl (0.5 g/mL), and extracted twice with 10 mL of ethyl acetate. The organic fractions were combined and concentrated to a final volume of approximately 0.25 mL under a gentle stream of nitrogen at room temperature. Organic extracts were stored at approximately -20°C for up to 2 days until analyzed by GC/MS. An aliquot of the aqueous phase was removed and stored at approximately 5°C until analyzed by LC/MS. Repeated analysis of stored samples confirmed sufficient stability of major metabolites for the duration of the experiments under these conditions. Although citronellyl nitrile is sufficiently volatile to allow quantitative analysis in the headspace vapor, it is approximately 2000 times less volatile than the extraction solvent ethyl acetate (vapor pressure, 0.04 mm Hg at 25°C for citronellyl nitrile versus 76 mmHg for ethyl acetate). Moreover, all of the synthetic oxidative metabolites of citronellyl nitrile were substantially less volatile than the parent compounds (vide infra). Thus, although recovery of test compounds and metabolites was not determined, it is unlikely that substantial loss of primary metabolites during concentration of extracts occurred.

- Metabolite Identification. Phase I metabolites of citronellyl nitrile were identified by GC/MS using a scan range of 29 to 300 amu. The GC oven was programmed as follows: initial oven temperature, 100°C; initial time, 1 min; temperature ramp, 2O°C/min; final oven temperature, 280°C; final time, 5 min. In some cases, the molecular weights of metabolites were confirmed by GC analysis with methane chemical ionization time of flight MS detection. Analyses were canied out using a Micromass GC-time of flight (Micromass Ltd., Manchester, UK). The column and GC conditions used were the same as those described above for EI-GC/MS.
Phase II metabolites were identified by time of flight LC/MS using negative electrospray ionization. Analyses were carried out using a Waters Alliance 2790 HPLC system (Waters, Milford, MA) coupled to a Micromass Q-ToF-II hybrid quadrupole time-of-flight mass spectrometer (Micromass Ltd.). Separation of metabolites was accomplished using a Zorbax SB-C18 RP, 2.1 x 150-mm, 5-µm particle size column (Agilent Technologies). The mobile phase was 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B with a flow rate of 0.3 mL/min. The HPLC was programmed with a linear gradient starting at 3% B increasing to 100% B over 30 min, holding 5 min, returning to 3% B at 35.1 min, and holding for 5 min.
- NMR Analysis. 1H NMR was performed on a 360-MHz Bruker AMX spectrometer (Bruker BioSpin, Bremmen, Germany) at 23°C with a dual 1H/19F probe. Samples were measured in CDCl3. One-dimensional 90°-pulse spectra were collected with 5-kHz sweep width, 10-pulse delay, 16 K data points, and four scans. Two-dimensional correlation spectroscopy spectra were recorded with 512 complex t1 increments, 8192 t2 points, and 16 scans for each free induction decay. Chemical shifts were referenced to tetramethylsilane.
Noncompartmental kinetic analysis was carried out using WinNonlin version 4.0 (Pharsight, Mountain View, CA). Hepatocyte intrinsic clearance values were calculated by dividing the model-independent clearance (CLz) by the number of cells in the reaction mixture.
CLh (mL/min/10E6 cells) = CLz/2.5 (10E6).
Values were then scaled to estimate whole animal intrinsic clearance (CLi) assuming a liver weight equivalent to 5% of body weight (50 g/kg) and a hepatocellularity of 1.28E8 cells/g liver for rodents (Seglen, 1976). The corresponding values for humans were a liver weight equivalent to 2.5% of body weight (25 g/kg) and a hepatocellularity of 1.37E8 cells/g liver (Arias et al., 1982). Equation 2 illustrates the calculation for rodents.
CLi (mL/min/kg) = CLh x (128[x 10E6]cells/g liver) x (50 g liver/kg body weight).

Results and discussion

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
- Metabolic Clearance: Clearance in mouse hepatocytes was approximately 2 times more rapid than in rat hepatocytes (Mouse: T1/2 = 1.28min, rat = 2.89min, human = 2.62 - 19.19min). Two of the three donors (HL1 and HL3) metabolized Citronellyl nitrile much more slowly than rodents, whereas the third donor (HL2) metabolized the compounds at rates comparable with rat.

- Biotransformation of Citronellyl nitrile in Rat, Mouse, and Human Hepatocytes: Only three phase I metabolites could be identified unequivocally by comparison to synthetic standards (C1, C2, and C4). Two additional peaks were tentatively assigned structures based on mass spectral similanty to synthetic standards or published spectra (C3 and C7). Four phase I metabolites of Citronellyl nitrile were common to all species examined.
6,7-Epoxycitronellyl nitriles (C1, C2) were identified by comparison to the synthetic standards. Interestingly, only one stereoisomer of 6,7-epoxycitronellyl nitrile was observed in mouse hepatocytes, whereas both stereoisomers were observed in rat and human hepatocytes in approximately equal amounts . C3 was tentatively assigned the structure of 5-hydroxycitronellyl nitrile
(5-hydroxy-3,7-dimethyl-oct-6-enenitrile). C3 was observed in human hepatocytes. The other metabolite common to all three species was
8-hydroxy-CN (C4). A metabolite unique to rat hepatocyte extracts was observed with a retention time of 10.24 min (C5) and could not be assigned a definitive structure based on its mass spectrum. In mouse, a novel metabolite (C6) eluted just after the CN epoxides (RT, 8.37 min). In human hepatocytes, a metabolite with a mass spectrum identical to synthetic 8-hydroxycitronellyl nitrile was detected at a slightly earlier retention time. This metabolite (C7) was tentatively identified as
9-hydroxycitronellyl nitrile. Two phase II metabolites were tentatively identified in the aqueous residue of rat hepatocyte extracts. Metabolite C8 had a molecular weight, consistent with a glutathione conjugate of 6,7-epoxycitronellyl nitrile. This metabolite was also detected in human hepatocytes. A second phase II metabolite of Citronellyl nitrile was detected (C9) with an apparent molecular weight, consistent with an
ether glucuronide of Citronellyl nitrile. Presence of 8-hydroxy-citronellyl nitrile (C4) in the organic extract suggests that this metabolite was a likely target for glucuronidation. Metabolite C9 was also detected in mouse hepatocyte incubations but not in human extracts. In human hepatocytes, a metabolite (C10) was tentatively identified as an
acylglucuronide of Citronellyl nitrile. The C8 position seems most likely based on steric considerations and on the presence of 8-hydroxycitronellyl nitnle in organic extracts.

Any other information on results incl. tables

In conclusion, Citronellyl nitrile was rapidly metabolized in hepatocytes from all species (Mouse: T1/2 = 1.28 min, rat = 2.89 min, human = 2.62 - 19.19 min). Within species intrinsic clearance was increased in the order human < rat < mouse. Major common pathways for biotransformation of citronellyl nitrile involved 1) epoxidation of the 6-alkenyl moiety followed by conjugation with glutathione, 2) hydroxylation of the terminal methyl group(s) followed by direct conjugation with glucuronic acid in rodents or further oxidation to the corresponding acid in human cells, and 3) hydroxylation of the allylic C5 position. No evidence for either phase I or phase II metabolism of the conjugated nitrile moiety was obtained.

Applicant's summary and conclusion