Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles; study acceptable as key study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Disposition of citral in male Fischer rats
Author:
Diliberto JJ, Usha G, Birnbaum LS
Year:
1988
Bibliographic source:
Drug Metab. Dispos. 16, 721-727

Materials and methods

Objective of study:
distribution
excretion
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Time course of distribution of 14C-label in tissues, blood, bile, urine, feces, expired air measured by liquid scintillation counting after single and repeated application; separation of unchanged and metabolized citral in blood and bile by HPLC (metabolites not identified)
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): citral and 14C-citral
- Analytical purity: >= 98%, purified by HPLC and concentrated into a biologically compatible solvent using a C18 reverse phase SDep_Pak cartridge
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components:
- Isomers composition: 74% geranial, 26% neral
- Specific activity (if radiolabelling): 10.7 mCi/mmol
- Locations of the label (if radiolabelling): C1 and C2
Radiolabelling:
yes
Remarks:
(1,2-14C)-citral

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, USA
- Age at study initiation: 2-3 month
- Weight at study initiation: 200-250 g
- Fasting period before study: no data
- Housing: individually
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/-2
- Humidity (%): 50 +/- 5
- Air changes (per hr): no data, air flow rate through the cages 0.3-0.4 L/min
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: oral gavage and intraveneous
Vehicle:
other: Ethanol/Emulphor EL-620/water: ratio 1:1:3 for oral application, ratio 1:1:8 for i.v. studies
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
radiolabeled citral was diluted with unlabeled citral to administer 1 µCi/rat for oral administration and 0.5 µCi/rat for i.v. administration

VEHICLE
- Concentration in vehicle: oral application 1, 10 or 100 mg/mL; i.v. application 2.5 mg/mL

Duration and frequency of treatment / exposure:
acute study: single treatment
multiple dosing study: oral pretreatment for 10 days with unlabelled citral at a dose of 5 mg/kg bw/day followed by single oral or i.v. dose of 5 mg/kg 14C-citral
Doses / concentrations
Remarks:
Doses / Concentrations:
oral application: 5, 50, 500 mg/kg/d
i.v. application: 5 mg/kg bw/d
No. of animals per sex per dose:
N ≥ 3
Details on study design:
- Dose selection rationale: 0.1, 1, and 10 % of the oral LD50 of 5000 mg/kg
Details on dosing and sampling:
PHARMACOKINETIC STUDY (distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air, blood, liver, kidneys, adrenals, thymus, spleen, brain, heart, lungs, testes, skin, adipose tissue, muscle, stomach contents, small intestine contents, large intestine contents, tail site (for i.v. application), bile
- Time and frequency of sampling:
excreta samples at 2, 4, 6, 8, 12, 16, 24, 32, 48, and 72 hrs;
tissue samples at sacrifice at 72 hrs p.a.
bile samples: at 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 min after dosing by cannulation of the common bile duct
blood samples: at 1, 2, 5, 10, 15, 20, 430, 45, 60, 90, 120, 150, 180, 210 and 240 min p.a. by cannulation of the jugular vein
air samples: the total air flow through the metabolism cages was continuously passed through two consecutive traps (charcoal trap and bubbler trap, see below)
- Quantitative measurements:
tissue radioactivity: after solubilisation with TEAH (tetraethyammonium hydroxide) followed by liquid scintillation counting
urine: liquid scintillation counting
faeces: after softening with Triton-X, homogenisation and solubilisation with TEAH, followed by liquid scintillation counting
expired air: activated charcoal filter traps for expired volatiles were extracted with ethanol, followed by liquid scintillation counting;
bubbler trap solutions containing ethanolamine and ethylene glycol monomethylether (3:7) to trap expired carbon dioxide were analyzed directly by liquid scintillation counting
bile samples: total radioactivity by direct liquid scintillation counting
blood samples: after solubilisation with TEAH and concentration on a Sep-Pak cartridge the eluate was used for liquid scintillation counting
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: bile and blood
- Time and frequency of sampling: see above
- From how many animals: 3
- Method type(s) for identification: bile samples were analyzed by HPLC to resolve into parent and metabolite fractions (no further identifcation of individual metabolites), quantitative analysis via radioactive labelling
- Limits of detection and quantification: not specified
Statistics:
computer based nonlinear regression analysis (RSI Bolt Beranek and Newman, Inc ., Cambridge, MA); additional two-tailed students t-test and z-statistics

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Rapid and complete absorption from gastrointestinal tract (91 - 95%). Citral was rapidly and completely absorbed after oral exposure. The difference between fecal excretion after oral and i.v. exposure indicated that 5 to 9% of the oral dose was not absorbed from the gastrointestinal tract and was eliminated via the feces.
Details on distribution in tissues:
amount in any tissue < 2%; highest concentrations in liver, muscle, blood, adipose tissue
relative amount in tissue independent of dose or route of administration
Details on excretion:
Excretion profiles independent from dose or route of administration:

Recovery after single 5 mg/kg oral dose:
24 hours: 67% with 45% in urine, 16% as exhaled 14CO2, 6% in feces, <1% as exhaled 14C-citral;
production of 14CO2 essentially ceased by 12 hrs;
72 hours: 83% (+- 10 %) with 51% in urine, 17% as exhaled 14CO2, 12% in feces, 3% in tissues, <1% as exhaled 14C-citral

Recovery after single 5 mg/kg i.v. dose:
12 hours: 57% with 47% in urine, 7% as 14CO2, 2% in feces, <1% as exhaled 14C-citral;
elimination essentially completed within 24 hrs
72 hours: 79% (+- 18 %) with 58% in urine, 8% as 14CO2, 7% via the feces, 6% tissues, <1% as exhaled 14C-citral

Elimination via bile after a single 5 mg/kg i.v. dose:
20% of the dose appeared in bile within 1 hr, with another 7% appearing by 4.5 hrs. The amount excreted in the bile was 4 times higher than that excreted in the feces within 3 days. HPLC of bile, even as early 5 min after treatment, demonstrated the complete absence of any unmetabolized citral.

Effect of multiple dosing:
In rats pretreated for 10 days (5 mg/kg bw/d orally), biliary excretion was increased to 34%, while excretion via urine, feces or expired CO2 was not affected. Therefore, repeated exposure did not alter the overall pattern of disposition.
Toxicokinetic parametersopen allclose all
Toxicokinetic parameters:
half-life 1st: 11 min for elimination from blood after i.v. application
Toxicokinetic parameters:
half-life 2nd: 43 min for elimination from blood after i.v. application
Toxicokinetic parameters:
half-life 3rd: 27 hr for elimination from blood after i.v. application
Toxicokinetic parameters:
half-life 1st: 1.8 h for elimination via urine after 5 mg/kg p.o.
Toxicokinetic parameters:
half-life 1st: 2.6 h for elimination of 14CO2 after 5 mg/kg p.o.
Toxicokinetic parameters:
half-life 1st: 11 h for elimination via faeces after 5 mg/kg p.o.

Metabolite characterisation studies

Metabolites identified:
not measured

Any other information on results incl. tables

Table 1: Distribution of citral-derived radioactivity in tissues and excreta after oral or i.v.exposure, 72 hr after administration (Values are means ± SE; n ≥ 3 )

Tissue or excreta

Test groups   

Oral

Preatreateda

5 mg/kg

 i.v.

500 mg/kg

50 mg/kg

5 mg/kg

 5 mg/kg

% of administered dose

 

Total tissuesb

5.21 ± 1.95

2.81 ± 0.77

3.17 ± 0.99

4.44 ± 1.00

6.31 ± 0.78

Blood

0.52 ± 0.23

0.19 ± 0.05

0.36 ± 0.11

0.13 ± 0.01

0.40 ± 0.14

Liver

0.94 ± 0.25

0.58 ± 0.18

0.70 ± 0.11

0.39 ± 0.04

0.94 ± 0.07

Kidneys

0.08 ± 0.02

0.04 ± 0.01

0.07 ± 0.01

0.08 ± 0.01

0.09 ± 0.01

Skin

1.00 ± 0.61

0.46 ± 0.08

0.64 ± 0.22

0.74 ± 0.12

1.35 ± 0.01

Adipose

0.16 ± 0.09

0.24 ± 0.06

0.44 ± 0.14

0.15 ± 0.01

0.79 ± 0.12

Muscle

0.67 ± 0.04

0.63 ± 0.00

0.53 ± 0.19

1.94 ± 0.59

1.93 ± 0.56

Stomach contents

0.16  ± 0.10

< 0.01

0.06 ± 0.04

0.00

0.05 ± 0.30

Small intestine contents

0.39  ± 0.15

0.17 ± 0.08

0.03 ± 0.01

0.28 ± 0.07

0.07 ± 0.02

Large intestine contents

0.65  ± 0.11

0.13 ± 0.06

0.11 ± 0.06

0.50 ± 0.11

0.12 ± 0.02

i.v. tail site

NAc

NA

NA

NA

0.12 ± 0.07

Urine

63.36 ± 10.23

47.92 ± 2.03

50.69 ± 5.19

52.96 ± 3.38

57.88 ± 15.02

Feces

13.22 ± 4.44

15.58 ± 2.89

11.99 ± 1.61

11.11 ± 1.56

6.84 ± 1.49

Expired CO2

12.70 ± 3.37

10.37 ± 0.72

16.62 ± 1.92

14.66 ± 0.73

7.92 ± 0.79

Expired Citral

0.16 ± 0.11

0.04 ± 0.01

0.48 ± 0.21

0.03 ± 0.01

0.33 ± 0.30

Total

93.65 ± 20.10

76.73 ± 6.42

82.95 ± 9.93

83.20 ± 6.78

79.28 ± 18.38

a Pretreated with 5 mg of citral/kg/day po for 10 days, then po with [14C]citral.

bTotal of collected tissues, including those listed except for dermal skin site. Tissues not individually listed contain 0.1% of the total dose.

cNA, not applicable.

Table 2: Toxicokinetic parameters for the elimination of citral-derived radioactivity (Estimate ± SE)

Excreta

Dose

[mg/kg]

Componenta

Pool size

[% total dose]

Decay rate

[hr-1]

Half life

[hr]

Urine

po

    5

1

47.29 ± 32.53

0.378 ± 0.124

1.8

2

0.96 ± 0.15

0.044 ± 0.006

15.8

    50

1

23.19 ± 0.83

0.252 ± 0.006

2.8

2

0.42 ± 0.09b

0.029 ± 0.006

23.9

    500

1

26.94 ± 12.59

0.161 ± 0.030

4.3

2

1.05 ± 0.52

0.015 ± 0.016

46.2

i.v. 5

1

57.97±0.12

0.208±0.001

3.3

2

0.64±0.16

0.046±0.008

15.1

CO2

po

    5

1

11.66 ± 0.75

0.271 ± 0.008

2.6

2

0.14 ± 0.01

0.042 ± 0.002

16.5

    50

1

10.16 ± 0.94

0.316 ± 0.012

2.2

2

0.09 ± 0.01b

0.084 ± 0,004b

8.3

    500

1

2.98 ± 0.42b

0.138 ± 0.016b

5.0

i.v. 5

1

2.09±0.30

0.280±0.058

2.5

2

0.07±0.02

0.064±0.012

13.8

Feces

po

    5

1

1.82 ± 0.89

0.063 ± 0.035

11.0

   50

1

1.11 ± 0.18

0.045 ± 0.011

15.4

   500

1

0.95 ± 0.12

0.037 ± 0.008

18.7

i.v. 5

1

0.67±0.21

0.093±0.035

7.5

a Component of the elimination phase: 1 = initial component of eliminiation phase; 2 = terminal component of elimination phase

bSignificantly different from 5 mg/kg po (z test, p < 0.01)

In summary, most of the citral-derived radioactivity was rapidly eliminated from the body with a whole body half-life of 8 hr after i.v. exposure. However, a small percentage tended to persist with a clearance half-life of 24 hrs.

Elimination from blood after i.v. administration:

< 25% of administered 14C-activity remained in the blood within 2 min p.a.; within 5 min several metabolites (not identified) and no citral were found in blood; elimination from blood was characterized by a three-exponential decay curve (pool size of the first, the second and the third phase was 26+-2%, 17+-1% and 4+-1% of the total dose, respectively).

Applicant's summary and conclusion