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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
year of publication: 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Objective of study:
toxicokinetics
Principles of method if other than guideline:
Investigation of in vivo pharmacokinetics in rats after intravenous administration
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-butyl acetate
Radiolabelling:
yes
Remarks:
Butyl-1-[14C]

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, NY, USA
- Age at study initiation: 9-12 weeks
- Weight at study initiation: 300-400 g
- Fasting period before study: no
- Housing: wire-mesh, stainless-steel cages
- Individual metabolism cages: yes/no
- Diet: certified rodent diet (Agway RMH 3000 for the preprobe study; PMI, Inc. Rodent 5002 Pellet for all other studies), ad libitum
- Water: domestic tap water, ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
intravenous
Vehicle:
physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- [14C] n-butyl acetate was dissolved in 0.9% NaCl


VEHICLE
- Justification for use and choice of vehicle (if other than water): due to complications observed when neat substance was used (see below)
- Concentration in vehicle: 5.5 mg/ml solution

Duration and frequency of treatment / exposure:
single exposure
Doses / concentrations
Remarks:
Doses / Concentrations:
- mean: 30.2 +/- 0.1 mg/kg (16.8 +/- 0.6 µCi per animal)
No. of animals per sex per dose:
- definite study: 32 male rats (corresponding to 4 rats per time point of blood/brain collection)
Control animals:
no
Positive control:
- none
Details on study design:
- Dose selection rationale: based on the results of a preprobe and probe study:

- preprobe study: i.v. administration of 1000 mg/kg of neat unlabelled n-butyl acetate over a time span of appr. 90-100 seconds to two animals did not result in mortality, one of the two rats was observed to be dragging his left hind limb beginning about two hours after the dose administration

- probe study: i.v. administration of 1000 mg/kg of neat radiolabelled n-butyl acetate over a time span of appr. 45 seconds resulted in sudden death of 3 out of the first 6 animals dosed; 7 additional animals were dosed, but over an appr. time span of 90 seconds; no further death or morbidity occurred, total radioactivity concentrations in whole blood were initially low following this i.v. administration and rose slowly during the 2 hour sampling period following dosing. The authors of the study concluded that "the bolus organic solvent dose apparently blocked circulation in the area of the tail vein, possibly due to denaturation of proteins contacted by it, leading to slow absorption of radiolabelled test substance from the tissue surrounding the injection site."

- repeated probe study: i.v. administration of 25-31 mg/kg of radiolabelled n-butyl acetate dissolved in 0.9% NaCl over a time span of appr. 45 seconds; the dose was maximized by preparing a near saturated dose solution and by administering injection volumes of 5.5 ml/kg; no mortality occurred; rapid systemic distribution and elimination of total radioactivity was observed which demonstrated the efficacy of the n-butyl acetate formulation in physiological saline; in the 60 min blood samples only polar metabolites were detectable and at 120 min the radiochemical concentration of all blood metabolites were below the limit of detection
Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood, brain; heparinized whole blood and brain homogenates were deproteinized using sodium tungstate and cupric sulfate and the supernatants were analyzed
- Time and frequency of sampling: as soon as possible following dosing (1.5 to 2.0 min) and at 2.5, 4.0, 7.0, 10, 15, 20, and 60 minutes following dose administration
- From how many animals: 4 animals per time point
- Method type(s) for identification: HP 1090 HPLC using a reverse phase column and an isocratic mobile phase and a radiochemical flow through detector

- Additionally samples of whole blood and brain homogenates were counted by liquid scintillation spectrometry to quantitate the total radiochemical concentration in the tissues

Results and discussion

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Tmax: 2.5-2.6 min in blood and brain
Test no.:
#1
Toxicokinetic parameters:
other: t1/2 alpha: 0.4 min (blood and brain)
Test no.:
#1
Toxicokinetic parameters:
other: t1/2 beta: 3.2 min (blood) and 4.7 min (brain)
Test no.:
#1
Toxicokinetic parameters:
other: elimination t1/2: 1.0-1.2 min
Test no.:
#1
Toxicokinetic parameters:
other: VD (apparent volume of distribution): 155.0 ml/kg

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
- the main metabolite identified in blood and brain was [14C] n-butanol with 52 and 79 µg equivalents/g tissue, respectively; these concentrations declined to undetectable levels after 20 min post dosing (for details see below)
- other metabolites detected in the blood and to only a minor degree in the brain included n-butyric acid (maximum of 5.7 equivalents/g whole blood at 7.4 minutes, followed by a slow decrease) and polar metabolites with a maximum level of 12.2 µg equivalents/g tissue at 4.2 min (probably citric acid cycle intermediates, glucuronide and sulfate conjugates which were not further identified)

Any other information on results incl. tables

- no morbidity was observed

- systemic distribution was rapid, maximal blood radiochemical concentrations occurred in the 2.6 min samples

- elimination of radioactivity was rapid and appeared to be biphasic: pharmacokinetic modeling of the blood data indicated an alpha phase half-life of 1.1 min, a beta phase half-life of 17.2 min, and an elimination t 1/2 of 3.2 min

- mean brain total radiochemical concentrations were similar to the blood, but with somewhat more rapid early elimination of radioactivity: pharmacokinetic modeling of the blood data indicated an alpha phase half-life of 0.9 min, a beta phase half-life of 12.8 min, and an elimination t 1/2 of 1.6 min

Tables: Concentrations of n-butyl acetate and its metabolites measured in rat whole blood and brain tissue. The values were calculated from the area percent of each peak in the HPLC chromatogram multiplied by the radiochemical concentration of the deproteinized sample divided by the specific activity of the dose solution. The total [14C] values were calculated from the radiochemical concentration of the whole blood or brain homogenate before deproteinization divided by the specific activity of the dose solution. The difference between the sum of the n-butyl Acetate and its metabolite concentrations and the total [14C]concentrations reflects the [14C] recovery efficiency of the deproteinization procedure. Unless otherwise indicated, values are the mean and standard deviation of four animals for each timepoint.

Whole Blood

Sampling
Time (min)

Polar
Metabolites
(µg equiv./g)

n-Butyric
Acid
(µg equiv./g)

n-Butanol
(µg equiv./g)

n-Butyl
Acetate
(µg equiv./g)

Total [14C]
(µg equiv./g)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

1.88

0.17

4.61

2.39

3.85

2.49

45.40

3.91

7.84

4.75

78.96

10.12

2.61

0.09

9.15

3.18

5.42

2.02

51.49

14.30

3.71

1.01

84.51

8.59

4.24

0.15

12.19

1.40

4.83

2.13

20.74

3.89

0.37a

0.09a

49.04

2.35

7.41

0.24

5.92

0.81

5.66

0.49

10.02

1.14

n.d.b

n.d.

33.93

1.42

10.19

0.38

4.48

0.92

4.41

0.81

5.35

1.84

n.d.

n.d.

25.37

2.86

15.27

0.21

2.64

0.14

3.90

0.42

1.76

0.36

n.d.

n.d.

18.75

0.93

20.44

0.52

2.31

0.63

3.81

0.44

1.00'

0.17

n.d.

n.d.

15.45

1.08

60.17

0.34

0.78d

n.a.e

1.91a

0.65a

n.d.

n.d.

n.d.

n.d.

6.27

0.74

Brain

Nominal
Sampling
Time (min)

Polar
Metabolites
(µg equiv./g)

n-Butyric
Acid
gequiv./g)

n-Butanol
(µg equiv./g)

n-Butyl
Acetate
(µg equiv./g)

Total [14C]
(gg equiv./g)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

1.88

0.17

n.d.b

n.d.

n.d.

n.d.

71.03

10.61

3.77

3.84

82.95

17.94

2.61

0.09

0.29d

n.a.e

n.d.

n.d.

69.57

10.19

2.13

1.11

82.94

12.94

4.24

0.15

1.05d

n.a.e

048d

n.a.e

27.73

2.58

n.d.

n.d.

37.21

4.03

7.41

0.24

1.61d

n.a.e

n.d.

n.d.

15.87

1.53

n.d.

n.d.

22.12

1.64

10.19

0.38

n.d.

n.d.

n.d.

n.d.

9.95

2.26

n.d.

n.d.

16.15

1.60

15.27

0.21

n.d.

n.d.

n.d.

n.d.

4.40

0.65

n.d.

n.d.

10.55

0.67

20.44

0.52

n.d.

n.d.

n.d.

n.d.

3.31a

0.35a

n.d.

n.d.

9.02

0.94

60.17

0.34

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

4.23

0.66

 

a) metabolite(s) detected in three of four replicate samples

b) n.d. = not detected

c) metabolite(s) detected in two of four replicate samples

d) metabolite(s) detected in one of four replicate samples

e) n.a. = not applicable

Summary of pharmacokinetic parameter estimates derived from nonlinearleast squares Fitting (Minsq, MicroMath Scientific Software, Salt Lake City, Utah,1992) of blood parent compound data after intravenous administration of [14C]n-butylacetate in male SD rats. The form of the equation is:

Ct = Dose/VD x e-Kelim t 

Parameter
(dimension)

n-Butyl Acetate

Kelim (min')

1.675

t1/2 (min)

0.414

VD(ml/kg)

155.0

VD: apparent volume of distribution

Kelim: elimination rate constant

Summary of pharmacokinetic parameter estimates derived from nonlinear least squares fitting (Minsq, MicroMath Scientific Software, Salt Lake City, Utah,1992) of blood and brain n-butanol concentrations after intravenous administration of[14C]n-butyl acetate in male SD rats.

Parameter
(dimension)

n-Butanol in blood

n-Butanol in brain

A

24.365

48.050

alpha (min')

1.890

1.574

t1/2 alpha(min)

0.367

0.440

B

65.447

105.472

beta (min')

0.215

0.147

t1/2 beta (min)

3.228

4.714

Kelim (min-1)

0.669

0.594

t1/2 (min)

1.036

1.167

Cmax (µg/g)

52.2

79.2

Tmax (min)

2.6

2.5

 


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
A 30 mg/kg i.v. dose of 14C-radiolabelled n-butyl acetate was very rapidly distributed via the circulatory system and into the brain, and at the same time is very rapidly hydrolyzed to n-butanol in the blood and brain. The elimination half-life for n-butyl acetate was similar in blood and brain, estimated to be 0.4 min. Hydrolysis of n-butyl acetate was calculated to be 99% complete in 2.7 min at this i.v. dose level.
Executive summary:

The rate of hydrolysis of n-butyl acetate(nBA) in male rats in vivo at the highest intravenous dose level possible was investigated. [14C] n-Butyl acetate was administered via a tail vein to 32 male Sprague Dawley rats at a mean of 30.2 mg/kg bodyweight (16.8 µCi/rat). Liquid scintillation analysis of the whole blood and brain tissue for total radioactivity following this dose revealed rapid systemic distribution of the dose and very rapid elimination from both whole blood and brain tissue. High performance liquid chromatography with radiochemical detection was used to separate and quantitate n-butyl acetate, its hydrolysis product n-butanol, and products of the subsequent oxidative and conjugative metabolism of n-butanol. These analyses indicated that n-butyl acetate was very rapidly eliminated from the blood (biphasic elimination; elimination t1/2 = 0.41 min), and was detected in brain tissue only at low concentrations (mean maximum of 3.8 µg equivalents/g at 1.9 min) in the first 2.5 min following dosing. n-Butanol was found at higher concentrations in both blood (Cmax = 52 µg equivalents/g at Tmax 2.6 min) and brain (Cmax = 79 µg equivalents/g at Tmax 2.5 min), but this was also rapidly eliminated in both tissues (biphasic elimination; t1/2, of 1.0 - 1.2 min) and was undetectable beyond 20 min post dosing. n-Butyric acid was present at low concentrations in blood (mean maximum of 5.7 µg equivalents/g at 7.4 min) and declined slowly following dosing; it was largely undetected in brain tissue. Early eluting, polar metabolites, presumably Krebs cycle intermediates of [14C]n-butanol and glucuronide and sulfate conjugates of [14C]n-butanol, were detected in the whole blood (mean maximum of 12.2 µg equivalents/g at 4.2 min), but were seen only in trace amounts in brain tissue. The hydrolysis of n-butyl acetate in blood and brain is estimated to be 99% complete by 2.7 min at this dose level (Deisinger et al., 1997; Barton et al., 2000).

This study is reliable without restriction (RL1).