Registration Dossier

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2007 to 07 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
adopted 4 April 1984
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: at least 8 weeks
- Weight at study initiation: 228 - 279 g (males), 164 - 218 g (females)
- Housing: individually in stainless steel, wire-mesh cages suspended above cage boards
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days with the exception of cannulated rats which were quarantined for at least 3 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The target radioactive dose was 30 μCi/250 g animal. The 14C-test substance was diluted with test substance to the appropriate specific activity for the selected dose level. The test substance, 14C-test substance and dose vehicle (0.5% methylcellulose) were weighed into a vial and mixed to a clear solution or homogeneous suspension. Dose preparations were prepared and stored refrigerated until use.

Duration and frequency of treatment / exposure:
single oral administration
Dose / conc.:
25 mg/kg bw/day
Remarks:
pilot material balance study and main pharmacokinetics study
Dose / conc.:
500 mg/kg bw/day
Remarks:
main pharmacokinetics study and metabolite profile study
No. of animals per sex per dose / concentration:
1 (pilot material balance study)
4 (main pharmacokinetics study ) + 1 additional rat per sex dosed at 500 mg/kg bw/day for analytical method development (metabolite profile)
3 (metabolite profile study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were based on the results of a toxicity study.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled: 14C exhaled volatiles and 14CO2, urine, faeces, blood, cage wash, residual feed (pilot material balance study), plasma and red blood cells (RBC) (main pharmacokinetics study)
- Time and frequency of sampling: 14C exhaled volatiles and 14CO2 for 48 h at 24 h intervals (e.g. 0-24 and 24-48 h), urine and feces were collected at 24 h intervals for a total of 168 h, cage wash was collected throughout the study, blood samples (plasma and RBC) were collected pre-dose and at 5, 15 and 30 minutes, 1, 2, 4, 8, 12, 24, and 30 h post dose
- From how many animals: 1 rat per sex and dose (pilot material balance study), 4 animals per sex and dose (main pharmacokinetics study)
- Other: The following tissues were collected for analyses at the end of the study: blood (plasma and RBC were analyzed separately for 14C content), fat liver, kidney, muscle, heart, lung, testes, ovaries, uterus, bone and bone marrow (analyzed separately for 14C content), brain, spleen, adrenals, pituitary, gastro-intestinal tract and contents (analyzed separately for 14C content), pancreas, skin sample, thyroid, thymus, bladder (urine in the bladder at the time of sacrifice was aspirated and placed in the terminal urine sample vial) and remaining carcass (pilot material balance study). Liquid scintillation counting was used for quantitation of radioactive residues.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: plasma (control and 500 mg/kg bw/day dose groups at 0.5 h timepoint), urine and feces ( 25 mg/kg bw/day dose group at 0-24 h intervals)
- Time and frequency of sampling: plasma was collected 0.5 h after test substance administration (metabolite profile study), urine and feces was collected at 24 h intervals for a total of 168 h (pilot material balance study)
- From how many animals: 3 samples per sex and dose (metabolite profile study), 1 sample per sex and dose (pilot material balance study)
- Method types for identification: quantification of radioactivity in plasma, urine, and feces was performed by HPLC with in-line radioactivity (LC/ARC). Observed radiochemical chromatographic peaks were identified by performing peak fractionation and LC/MS. Each of the prepared samples was also analyzed by LSC to verify adequate extraction recoveries

Clinical Observations and Mortality
Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least once daily throughout the study.

(For further details on study design please refer to table 1 in the "Any other information and methods incl. tables" section.)




Statistics:
Group data are represented as a mean ± SD.
Preliminary studies:
The test substance was rapidly eliminated after single oral gavage administration. No 14C residues were detected in exhaled breath. These results suggest that the position of the radiolabeled carbon was stable to metabolic biotransformation. Excretion occurred via the urine and feces in approximately equal proportions and was substantially complete within the first 24 h after dosing. Tissue 14C residues, with the exception of 0.039% in the male rat carcass, were below the limit of detection by 168 h after dosing. (For details on Toxikokinetic parameters please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)
Type:
absorption
Results:
The results indicated rapid absorption of the test substance.
Type:
distribution
Results:
Test substance is predominantely found in plasma. Tissue 14C residues, with the exception of male rat carcass ( 0.039%), were below the limit of detection by 168 h after dosing.
Type:
metabolism
Results:
No biotransformation was evident under the condition of single dose administration at 25 or 500 mg/kg bw in plasma urine and feces.
Type:
excretion
Results:
Radioactivity was not detected in respired breath (exhaled volatiles or CO2). Excretion occurred in both the urine and feces and was substantially complete in the first 24 h after dosing.
Details on absorption:
The results indicated rapid absorption with peak concentrations in plasma at 0.4 to 1 h after dosing. The mean Tmax values in red blood cells were 0.3 to 1 h. The peak concentrations in red blood cells were 0.33 to 0.48 fractionally lower than those observed in plasma indicating very limited potential for binding within red blood cells. The red blood cell concentration data were insufficient for calculation of pharmacokinetic parameters. The mean peak concentrations in plasma (3.8-5.0 μg equiv/g at the low dose and 57.3-61.6 μg equiv/g at the high dose, respectively) were approximately proportional to the 20-fold increase in dose from 25 to 500 mg/kg bw. Near direct proportionality was observed for the AUCINF values suggesting linear first-order kinetic processes for uptake and elimination. (For details on Toxikokinetic parameters please refer to table 4 and 5 in the "Any other information on results incl. tables" section.)
Details on distribution in tissues:
The peak concentrations in red blood cells were 0.33 to 0.48 fractionally lower than those observed in plasma indicating very limited potential for binding within red blood cells. The test substance is distributed within plasma. By 168 h after dosing, 14C residues were not detected in any of the various tissues collected to evaluate tissue distribution. One exception was the male rat carcass which contained 0.039% of the dose. Radioactivity detected in cage wash and residual feed was 1.4% and 0.4% of the administered dose for the male and female rat, respectively. The overall material balance was 69.9% for the single male rat and 107.9% for the single female rat. (Please refer to table 2 in the "Any other information on results incl. tables" section.)



Details on excretion:
Radioactivity was not detected in respired breath as either exhaled volatiles or CO2, and thus collection was stopped at 48 h. Excretion occurred in both the urine and feces and was substantially complete in the first 24 h after dosing. By 168 h after dosing, the individual male and female rat urine contained 36.3% and 55.8%, respectively; and the percentages were approximately equal to the 32.1% and 51.7% excreted in the feces. Radioactivity detected in cage wash and residual feed was 1.4% and 0.4% of the administered dose for the male and female rat, respectively. The overall material balance was 69.9% for the single male rat and 107.9% for the single female rat. In plasma, the terminal elimination half-lives were 5.6 to 5.7 h for male and female rats with no difference between the low (25 mg/kg bw) or high (500 mg/kg bw) dose levels. Plasma 14C residues were quantifiable for up to 30 h after dosing. (Please refer to table 2, 3, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15 and 16 in the "Any other information on results incl. tables" section.)

Key result
Toxicokinetic parameters:
half-life 1st: Plasma: 5.6 h in males and 5.7 h in females (25 and 500 mg/kg bw)
Remarks:
The half-lives were unchanged with the 20-fold increase in dose.
Key result
Toxicokinetic parameters:
Cmax: Plasma: 3.8 and 5.0 μg equiv/g in male and female rats, respectively (25 mg/kg bw dose) 57.3 and 61.6 μg equiv/g for male and female rats, respectively (500 mg/kg bw dose)
Key result
Toxicokinetic parameters:
Cmax: Red blood cells: 1.3 and 2.0 μg equiv/g in males and females, respectively (25 mg/kg bw/day) 27.2 and 28.7 in males and females, respectively (500 mg/kg bw/day)
Key result
Toxicokinetic parameters:
Cmax: RBC/Cmax plasma ratios: 0.33 and 0.4 in males and females, respectively (25 mg/kg bw/day) 0.48 and 0.47 in males and females, respectively (500 mg/kg bw/day)
Remarks:
(very limited potential for uptake and binding in the red blood cell)
Key result
Toxicokinetic parameters:
AUC: Plasma: 7.0 and 9.0 hr*μg/g in males and females, respectively (25 mg/kg bw dose) 150.8 and 168.4 hr*μg/g in males and females, respectively (500 mg/kg bw dose)
Key result
Toxicokinetic parameters:
Tmax: Plasma: 0.5 h and 0.4 h in males and females, respectively (25 mg/kg bw dose) 6.0 h and 1.0 h in males and females, respectively (500 mg/kg bw dose)
Toxicokinetic parameters:
Tmax: Red blood cells: 0.5 h and 0.3 h in males and females, respectively (25 mg/kg bw dose) 6.0 h and 1.0 h in males and females, respectively (500 mg/kg bw dose)
Metabolites identified:
no
Details on metabolites:
No metabolite was evident under the condition of single dose administration of 25 or 500 mg/kg bw in plasma at 0.5 h timepoint and urine or feces 0-24 h time interval (pilot study). Only test substance, as parent chemical was confirmed in all 3 matrices by radiochemical and mass spectral analysis. Biotransformation of the test substance was absent in rats under conditions of single oral gavage at the 25 and 500 mg/kg bw doses. One expected metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, was not observed. This metabolite has been quantified in rat plasma at Day 56 of a 90-day toxicity study by dietary administration at concentrations corresponding to 100 to 945 mg/kg bw/day for male rats and 129 to 1268 mg/kg bw/day in female rats. Although not identified under conditions of the current study, the metabolite 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine appears to be formed by de-carboxylation of the test substance upon repeated daily uptake of the test substance. Under the conditions of this study, no metabolism of the test substance was observed.

 Table 2: Material balance for rats administered a single oral dose of14C-test substance (25 mg/kg bw)

 

 

Percent of dose

Sample

Hour

Male

Female

 

 

101M

102F

urine

168 h

36.304

55.761

feces

168 h

32.145

51.688

exhaled a

48 h

<LOD

<LOD

cage wash

168 h

1.209

0.358

residual feed

168 h

0.197

0.076

tissues

168 h

<LOD

<LOD

carcass

168 h

0.039

<LOD

 

Total

69.9

107.9

a Includes data from 3 traps (CO2, volatiles, and water).

 LOD limit of detection

LOQ limit of quantitation

Table 3: Percent recovery in excreta of rats administered a single oral dose of14C-test substance (25 mg/kg bw)

Percent of dose 

 

 

 

Cumulative

percent of dose a

Male Female

 

Male

Female

Male

Female

Sample

Hour

101M

102F

101M

102F

urine

Pre-dose

 <LOD

<LOD

0

0

urine

24 h

35.8876

53.7113

35.888

53.711

urine

48 h

0.2835

1.5093

36.171

55.221

urine

72 h

0.0893

0.5271

36.260

55.748

urine

96 h

0.0184

0.0086

36.279

55.756

urine

120 h

0.0048

0.0025

36.284

55.759

urine

144 h

0.0164

0.0016

36.300

55.760

urine

168 h

0.0041

0.0008

36.304

55.761

feces

Pre-dose

 <LOD

<LOD

0

0

feces

24 h

31.1368

47.9955

31.137

47.996

feces

48 h

0.84

3.6031

31.977

51.599

feces

72 h

0.1249

0.0804

32.102

51.679

feces

96 h

0.014

0.0068

32.116

51.686

feces

120 h

0.0062

0.0023

32.122

51.688

feces

144 h

0.0176

<LOQ

32.140

51.688

feces

168 h

0.0054

<LOD

32.145

51.688

a Pre-dose time point assigned zero hour and concentration for graphical representation

 LOD limit of detection

LOQ limit of quantitation

Table 4: Concentration (μg equiv/g) in plasma and red blood cells following a 25 or 500 mg/kg bw single oral dose of14C-test substance

 

25 mg/kg bw

500 mg/kg bw

Hour

Male

Female

Male

Female

Plasma

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Pre-dose

NAa

NA

NA

NA

NA

NA

NA

NA

5 m

1.13

0.32

2.58

0.60

3.98

1.22

6.69

3.58

15 m

3.09

0.85

4.86

1.13

28.37

5.41

35.57

14.08

30 m

3.01

0.20

4.35

1.25

54.40

18.45

54.28

20.60

1 h

2.74

1.52

2.91

0.61

51.17

8.27

52.92

14.50

2 h

1.14

0.50

1.57

0.82

29.68

4.30

42.77

7.73

4 h

0.13

0.05

0.13

0.02

4.66

1.20

3.69

1.66

8 h

0.07

0.02

0.11

0.07

2.35

0.32

1.84

0.75

12 h

0.05

0.04

0.05

0.04

1.36

0.56

1.45

0.34

24 h

NA

NA

NA

NA

0.46

NA

0.43

NA

30 h

0.005

0.002

0.01

0.002

0.16

0.02

0.13

0.04

Red blood cells

Pre-dose

NA

NA

NA

NA

NA

NA

NA

NA

5 m

0.38

0.10

0.88

0.17

1.90

NA

2.60

1.10

15 m

1.07

0.21

1.99

0.66

11.46

2.05

14.69

5.96

30 m

0.99

0.09

1.76

0.60

25.68

8.47

23.47

9.69

1 h

0.95

0.49

1.14

0.26

23.96

4.74

24.23

7.07

2 h

0.43

0.19

0.60

0.30

13.97

2.57

19.72

4.03

4 h

0.07

NA

NA

NA

1.78

0.52

2.06

NA

8 h

0.03

NA

0.06

NA

1.07

NA

0.97

NA

12 h

0.04

NA

0.04

NA

0.73

NA

NA

NA

24 h

NA

NA

NA

NA

NA

NA

NA

NA

30 h

0.004

NA

NA

NA

NA

NA

NA

NA

a Pre-dose samples and others with mean values designated NA had individual animal concentrations that were <LOD

  NA not applicable

Table 5: Pharmacokinetic parameters in plasma and red blood cells following a 25 or 500 mg/kg bw single oral dose of14C-test substance

Parameter

25 mg/kg bw

500 mg/kg bw

Plasma

Male

Female

Male

Female

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Elimination half-life (h)

5.6

0.5

5.7

0.4

5.6

0.3

5.7

0.7

Area-under-the-curve

(AUCINF, hr*µg/g)

7.0

1.4

9.0

1.9

150.8

28.7

168.4

26.2

AUCINF/dose (hr*kg*µg/g/mg)

0.3

0.1

0.4

0.1

0.3

0.1

0.3

0.1

Peak concentration

(Cmax, µg eq/g)

3.8

0.9

5.0

1.2

57.3

14.2

61.6

13.0

Half peak concentration

(Cmax/2, µg) a

1.9

 

2.5

 

28.7

 

30.8

 

Time of Cmax (Tmax, h)

0.5

0.4

0.4

0.1

0.6

0.3

1.0

0.7

Time of Cmax/2 (Tmax/2, hr a

1.4

 

1.2

 

2.0

 

2.3

 

Red blood cells (RBC) b

Peak concentration (µg eq/g)

1.3

0.3

2.0

0.7

27.2

6.2

28.7

4.7

Time of Cmax (Tmax, h)

0.5

0.4

0.3

 

0.1

0.6

0.3

1.0

Ratio (Cmax RBC)/(Cmax plasma) a

0.33

 

0.40

 

0.48

 

0.47

 

a Calculated from Mean value, SD not given.

b Only Cmax and Tmax are presented. Time course data were insufficient to calculate other kinetic parameters.

AUCINF area under the concentration vs. time curve, extrapolated to infinity

AUCINF/D AUCINF, normalized to dose

 

Table 6: Concentration (μg equiv/g) in plasma of rats administered 0 or 500 mg/kg bw single oral dose of

14C-test substance

Dose

 

 

Plasma concentration (µg equiv/g)

Plasma concentration (µM)a

(mg/kg bw)

Sex

Hour

Mean

SD

Mean

SD

0 b

Male

0.5

<LOD

NA

<LOD

NA

 

Female

0.5

<LOD

NA

<LOD

NA

500 c

Male

0.5

53.4

17.6

250

82

 

Female

0.5

63.3

21.7

296

102

a Calculated using MW = 213.62 g/mol

b Control sample (n =1 per sex)

c Treated samples used for metabolite profiling (n = 3 per sex)

 

Table 7: Summary of retention times for reference standards and components in plasma from rats administered14C-test substance

 

 

Reference Standard HPLC/14Ca

Sample Summary HPLC/14C

[M+H]

Peak Retention

Time

Fraction Collection Window

Minimum Retention Time

Maximum Retention Time

 

(dalton)

(minutes) (minutes)

(minutes)

(minutes) b

 (minutes) b

Test substance

214

5.02

4.51-5.25

5.02

5.02

Metabolite (INLXT69)

170

N/Ac

5.26-6.00

NId

NI

a 14C retention time for radiolabeled 14C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not 14C radiolabeled.

d Not identified in the matrix.

 

Table 8:Summary of components identified in plasma of rats administered14C-test substance (A-male, B-female)

Component

M+H

(Da) a

Peak Fraction Collection Window (min)

Dose Group b

Mass Spectral Criteria Figure

 

Test substance

214

4.51-5.25

A,B

Spectral and retention time

match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed for the respective dose group: A – 601M-603M, 1F-603F.

 

Table 9: Summary of test substance parent concentrations in plasma

Time (minutes)

Subject

oncentration in plasma (µM) Test substance - Parent

30

601M

254

 

602M

108

 

603M

296

 

Mean

219

 

SD

99

30

601F

231

 

602F

358

 

603F

165

 

Mean

251

 

SD

98

 

Table 10: Summary of retention times for reference standards and components in urine from rats administered14C-test substance

 

 

Reference Standard HPLC/14C a

Sample Summary HPLC/14C

[M+H]

Peak Retention Time

Fraction Collection Window

Minimum Retention Time

Maximum Retention Time

 

(dalton)n)

(minutes)

(minutes)

(minutes) b

(minutes) b

Test substance

214

5.02

4.51-5.25

5.02

5.02

Metabolite

170

N/Ac

5.26-6.00

NId

NI

a 14C retention time for radiolabeled14C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not14C radiolabeled.

d Not identified in the matrix.

 

Table 11: Summary of components identified in urine of rats administered14C-test substance (A-male, B-female)

 

[M+H] (dalton) a

Peak fraction collection window (minutes)

Dose roup b

Mass spectral criteria

Test substance

214

4.51-5.25

A, B

Spectral and retention time match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed in the 24-hour dose group: A – 101M, B – 102F.

 

Table 12: Summary of components identified in urine of rats administered with 14C-test substance as percent of administered dose

Identified component

14C Retention time (min)

Component as percent of dose

500 mg/kg bw

 

Minimum

Maximum

Male

Female

Test substance

5.02

5.02

35.9

53.7

Identified

 

 

35.9

53.7

Not identified

 

 

0 a

0

Total

 

 

35.9

53.7

a Zero indicates not identified (detected)

 

Table 13: Summary of retention times for reference standards and components in feces from rats administered14C-test substance

 

 

Reference Standard HPLC/14C a

Sample Summary HPLC/14C

 

[M+H]

Peak Retention

Time

Fraction Collection Window

Minimum Retention Time

Maximum Retention Time

 

(dalton) aa

(minutes)

(minutes)

(minutes) b

(minutes) b

Test substance

214

5.17

4.51-5.25

5.18

5.33

Metabolite

170

N/A c

5.26-6.00

NI d

NI

a 14C retention time for radiolabeled14C-test substance reference standard spiked into the sample matrix.

b Retention time of base peak if tailing occurred and resulted in multiple integrations.

c Not applicable since the metabolite is not14C radiolabeled.

d Not identified in the matrix.

 

Table 14: Summary of components identified in feces of rats administered14C-test substance (A-male, B-female)

 

[M+H] (dalton) a

Peak fraction collection window (minutes)

Dose group b

Mass spectral criteria

Test substance

214

4.51-5.25

A, B

Spectral and retention time match with reference standard.

a Most abundant ion in molecular ion cluster

b The structural identification was confirmed in the 24-h sample dose group: A – 101M, B - 102F.

 

Table 15: Summary of components identified in feces of rats administered with14C-test substance as percent of administered dose

 

14C Retention time (min)

Component as percent of dose

500 mg/kg bw

 

Minimum

Maximum

Male

Female

Test substance

5.18

5.33

31.1

48.0

Identified

 

 

31.1

48.0

Not identified

 

 

0 a

0

Total

 

 

31.1

48.0

a Zero indicates not identified (detected)

 

Table 16: Summary of components quantified in urine and feces of rats administered 14C-test substance

Dose

mg/kg bw

Proposed or identified component

Parent or component as percent of dose

 

25

 

Urine

Feces

Urine + Feces

 

Male

Female

Male

Female

Male

Female

Test substance

35.9

53.7

31.1

48.0

67.0

101.7

Identified

35.9

53.7

31.1

48.0

67.0

101.7

Not identified

0.0

0.0

0.0

0.0

0.0

0.0

Total

35.9

53.7

31.1

48.0

67.0

101.7

Description of key information

The target substance is rapidly absorbed after oral ingestion and is mainly distributed within the plasma.. Based on physicochemical and toxicity data, it can be anticipated that the test substance is also absorbed via the dermal and inhalative route. However, dermal absorption is anticipated to be rather low. Under the conditions of the basic toxikokinetic study in rats no metabolites were identified. However, under other study conditions a metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, was observed in rats and dogs. The metabolite appears to be formed by de-carboxylation of the test substance upon repeated daily uptake of the test substance. In rats, the test substance was only excreted as parent chemical at approximately equal rates in urine (36% males, 56% females) and feces (32% males and 52% females). Total recovery of the test substance in urine, feces, cage wash, residual feed, tissues, and the remaining carcass was 69.9% and 107.9% for male and female rats, respectively. 14C residues of the test substance were not observed in exhaled air and tissues, except for a minor percentage in the carcass of males rats.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

A GLP-compliant toxikokinetik studyaccording to OECD 417 and GLP was performed in Sprague Dawley rats (Crl:CD(SD)) (2010 a). Main focus of this study was to obtain preliminary data on the material balance (route, rate, and extent of excretion, metabolic profile in excreta, and tissue burden of14C residues) and to establish definitive data on the pharmacokinetics and metabolic profile of14C residues in plasma. In this study, radiolabelled14C-test substance and unlabelled test substance were prepared in 0.5% methylcellulose and administered to male and female rats by single oral gavage. The radioactivity administered per rat ranged from 27 to 38 μCi for male rats and 19 to 27 μCi for female rats at a dose volume of approximately 4 mL/kg body weight (bw).

In a preliminary material balance experiment, a low (25 mg/kg bw) dose was administered to one male and one female rat followed by collection of exhaled breath for 0-48 h and urine and feces for 0-168 h, each at 24 h intervals. The distribution of14C residues in tissues including the carcass were evaluated at 168 h after dosing.

In the main pharmacokinetic study14C-test substance was administered to male and female rats at 25 or 500 mg/kg bw (4 rats per sex per dose level). Blood was collected for time course measurement of14C residues in plasma and red blood cells up to 30 h after dosing.

A third experimental group included evaluation of test substance metabolism in the plasma of male and female rats at 0.5 h after administration of a single high (500 mg/kg bw) dose using 3 rats per sex. The profile of metabolites was characterized in plasma, urine and feces by HPLC mass spectrometry in conjunction with in-line HPLC14C radio chromatography retention match using available reference standards for the test substance labelled and unlabelled and the metabolite.

 

In the preliminary material balance study a total recovery of the test substance in urine, feces, cage wash, residual feed, tissues, and the remaining carcass was 69.9% and 107.9% for male and female rats, respectively. No14C residues were detected as exhaled volatiles or CO2throughout a period of 48 h, indicating stability of the14C label within the parent molecular structure. The majority of the dose was recovered in the urine and feces during the first 24 h collection. By 168 h, excretion accounted for 36% and 32% in males and 56% and 52% in females in urine and feces, respectively. Analysis of14C residues in tissues were below detection by 168 h except for the low percentage (0.039%) detected in the male carcass.

Pharmacokinetic data were obtained for plasma and red bloods cells. The results indicated rapid absorption with peak concentrations in plasma (Tmax) at 0.4 to 1 h after dosing at 25 and 500 mg/kg bw. The mean Tmax values in red blood cells were 0.3 to 1 h. The peak concentrations (Cmax) in red blood cells were 1.3 to 2.0 µg eq/g at low dose and 27.2 to 28.7 µg eq/g at high dose and thus lower than those observed in plasma indicating very limited potential for binding within red blood cells. The red blood cell concentration data were insufficient for calculation of other pharmacokinetic parameters. In plasma, peak concentrations (Cmax) were 3.8 to 5.0 µg eq/g at low dose and 57.3 to 61.6 µg eq/g at high dose and were approximately proportional to the 20-fold increase in dose from 25 to 500 mg/kg bw. Near direct proportionality was observed for the Area-under-the-curve concentration vs. time curve, extrapolated to infinity (AUCINF) which was 7.0 to 9 h*µg/g in males and females of the low dose group and 150.8 to 168.4 h*µg/g in males and females of the high dose group. Plasma14C residues were quantifiable for up to 30 h after dosing. The terminal elimination half-lives in plasma were 5.6 to 5.7 h for male and female rats with no difference between the low (25 mg/kg bw) or high (500 mg/kg bw) dose groups.

Analysis of metabolites (14C residues) in plasma, urine and feces revealed that only test substance, as parent chemical was confirmed in all 3 matrices by radiochemical and mass spectral analysis. The results in this study indicated that biotransformation of test substance was absent in rats under conditions of single oral gavage at the 25 and 500 mg/kg bw doses. A metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, which has been quantified in plasma of rats administered test substance by dietary exposure was not observed under the conditions of this study.

 

Taken together, the test substance administered by single oral (gavage) administration at 25 and 500 mg/kg bw was rapidly absorbed and excreted unchanged at equally ratios in urine and feces within the 24 h.14C residues were not observed in exhaled air (0-48 h) and tissues (collected after 168 h), except for a minor percentage in the carcass of males rats. Pharmacokinetic data in plasma indicated rapid uptake and terminal elimination (T1/2 = 5.6-5.7 hours) with peak concentration and AUCINF values that were proportional with dose. Analysis of the metabolite profile in plasma at 0.5 h and in urine and feces at 0-24 h after dosing revealed only the test substance as parent chemical under the conditions of the rat study.

 

Anaylsis of test substance and its metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, in plasma samples were also included in 90 days-repeated dose toxicity studies in mice (2011 h) and dogs (2011 i).

In the mouse study, animals were fed with diet containing 300, 1000, 3000 or 7000 ppm test substance (corresponding to 47, 154, 459, and 1088 mg/kg bw/day in males and 61, 230, 649, and 1623 mg/kg bw/day in females, respectively). Blood of selected animals (5/sex/dose level) was collected for determination of plasma concentrations of the test substance and one of its metabolites on Day 60. Analysis revealed that the average concentration of the test substance in plasma was slightly higher in males than in females at all dose levels, with the highest concentrations reaching 2484 and 2313 ng/mL at 7000 ppm in males and females, respectively. There was no apparent plateau for the test substance plasma concentrations versus the dietary intake levels. In this study the analysed metabolite5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, was below the limit of quantitation (10 ng/mL) in all examined mouse plasma samples.

In the dog study, 4 animals per dose and sex were fed diet containing test substance concentrations of 250, 1250, 5000 and 15,000 ppm (corresponding to 6.46, 33.31, 126.23 and 425.71 mg/kg bw/day in males and 7.02, 37.94, 124.12, and 387.53 mg/kg bw/day in females, respectively). In week 9, blood was collected for determination of plasma concentrations of the test substance and its metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine. Analysis revealed that in both male and female dogs, the parent test substance was the most abundant analyte in plasma, with the highest concentrations reaching 63,000 and 82,575 ng/mL for 15,000 ppm males and females, respectively. In contrast, the plasma levels of the metabolite were significantly lower for this dose group with concentrations of approximately 12 ng/mL for males and females. This value was just above the limit of quantitation. The concentration of test substance in plasma was higher in females than in males at all dose levels, which may suggest greater dietary absorption by female dogs. There was no apparent plateau for the test substance plasma concentrations versus the dietary intake levels.

 

There are no studies available in which inhalation or dermal absorption were investigated. In accordance with Annex VIII, Column 1, Item 8.8 of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), dermal and inhalative absorption are assessed to the extent that can be derived from the relevant available information on physicochemical and toxicological characteristics.

 

The test substance is a solid with a molecular weight 213.62 g/mol. The substance has a low vapour pressure of 6.9215E-6 Pa at 20 °C and 4.9113E-6 Pa at 25 °C. The log Pow was determined to be -1.12 at 20 °C in Milli-Q water and the water solubility was shown to be 2.81 g/L at 20 °C. The median particle size distribution measured with laser scattering/diffraction was D50 = 11.8 µm, D10 = 2.8 µm, D90 = 48.6 µm. The surface tension was determined to be 72.1 nN/m at 20 °C and 885 mg/L, thus the substance is not surface active.

 

Absorption

Absorption is a function of the potential of a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

 

Oral absorption

Experimtal data show that the test substance is rapidly absorbed after oral administration (2010 a). In addition, signs of systemic toxicity reflected in lower body weights, reduced weight gain and food efficiency were visible in animal studies in rats and rabbits with repeated dose administration via food or gavage thereby pointing to systemic bioavailability (2011 g, 2011 o, 2011 m). Further, analysis of test substance in plasma in rats, mice and dogs, demonstrated that the test substance, as parent compound, was detectable in plasma of all species, indicating that the substance was absorbed after oral administration (2010 a, 2011 g, h, 2011 i).

 

Dermal absorption

There are no experimental data available on dermal absorption of the test substance. On the basis of the following considerations, the dermal absorption of the substance is considered to be rather limited. The test substance is a solid, which does not favour dermal absorption, since dry particulates will have to dissolve into the surface moisture of the skin before uptake can begin. However, the molecular weight of the substance indicates possible dermal uptake (100 g/mol < test substance (213.62 g/mol) < 500 g/mol). Furthermore, the log P of -1.12 indicates that substance is not likely to be sufficiently lipophilic to cross the stratum corneum and also the water solubility of 2.81 mg/L points to a rather low absorption potential through the skin. Since the surface tension of the test substance in an aqueous solution is > 10 mN/m, the substance is not a surfactant and thus dermal uptake is not enhanced.

The dermal permeability constant Kp of the substance was estimated to be 1.79e-005 cm/h using DermwinTM and taking into account the log Pow and molecular weight. Furthermore, the maximum flux Imax (Imax = Kp [cm/h] x water solubility [mg/cm³]) was calculated to be 1.79e-005cm/h*2.81 mg * 10-3/cm3= 0.05 ng/cm²/h. This flux value can be assigned to a possible but low dermal absorption.

For estimating the dermal permeability coefficient (Kp), the EPA developed an algorithm based on the Kow and molecular weight (U.S. EPA/ORD 1992, 2007). Using the molecular weight of 213.62 and the Kow at pH 7 of 0.0033 (Table 1), the estimated dermal permeability coefficient for the target substance is about 0.0000016 (0.00000043 to 0.0000061) cm/h. A similar algorithm, also based on molecular weight and Kow, was developed for estimating first-order dermal absorption rates. Applying the above values for the molecular weight and Kow of aminocyclopyrachlor, the estimated first-order dermal absorption rate coefficient for aminocyclopyrachlor is estimated at 0.00052 hour-1 with a 95% confidence interval of 0.00011 to 0.0025/h (Durkin, 2012).

 

The repeated-dose dermal toxicity study performed in rats (2008 a) contributes the assumption that the test substance is only poorly absorbed following topical application since there were no systemic signs of toxicity observed, which is in contrast to the observed effects after oral application (please refer to effects described above under “oral absorption”). In an acute dermal toxicity study in rats, a LD 50 > 5000 mg/kg bw was determined (2011 c). However, in the absence of adverse effects neither after oral nor dermal exposure, no conclusion can be drawn regarding dermal absorption, as absence of effects could be due to either low hazardous properties of the test substance or limited dermal absorption. As the test substance was not irritant to skin (2011 d), dermal absorption will not be enhanced due to damage to skin surface. In summary, based on the physicochemical and toxicity data, it can be anticipated that dermal absorption of the test substance is most probably rather low.

 

Inhalative absorption

There are no data available on inhalative absorption of the test substance. Substances that can be inhaled include gases, vapours, liquid aerosols (both liquid substances and solid substances in solution) and finely divided powders/dusts (ECHA, 2017). In general, depending on the physicochemical properties, test substances may deposit in the respiratory tract followed by absorption and/or transport out of the respiratory tract with possible subsequent uptake via the oral route. Since the test substance has a very low vapour pressure of 6.9215E-6 Pa at 20 °C and 4.9113E-6 Pa at 25 °C, it is of low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours, gases, or mists is not significant. However, in humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2017). Based on the median particle size distribution (D50 = 11.8 µm, D10 = 2.8 µm, D90 = 48.6 µm), test substance particles may reach the alveolar region once they have been inhaled. The moderate Log P of -1.12 favours absorption across the respiratory tract epithelium by passive diffusion. The relatively low water solubility of 2.8.1 mg/kg bw, together with a small particle size may enhance penetration to the lower respiratory tract.

In the acute inhalation toxicity study (2011 b), slight reductions of body weight were observed on the day following test substance exposure. In addition, one female rat had discharge from the eyes on the day of exposure (day 0) and 3 males and 2 females demonstrated stained skin/fur on days 0 and 1. No clinical observations were determined after 2 days of exposure. However, as it is unclear whether these signs were related to systemic availability of the test-substance, no conclusion can be drawn in regard to toxicokinetic behaviour following inhalation. In conclusion, the physicochemical and toxicity data indicate possible inhalative absorption once the test substance is inhaled.

Overall, the toxicokinetic studies performed with the target substance indicate a rapid absorption of the test substance after oral administration. Based on physicochemical and toxicity data, it can be anticipated that the test substance is also absorbed via the dermal and inhalative route. However, dermal absorption is anticipated to be rather low. After oral uptake, the test substance was distributed in plasma and to a minor extent in red blood cells. In contrast the test substance was not observed in exhaled air or any tissues, with the exception of a very limited amount found in the carcass of male rats.

No metabolite was evident after a single dose administration of 25 or 500 mg/kg bw in plasma, urine or feces. Only test substance, as parent chemical was confirmed in all 3 matrices by radiochemical and mass spectral analysis. However, a metabolite, 5-chloro-2-cyclopropyl-pyrimidin-4-ylamine, was detectable at concentrations of approximately 12 ng/mL, which is just above the limit of quantitation, in a repeated dose toxicity study in dogs (2011 i). In addition, the metabolite has been quantified in rat plasma at Day 56 of a 90-day toxicity study by dietary administration at concentrations corresponding to 100 to 945 mg/kg bw/day for male rats and 129 to 1268 mg/kg bw/day in female rats. Thus, although not identified under conditions of the available toxicokinetik study in rats, a metabolite appears to be formed by de-carboxylation of the test substance upon repeated daily uptake of the test substance (2010 a).

In the rats, excretion of the test substance occurred via the urine and feces in approximately equal proportions and was substantially complete within the first 24 h after dosing (2010 a).

 

References:

Durkin, P.R, Aminocyclopyrachlor Human Health and Ecological Risk Assessment, SERA TR-056-01-03a, 2012