Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 December 2016 till 04 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Objective of study:
absorption
excretion
toxicokinetics
Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
Version July 22, 2010
Deviations:
no
Remarks:
Tissue distribution and metabolism were not investigated as these were not objectives of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N'-{3-[(3-aminopropyl)amino]propyl}-N,N-di-C16- C18 (evennumbered) alkyl propane-1,3-diamine
EC Number:
941-593-4
Cas Number:
1623405-26-4
Molecular formula:
Not applicable UVCB
IUPAC Name:
N'-{3-[(3-aminopropyl)amino]propyl}-N,N-di-C16- C18 (evennumbered) alkyl propane-1,3-diamine
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Purity: 100% (UVCB substance)
- Purity test date: 08 July 2016
- Batch No.: 1330539
- Expiration date of the batch: 04 August 2018
- Appearance: Viscous liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- pH: 8.5-10.5 at concentration of 1%
- Specific gravity/density: 0.864 at 20°C
Specific details on test material used for the study:
Non-radiolabeled Test Item: di-C16-C18 (evennumbered) alkyl tripropylenetetramine, batch 1330539

Radiolabeled test item:
Identification: [octadecyl-1-14C]Tetrameen 2HT Main component
Appearance: Light brown waxy solid
Batch 9448JRD062-1
Radiochemical purity: 93.4%
Chemical purity: Not indicated
Test substance storage: In freezer (≤ -15°C)
Stable under storage conditions: Not indicated
Specific activity: 2025 MBq/mmol, 2.91 MBq/mg
Supplier: Selcia Limited, Fyfield Business and Research Park, Fyfield Road, Ongar, Essex, CM5 0GS, UK
Total activity received: 1382.5 MBq
Date received: 09 November 2016

The guidelines indicated that radiochemical purity should be ideally greater than 95%, but despite considerable efforts it was not possible to attain a greater radiochemical purity than 93.4%.
The radiochemical purity was determined by the supplier and was also checked at Charles River Den Bosch before the start of the experiment.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Rat strain was Crl:WI(Han) (outbred, SPF-Quality), obtained from Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France. Young adult animals of 8-12 weeks old were used. Environmental controls for the animal room were set to maintain 18 to 24°C (range of actual daily mean: 20.2-20.6°C), a relative humidity of 40 to 70% (range of actual daily mean 37-48%), approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Animals were acclimatized for at least 5 days under laboratory conditions. Animals were dosed orally at least twice during the acclimatization period with milli-Q in order to train the animals. For bile cannulated animals the acclimatization period was kept as short as possible and no oral training was performed. The day prior to dosing and following radioactive dose administration, the rats of Groups 1-2 were individually housed in stainless steel metabolism cages with a Plexiglas lid. A stream of air, which was aspired by a vacuum source, was successively passed through the metabolism cage, and through 4 glass gas absorption towers. The first two gas absorption towers contained a 1.5 M NaOH solution, to collect expired CO2 and water soluble volatiles. The next two gas absorption towers contained approximately 150 mL of Cellosolve to collect residual organic volatiles. The urine and feces collection assembly of the metabolism cage was cooled using dry ice. Following radioactive dose administration, the rats of the bile groups (Group 5) were individually housed in stainless steel metabolism cages (LxWxH = 18.5x19x20 cm) with a grid. Following radioactive dose administration, animals in Groups 3 and 4 were housed individually in Makrolon cages (type MII), equipped with a bottom grid and paper bedding. Throughout the study, except for approximately 18 hours prior to and for approximately four hours after dose administration, animals had free access to pelleted rodent diet and to tap-water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Oral gavage using a stainless steel stomach tube. Dose volume 5 mL/kg body weight.
Formulation dose group 1, 2, 3 and 4:
An empty container with stirring device was weighed and 19.2 mg of 208027/A and 5.6017 g of 206646/B were added. Subsequently, 20.7360 g of corn oil was added and the formulation was stirred until complete dissolution. The resulting formulation was a yellow suspension.
Formulation dose group 5
An empty container with stirring device was weighed and 5.6 mg of 208027/A and 1.6002 g of 206646/B were added. Subsequently, 5.8953 g of corn oil was added and the formulation was stirred until complete dissolution. The resulting formulation was a yellow suspension.
Duration and frequency of treatment / exposure:
Groups 1, 3 and 5 were dosed once on Day 1. Groups 2 and 4: Rats were treated with unlabeled test item for 7 days prior to administration of a single radiolabeled dose.
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
The dose level of 1000 mg/kg body weight was selected based on the OECD-422 Combination 28-Day Study (Test Facility Study No. 509313/509314) where no clinical signs were noted in the animals. In addition, 1000 mg/kg body weight is the limit dose according to the OECD-417 guidelines.
The radioactive dose administered to each animal was calculated from the weight of the dose administered and the mean radioactive concentration of the formulations. Animals in Groups 1 - 4 received an average dose ranging between 951 - 968 mg/kg di-C16-C18 (evennumbered) alkyl tripropylenetetramine. Animals in Group 5 received a slightly higher effective average radioactive dose of 977 mg/kg di-C16-C18 (evennumbered) alkyl tripropylenetetramine.
No. of animals per sex per dose / concentration:
4 male animals for each of groups 1, 2, 3, 4 and 5.
Positive control reference chemical:
No, not relevant.
Details on study design:
Please refer to attached background material, which starts with a Table providing an outline of the study design.
Details on dosing and sampling:
Please refer to attached background material, which starts with a Table providing an outline of the study design including the duration of sampling. ADME group (Groups 1-2): The duration was 168 hours. Toxicokinetics group (Group 3-4): The duration was 24 hours. Bile-cannulated group (Group 5): The duration was 48 hours as almost 100% of the labeled test item was already excreted via feces within 48 hours in Group 1.

SAMPLING FOR 14C-ANALYSIS
Mass-balance groups (Groups 1 and 2):
Urine was collected over the following time intervals: 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168h. Feces was collected over the following time intervals: 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168h. Volatiles were collected over the following time intervals: 0-24 and 24-48h.
Bile group (Group 5):
Urine was collected over the following time intervals: 0-6, 6-24 and 24-48h. Bile was collected over the following time intervals: 0-6, 6-24 and 24-48h. Feces was collected over the following time intervals: 0-24 and 24-48h.
At termination, the interior of the metabolism cages were rinsed with methanol/water (1:1, v/v). Volatiles were collected for 48 h. Cellosolve-traps were omitted, since the daily amount collected in these traps was negligible (< 0.4% of the administered dose).
For animals in Group 3 and 4, urine, feces, bile, volatiles and cage washings were not sampled for analysis.
At the end of the collection period, the animals were euthanized and the carcass was stored at ≤-15ºC for total 14C analysis.

Toxicokinetic Blood Sampling
Approximately 0.3 mL blood samples were taken from the jugular vein and collected into
tubes containing Li-heparin as anticoagulant. Samples were taken on each of the following time points:
Group 3: 1, 2, 4, 7 and 24 hours after dosing.
Group 4: predose, 1, 2, 4, 7 and 24 hours after the last dosing.
A sub sample of approximately 0.1 mL of the blood was removed for total radioactivity analysis and the remaining blood was centrifuged to obtain the plasma.
Statistics:
Standard calculation of mean, standard deviation and coefficient of variation.

Results and discussion

Preliminary studies:
Not applicable.
Main ADME resultsopen allclose all
Type:
absorption
Results:
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) after a single oral dose of 1000 mg/kg bw (group 1) was 0.663% AR.
Type:
absorption
Results:
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) after a repeated oral dose of 1000 mg/kg bw (group 2) was 0.748% AR.
Type:
excretion
Results:
The mean total excretion after a single oral administration of 1000 mg/kg bw (group 1) was 108% after 7 days (0.15% in urine and 108% in faeces). Excretion in bile of bile-cannulated rats (group 5) was 0.006-0.007%.
Type:
excretion
Results:
The mean total excretion after repeated oral administration of 1000 mg/kg bw (group 2) was 101% after 7 days (0.15% in urine and 101% in faeces).
Type:
other: Toxicokinetic evaluation of radioactivity levels in blood and plasma
Results:
After oral administration the measured radioactivity levels in blood and plasma were low, the majority of the measured values were below the lower limit of quantification. As a result, no reliable toxicokinetic evaluation could be performed.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Please refer to attached background material, which provides a Table with a summary of radioactivity data (Text Table 2).

The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose).

BILE-CANNULATED RATS (GROUP 5)
One animal (animal 18) died within 24 hours, this animal was excluded from the radioactive concentration calculations in the bile-cannulated group. Animal R3 showed a different radioactivity profile compared to the other two animals in the bile-cannulated group; higher total remaining amount in the carcass and lower amount in the feces. To visualize the difference between animal R3 and the other animals, the average value was calculated with and without animal R3. These values are presented between brackets below and in Text Table 2 in the attached background document.
After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 81% (96%) after 48 hours. Excretion via urine and bile were both minor routes of excretion, i.e. 0.03% (0.03%) for urine and <0.01% (<0.01%) for bile after 48 hours. These results indicate that the radioactivity excreted in the feces after oral administration consists of unabsorbed compound. In the bile-cannulated group, the average total remaining radioactivity in carcass inclusive blood and tissues was 15.9% (3.5%). Average total recovery of radioactivity after a single oral administration was 97% (100%) in the bile-cannulated group.
The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 16% (4%). The relative high remaining radioactivity in the carcass and as a result the high oral absorption was caused by animal R3, where the total remaining amount in the carcass was 40.7%. This is likely due to radioactivity still being present in the GI tract, as the radioactivity in feces was much lower compared to the other two animals (49.8%). The radioactivity excreted via urine and bile was very low for this animal, comparable to the other two animals, therefore this radioactivity remaining in the GI tract is considered unabsorbed.



Details on distribution in tissues:
Not applicable.
Details on excretion:
Please refer to attached background material, which provides a Table with a summary of radioactivity data (Text Table 2).

After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Average total recovery of radioactivity after a single oral administration was 109% and 102% after repeated administration.

BILE-CANNULATED RATS (GROUP 5)
After oral administration of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 81% (96%) after 48 hours. Excretion via urine and bile were both minor routes of excretion, i.e. 0.03% (0.03%) for urine and <0.01% (<0.01%) for bile after 48 hours. These results indicate that the radioactivity excreted in the feces after oral administration consists of unabsorbed compound. In the bile-cannulated group, the average total remaining radioactivity in carcass inclusive blood and tissues was 15.9% (3.5%). Average total recovery of radioactivity after a single oral administration was 97% (100%) in the bile-cannulated group.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
Not applicable.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
Not applicable.

Any other information on results incl. tables

CLINICAL OBSERVATIONS

No mortality occurred, except for one animal (no. 18) in the bile-cannulated group that was found dead approximately 24 hours after radioactive dose administration. Piloerection and no feces production were observed. No macroscopic findings were observed at necropsy. This animal was excluded from the radioactive concentration calculations in the bile-cannulated group.

Animal R3 in the bile-cannulated group showed decreased food consumption and feces production compared to the other bile-cannulated animals, possible indicating a deteriorated health state of animal R3. Piloerection was noted after radioactive dose administration in almost all animals for a maximum of seven days. In addition, salivation, red discoloration of the feces and chromodacryorrhoea and rales were noted in the repeated dose groups after radioactive dose administration.

Table: Summary Radioactivity

Groups

Radioactivity

% of administered dose presented as mean ± SD

Urine

Feces

Bile

Total excretion

Carcass, incl. blood and tissues

Total recovery

Minimal oral absorption

Mass-balance

Group 1 - Single Dose

0.150±0.060

108.0±10.2

n/a

108.3±10.3

0.328±0.041

108.6±10.2

0.663±0.057

Mass-balance

Group 2 - Repeated Dose

0.148±0.052

100.8±4.24

n/a

101.1±4.15

0.406±0.099

101.5±4.11

0.748±0.242

Bile-cannulated

Group 51

0.033±0.009

(0.028)

80.7±26.9

(96.2)

0.006±0.001

(0.007)

80.7±26.9

(96.2)

15.9±21.5

(3.53)

96.7±5.41

(99.7)

16.0±21.5

(3.56)

1:            Values between brackets in Group 5 are the mean values without animal R3, as explained below in the bile-cannulated section of the summary.

n/a:       not applicable

Applicant's summary and conclusion

Conclusions:
After oral administration at 1000 mg/kg bw of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Excretion via bile in bile-cannulated rats was ≤0.007%. The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose). After oral administration the majority of the measured values were below the lower limit of quantification and as a result, no reliable toxicokinetic evaluation could be performed.
Executive summary:

Male Wistar rats (4 per group) received either a single or a repeated oral dose of 1000 mg/kg bw of of [octadecyl-1-14C]Tetrameen 2HT Main component. The duration of sample collection following dosing was 168 hours for the mass balance groups 1 (single dose) and 2 (repeated dose), 24 hours for the toxicokinetics groups 3 (single dose) and 4 (repeated dose) and 48 hours for the bile-cannulated group, which received a single dose.

After administration of the radiolabeled dose in the mass-balance groups, urine was collected in 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours intervals. Feces were collected in 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours intervals. Volatiles were collected in 0-24 and 24-48 hours intervals. Animals were euthanized at the end of the collection period and cage washings were collected. The carcass was stored for total 14C analysis. Total radioactivity in urine, feces, volatiles, cage washings and carcass was determined.

In the toxicokinetic groups, blood was sampled from the jugular vein at pre-dose (Group 4 only) and 1, 2, 4, 7 and 24 hours after the radiolabeled dosing. Total radioactivity concentrations were determined.

In the bile-cannulated group, urine and bile were collected in 0-6, 6-24 and 24-48 hours intervals and feces were collected in 0-24 and 24-48 hours intervals. Animals were euthanized at the end of the collection period and cage washings were collected. The carcass was stored for total14C analysis. Total radioactivity in urine, bile, feces, cage washings and carcass was determined.

After oral administration at 1000 mg/kg bw of [octadecyl-1-14C]Tetrameen 2HT Main component, the majority of the radioactivity was excreted via feces, i.e. 108% (single dose) and 101% (repeated dose) after 168 hours. Excretion via urine was a minor route of excretion. Urinary excretion accounted for 0.15% (single and repeated dose) after 168 hours. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was on average 0.3% (single dose) and 0.4% (repeated dose), indicating no accumulation of radioactivity after 168 hours. Excretion via bile in bile-cannulated rats was ≤0.007%. The calculated oral absorption (accumulate urine, volatiles and carcass recoveries) of [octadecyl-1-14C]Tetrameen 2HT Main component after oral dosing was on average 0.7% (single dose) and 0.8% (repeated dose). After oral administration the majority of the measured values in blood and plasma were below the lower limit of quantification and as a result, no reliable toxicokinetic evaluation could be performed.

In conclusion, [octadecyl-1-14C]Tetrameen 2HT Main component administered orally was barely absorbed, not quantifiable in plasma and blood and mainly excreted unabsorbed via the feces.

(See graph cumulative excretion)

  • The repeated dose group shows no differences to single dose group.
  • The low inter-animal variability, including single & repeated dose groups, supports that these absorption characteristics are mostly caused by physico-chemical properties with little biological influence, suggesting also a low intra-species variability.
  • Considering radio-chemical purity of 93.4%, it is most likely that all this information is applicable to both the a.i as the impurities (conservative approach, as likely the impurities will be lower mw substances and likely to have a somewhat higher bioavailability)
  • The bile data shows that there is no entero-hepatic circulation taking place
  • The apparently higher absorption of bile group relates to higher carcass levels. Considering lower levels in urine it is actually indicating a lower level of systemic absorption. This means that carcass levels relate to unabsorbed remnants in intestines. Furthermore, bile group is only different to other with the evaluation of excretion via bile. They cannot be expected to show otherwise different absorption as non-surgically treated animals. (Besides, bile suggested to increase the uptake of fatty substances; bile cannulated animals do not produce bile for GI tract anymore, and if anything, absorption would possibly be even lower.)