Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
other: Prediction of in vivo exposure in the male and female rat
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
There are currently no specific regulatory guidelines that cover in vitro toxicokinetics. However, this type of work is exemplified in OECD 417 (22nd July 2010) Guideline for the testing of chemicals, Toxicokinetics, Supplemental approaches, use of in vitro information (paragraphs 59-61), and use of toxicokinetic modelling (paragraph 65). The work carried out also meets the spirit of the European Chemical Agency Guidance for the Implementation of REACH. i.e. Guidance on information requirements and chemical safety assessment, chapter R.7C: endpoint specific guidance (ECHA-12-G-23-EN), sections R.7.12.2.2 generating and integrating TK information, in silico methods – kinetic modelling, and Appendix R.7.12-2 prediction of toxicokinetics integrating information generated in silico and in vitro. All experimental work was carried out against appropriate well recognised and established control compounds detailed in the methods and the study data only reported when the control data fell within acceptable predefined limits.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
452-330-3
EC Name:
-
Cas Number:
314020-40-1
Molecular formula:
C14H20N2O2
IUPAC Name:
2-(2,6-diethyl-4-methyl-phenyl)propanediamide
Test material form:
solid: particulate/powder
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Vehicle:
DMSO

Results and discussion

Any other information on results incl. tables

MICROSOMAL INTRINSIC CLEARANCE

The in vitro CLint of the test substance in male Wistar rat microsomes was 1.45 ± 3.25 µL/min/mg protein. This was characterised by 9.6% depletion over 45 minutes. The in vitro CLint in female Wistar rat microsomes was 1.79 ± 3.85 µL/min/mg protein, characterised by 9.1% depletion over 45 minutes. In the absence of NADPH, there was approximately 4% depletion over 45 minutes in male microsomes and approximately 4.2% depletion over 45 minutes in female microsomes, indicating no non NADPH dependent metabolism. In the absence of microsomes, there was <1 % loss of the test substance over 45 minutes suggesting no potential chemical instability of the molecule in this matrix.

 

HEPATOCYTE INTRINSIC CLEARANCE

The in vitro CLint of the test substance in male Wistar rat hepatocytes was 1.54 ± 1.17 µL/min/10^-6 cells characterised by no depletion over 60 minutes. The in vitro CLint in female Wistar rat hepatocytes was 0.182 ± 0.651 µL/min/10^-6 cells characterised by 2.6% depletion over 60 minutes. In the absence of live hepatocytes, there was no loss of CA3250 at the 60 minute time point, suggesting no potential chemical instability of the molecule in this matrix. Due to the negligible intrinsic clearance in hepatocytes it was considered appropriate to use the microsomal data for modelling purposes.

 

PLASMA PROTEIN BINDING

The fraction of test substance unbound in plasma (fup) was 0.893 ± 0.142 in male plasma and in female plasma was 0.612 ± 0.0452. The recovery of test substance in the incubations was 82.6 and 93.4% respectively.

 

CACO-2 PERMEABILITY

The apparent permeability (Papp) of test substance across Caco-2 cell monolayers was 25.7 x 10^-6 cms-1 ± 1.86 x 10-6cms -1. The recovery of test substance from the incubation media was 92.9%.

 

CLOE PK™ - PK SIMULATION

The following parameters were used for Cloe PK™ modelling:

Molecular weight: 248.32

LogP: 1.1

pKa: Neutral

Aqueous solubility at pH 7.96 (g/L): 1.6

Fup: 0.893 for males 0.612 for females

Microsomal CLint (µL/min/mg): 1.45 for males 1.79 for females

Papp (cms-1 ): 25.7 x 10^-6

Certain physiological assumptions were made within the model such as body weight, hepatic blood flow, hepatic mass fraction and microsomal protein content.

 

TABLE 1: Median predicted pharmacokinetic parameters derived using Cloe PKTM simulating intravenous administration at doses of 15, 150 and 1000 mg/kg in male and female rats

 

Parameter

15 mg/kg

 

150 mg/kg

 

1000 mg/kg

 

 

Male

Female

Male

Female

Male

Female

Cmax (µg/mL)

760

870

7600

8700

51000

58000

AUC (µg.h/mL)

63

81

630

810

4200

5400

T ½ (h)

33

28

33

28

33

28

Vd (L/kg)

11

6.8

11

6.8

11

6.8

Total CL (mL/min/mg)

4

3.1

4

3.1

4

3.1

MRTinf (h)

22

19

22

19

22

19

Vss (L/kg)

5.4

3.7

5.4

3.7

5.4

3.7

Elimination rate (h-1)

0.021

0.024

0.021

0.024

0.021

0.024

 

TABLE 2: Median predicted pharmacokinetic parameters derived using Cloe PK™ simulating oral administration at doses of 15, 150 and 1000 mg/kg in male and female rats

 

Parameter

15 mg/kg

 

150 mg/kg

 

1000 mg/kg

 

 

Male

Female

Male

Female

Male

Female

Cmax (µg/mL)

4.2

5.8

41

57

140

190

Tmax (h)

2.5

2.5

2.4

2.4

3.3

3.3

AUC (µg.h/mL)

60

78

590

780

2500

3200

T ½ (h)

34

29

33

29

36

31

Vd (L/kg)

11

6.9

11

6.9

12

7.5

Elimination rate (h-1)

0.021

0.024

0.021

0.024

0.019

0.023

Bioavailability (%)

0.93

0.94

0.93

0.93

0.57

0.57

Fraction absorbed

1

1

0.99

0.99

0.61

0.61

 

Applicant's summary and conclusion

Conclusions:
Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex Discovery Ltd. together with their commercially accepted physiologically based pharmacokinetic model (Cloe PK™), the test substance, when administered in vivo to male and female rats, is predicted to have a good fraction absorbed across the gastrointestinal tract following doses of 15, 150 or 1000 mg/kg (approx. 99% at lower doses reducing to 61% at 1000 mg/kg). This translates to a systemic oral bioavailability of 57-100% in male and female rats over this dose range. In general in vivo the test substance would be expected to be a low clearance compound that distributes widely into tissues with a mean residence time in the body of around 19-22 hours.
Executive summary:

This study was designed to assess the in vivo pharmacokinetic behaviour of the test substance via an in vitro to in vivo extrapolation (IVIVE) to help put into context the findings of toxicology studies carried out separately to this report. In vitro metabolic stability, plasma protein binding and Caco-2 permeability were determined by Cyprotex Discovery Ltd., Macclesfield, UK and the data used in the physiologically based pharmacokinetic (PBPK) model, Cloe PKTM, to simulate the predicted in vivo exposure in male and female rat at 100, 300 and 1000 mg/kg. Physical chemistry data including solubility, logP and pKa was provided by Syngenta for the purposes of modelling and simulation.

The in vitro CLint of the test substance in male Wistar rat microsomes was 1.45 ± 3.25 µL/min/mg protein. This was characterised by 9.6% depletion over 45 minutes. The in vitro CLint in female Wistar rat microsomes was 1.79 ± 3.85 µL/min/mg protein characterised by 9.1% depletion over 45 minutes. The in vitro CLint of the test substance in male Wistar rat hepatocytes was 1.54 ± 1.17 µL/min/10^-6 cells characterised by no depletion over 60 minutes. The in vitro CLint in female Wistar rat hepatocytes was 0.182 ± 0.651 µL/min/10-6 cells characterised by 2.6% depletion over 60 minutes. Due to the negligible intrinsic clearance in hepatocytes it was considered appropriate to use the microsomal data for modelling purposes. The fraction of test substance unbound in plasma (fup) was 0.893 ± 0.142 in male plasma and in female plasma was 0.612 ± 0.0452. The recovery of test substance in the incubations was 82.6 and 93.4%, respectively. The apparent permeability (Papp) of test substance across Caco-2 cell monolayers was 25.7 x 10^-6 cms^-1 ± 1.86 x 10-6cms^-1. The recovery of test substance from the incubation media was 92.9%. The predicted median pharmacokinetic parameters for the test substance using Cloe PKTM simulating oral administration at doses of 15, 150 and 1000 mg/kg in male and female rats is summarised below.

 

Parameter

15 mg/kg

 

150 mg/kg

 

1000 mg/kg

 

 

Male

Female

Male

Female

Male

Female

Cmax (µg/mL)

4.2

5.8

41

57

140

190

Tmax (h)

2.5

2.5

2.4

2.4

3.3

3.3

AUC (µg.h/mL)

60

78

590

780

2500

3200

T ½ (h)

33

28

33

28

33

28

Vd (L/kg)

11

6.8

11

6.8

11

6.8

CL (mL/min/kg)

4

3.1

4

3.1

4

3.1

MRTinf (h)

22

19

22

19

22

19

Vss (L/kg)

5.4

3.7

5.4

3.7

5.4

3.7

Bioavailability (%)

0.93

0.94

0.93

0.93

0.57

0.57

Fraction absorbed

1

1

0.99

0.99

0.61

0.61

 

Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex Discovery Ltd. together with their commercially accepted physiologically based pharmacokinetic model (Cloe PK), the test substance, when administered in vivo to male and female rats, is predicted to have a good fraction absorbed across the gastrointestinal tract following doses of 15, 150 or 1000 mg/kg (approx. 99% at lower doses reducing to 61% at 1000 mg/kg). This translates to a systemic oral bioavailability of 57-100% in male and female rats over this dose range. In general in vivo the test substance would be expected to be a low clearance compound that distributes widely into tissues with a mean residence time in the body of around 19-22 hours.