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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Objective of study:
metabolism
toxicokinetics
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.36 (Toxicokinetics)
Version / remarks:
1988
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Name of test material: DOPA-Glycinate
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: CD / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., Sulzfeld, Germany
- Age at study initiation: 7 weeks (males), 9-12 weeks (females)
- Weight at study initiation: 228–252 g (males), 210–288 g (females)
- Fasting period before study: no
- Housing: individually
- Individual metabolism cages: no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5-8 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: Oral - gavage (single administration: Group 1, 2 and 3; repeated administration: group 5); intravenous (single administration: group 4)
Vehicle:
other: Oral (group 1, 2, 3 und 5): 0.8 % aqueous hydroxypropyl-ethyl-cellulose gel; Intravenous (group 4): 0.9 % NaCl solution
Duration and frequency of treatment / exposure:
single oral administration: Group 1, 2 and 3
repeated oral administration: group 5; once daily for 13 d
single intravenous administration: group 4
Doses / concentrations
Remarks:
Doses / Concentrations:
30 mg/kg bw and 100 mg/kg bw of test substance (each dose contained 0.49 mg 14C-labelled test item/kg bw corresponding to a radioactive dose of 700 kBq/kg bw).
No. of animals per sex per dose:
Group 1 (low dose): 8 females
Group 2 (low dose): 4 males and 4 females
Group 3 (high dose): 4 males and 4 females
Group 4 (low dose): 8 females
Group 5 (repeated low dose): 4 females
Control animals:
no
Details on study design:
Pilot study:
In order to determine the high dose level for the main study an orientating acute oral toxicity test and a preliminary excretion experiment to determine the relevance of excretion via expired air were carried out. Based on the results of these preliminary studies the following decisions were made for main study:
A high dose level of 100 mg/kg bw was used in the main study;
Excretion of test substance via expired air was significant and thus measured during main study.
Statistics:
Pharmacokinetic evaluation of plasma data using program TopFit 2.11

Results and discussion

Preliminary studies:
The following no-effects levels were determined in an orientating experiment:
NOEL rat, i.v. = 125 mg/kg bw; NOEL rat, p.o = 400 mg/kg bw.
The administered radioactivity was distributed as follows: Approximately 55 %/19 % (p.o./i.v.) in the faeces, 6 %/23 % in urine, 0.6 %/0.9 % in cage wash, and 12 % (p.o.) in expired air. The mean of the highest plasma concentration was 0.04 %/0.25 % (p.o./i.v.). The fraction of radioactivity excreted in expired air indicated that this route of elimination is relevant.
Main ADME resultsopen allclose all
Type:
absorption
Results:
34% oral absorption
Type:
distribution
Results:
throughout the body, with the highest levels in the residual carcass, in the liver, the kidneys, and the adipose tissue
Type:
metabolism
Results:
mainly oxidation (hydroxylation), to a minor extent dehydrogenation and acetylation
Type:
excretion
Results:
excreted rapidly within 24 hours; main elimination pathway: via the faeces, followed by expired air and urine

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Oral absorption: The systemic bioavailability of 14C labelled test substance was 34 % following oral administration, as derived from the comparison of the AUC0–∞ (see Table A6.2- 2).
Details on distribution in tissues:
Radioactivity was distributed throughout the body, with the highest levels in the residual carcass, in the liver, the kidneys, and the adipose tissue. Nevertheless, distribution was quite uniform across all examined organs with no tissue storage in brain, heart, ovaries and spinal cord. There was no significant difference in the distribution of radioactivity in the tissues after single or repeated administration of 30 mg test substance/kg bw.
Results of tissue distribution are summarised in the Table A6.2- 3.
Details on excretion:
The vast majority of the administered radioactivity was excreted rapidly within 24 hours.
The main elimination pathway was via the faeces, followed by expired air and urine in all test groups. See Table A6.2- 1 for details.
After 168 hours approximately 60% of the radioactivity administered had been excreted via the faeces in the male and female animals, approximately 18% was excreted via expired air (approx. 15% in the males, 20% in the females), and approximately 9% was recovered in the urine. Less than 20% was found in organs and carcass.
There were no relevant differences in the mean radioactive recovery after 24 or 168 hours from urine and cage wash between the low, high, and repeated dose groups (see Table A6.2- 1).

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
In urine, the parent compounds N-C12 Gly, N’-C12 Gly, N’-C12 di Gly, C12 PDA and several metabolites were identified by mass spectrometry. Oxidation (hydroxylation) of the parent and some other metabolites like dehydrogenated and acetylated compounds, especially for C12 PDA, were identified. The most abundant compounds in urine were the oxidation products.
In plasma, only the unchanged parent compounds N-C12 Gly, N’-C12 Gly, N’-C12 di Gly, C12 PDA could be identified by mass spectrometry. For the radioactivity counting only N-C12 Gly, N’-C12 Gly were identified. The most abundant compounds in plasma were the parent substance N-C12 Gly and N’-C12 Gly.
In faeces, N-C12 Gly, N’-C12 Gly, N’-C12 di Gly, C12 PDA and several metabolites were identified by mass spectrometry. Oxidation (hydroxylation) of the parent and some other metabolites, like dehydrogenated and acetylated modifications, was demonstrated. The most abundant compounds in faeces were the oxidation products.

Any other information on results incl. tables

 

Toxic effects, clinical signs

In the main experiment one male animal died prematurely and was found dead on test day 6. This death was not test item-related, but caused by a congested and blocked absorber for CO2determination.

The faeces of all rats showed a normal consistency during the entire experimental period.

No other effects were reported.

Recovery of labelled compound

Total recovery of administered radioactivity as determined in the excretion studies ranged from 97 to 103 % (rounded, seeTable A6.2-1).

Pharmacokinetics

After administration of 30 mg/kg bw (low dose) of [14C]-labeled test substancevia the oral and intravenous route the following toxicokinetic parameters were established:

Plasma half-life:

approx. 74 h both in the p.o. and i.v. group

 

 

 Table A6.2-1:Excretion: Mean recovery of radioactivity (%) after administration of [14C]-labelled test substance

 

Group 2:

Single low dose
30 mg/kg bw

Group 3:

Single high dose
100 mg/kg bw

Group 5:

Repeated low dose
30 mg/kg bw

 

Male

Female

Male

Female

Female

Number of animals

4

4

4

4

4

Urine (0–24 h)

6.88
± 0.56

6.65
± 0.42

7.92
± 0.66

7.77
± 0.72

6.62
± 0.88

Urine (0–168 h)

8.59
± 2.52

8.39
± 2.42

9.84
± 2.90

10.03
± 2.83

8.92
± 2.39

Faeces (0–24 h)

51.06
± 3.26

51.72
± 3.28

49.03
± 2.82

44.01
± 3.89

42.35
± 4.04

Faeces (0–168 h)

59.32
± 19.16

59.47
± 18.89

58.55
± 18.13

57.68
± 16.24

54.17
± 15.55

Expired air (0–24 h)

10.13
± 2.72

12.74
± 0.77

9.85
± 1.20

13.00
± 2.21

7.84
± 1.42

Expired air (0–168 h)

15.39
± 3.58

21.49
± 4.44

15.57
± 3.46

21.53
± 4.53

13.76
± 2.68

Cage wash (0–168 h)

0.31
± 0.08

0.28
± 0.06

0.15
± 0.03

0.34
± 0.07

0.40
± 0.06

Carcass and organs

n.e.

13.33*
± 4.24*

n.e.

n.e.

19.22
± 6.20

Total

 

102.96

 

 

96.47

Data expressed as mean % of total dose (dpm) administered ± standard deviation

*          Values derived from test group 1 with 8 female test animals administered orally a single low dose of test substance

n.e.:    not examined

 

 

Table A6.2-2:Toxicokinetic parameters for [14C]-labelled test substance.

Dosage

Route of administration

Noncompartmental analysis

Cmax#
[Bq/mL]

tmax#
[h]

t1/2
[h]

AUC0-120 h
[Bq*h/mL]

AUC0-¥h
[Bq*h/mL]

AUC/dose##
[g*h/mL]

30 mg/kg bw

p.o.

47.5

6.00

76.62

2733.30

4126.83

6.87

 

i.v.

606.5

-

71.28

9304.88

12121.16

16.76

#         Values obtained from the analytical results, all other values calculated by toxicokinetic analysis

##       Dose in Bq/kg b.w. (mean values per group)

i.v.      intravenous administration

p.o.     oral administration

 

Table A6.2-3:Distribution:Mean recovery of radioactivity (%) in organs at termination

 

Group 1:

Single oral dose
30 mg/kg bw

Group 4:

Single intravenous dose
30 mg/kg bw

Group 5:

Repeated oral dose
30 mg/kg bw

 

Female

Female

Female

Number of animals

8

8

4

Adipose tissue

0.07
± 0.01

0.11
± 0.05

0.15
± 0.06

Brain

0.02
± 0.00

0.07
± 0.01

0.02
± 0.01

Carcass

12.13
± 3.94

19.06
± 2.16

17.72
± 2.72

Heart

0.02
± 0.01

0.09
± 0.02

0.03
± 0.01

Kidneys

0.17
± 0.03

0.75
± 0.19

0.22
± 0.04

Liver

0.91
± 0.21

3.72
± 0.68

1.06
± 0.50

Ovaries

0.01
± 0.01

0.05
± 0.01

0.02
± 0.01

Spinal cord

0.00
± 0.00

0.01
± 0.00

0.00
± 0.00

Total

13.33
± 4.24

23.86
± 6.62

19.22
± 6.20

Data expressed as mean % of total dose (dpm) administered ± standard deviation

 

Figure A6.2-1:Proposed pathway of the metabolism of N-C12 Gly

see attached Image file

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
The systemic bioavailability of 14C labelled DOPA-Glycinate was 34% following oral administration.
The substance was distributed throughout the body, with the highest levels in the residual carcass, in the liver, the kidneys, and the adipose tissue.
The plasma half-life was determined to be approx. 74 h.
The substance is mainly metabolised by oxidation (hydroxylation), and to a minor extent dehydrogenation and acetylation.
The vast majority of the administered radioactivity was excreted rapidly within 24 hours. The main elimination pathway was via the faeces, followed by expired air and urine.
Executive summary:

Absorption, distribution, metabolism and excretion of [14C]- DOPA-Glycinate (20% a.i.) were investigated in the rat according to the OECD test guideline 417. The test substance was administered orally by gavage at two single dose levels of 30 and 100 mg/kg bw, singly intravenous and orally at one repeated low dose level.

Upon oral administration, radioacitivity was predominantly eliminated via faeces (approx. 60 %) and via expired air (approx. 18 %). A lower proportion was excreted via urine (approx. 9 %) and less than 20 % of the administered dose was found in the carcass. No significant differences of absorption and elimination of test substance regarding high or low dose, repeated administration or sex could be indentified. The recovery of radioactivity was greatest in the residual carcass (12–19 %), followed by the liver (approx 4 %). In all other tissues the radioactive recovery was low (≤ 1.00 %) and quite uniformly distributed, with no tissue storage in brain, heart, ovaries and spinal cord. There was no relevant difference in the tissue distribution of radioactivity between animals treated orally or intravenously with a single or repeated dose of the test substance. The elimination half-life was approx. 74 hours both in the p.o. and i.v. group.

The systemic bioavailability, assessed by comparison of plasma radioactivity after oral and intravenous administration, was 34%.

In plasma, only parent substances but no transformation products could be identified. A first-pass effect following intestinal absorption is therefore unlikely. In urine and faeces, parent substances as well as transformation products were detected. Metabolites in both urine and faeces were characterised by oxidation (hydroxylation) of the parent and some other metabolites like dehydrogenated and acetylated compounds. The most abundant compounds in urine were the oxidation products.