tris[3-bromo-2,2-bis(bromomethyl)propyl] phosphate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and appropriate guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Principles of method if other than guideline:

not relevant

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-060-1

EC Name:

-

Cas Number:

19186-97-1

Molecular formula:

C15 H24 O4 P Br9

IUPAC Name:

tris[3-bromo-2,2-bis(bromomethyl)propyl] phosphate

Constituent 2

Reference substance name:

4130601

IUPAC Name:

4130601

Details on test material:

Distribution of radiolabel: 50% of the 14C label is at the central carbon of the neopentyl group, while the remaining 50% of the label is equally distributed between the three bromomethyl groups
Appearance: Solution in chloroform : ethanol (18 : 1)
Storage conditions: At <-15deg C in the dark
Specific activity: 60 mCi/mmol (58.8 microCi/mg = 2.18 MBq/mg)
Batch number: 248-020-060
Radiochemical purity: 99.4% (2 October 2002; Moravek Biochemicals Inc, USA)
Date received: 11 October 2002
Supplier: Moravek Biochemicals Inc, CA, USA

Non-radiolabelled test substance

Identity: FR 370
Appearance: Off-white powder
Storage conditions: Room temperature
Batch number: 141476
Expiry date: 7 November 2003
Chemical purity: 99.8% (by GC)
Date received: 12 November 2001
Huntingdon Life Sciences Hazard Class: 1
Supplier: Study Sponsor

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of seventy-eight (78; 39 male and 39 female) Sprague-Dawley CD strain rats (Crl : CD BR) were obtained from Charles River UK Ltd (Margate, England) for use in this study. The estimated age at the time of dosing was about 6 – 7 weeks (males) and 7 – 8 weeks (females), when their bodyweights were in the range 194 – 200 g (Group 1), 192 – 217 g (Group 2), 181 – 220 g (Group 3), 189 – 240 g (Group 4), 193 – 220 g (Group 5) and 200 – 219 g (Group 6).
After arrival at Huntingdon Life Sciences, the rats were subjected to a physical examination to accepted animal husbandry procedures to ensure their suitability for inclusion in this study. The rats were allowed an acclimatisation period of at least 5 days in the animal housing unit of the Department of Drug Metabolism, Huntingdon Life Sciences prior to treatment with the test compound, during which time they were housed in standard stainless-steel battery cages (maximum of six rats per cage). For the excretion balance experiments, the animals were transferred to individual glass metabolism cages specifically designed to allow the separate collection of urine, faeces and expired air the day before administration of the radiolabelled test substance. No rats were housed with rats of another study or sex.

Before dosing, the rats were randomly allocated individual identification in the form of indelible tail markings. After dosing the rats were either returned to their battery cages (pre-test observation, pharmacokinetic and tissue distribution studies) or individual glass metabolism cages (excretion study).

The rats were offered a complete dry diet (VRF1C diet; manufactured by Usine d’Alimentation Rationelle; batch nos. 20713 and 20723) ad libitum. Drinking water obtained from the local water supply was also available ad libitum. The diet and water supplied to the animals are routinely analysed for quality. There were no known contaminants in the diet or water that were considered to have affected the integrity or outcome of the study.

During the experimental period, the ambient air temperature in the animal housing unit was normally maintained at 21 plus minus 2degC and the relative humidity within the range 55 plus minus 5%; however, the relative humidity in the room in which the rats were housed fell below the stated acceptable range on a number of occasions. Both temperature and humidity were continuously monitored and automatically recorded. Lighting was controlled in a 12-hour light/dark cycle throughout the study, and the animal rooms were ventilated with frequent air changes.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Method of administration or exposure: single doses; gastric gavage

Duration and frequency of treatment / exposure:

Pre-test observation (Group 1): Four rats (2 male (1M and 2M), 2 female (3F and 4F)) received single oral high level (1000 mg/kg) doses of non-radiolabelled FR 370 and were observed for up to 48 hours. No samples were collected and after 48 hours the animals were sacrificed.
Excretion/balance (Groups 2 and 3): Eight rats (4 male, 4 female) received single low level (50 mg/kg) oral doses (Group 2, 5M – 8M and 9F – 12F) or single high level (1000 mg/kg) oral doses (Group 3, 13M – 16M and 17F – 20F). Urine was collected separately from each animal into solid CO2 cooled receivers during 0 – 6, 6 – 24 and 24 hour intervals up to 120 hours.
Plasma/whole-blood kinetics (Groups 4 and 5): Twenty four rats (12 male, 12 female) received single low level (50 mg/kg) oral doses (Group 4; 21M – 24M, 25F – 28F, 29M – 32M, 33F – 36F, 37M – 40M, 41F – 44F) or single high level (1000 mg/kg) oral doses (Group 5; 45M – 48M, 49F – 52F, 53M – 56M, 57F – 60F, 61M – 64M, 65F – 68F), with each group of 12 animals divided into 3 sub-groups. Blood samples (ca 400 L) were collected from a tail vein into heparinised tubes at the following times: pre-dose, 0.25, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, 120 and 168 hours post-dose.
Tissue distribution (Group 6): After administration of single oral doses of 14C FR 370 at a target dose level of 50 mg/kg to 10 rats (5 male (69M – 73M), 5 female (74F – 78F)), animals were sacrificed by exsanguination (cardiac puncture under isoflurane/oxygen anaesthesia) followed by cervical dislocation singly (1 male, 1 female) at each of the following times: 3, 6, 12, 24 and 120 hours post-administration.

Doses / concentrations

Remarks:

Doses / Concentrations:
see above

No. of animals per sex per dose / concentration:

Male: 2 animals at CA 1000 mg/kg
Male: 4 animals at CA 50 mg/kg
Male: 4 animals at CA 1000 mg/kg
Male: 12 animals at CA 50 mg/kg
Male: 12 animals at CA 1000 mg/kg
Male: 5 animals at CA 50 mg/kg
Female: 2 animals at CA 1000 mg/kg
Female: 4 animals at CA 50 mg/kg
Female: 4 animals at CA 1000 mg/kg
Female: 12 animals at CA 50 mg/kg
Female: 12 animals at CA 1000 mg/kg
Female: 5 animals at CA 50 mg/kg

Control animals:

no

Positive control reference chemical:

n/a

Details on study design:

Single oral doses of 14C FR 370 were administered to male and female albino rats at dose levels of 50 mg/kg (low dose) and 1000 mg/kg (high dose) and concentrations of radioactivity were measured in whole-blood, plasma and excreta to obtain information concerning the absorption, pharmacokinetics and rates and routes of excretion of total test compound related material (radioactivity). In addition, single oral doses of 14C FR 370 were administered to male and female albino rats at a dose level of 50 mg/kg (low dose) and concentrations of radioactivity were measured in selected tissues and organs to obtain information concerning the distribution of the test compound. The study groups, animal numbers, dose levels and study aspect (ie excretion/balance, pharmacokinetics or tissue distribution phase) are tabulated below.

Group No. Dose level No. of animals Experimental type Animal
identification
nos.
Male Female
1 Single, oral high 2 2 Pre-test observation 1-2M, 3-4F
2 Single, oral low 4 4 Main excretion/balance 5-8M, 9-12F
3 Single, oral high 4 4 Main excretion/balance 13-16M, 17-20F
4 Single, oral low 12 12 Plasma and whole blood kinetics 21-24M, 25-28F, 29-32M, 33-36F, 37-40M, 41-44F
5 Single, oral high 12 12 Plasma and whole blood kinetics 45-48M, 49-52F, 53-56M, 57-60F, 61-64M, 65-68F
6 Single, oral low 5 5 Tissue distribution 69-73M, 74-78F

Metabolite profiling of urine and faeces was also conducted.

The dose levels and routes of administration used were specified by the Study Sponsor to best complement existing toxicology studies with the test compound.

Details on dosing and sampling:

SAMPLE COLLECTION

Pre-test observation (Group 1)

Four rats (2 male (1M and 2M), 2 female (3F and 4F)) received single oral high level (1000 mg/kg) doses of non-radiolabelled FR 370 and were observed for up to 48 hours. No samples were collected and after 48 hours the animals were sacrificed.

Excretion/balance (Groups 2 and 3)

Eight rats (4 male, 4 female) received single low level (50 mg/kg) oral doses (Group 2, 5M – 8M and 9F – 12F) or single high level (1000 mg/kg) oral doses (Group 3, 13M – 16M and 17F – 20F). Urine was collected separately from each animal into solid CO2 cooled receivers during 0 – 6, 6 – 24 and 24 hour intervals up to 120 hours. Faeces was collected separately at 24 hour intervals during 120 hours post-administration. Any 14CO2 present in the expired air of the rats was trapped in a mixture of 2-ethoxyethanol : ethanolamine (3 : 1, v/v) and was monitored at 0 – 6, 6 – 24 and 24 – 48 hours. Trapping of expired air was terminated after 48 hours since no radioactivity was detected at this time. At the end of each 24-hour sample collection period, the interior of each metabolism cage was washed with distilled water (ca 100 mL) and the washings retained. At 120 hours, the rats were killed (cervical dislocation) and the carcasses retained. The interiors of each metabolism cage was washed successively with distilled water and acetone (ca 100 mL of each) and the washings retained. The intact gastrointestinal tract was removed from each carcass and retained for radioactivity measurements.

Plasma/whole-blood kinetics (Groups 4 and 5)

Twenty four rats (12 male, 12 female) received single low level (50 mg/kg) oral doses (Group 4; 21M – 24M, 25F – 28F, 29M – 32M, 33F – 36F, 37M – 40M, 41F – 44F) or single high level (1000 mg/kg) oral doses (Group 5; 45M – 48M, 49F – 52F, 53M – 56M, 57F – 60F, 61M – 64M, 65F – 68F), with each group of 12 animals divided into 3 sub-groups. Blood samples (ca 400 microL) were collected from a tail vein into heparinised tubes at the following times: pre-dose, 0.25, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 72, 96, 120 and 168 hours post-dose. Aliquots (2  50 L) of whole-blood were taken for combustion analysis. Plasma was obtained by centrifugation (ca 4000 rpm for 3 minutes) of the remaining whole-blood, and aliquots (2  50 L plus 0.5 mL of distilled water) were taken for measurement of radioactivity concentrations; residual plasma was retained and stored at ca 20degC, but blood cells were discarded.

Tissue distribution (Group 6)

After administration of single oral doses of 14C FR 370 at a target dose level of 50 mg/kg to 10 rats (5 male (69M – 73M), 5 female (74F – 78F)), animals were sacrificed by exsanguination (cardiac puncture under isoflurane/oxygen anaesthesia) followed by cervical dislocation singly (1 male, 1 female) at each of the following times: 3, 6, 12, 24 and 120 hours post-administration. Terminal blood samples (ca 6 – 8 mL) were transferred to heparinised tubes and a portion (ca 1 mL) retained as whole-blood for measurement of radioactivity concentrations. The remaining whole-blood was centrifuged (ca 4000 rpm for 10 minutes) to separate the plasma and blood cells for measurement of radioactivity concentrations.

After sacrifice, the following organs/tissues were removed or sampled (as appropriate) from each carcass:

Bone (femur) Lungs
Brain Muscle (skeletal)
Fat (abdominal) Spleen
Heart Testes
Kidneys Uterus
Liver Gastrointestinal tract

The remaining carcass was also retained for analysis.

All samples generated during the course of the study were stored at about -20degC pending analysis, apart from whole-blood which was stored at about +4deg C.



RADIOACTIVITY MEASUREMENT AND SAMPLE PROCESSING

Determination of radioactivity

Radioactivity was measured in all cases by liquid scintillation counting (LSC), using a 1219 Rackbeta or Wallac 1409 (Wallac Oy, Turku, Finland) automatic liquid scintillation analyser. After the optimal channel settings had been selected, quench correction curves were prepared from radiochemical standards (traceable to a national standard) using the spectral quench parameter of the external standard. The coefficients of a quadratic quench curve function were calculated by computer and entered in the analyser data processors which automatically calculated disintegration rates. The validity of each of the calibration curves was checked at intervals of about 10 working days.

The mode of counting was generally pre–set at 10 minutes or until 40,000 counts had been accumulated (minimum counting time of 30 seconds) or for samples which contained relatively large quantities of radioactivity, for 4 minutes or until 9 X 105 had been accumulated. A solvent/scintillator background disintegration rate was measured for every batch of samples and subtracted from each sample disintegration rate. Radioactivity in amounts less than twice that of background levels (typical background was about 25 40 dpm) in the sample under investigation was considered to be below the limit of accurate quantification (ND).

Sample processing

Triplicate aliquots of diluted dose solution (1 mL by weight or volume), diluted dose accuracy checks (100 microL) and HPLC eluates collected during radiochemical purity measurements (1 mL) were mixed with Ultima Gold scintillator (7 mL) prior to LSC analysis.

Urine and Cage washes

Samples were mixed by inversion/shaking and the weight recorded. Replicate aliquots of urine (ca 100 – 500 mg) and cage washings (ca 1 g for aqueous cage wash, ca 0.8 g for acetone cage wash) were mixed with Ultima Gold scintillator (7 mL) prior to LSC analysis.

Faeces extracts

Each faeces sample was extracted with pentane (mixed isomers; 15 mL). After the addition of the solvent each sample was then whirlimixed for ca 1 minute and sonicated for ca 10 minutes. The samples were then centrifuged (4000 rpm/10 min) and the supernatant decanted into a pre-weighed vial and the extract weight recorded. This process was repeated using dichloromethane (1 x 15 mL) and acetonitrile : water (80 : 20, v/v; 2 X 15 mL). The final faeces residue was allowed to air-dry for ca 48 hours. Replicate aliquots of each faeces extract (typically ca 30 – 1300 mg) were mixed with Ultima Gold scintillator (7 mL) prior to LSC analysis.

Faeces residues and Cage debris

After weighing the air-dried faeces residue, water was added and the samples mixed thoroughly to a smooth paste before aliquots (2  ca 250 mg) were taken for combustion. Some cage wash samples contained debris (primarily food residues). This debris was removed from the cage wash sample by centrifugation (4000 rpm/10 min), and then processed as above for the faeces residue.

Blood fractions

From the tissue distribution phase, duplicate portions (ca 0.25 g) of whole-blood, plasma and blood cells were taken for combustion analysis. From the kinetics experiments, duplicate portions of whole-blood (50 L) were taken for combustion analysis. The remaining whole-blood was centrifuged (4000 rpm/3 minutes) and duplicate aliquots of plasma (50 L plus 0.5 mL of distilled water) were mixed with Ultima Gold scintillator (7 mL) prior to LSC analysis.

Tissues/organs

The systemic tissues (whole organs or dissected portions thereof) were weighed then prepared for combustion as follows: gastrointestinal tract, brain, spleen, testes, heart, kidney and lungs, together with portions of fat (abdominal), muscle (skeletal) and bone (femur) taken from the bulk sample provided were homogenised by scissor mincing or by the addition of distilled water and homogenising using a laboratory homogeniser (Ultra-Turrax, Janke and Kunkel, IKAF-Labortechnik, Stauffen, Germany). Livers were homogenised using the Ultra-Turrax homogeniser without the addition of water. The uterus samples were combusted in toto.

Carcasses

Rat carcasses were solubilised by digestion at ca 56deg C for between 20 – 48 hours in a solution prepared from sodium hydroxide (80 g), distilled water (600 mL), methanol (300 mL) and Triton X-405 (100 mL). After digestion, replicate aliquots (2 x ca 1 g) were mixed with distilled water (1 mL) and Ultima Gold scintillator (14 mL) prior to LSC analysis.

Expired air

Samples were mixed by inversion/shaking and the weight recorded. Replicate aliquots (ca 2 x 1 g) were mixed with Ultima Gold scintillator (7 mL) prior to LSC analysis.

Combustion analysis

Portions of the homogenised tissues, faeces residues, cage debris, whole-blood, plasma and blood cells were taken from the bulk samples for combustion. With the exception of the fat and bone (ca 100 mg) and uterus (in toto), sample weights for combustion were typically 0.25 g. Samples for combustion were placed on cellulose pads within combustocones (Canberra Packard Ltd) and burned in oxygen using an Automatic sample oxidiser (Model 307; Canberra Packard Ltd). The products of combustion were absorbed in Carbosorb (about 9 mL) and mixed with Permafluor E+ scintillator (about 12 mL) for measurement of radioactivity.

The efficiency of the sample oxidiser was determined at regular intervals (normally with every batch of 20 – 30 samples) by combusting a radiochemical standard (Spec-Chec-14C; Canberra Packard Ltd). The results of these checks indicated that the oxidiser combustion efficiency generally exceeded 97%, with the exception of the whole-blood from low dose animals 35F, 36F, 40M, 41F and 42F, where the instrument failed and no data was obtained from these samples. Results from these samples are reported as NS (no sample; sample lost on combustion). Where data was obtained, all results are corrected to 100% for combustion efficiency.

METABOLITE PROFILING

HPLC analysis

Radioactive components in the faeces extracts (low dose and high dose) and urine samples (low dose only) from rats dosed orally with 14C FR 370 were separated by HPLC with on-line UV and radioactivity detection. Due to the relatively low levels of radioactivity in the urine samples, quantification of the separated metabolites was performed by fraction collection with liquid scintillation counting of 1-minute eluate fractions for the duration of each chromatogram (40 minutes). The performance of the HPLC system was checked by injection of either a solution of parent compound and reference FR 513 or injection of a solution of 14C FR 370, FR 370 and FR 513, and their retention time and peak shape confirmed prior to sample analysis. Every sample was co-injected with a standard mix solution of FR 370 and FR 513 to aid the identification of the radioactive components.

Proportions of the principle radioactive components separated and measured as described above are expressed as % sample radioactivity and % dose.

The following samples were subjected to metabolite profiling by HPLC:

Urine: For both sexes, 0 – 48 hour samples from each animal (pooled proportionally by weight) at the low dose level were analysed before and after incubation with beta-glucuronidase/sulphatase. Exceptions to the pools were the 0 – 6 hour urine samples from animals 8M and 9F as the radioactivity levels in these samples were ND (not detected).

Faeces: For both sexes, 0 – 48 hour faeces extracts from each animal (pooled proportionally by weight) at both the low and high dose levels were analysed.

Urine (low dose only)

Pooled urine samples were centrifuged after spiking with cold references, then directly injected onto the chromatograph.

Faeces extracts (low and high dose)

A sub-sample of the pooled faeces extracts (1 – 4) was concentrated under nitrogen and the residue reconstituted in tetrahydrofuran (2 mL) : water (0.25 mL). A sub-sample of the concentrated faeces extract was spiked with cold references, prior to injection onto the chromatograph.

Deconjugation experiments

A solution of beta-glucuronidase with arylsulphatase activity (type H-1 from Helix pomatia) was prepared in 0.2M sodium acetate buffer (pH 5.0) at a concentration of 2000 Sigma units/mL.

Samples of the pooled 0 – 48 hour urine sample were mixed with an equal volume of the beta-glucuronidase/arylsulphatase solution, then incubated for ca 17 hours at ca 37degC.


The following control incubations were performed in urine matrix:

a) Control (sample plus 0.2M sodium acetate buffer (pH 5.0) alone)
b) Enzyme inhibitor control (sample plus enzyme plus D-saccharic acid-1,4-lactone)

The above controls were incubated at ca 37degC as for the study samples, then further incubated with either phenolphthalein glucuronic acid or p-nitrocatchol sulphate for about 1 hour, and tested for colour response with 1M sodium hydroxide solution.

Statistics:

no

Results and discussion

Preliminary studies:

see attached document on results

Main ADME results

Main ADME results 1

Type:

absorption

Results:

only about 1% of an oral dose of 14C FR 370 administered as a suspension in corn oil was absorbed by rats

Main ADME results 2

Type:

metabolism

Results:

up to eight metabolites were detected, indicating that the small amount of14C‑FR‑370 that was absorbed had undergone extensive metabolism, but no individual metabolite accounted for greater than 0.29% of the recovered dose. 

Main ADME results 3

Type:

excretion

Results:

The excretion results obtained in this study therefore suggest that less than 1% of an oral dose of 14C FR 370 was absorbed by rats

Toxicokinetic / pharmacokinetic studies

Details on absorption:

see attached document on results

Details on distribution in tissues:

see attached document on results

Details on excretion:

see attached document on results

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

see attached document on results

Any other information on results incl. tables

1. The plasma and whole-blood levels of radioactivity,
excretion of radioactivity and metabolite profiles were
investigated following single oral doses of 14C-FR-370
administered as a suspension in corn oil at dose levels of
50 and 1000 mg/kg to male and female albino rats. In
addition, the tissue distribution of radioactivity was
investigated following single oral doses of 14C-FR-370 at 50
mg/kg only to male and female rats.


2. At the low dose level (50 mg/kg) in male rats, the
maximum mean concentration of radioactivity in plasma
occurred 12 hours after oral administration of 14C-FR-370,
when it represented 1821 ng equivalents FR-370/ml.
Concentrations declined thereafter to 215.2 ng
equivalenta/ml at 120 hours and were not detectable (ND) at
168 hours post-dose (the final sampling time). In female
rats, the maximum mean concentration of radioactivity in
plasma occurred 6 hours after the oral dose of 14C-FR-370,
when it represented 745.2 ng equivalents FR-370/ml.
Thereafter, mean concentrations declined to 215.4 ng
equivalents/ml at 72 hours post-dose and were not detectable
after this time. Total radioactivity concentrations in
plasma were greater than those in whole-blood at all times.
This observation is supportive of a lack of affinity of FR-
370, or related material, with blood cells.


3. Concentrations of radioactivity in plasma and whole-
blood, in the high dose group (1000 mg/kg), were generally
below the limit of detection due to the much reduced
specific activity of the test compound. As a result it was
not possible to draw meaningfull comparisons between the two
dose levels. The available data indicate that plasma
concentrations were only ca 2-3 times higher than those
obtained at the 50 mg/kg dose level at 3 and 6 hours post-
administration, respectively. These limited data suggets
that the absorption rate is significantly lower than what
might jave been expected based upon a 20-fold increase in
dose level.


4. Following low dose (50 mg/kg) oral administrationof 14C-
FR-370 the mean total radioactivity recovered in excreta and
expired air at 120 hours was 99.40% (male) and 100.90%
(female). Radioactivity  in the animal carcass and
gastrointestinal tract provided an additional 0.70% (male)
and 0.52% (female) contributing to a mean overall
radioactivity recovery of 100.10% dose (male) and 101.41%
dose (female). The faeces was the  major excretory route in
rats following oral dosing with mean totals of 98.33% and
99.74% of the dose in males and females respectively. The
corresponding values in urine being 0.76% and 0.87%, in
expired air 0.12% and 0.09% and that associated with cage
washings being 0.19% and 0.20% for males and females
respectively. Excretion of radioactivity was rapid, as 85%
(male) and 81% (female) of the total faecal radioactivity
was recovered by 24 hours post-dose, increasing to >99%
(male) amd 99% (female) by 48 hours psot-dose. For urine,
76% (male) and 77% (female) of the toatl urinary
radioactivity was recovered by 24 hours post-dose,
increasing to 91% (male) and 93% (female) by 48 hours post-
dose. For both males and females, excretion of radioactivity
was essentially complete by 120 hours post-administration
as totals of less than 0.1% of the dose were present in the
final daily samples collected.


5.Following high dose (1000 mg/kg) oral administration of
14C-FR-370 the mean total radioactivity recovered in excreta
at 120 hours was 98.92% (male) and 97.49% (female).
Radioactivity in the animal carcass and gastrointestinal
tract provided an additional 0.05% dose (male), whereas in
females both were below the limit of detection, providing a
mean overall radioactivity recovery pf 98.97% dose (male)
and 97.49% dose (female). The faecs was the amjor excretory
route in rats following oral dosing with mean totals of
98.70% and 97.31% of the dose in males and females
respectively. The corresponding values in urine being 0.14%
and 0.11%, and that associated wuth cage washing being 0.09%
and 0.07% for males and females respectively. Radioactivity
levels in the expired air were below the limit of detection
for males and represented 0.01% of the dose for females.
Excretion of radioactivity was rapid as 87% (male) and 76%
(female) of the total faecal radioactivity was recovered by
24 hours post-dose, increasing to 97% (male) and 97%
(female) by 48 hours post-dose. For urine, 71% (male) and
82% (female) of the total urinary radioactivity was
recovered by 24 hours post-dose, increasing to 86% a9male)
and 91% (female) by 48 hours post-dose. For both males and
females, excretion of radioactivity was essentially complete
by 120 hours post-administration, as less than 0,02% of the
dose were present in the final daily samples collected.


6. After a single oral administration of 14C-FR-370 at 50
mg/kg to male and female rats, radioactivity was widely
distributed throughout the animal body. Greatest
concentrations in most systemic tissues occurred at times
consistent with the plasma Tmax, i.e. either 12 or 6 hours
post administration for males and females respectively.
Highest concentrations of radioactivity at these times were
presnet in the gastrointestinal tract and liver (the
principle excretory organs for this compound), although high
levels were also detected in fat, probably reflecting the
lipophilic nature of the parent compound and/or its
metabolites. Lowest tissue radioactivity concnetrations were
associated with brain, indicating that test-compund related
material did not readily penentrate the blood-brain
barrier. in addiiton, whole-blood radioactivity
concentrations were generally only about half of those in
plasma, and only low levels of radioactivity were detected
in blood cells, indicating that association of test-compound
related material with the blood cells was not extensive.


7. Chromatographic analysis revealed that essentially all of
the radioactivity excreted in the faeces was unchanged FR-
370, with one minor unidentified metabolite (RM7),
representing ca 2% of the sample radioactivity detected.
Since nearly all of the faecal radioactivity was excreted
during 48 hours after dosing, these data indicate that
parent drug in faeces represented unabsorbed FR-370. Only ca
1% of the dose was recovered in urinefollowing low dose (50
mg/kg) oral administration, although up to eight
metabolites were detected, indicating that the small amount
of 14C-FR-370 that was absorbed had undergone extensive
metabolism, but no individual metabolite accounted for
greater than 0.29% of the recovered dose. Following enzyme
hydrolysis, FR-513 was tentatively identified by
chromatographic retention only, indicating the presence of a
glucuronide and/or sulphate conjugation thereof in urine.


8. It may be concluded, therefore, that only about 1% of an
oral dose of 14C-FR-370 administered as a suspension in corn
oil was absorbed by rats, this being the proportion of the
radioactive dose excreted in urine. Absorption may have been
more extensive than this if biliary excretion had occurred,
but this is not considered to be likley in the present case
as essentially all of the faecal radioactivity was
recovered within 48 hours of dosing, which was in the form
of unchanged FR-370.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The results of this study have shown that a single 50 mg/kg oral dose of 14C FR 370 administered as a suspension in corn oil was slowly absorbed by rats. Plasma and whole-blood concentrations peaked at 3 hours after dosing, before declining then rising again, with maximum concentrations of radioactivity attained at 12 and 6 hours post-dose in males and females, respectively. After reaching peak values, radioactivity concentrations declined in an apparently monoexponential manner and were quantifiable at 120 and 72 hours post-dose in males and females, respectively. These results may indicate that following slow absorption of radioactivity into the systemic circulation one or more metabolites with a smaller volume of distribution than the parent compound are formed which results in a second peak in the plasma concentration-time profile.

Following single 1000 mg/kg oral doses of 14C FR 370 to rats, concentrations of radioactivity in plasma and whole-blood were generally below the limit of detection (due to the much reduced specific activity of the test compound). It was not possible to draw meaningful comparisons across the two dose levels, although the data indicate that plasma concentrations were ca 2-3 times higher than those obtained at the 50 mg/kg dose level at 3 and 6 hours post-administration, respectively. Therefore, absorption rates were considerably lower than expected for a 20-fold increase in dose level.

After a single oral dose of 14C FR 370 at 50 mg/kg, radioactivity was rapidly excreted by both male and female rats. Only ca 0.8% of the radioactive dose was excreted in urine during 5 days, whereas ca 99% of the dose was excreted in faeces essentially all of which was excreted within 48 hours of dose administration. A further ca 0.1% of dose was found in the expired air of these rats. At the 1000 mg/kg dose level, only ca 0.1% of the radioactive dose was excreted in urine during 5 days, whereas ca 98% of the dose was excreted in faeces, essentially all of which was excreted within 48 hours of dose administration. No radioactivity was detected in the expired air of these rats, confirming that the radiolabel was incorporated in a metabolically stable position of the test compound molecule.

After a single oral administration of 14C FR 370 at 50 mg/kg to male and female rats, radioactivity was widely distributed throughout the animal body. Greatest concentrations in most systemic tissues occurred at times consistent with the pharmacokinetic Tmax, i.e. either 12 or 6 hours post-administration for male and female rats respectively. Highest concentrations of radioactivity at these times were present in the gastrointestinal tract and liver (the principle excretory organs for this compound), although high levels were also detected in fat at later sacrifice times, presumably reflecting the lipophilic nature of the parent compound and/or its metabolites. Lowest tissue radioactivity concentrations were associated with brain, indicating that test-compound related material did not readily penetrate the blood-brain barrier. In addition, whole-blood radioactivity concentrations were generally only about half of those in plasma, and only low levels of radioactivity were detected in blood cells, indicating that association of test-compound related material with the blood cells was not extensive.

Chromatographic analysis revealed that essentially all of the radioactivity excreted in faeces was unchanged FR 370, with one minor unidentified metabolite (RM7), representing ca 2% of the sample radioactivity detected. In urine, up to eight metabolites were detected indicating that the small amount of 14C FR 370 that is absorbed into the systemic system does undergo metabolism, although no individual metabolite accounted for greater than 0.29% of the recovered dose. Following enzyme hydrolysis, FR-513 was tentatively identified indicating the presence of a glucuronide and/or sulphate conjugation of FR-513 in urine.

The excretion results obtained in this study therefore suggest that less than 1% of an oral dose of 14C FR 370 was absorbed by rats, this being the proportion of the radioactive dose excreted in urine. Absorption may have been more extensive than this if biliary excretion of FR 370 derived material occurred, but this is not likely to have been extensive as essentially all of the faecal radioactivity was excreted within 48 hours of dosing, and most of the faecal radioactivity was in the form of unchanged FR 370.
Overall:
no obvious side effects were observed in any of the rats dosed orally with C-FR-370 at target dose levels of either 50 mg/kg or 1000 mg/kg

Executive summary:

The plasma and whole-blood levels of radioactivity, excretion of radioactivity and metabolite profiles were investigated following single oral doses of¹⁴C‑FR‑370 administered as a suspension in corn oil at dose levels of 50 and 1000 mg/kg to male and female albino rats. In addition, the tissue distribution of radioactivity was investigated following single oral doses of¹⁴C‑FR‑370 at 50 mg/kg only to male and female rats.

At the low dose level (50 mg/kg) in male rats, the maximum mean concentration of radioactivity in plasma occurred 12 hours after oral administration of¹⁴C‑FR‑370, when it represented 1821 ng equivalents FR‑370/ml. Concentrations declined thereafter to 215.2 ng equivalents/ml at 120 hours and were not detectable (ND) at 168 hours post-dose (the final sampling time). In female rats, the maximum mean concentration of radioactivity in plasma occurred 6 hours after the oral dose of¹⁴C‑FR‑370, when it represented 745.2  ng equivalents FR‑370/ml. Thereafter, mean concentrations declined to 251.4 ng equivalents/ml at 72 hours post-dose and were not detectable after this time. Total radioactivity concentrations in plasma were greater than those in whole-blood at all times. This observation is supportive of a lack of affinity of FR‑370, or related material, with blood cells.

Concentrations of radioactivity in plasma and whole-blood, in the high dose group (1000 mg/kg), were generally below the limit of detection due to the much reduced specific activity of the test compound. As a result it was not possible to draw meaningful comparisons between the two dose levels. The available data indicate that plasma concentrations were onlyca 2-3 times higher than those obtained at the 50 mg/kg dose level at 3 and 6 hours post-administration, respectively. These limited data suggest that the absorption rate is significantly lower than what might have been expected based upon a 20-fold increase in dose level.

Following low dose (50 mg/kg) oral administration of¹⁴C‑FR‑370 the mean total radioactivity recovered in excreta and expired air at 120 hours was 99.40% (male) and 100.90% (female). Radioactivity in the animal carcass and gastrointestinal tract provided an additional 0.70% dose (male) and 0.52% dose (female) contributing to a mean overall radioactivity recovery of 100. 10% dose (male) and 101.41% dose (female). The faeces was the major excretory route in rats following oral dosing with mean totals of 98.33% and 99.74% of the dose in males and females respectively. The corresponding values in urine being 0.76% and 0.87%, in expired air 0.12% and 0.09% and that associated with cage washings being 0.19% and 0.20% for males and females respectively. Excretion of radioactivity was rapid, as 85% (male) and 81% (female) of the total faecal radioactivity was recovered by 24 hours post-dose, increasing to >99% (male) and 99% (female) by 48 hours post-dose. For urine, 76% (male) and 77% (female) of the total urinary radioactivity was recovered by 24 hours post-dose, increasing to 91% (male) and 93% (female) by 48 hours post-dose. For both males and females, excretion of radioactivity was essentially complete by 120 hours post-administration as totals of less than 0.1% of the dose were present in the final daily samples collected.

Following high dose (1000 mg/kg) oral administration of¹⁴C‑FR‑370 the mean total radioactivity recovered in excreta at 120 hours was 98.92% (male) and 97.49% (female). Radioactivity in the animal carcass and gastrointestinal tract provided an additional 0.05% dose (male), whereas in females both were below the limit of detection, providing a mean overall radioactivity recovery of 98.97% dose (male) and 97.49% dose (female). The faeces was the major excretory route in rats following oral dosing with mean totals of 98.70% and 97.31% of the dose in males and females respectively. The corresponding values in urine being 0.14% and 0.11%, and that associated with cage washings being 0.09% and 0.07% for males and females respectively. Radioactivity levels in the expired air were below the limit of detection for males and represented 0.01% of the dose for females. Excretion of radioactivity was rapid as 87% (male) and 76% (female) of the total faecal radioactivity was recovered by 24 hours post-dose, increasing to 97% (male) and 97% (female) by 48 hours post-dose. For urine, 71% (male) and 82% (female) of the total urinary radioactivity was recovered by 24 hours post-dose, increasing to 86% (male) and 91% (female) by 48 hours post-dose. For both males and females, excretion of radioactivity was essentially complete by 120 hours post-administration, as less than 0.02% of the dose were present in the final daily samples collected.

 

    After a single oral administration of¹⁴C‑FR‑370 at 50 mg/kg to male and female rats, radioactivity was widely distributed throughout the animal body. Greatest concentrations in most systemic tissues occurred at times consistent with the plasma T_max,i.e.either 12 or 6 hours post-administration for males and females respectively. Highest concentrations of radioactivity at these times were present in the gastrointestinal tract and liver (the principle excretory organs for this compound), although high levels were also detected in fat, probably reflecting the lipophilic nature of the parent compound and/or its metabolites. Lowest tissue radioactivity concentrations were associated with brain, indicating that test-compound related material did not readily penetrate the blood-brain barrier. In addition, whole-blood radioactivity concentrations were generally only about half of those in plasma, and only low levels of radioactivity were detected in blood cells, indicating that association of test-compound related material with the blood cells was not extensive.

 

   Chromatographic analysis revealed that essentially all of the radioactivity excreted in faeces was unchanged FR‑370, with one minor unidentified metabolite (RM7), representingca2% of the sample radioactivity detected. Since nearly all of the faecal radioactivity was excreted during 48 hours after dosing, these data indicate that parent drug in faeces represented unabsorbedFR‑370. Onlyca1% of the dose was recovered in urine following low dose (50 mg/kg) oral administration, although up to eight metabolites were detected, indicating that the small amount of¹⁴C‑FR‑370 that was absorbed had undergone extensive metabolism, but no individual metabolite accounted for greater than 0.29% of the recovered dose. Following enzyme hydrolysis, FR-513 was tentatively identified by chromatographic retention only, indicating the presence of a glucuronide and/or sulphate conjugation thereof in urine.

 

     It may be concluded, therefore, that only about 1% of an oral dose of¹⁴C‑FR‑370administered as a suspension in corn oil was absorbed by rats, this being the proportion of the radioactive dose excreted in urine. Absorption may have been more extensive than this if biliary excretion had occurred, but this is not considered to be likely in the present case as essentially all of the faecal radioactivity was recovered within 48 hours of dosing, which was in the form of unchangedFR‑370.