Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-January 2014 to 1-February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Radiolabelled material
Chemical name: [14C] EXP1200078
Source: Quotient Bioresearch (Radiochemicals) Ltd
Batch number: CFQ42196
Specific activity: 1.77 µCi/mg or 65.5 kBq/mg
Physical form: Liquid
Storage conditions: Ambient

Non-radiolabelled material
Chemical name: EXP1200078
Source: Infineum
Batch number: E00007-285
Physical form: Very dark brown (almost black) viscous liquid
Purity: Classed as UVCB (Unknown or Variable composition, Complex reaction products or Biological materials) therefore absolute purity cannot be defined.
Expiry: End-2015
Storage conditions: Ambient
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Male and female Sprague Dawley rats were used in this study. On receipt, from Charles River UK Ltd. (Margate, Kent, UK), the animals were subjected to health examination prior to acceptance onto the study.
Sex:
male/female
Details on test animals and environmental conditions:
Animals (n=61) used in this study were between 7 and 13 weeks old and in the weight range 183-342 g at the time of first dosing. Each rat was given a unique identity number and identified by unique tail markings (with indelible ink).

Animals were housed in polypropylene cages in a thermostatically monitored room and exposed to 12 hours fluorescent lighting and 12 hours dark per day. The temperature and relative humidity of the holding room was recorded continually throughout the experiment and were generally within the ranges 21 ± 2 degrees C and 55 ± 10%, respectively. Animals were equilibrated under standard animal house conditions for at least 5 days prior to dosing. The health status of the animals was monitored throughout this period and the suitability of each animal for experimental use was confirmed before use.

At least one day prior to dosing, the animals for use in Phases 1 (stage 2), 4, 5 and 6 (excretion balance) and at least two days prior to dosing (biliary excretion animals) were transferred to individual metabolism cages specially designed for the separate collection of urine, faeces and expired air (if applicable) under the above environmental conditions prior to administration (for acclimatisation) and remained therein for the duration of the study except for dosing. Animals were returned to their individual metabolism cages immediately after completion of this procedure.

The animals were fed a pellet diet (RM1 (E) SQC, Special Diets Services, Witham, Essex, UK) and water was available ad libitum throughout the holding, acclimatisation and post dose periods.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
paraffin oil
Details on exposure:
Described below in "Details on study design."
Duration and frequency of treatment / exposure:
Described below in "Details on study design."
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control for all study phases
Dose / conc.:
800 mg/kg bw/day
Remarks:
Phase 1 and 2 preliminary experiments, only. Dilution error, so solution should have been 1000 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Remaining study phases for the high-dose group.
No. of animals per sex per dose:
Described below in "Details on study design."
Control animals:
yes, concurrent vehicle
Positive control:
None
Details on study design:
Phase 1: Preliminary studies / Preliminary pharmacokinetic experiment
After a single oral dose of [14C]-Test Material (ca. 800 mg/kg) to one male and one female rat, serial blood samples (for plasma preparation) were collected from a tail vein at the following times:
30 minutes and 1, 2, 4, 6, 8, 12, 24 and 48 hours post dose administration
At 48 hours after the dose administration the rats were killed by cervical dislocation.

Phase 1: Preliminary studies / Preliminary excretion experiment
After a single oral dose of [14C]-Test Material (ca. 700-800 mg/kg) to one male and one female rat, urine (collected in a container cooled in solid carbon dioxide), faeces and expired air were collected separately at the following intervals after dosing:
Urine: Predose, 0–6, 6 – 24 and 24–48 hours
Faeces: redose, 0–24 and 24–48 hours
Expired air: Predose, 0–24 and 24–48 hours
At 48 hours after dose administration, the rats were killed by cervical dislocation.

Phase 2: Pharmacokinetic study in male and female rats with single oral dose.
After a single oral dose of [14C]-Test Material at a dose level of either 100 mg/kg or 1000 mg/kg to two groups of three male and three female rats (one group per dose level), concentrations of radioactivity were measured in whole blood and plasma at pre dose and at the following times after dosing:
30 minutes and 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours

Blood samples were collected from three male and three female rats (per dose group), either serially via a tail vein or terminally via cardiac puncture under anaesthetic. After the final sampling time, the animals were killed by cervical dislocation. All blood samples were collected into heparinised containers. A portion of each blood sample was retained as whole blood whilst the remainder was centrifuged for harvesting of plasma into clean neutral tubes, blood cells were discarded.

Phase 3: Pharmacokinetic study in male rats (repeat oral dose)
A single oral dose of [14C]-Test Material was administered once a day for seven consecutive days at a dose level of 1000 mg/kg/day to two groups of three male rats.
For group 1, concentrations of radioactivity were measured in whole blood and plasma at pre dose, once a day during dosing (4 hours post dose administration) and a terminal sample at 4 hours after the final dose.

For group 2, concentrations of radioactivity were measured in whole blood and plasma at pre first and last dose and at the following times after the final dose:
30 minutes and 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours

Blood samples were collected either serially via a tail vein or terminally via cardiac puncture under anaesthetic. After the final sampling time, the animals were killed by cervical dislocation. All blood samples were collected into heparinised containers. A portion of terminal blood sample was retained as whole blood whilst the remainder was centrifuged for harvesting of plasma into clean neutral tubes, blood cells were discarded.

Phase 4: Excretion Balance study in male and female rats (single oral dose)
Two groups of four male and four female rats (one group per dose level) were administered a single oral dose of [14C]-Test Material at a dose level of either 100 mg/kg or 1000 mg/kg. Urine and faeces were collected separately as described below.

At 168 hours animals were killed by cervical dislocation and the carcasses retained at approximately –20°C prior to analysis.

The radioactivity associated with each sample was determined by quantitative radiochemical analysis (QRA).

Urine, cooled by solid carbon dioxide, was collected into individual, pre-weighed containers at the following intervals:

Pre-dose, 0–6, 6-12, 12-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours

The weight of each urine sample was recorded and duplicate weighed aliquots were taken for direct QRA. Prior to and following analysis, urine samples were retained at approximately -20°C.

Faeces were collected into individual pre-weighed containers at the following intervals:

Pre-dose, 0–24, 24–48, 48–72, 72-96, 96-120, 120–144 and 144–168 hours

The weight of each faeces sample was recorded and an appropriate volume of water was added prior to homogenisation. Each homogenate weight was recorded and duplicate weighed aliquots of each homogenate were taken for oxidation prior to QRA. Prior to and following analysis, faeces samples were retained at approximately -20°C.

Carcasses were solubilised individually, in ca. 1L of 2M NaOH in Water:Methanol:Triton X-405 (6:3:1, v:v:v) at ca. 55°C for ca. 24-48 hours. The total weight of each solubilised carcass was recorded and duplicate weighed aliquots of each sample were taken for direct QRA. Prior to solubilisation, carcasses were stored at approximately -20°C. After solubilisation digests were retained at room temperature.

The washings (water followed by methanol) from each metabolism cage were separately collected into pre-weighed plastic containers. The weight of each cage wash sample was recorded and duplicate weighed aliquots were taken for direct QRA. Cage wash samples were retained at room temperature.

Phase 5: Excretion Balance study in male rats (repeat oral dose)
A single oral dose of [14C]-Test Material was administered once a day for seven consecutive days at a dose level of 1000 mg/kg/day to a group of four male rats. Urine and faeces were collected separately at 24 hour intervals for days 1-6. On day 7, samples were collected as described previously.

Phase 6: Biliary Excretion study in male and female rats (single oral dose)
After a single oral dose of [14C]-Test Material to two groups of three male and three female rats with cannulated bile ducts (one group per dose level of either 100 mg/kg or 1000 mg/kg), bile, urine and faeces were collected separately as described below.

At 48 hours animals were killed by cervical dislocation and the carcasses retained at approximately -20°C prior to analysis.

Bile collection intervals were as follows:
Pre-dose, 0-1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-24 and 24-48 hours
Bile was collected into individual pre-weighed plastic containers (cooled in solid carbon dioxide), and the mass of each sample recorded. Samples were retained at approximately –20°C pending analysis.

Urine collection intervals were as follows:
Pre-dose, 0–6, 6-24 and 24-48 hours
Urine was collected into individual pre-weighed plastic containers (cooled in solid carbon dioxide), and the mass of each sample recorded. Samples were retained at approximately –20°C pending analysis.

Faeces collection intervals were as follows:
Pre-dose, 0–24 and 24-48 hours
Faeces were collected into individual pre-weighed plastic containers and the mass of each sample recorded. Prior to analysis each faeces sample was homogenised to a paste in water and the homogenate weight recorded. Samples were retained at approximately –20°C pending analysis.

Carcasses were weighed and solubilised at about 55°C for about 24 – 48 hours in a solution of NaOH (80g) and Triton X-405 (100 mL) in distilled water (600 mL) diluted to 1 litre with methanol. Aliquots of the digests were mixed with HionicFluor scintillant prior to determination of radioactive content by LSC. Carcass digests were retained at room temperature.

Cage washes (water followed by methanol) from each metabolism cage were separately collected into pre-weighed plastic containers. The weight of each cage wash sample was recorded and duplicate weighed aliquots were taken for direct QRA. Cage wash samples were retained at room temperature.

Phase 7: Tissue distribution
A group of six male Sprague Dawley rats received single oral doses of [14C]-Test Material at 1000 mg/kg, as described in previous section.

One rat was killed by overdose of CO2 at 1, 4, 12, 24, 72 and 168 hours post dose. All carcasses were immediately snap frozen in a hexane/solid CO2 mixture and retained.
Each carcass was subjected to whole-body autoradiography using procedures based on the work of Ullberg (Acta. Radiol. Suppl 118, 22-31, 1954).
Sections were presented at up to five different levels of the rat body to include as many tissues as possible.
Level 1: ex-orbital lachrymal gland
Level 2: eye/intra-orbital lachrymal gland
Level 3: Harderian gland/adrenal gland
Level 4: thyroid gland
Level 5: brain and spinal cord
Distribution of radioactivity was determined and quantified using a Fuji FLA-5100 fluorescent image analyser and associated Tina and SeeScan Software. Tissue concentrations of radioactivity were determined using calibrated autoradiographic microscales produced by GE Healthcare. A standard curve was produced using these microscales from which tissue concentrations of radioactivity were determined (nCi/g). For calculation of the weight equivalent/g data, the nCi/g data was divided by the relevant specific activity (nCi/µg).

Prior to analysis, samples were stored at approximately -20°C (carcass) or ambient temperature (sections after freeze drying). After analysis carcass remains were disposed of. Sections were stored at ambient temperature.

Phase 8: Chromatographic Analysis Experiment
Chromatographic analysis was performed on a representative faeces and a bile sample obtained in the study. A method was established at Quotient Bioresearch (Rushden) Limited for the extraction and recovery of radioactivity from selected samples of faeces. Extraction of radioactivity from bile was not required.

Faeces extract, predose faeces spiked with formulation extract, bile sample and bile sample spiked with formulation were analysed by HPLC for the qualitative evaluation of the radiolabelled test compound using on-line radiodetection.

As the test compound was classed as UVCB (Unknown or Variable composition, Complex reaction products or Biological materials) qualitative analysis only was performed in this study. Initial
qualitative evaluation of the radiolabelled test compound of a faeces extract and a bile sample by HPLC analysis confirmed considerable differences between the samples and dose formulation. Quantitative analysis was not considered appropriate, or indeed feasible, as no reference standards are available for UVCB materials of this nature. As no reference standards were available, further characterisation of the differences observed between dose formulation chromatography and that observed in faeces and bile samples could not be accomplished.
Details on dosing and sampling:
Described above in "Details on study design."
Statistics:
Basic statistics only - no further descriptions within document

Results and discussion

Preliminary studies:
Preliminary studies for Phase 1 and 2 where conducted at a single oral dose of ~ 800 mg/kg of test article. For each Study phase, one male and one female were employed to minimize animal usage.

In Phase 1 plasma clearance was examined, and clearance was similar in both the male and female. Most of the 14C-test material was cleared withing 48 hours. (Attached Table 1).

Phase 2 examined recovery of radioactivity in one male and one female rat after a single oral dose of ~ 800 mg/kg. There was no detectable isotope/test article in expired air. The urine accounted for less than 2% of dose in both species. For both species, >= 90% of the radioactive test article was recovered in the feces. (Attached Table 2)

Based on these results, the main study was only conducted in male rats.
Main ADME resultsopen allclose all
Type:
absorption
Results:
Maximum plasma concentrations attained at 2-4 hours after a single dose. Apparent terminal half-lives estimated to be 17.5 (male) and 14.2 hours (female). After 7 consecutive doses terminal half-life estimated as 24.9 hours (male).
Type:
distribution
Results:
Test material only found in gastro-intestinal tract and excretory tissues via whole body autoradiography. By 24-hours radioactivity only found in fatty tissue, secretory glands and lung. No residual radioactivity detected in any tissue after 168-hours.
Type:
metabolism
Results:
Material is UVCB and no suitale standards exist, and the material could not be characterized by any chromatographic methodology attempted.
Type:
excretion
Results:
Isotope not recovered in expired air at either dose level. Bile + urine were ~ 10 and 5% with ~ 90% recovered from fecal material after single dose (100 or 1000 mg/kg). Fecal and urinary results were identical after 7 repeated doses of 100 or 1000 mg/kg.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Following single oral doses (100 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, mean maximum concentrations of 5.70 and 3.81 µg equivalents/mL, respectively were measured in plasma at 2 hours post dose, which would suggest low absorption and/or rapid clearance of test material. Concentrations of radioactivity in plasma initially declined rapidly and subsequently more slowly. The apparent terminal half-lives were estimated at 21.1 and 20.9 hours respectively. Similar rates of elimination were observed in both sexes, indicating no notable gender differences.

At the higher dose administration rate, 1000 mg/kg of [14C]-Test Material to male and female Sprague Dawley rats, mean maximum concentrations of 44.6 and 29.8 µg equivalents/mL respectively, were measured in plasma at 4 hours post dose, again reflecting low absorption and/or rapid clearance of test material. The profile of radioactivity decline was similar to that observed at the lower dose rate. The apparent terminal half-lives were estimated at 17.5 and 14.2 hours, respectively. Similar rates of elimination were observed in both sexes, indicating no notable gender differences.

Mean maximum concentration of 50.7 µg equivalents/mL was measured in plasma at 4 hours post dose 7 following seven consecutive daily oral doses (1000 mg/kg/day) of [14C]-Test Material to male Sprague Dawley rats. Radioactivity initially declined rapidly and subsequently more slowly. The apparent terminal half-life was estimated at 24.9 hours post last dose.

The systemic exposure to the total radioactivity (AUC0-72) was also comparatively low for all dose groups (<90 µg equivalents.h/mL (100 mg/kg dose group) and <870 µg equivalents.h/mL (1000 mg/kg dose groups)). The apparently low absorption and rapid elimination of radioactivity is further supported by the excretion balance results obtained.

Overall the pharmacokinetics of total radioactivity indicated dose proportionality (AUC) with increasing dose from 100 to 1000 mg/kg with no notable indications of accumulation or change in plasma profile following repeated dose administration (during 7 days) at 1000 mg/kg.

See attached Tables 3 to 15 & Figures 2 to 4.
Details on distribution in tissues:
Whole body autoradiography was used to determine tissue distribution. Since the test article was an UVCB, the composition was much too complex without standards to differentiate and quantitate all the possible species present. See attached Figure 1 which is a HPLC chromatogram of the native test article.

The lower limit of accurate quantification (LLOQ) was 5.05 µg equivalents/g. Values below this limit are described as ‘BLQ’ (below limit of quantification) in the following text and associated tables. The upper limit of accurate quantification was 8680 µg equivalents/g; values above this limit have been reported and are identified (†) in the relevant tables and text.

Radioactivity at 1 hour post dose (the first sampling time point) was quantifiable in approximately half the tissues measured and generally confined to components of the gastro-intestinal tract and tissues associated with elimination following single oral doses (1000 mg/kg) of [14C]-Test Material to male Sprague Dawley rats. Highest concentrations were observed in contents of the caecum, small intestine and stomach, (2100 - 8510 µg equivalents/g), with notable concentrations measured in the liver (108 µg equivalents/g) kidney (31.9 µg equivalents/g) and urinary bladder contents (21.6 µg equivalents/g). Concentrations in the other tissues analysed at this time were at levels below the limit of detection (BLQ), indicating limited tissue distribution of test material.

Peak tissue concentrations generally occurred at 1 and 4 hours after dose administration which compares well to maximum plasma concentrations measured at this time. At 4 hours, greatest concentrations (excluding components of the GI tract and urinary bladder) were measured in the liver, adrenal gland, kidney and brown fat (98.1, 55.7, 37.6 and 24.5 µg equivalents/g respectively). The majority of radioactivity remained associated with contents of the GI tract, with radioactivity in remaining tissues close to or below the limit of quantification.

Radioactive concentrations were generally in decline thereafter with a small rise in some tissue concentrations (fatty tissue, secretory glands, lung) observed at 24 hours post-dose. Greatest concentrations at this time were measured in the liver, brown fat, Harderian gland and kidney (47.7, 18.3, 15.9 and 10.9 µg equivalents/g respectively). Almost half of the tissues analysed were BLQ at this time.

At 72 hours after dose administration, radioactivity was measureable only in the GI tract, liver and brown fat, and by 168 hours post-dose, radioactivity concentrations were at levels below the limit of detection in all tissues, which further supports the data obtained during the excretion balance phase to show that elimination of total radioactivity is essentially complete by this time point.

Attached Tables 25 & 26; Figures 5 to 10.
Details on excretion:
Preliminary data are in Table 2. Data from the main phase is in Table 16 - Table 20 and Figures 1 & 11 (separate attachments).

Expired air was not collected during the main phases since there was no detection of radioactivity during the preliminary experiment, indicating that the radiolabel was placed in a metabolically-stable position.

Following single oral doses (100 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, radioactivity was excreted predominantly via the faecal route (mean total of 90.28 and 93.43% of administered radioactivity in male and female rats, respectively), with the majority of this radioactivity recovered during the first 24 hours after dosing (84.6 and 84.2% in male and female rats, respectively). Notably smaller proportions of administered radioactivity were recovered in the urine (mean total of 0.95 and 1.44% of administered radioactivity in male and female rats, respectively). Mean total radioactivity concentrations measured in the cage washings and carcasses were 0.20% and 0.23% administered radioactivity in male and female rats respectively. The mean total recovery after 168 hours was 91.41% in male rats and 95.09% in female rats.

As observed in the lower dose group, radioactivity was again excreted predominantly via the faecal route (mean total of 92.13 and 90.03% of administered radioactivity in male and female rats, respectively), with the majority of this radioactivity recovered during the first 24 hours after dosing (86.0 and 76.1% in male and female rats, respectively) following single oral doses (1000 mg/kg) of [14C]-Test Material. Notably smaller proportions of administered radioactivity were recovered in the urine (mean total of 0.65 and 1.72% of administered radioactivity in male and female rats, respectively). Mean total radioactivity concentrations measured in the cage washings and carcasses were 0.08 and 0.36% administered radioactivity in male and female rats, respectively. The mean total recovery after 168 hours was 92.86% in male rats and 92.12% in female rats.

Radioactivity was again excreted predominantly via the faecal route (mean total of 91.22% of administered radioactivity) following seven consecutive daily oral doses (1000 mg/kg/day) of [14C] Test Material to male Sprague Dawley rats. Notably smaller proportions of administered radioactivity were recovered in the urine (mean total of 0.64% of administered radioactivity). Mean total radioactivity concentrations measured in the cage washings and carcasses were <0.4% administered radioactivity. The mean total recovery at Dose administration 7 + 168 hours was 92.19%.

Excretion of administered radioactivity during 168 hours following both single and repeated oral administration of [14C]-Test Material was almost exclusively via the faecal route in both sexes, again indicating no notable gender difference. Although greater than 90% recovery of radioactivity was not achieved for all animals, this can potentially be explained by the fact that the majority of the faecal radioactivity is eliminated in one sample and therefore obtaining a truly homogeneous sample for determination of radioactive content is challenging. Urine accounted for a smaller proportion of radioactivity (< 2.5 % administered dose for each animal) with remaining radioactivity measured in the cage washings and carcass. The mean total recovery of radioactivity ranged from 82 - 102% (n = 20 animals). The majority of radioactivity was recovered during 0-24 hours post dosing, reflecting the rapid elimination of test material. After each dosing phase, the very low proportion of the dose associated with the carcasses indicated that the elimination of radioactivity was essentially complete after 7 days.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Following single oral doses (100 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, radioactivity was rapidly excreted, predominantly via the faecal route (mean total of 72.89 and 78.91% of administered radioactivity, respectively) during 0-48 hours after dose administration. Smaller proportions of administered radioactivity were recovered in the bile (9.57 and 11.13% administered radioactivity, respectively), suggesting limited excretion of absorbed labelled test material via the liver. Notably smaller proportions of administered radioactivity were recovered in the urine (mean total of 0.39 and 0.74% of administered radioactivity in male and female rats, respectively). Mean total radioactivity measured in the cage washings were ca. 1% and 1.56–3.43% administered radioactivity was recovered from the carcass. The mean total recovery after 48 hours was 87.07 and 93.38% in male and female rats, respectively.

Following single oral doses (1000 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, radioactivity was again rapidly excreted predominantly via the faecal route (mean total of 88.92 and 86.28% of administered radioactivity, respectively) during 0-48 hours after dose administration. A mean total of 0.20% (male rats) and 0.32% (female rats) of administered radioactivity were recovered in urine. In bile, 5.33 and 5.51% of the mean total administered radioactivity was observed in male and female animals, respectively, again suggesting limited excretion of labelled test material via the liver. Mean total radioactivity proportions recovered in the cage washings were <1% administered radioactivity and a mean total of <1% radioactivity was recovered from the carcass. The mean total recovery after 48 hours was 95.32 and 93.22% in male and female rats, respectively.

It can be considered that the total of the administered dose eliminated in the bile, plus the dose excreted in the urine, corresponds to the proportion of the dose absorbed. In this case, approximately 10% and 5% dose after dosing at 100 and 1000 mg/kg respectively. The remaining dose, associated with faecal elimination may be considered to be associated with unabsorbed dose.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Biliary Excretion Balance results are in Tables 21 - 24 and Figures 1 & 12.

Following single oral doses (100 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, radioactivity was rapidly excreted, predominantly via the faecal route (mean total of 72.89 and 78.91% of administered radioactivity, respectively) during 0-48 hours after dose administration. Smaller proportions of administered radioactivity were recovered in the bile (9.57 and 11.13% administered radioactivity, respectively), suggesting limited excretion of absorbed labelled test material via the liver. Notably smaller proportions of administered radioactivity were recovered in the urine (mean total of 0.39 and 0.74% of administered radioactivity in male and female rats, respectively). Mean total radioactivity measured in the cage washings were ca. 1% and 1.56–3.43% administered radioactivity was recovered from the carcass. The mean total recovery after 48 hours was 87.07 and 93.38% in male and female rats, respectively.

Following single oral doses (1000 mg/kg) of [14C]-Test Material to male and female Sprague Dawley rats, radioactivity was again rapidly excreted predominantly via the faecal route (mean total of 88.92 and 86.28% of administered radioactivity, respectively) during 0-48 hours after dose administration. A mean total of 0.20% (male rats) and 0.32% (female rats) of administered radioactivity were recovered in urine. In bile, 5.33 and 5.51% of the mean total administered radioactivity was observed in male and female animals, respectively, again suggesting limited excretion of labelled test material via the liver. Mean total radioactivity proportions recovered in the cage washings were <1% administered radioactivity and a mean total of <1% radioactivity was recovered from the carcass. The mean total recovery after 48 hours was 95.32 and 93.22% in male and female rats, respectively.

The presence of radioactivity in bile is further supported by the detection of radioactivity in liver tissues analysed by quantitative whole-body autoradiography.

It can be considered that the total of the administered dose eliminated in the bile, plus the dose excreted in the urine, corresponds to the proportion of the dose absorbed. In this case, approximately 10% and 5% dose after dosing at 100 and 1000 mg/kg respectively. The remaining dose, associated with faecal elimination may be considered to be associated with unabsorbed dose.
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Cmax: 5.70 ug equiv/mL
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Key result
Test no.:
#1
Toxicokinetic parameters:
C(time): 2 hours
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 21.1 hours
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
AUC: 0-72h; 89.56 ug equiv.h/mL
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
AUC: 0-infinity: 96.27 ug equiv.h/mL
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
other: Rate constant: 0.03
Remarks:
plasma, male rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
Cmax: 3.81 ug equiv/mL plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Key result
Test no.:
#1
Toxicokinetic parameters:
C(time): 2 hours plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Key result
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 20,9 hours plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
AUC: 0-72h; 55.00 ug equiv.h/mL plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
AUC: 0-infinity: 59.67 ug equiv.h/mL plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Test no.:
#1
Toxicokinetic parameters:
other: : Rate constant: 0.03 plasma
Remarks:
female rat, 100 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
Cmax: 44.59 ug equiv/mL plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Key result
Test no.:
#2
Toxicokinetic parameters:
C(time): 4 hours plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Key result
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: 17.5 hours plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
AUC: 0-72h; 865.6 ug equiv.h/mL plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
AUC: 0-infinity: 905.3 ug equiv.h/mL plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
other: Rate constant: 0.04 plasma
Remarks:
male rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
Cmax: 29.83 ug equiv/mL plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Key result
Test no.:
#2
Toxicokinetic parameters:
Tmax: 4 hours plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Key result
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: 14.2 hours plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
AUC: 0-72h; 456.7 ug equiv.h/mL plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
AUC: 0-infinity: 466.3 ug equiv.h/mL plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#2
Toxicokinetic parameters:
other: Rate constant: 0.05 plasma
Remarks:
female rat, 1000 mg/kg, oral gavage, single dose
Test no.:
#3
Toxicokinetic parameters:
Cmax: 50.74 ug equiv/mL plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days
Key result
Test no.:
#3
Toxicokinetic parameters:
Tmax: 4 hours plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days
Key result
Test no.:
#3
Toxicokinetic parameters:
half-life 1st: 24.9 hours plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days
Test no.:
#3
Toxicokinetic parameters:
other: 0-72h; 765.9 ug equiv.h/mL plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days
Test no.:
#3
Toxicokinetic parameters:
other: AUC: 0-infinity:840.9 ug equiv.h/mL plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days
Test no.:
#3
Toxicokinetic parameters:
other: Rate constant: 0.03 plasma
Remarks:
male rat, 1000 mg/kg/day, oral gavage, seven consecutive days

Metabolite characterisation studies

Metabolites identified:
not measured
Remarks:
EXP1200078 is an UVCB material as characterized and described.
Details on metabolites:
Attempts were made to perform chromatographic analyses on EXP1200078, but all efforts failed. Only small amounts of material were absorbed from the oral gavage dose (near limits of radiochemical detection), and chromatographic analyses could not be developed to handle the test material. Furthermore, there was no material available that could be used for any chromatographic standards.

Any other information on results incl. tables

Table Content
1 Preliminary Phase: Concentrations of radioactivity in plasma from male and female Sprague Dawley rats following a single oral administration of [14C]-Test Material at ca. 800 mg/kg
2 Preliminary Phase: Recovery of radioactivity from male and female Sprague Dawley rats following a single oral administration of [14C]-Test Material at ca. 700-800 mg/kg
3 Concentration of radioactivity measured in whole blood obtained from male Sprague Dawley rats following a single oral administration of [14C]-Test Material at 100 mg/kg
4 Concentration of radioactivity measured in whole blood obtained from female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 100 mg/kg
5 Concentration of radioactivity measured in plasma obtained from male Sprague Dawley rats following a single oral administration of [14C]-Test Material at 100 mg/kg
6 Concentration of radioactivity measured in plasma obtained from female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 100 mg/kg
7 Concentration of radioactivity measured in whole blood obtained from male Sprague Dawley rats following a single oral administration of [14C]-Test Material at 1000 mg/kg
8 Concentration of radioactivity measured in whole blood obtained from female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 1000 mg/kg
9 Concentration of radioactivity measured in plasma obtained from male Sprague Dawley rats following a single oral administration of [14C]-Test Material at 1000 mg/kg
10 Concentration of radioactivity measured in plasma obtained from female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 1000 mg/kg
11 Concentrations of radioactivity in whole blood from male Sprague Dawley rats following seven consecutive oral doses of [14C]-Test Material at 1000 mg/kg/day
12 Concentrations of radioactivity in plasma from male Sprague Dawley rats following seven consecutive oral doses of [14C]-Test Material at 1000 mg/kg/day
13 Pharmacokinetic parameters of mean total radioactivity measured in plasma obtained from male and female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 100 mg/kg
14 Pharmacokinetic parameters of mean total radioactivity measured in plasma obtained from male and female Sprague Dawley rats following a single oral administration of [14C]-Test Material at 1000 mg/kg
15 Pharmacokinetic parameters of mean total radioactivity measured in plasma obtained from male Sprague Dawley rats following seven consecutive oral doses of [14C]-Test Material at 1000 mg/kg/day
16 Recovery of radioactivity from male Sprague Dawley rats following a single oral dose administration of [14C]-Test Material at 100 mg/kg
17 Recovery of radioactivity from female Sprague Dawley rats following a single oral dose administration of [14C]-Test Material at 100 mg/kg
18 Recovery of radioactivity from male Sprague Dawley rats following a single oral dose administration of [14C]-Test Material at 1000 mg/kg
19 Recovery of radioactivity from female Sprague Dawley rats following a single oral dose administration of [14C]-Test Material at 1000 mg/kg
20 Recovery of radioactivity from male Sprague Dawley rats following seven consecutive oral dose administrations of [14C]-Test Material at 1000 mg/kg/day
21 Recovery of radioactivity from male Sprague Dawley bile-duct cannulated rats following a single oral dose administration of [14C]-Test Material at 100 mg/kg
22 Recovery of radioactivity from female Sprague Dawley bile-duct cannulated rats following a single oral dose administration of [14C]-Test Material at 100 mg/kg
23 Recovery of radioactivity from male Sprague Dawley bile-duct cannulated rats following a single oral dose administration of [14C]-Test Material at 1000 mg/kg
24 Recovery of radioactivity from female Sprague Dawley bile-duct cannulated rats following a single oral dose administration of [14C]-Test Material at 1000 mg/kg
25 Concentrations of radioactivity in tissues (expressed as µg equivalents/g) following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
26 Concentrations of radioactivity in tissues (expressed tissue: blood ratio) following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats

Figure Content
1 Representative HPLC Radiochromatogram of [14C]-Test Material dose formulation
2 Plot of mean plasma radioactivity concentrations in male and female Sprague Dawley rats following single oral administration of [14C]-Test Material at 100 mg/kg
3 Plot of mean plasma radioactivity concentrations in male and female Sprague Dawley rats following single oral administration of [14C]-Test Material at 1000 mg/kg
4 Plot of mean plasma radioactivity concentrations in male Sprague Dawley rats following seven consecutive oral doses of [14C]-Test Material at 1000 mg/kg/day
5 Distribution of radioactivity in tissues at 1 hour following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
6 Distribution of radioactivity in tissues at 4 hours following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
7 Distribution of radioactivity in tissues at 12 hours following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
8 Distribution of radioactivity in tissues at 24 hours following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
9 Distribution of radioactivity in tissues at 72 hours following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
10 Distribution of radioactivity in tissues at 168 hours following a single oral administration of [14C]-Test Material (nominal 1000 mg/kg) to male albino rats
11 Representative HPLC radio-chromatograms of faeces collected following a single oral administration of [14C]-Test Material
12 Representative HPLC radio-chromatograms of bile collected following a single oral administration of [14C]-Test Material

Applicant's summary and conclusion

Conclusions:
1. The 14C-labelled Test Material has limited absorption after oral administration, with approximately 10% administered dose absorbed at 100 mg/kg and approximately 5% administered dose absorbed at 1000 mg/kg in both males and females;

2. Absorbed dose distributes rapidly to many tissues albeit at relatively very low concentrations. The tissues containing most radioactivity are those associated with absorption (i.e GI Tract) and elimination (liver and kidney);

3. There is less than 1% dose excreted in the urine and the remainder excreted in the faeces. Excretion of the dose is essentially complete after 7 days;

4. In both males and females, approximately 10% of the dose (100 mg/kg) and approximately 5% of the dose (1000 mg/kg) is eliminated via the bile into the faeces. Elimination of the absorbed dose therefore occurs via the bile;

5. Profiles of the radioactivity in the bile and faeces were qualitatively different to those of the 14C-labelled Test Material indicating that there were probably some associated in-vivo interaction processes during the absorption, distribution and excretion phases of the material;

6. Since the test material was primarily in the gut, associated in-vivo interactions may have been due to gut microflora rather than mammalian metabolism.
Executive summary:

All the results suggest that that this UVCB test material has poor absorption characteristics. A very small amount of the test material is absorbed via the GI tract, but that quantity of material undergoes rapid elimination primarily through bile and faeces. Even repeat oral doses of test article do not result in any greater absorption or retention of material at the highest dose (1000 mg/kg/day). Metabolites could not be identified because of the UVCB nature of the test material, and the use of quantitative whole body autoradiography demonstrated that there is was very little distribution and retention in all tissues. All these results demonstrate very low hazard via the oral route up to the tested dose of 1000 mg/kg/day.