Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2014 to 15 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & ICH test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline" below.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH M3 (R2): Note for guidance on non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, December 2009.

Deviations:

yes

Remarks:

See "Principles of method if other than guideline" below.

Principles of method if other than guideline:

List of protocol deviations
1 Temporary deviations from the daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2 The animals in Group 1, 2, 4 and 5 were not dosed within 2 hours after the preparation of the formulation, but in maximally 3.5 hours.
Evaluation: The radiochemical purity of the formulations were checked before and after administration and were good. Therefore this deviation has not affected the study integrity.
3 The faeces collected of Group 3 in the time interval 6-24 hours was accidently left at room temperature for several hours instead of freezing the samples directly after collection.
Evaluation: This has no effect on the amount of radioactivity measured but since the effect of this error is not known on the metabolite identification it was decided to make two pools instead of one for metabolite identification in order to determine the effect. During metabolite identification the two different time intervals of the Group 3 displayed the same pattern, hence concluding that the storage at room temperature for several hours had no effect on the samples and no effect on the integrity.
4 On 5 May animal number 27 received food approximately 4 hours later than planned.
Evaluation: the deviation was accidental and it is not expected that the later feeding time has an effect on the distribution of the compound.
5 A partial area from 0 to 10 hours (AUC0-10) after administration has been calculated in the TK calculations.
Evaluation: this is a correct way to adjust for the difference in the observed tlast values between males and females. In that way a comparison is made over an identical time frame.

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

EC Number:

700-485-5

Cas Number:

939402-02-5

Molecular formula:

Mixture of 4 components, the molecular formulae of which are: C33 H45 O3 P, C38 H55 O3 P, C43 H65 O3 P and C48 H75 O3 P

IUPAC Name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

Test material form:

liquid: viscous

Details on test material:

Radiolabelled [phenol-U-14C] DVS005 (Weston 705T) (Substance 205606/A)
Identification: [phenol-U-14C]DVS005
Description: Colourless, viscous oil
Batch: 7717PXM004-1
Radiochemical purity: 79.7%
Chemical purity: 78.2%
Test substance storage: In freezer (≤ -15°C) in the dark container flushed with nitrogen
Expiry date: 28 March 2015 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
Specific activity: 1.51 MBq/mg
Total activity: 372.4 MBq
Supplier: Selcia Limited, Fyfiled Business and Research Park, Fyfield Road, Ongar, Essex, CM5 0GS, UK
Date received: 28 March 2014

Non-radiolabelled DVS005 (Weston 705) (Substance 205011/B)
Identification: DVS 005 (Weston 705T)
CAS Number: 939402-02-5
Description: Clear colourless viscous liquid (determined at WIL Research Europe B.V.)
Batch: MW3N14T701
Purity/Composition: 100%
Test substance storage: In refrigerator (2-8°C) under nitrogen, desiccated
Stable under storage conditions until: 13 December 2014 (expiry date)

Study specific test substance information
Analysis of stability, homogeneity and concentration of the test substance under test conditions were not performed as part of this study.
Purity/composition correction factor required: No
Hygroscopic: Yes, desiccated
Volatile: No
Reactivity: Reactive to moisture
Test substance handling: Flush container with nitrogen after handling
Specific Gravity / Density: 1.0180 (25/15.5°C)
pH: 6.5-7.5
Stability at higher temperatures: Yes, maximum temperature: 70°C
Stability in the following vehicles:
-Water: No
-1% Aq. Carboxymethyl cellulose: No
-Propylene glycol: No
-Polyethylene glycol: No
-Methyl ethyl ketone: Yes
-Ethanol: No
-Acetone: Yes
-Olive oil: Yes

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Females were nulliparous and non-pregnant.
Rationale: The rat is a species commonly used for toxicokinetic studies and is recognized by international guideline (e.g., EPA, OECD, EEC, FDA, MHW) as a recommended test system for toxicity studies.
Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: 20 males and 20 females.
Number per group: 4 animals/sex/group.
Age/weight at start of study: Young adult animals of 9-13 weeks old were used. Animals were approximately 200 gram in case of blood sampling. Females were non-pregnant and nulliparous.
Identification: Tail mark using indelible ink.
Allocation: By computer-generated random algorithm according to bodyweight, with all animals within ± 20% of the sex mean.
Health inspection: At least upon receipt of the animals.

Animal husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle (lights are on 07:00-19:00). Any variations to these conditions were maintained in the raw data and were considered to have no effect on the outcome of the study.

Accommodation: Upon receipt from the supplier, animals were group-housed in Macrolon cages (type MIV, height 18 cm) containing sterilized sawdust bedding (Lignocel S 8-15, JRS – J. Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). In addition, paper was provided as nest material (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
The day prior to dosing and following radioactive dose administration, the rats of ADME groups were individually housed in stainless steel metabolism cages (LxWxH = 18.5x19x20 cm) with a grid. The urine and faeces collection assembly of the metabolism cage was cooled using dry ice.
Animals of the PK groups were housed individually in Macrolon cages (type MII), equipped with a bottom grid and paper bedding.

Diet and water: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germnay) and to tap-water.

Diet, water bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.
There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Stock solutions of labelled DVS005 (Weston 705T) were prepared in acetone. The dosing formulations were prepared in olive oil with the following nominal concentration and specific activity:
Group 1, 3 and 4: 20 mg/mL with a specific activity of 0.1 MBq/mg
Group 2 and 5: 200 mg/mL with a specific activity of 0.01 MBq/mg
A weighed amount of unlabelled DVS005 (Weston 705T) was placed into an empty glass container. A measured amount of [phenol-U-14C]DVS005 (Weston 705T) was added. Acetone was evaporated using a gentle flow of nitrogen. Olive oil was added to obtain the desired concentration and specific activity.
All radiolabelled formulations were prepared immediately prior to dosing and stored at ambient temperature. Before and during the treatment procedure the formulations were kept on a magnetic stirring device.
No correction was made for the purity/composition of the test substance, a correction was made for the specific gravity of the vehicle (olive oil = 0.911 g/mL) and the unlabelled test substance (=1.018 g/mL).

Duration and frequency of treatment / exposure:

Rats were dosed by oral gavage with a single dose of 100 or 1000 mg/kg DVS005 (Weston 705T) or a repeated dose (14 days) of 100 mg/kg DVS005 (Weston 705T). Group 1, 2, 4 and 5: single dose, Group 3: Once daily in the morning, for 14 days, approximately the same time each day with a maximum of 6 hours difference between the earliest and the latest dose. On Day 15 the animals were dosed with radiolabeled DVS005.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 and 1000 mg/kg.
Each oral dose of DVS005 contained approximately 10 MBq/kg of radioactivity.

No. of animals per sex per dose / concentration:

4 animals/sex/group

Control animals:

no

Positive control reference chemical:

Positive control not required for this study.

Details on study design:

Rationale for dosing method: The route was similar to the route used in toxicity studies with the compound.
Rationale for dose levels: Non-toxic dose levels, based on the 90 day toxicity study (0445-0520)

Details on dosing and sampling:

Observations and samplings
Mortality/Viability: At least twice daily.
Clinical signs: At least once daily. The time of onset and duration were recorded.
Body weights: On the day of treatment with [phenol-U-14C]DVS005 and prior to sacrifice (to ensure the suitability of the selected dose level). In case of repeated dosing body weights were measured once weekly for “cold” dosing.

Sample collection

Urine, faeces and cage washings
In the metabolism cages, urine and faeces were collected over the following time intervals:
pre-dose (-24-0 h), 0-6, 6-24 and 24-48 after dosing with radiolabelled DVS005.
Urine and faeces were freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation. Urine and faeces samples were weighed and analysed immediately or stored at ≤ -75°C prior to analysis. At termination, the interior of the metabolism cages was rinsed with methanol/water (1:1, v/v). The cage rinse was weighed and analysed immediately or stored at ≤ -15°C until analysis.
For animals in groups 4 and 5, urine and faeces were not sampled for analysis.

Toxicokinetic blood sampling
Blood samples were collected from the tail vein of rats of PK groups according to the following schedule based upon results from pilot study 505497 (detailed in table form – see Any other information). The amount of sample collected was approximately 300 μL per sampling time point.
To facilitate blood sampling animals were transferred to an incubator set at 40°C approximately half an hour prior to blood sampling. The animals remained in the incubator until the time of blood sampling. Water was available in the incubator. The rats were maintained in restrainers during all blood sampling times. At other times, rats were housed individually in Macrolon cages and allowed food and water ad libitum. After the final blood sample was collected, the animals were euthanized by an O2/CO2 (approximately 60% / 40%) inhalation procedure.
All blood samples were put into tubes containing Li-heparin and placed on melting ice immediately after sampling. The blood was centrifuged within 1 hour to obtain the plasma. Plasma was transferred into labelled tubes and temporary stored on dry ice until storage at ≤ -75°C prior to analysis. Based on the non-GLP pilot (505497) study there was no distribution to the red blood cells, therefore only plasma kinetics was performed.
Blank plasma (5 mL) were obtained from spare animals (max 2 animals). Blood samples were collected by dorsal aorta puncture under isoflurane inhalation anaesthesia, causing the animals to be exsanguinated. Blood was centrifuged to obtain plasma. Animals were discarded after the blood sampling.

Necropsy
At the designated time of euthanasia each animal in the mass-balance groups (Group 1-3) was deeply anaesthetized using isoflurane by inhalation. The abdomen was opened with a midline incision. By means of dorsal aorta puncture the maximum possible amount of blood was withdrawn, causing the animals to be exsanguinated. Care was taken to avoid contamination of other organs with blood. Sampled blood were transferred into tarred tubes containing Li-heparin and weighed. A weighed subsample of approximately 1 mL of the heparinized blood was removed for total 14C analysis. The remaining blood was centrifuged to obtain the plasma which was weighed and stored at ≤ -15°C. Following the removal of the blood, the following tissues and organs were harvested:
Spleen
Gonads (Females: uterus and ovaries; Males: prostate and testis: all collected separately)
Abdominal fat
Thyroid
Liver
Kidney
Brain
GI tract (including contents)
Skin (shaved)
Heart
Lung
Muscle
Adrenals
Bone
Residual carcass
Samples from muscle and bone were collected from the left hind leg. The weight of each tissue and blood sample was recorded at the time it was harvested. Tissues and carcass were weighed and stored at ≤ -15°C prior to analysis.

Sample processing for radioactivity
Urine, faeces and cage washings
Pre-dose samples were not processed for radioactivity measurements.

Faeces: Each sample was homogenized with an equal weight of methanol (if sample size allowed). Triplicate aliquots of approximately 400 mg of the homogenate (if sample size allowed) were transferred into combustion cones, allowed to air-dry and combusted in a PerkinElmer, Model 307 (PerkinElmer Life and Analytical Sciences, Meriden, USA) oxidizer. The combusted samples were trapped in Permafluor E+ and Carbo-sorb E (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) present in liquid scintillation vials and the amount of radioactivity determined.

Urine and cage washings: One weighed aliquot of 1 mL (or less, in case of expected high concentration test substance or insufficient sample volume) of urine or 2 mL of the other liquids were transferred to a liquid scintillation counting vial and weighed. The amount of radioactivity was determined using Ultima Gold as the scintillation fluid.

Blood, plasma and tissues
Blood (collected at necropsy): Group 1-3: Duplicate aliquots of approximately 100 μL of blood were solubilized using Solvable. Following discoloration by the addition of a H2O2 solution, the amount of radioactivity in each aliquot was measured by LSC using Ultima Gold as scintillation fluid.

Plasma (collected at necropsy): Groups 1-3: For analysis, one aliquot of approximately 100 μL was transferred to a liquid scintillation counting vial and the amount of radioactivity determined. Ultima Gold was used as the scintillation fluid.

Bone and (treated) skin: Skin samples were solubilized in a 6N NaOH solution. Following neutralization using HCl, triplicate weighed aliquots of 750 μL of the solubilized sample were transferred to a liquid scintillation counting vial.
The bone sample was solubilized in a 6N HCl solution. Following neutralization using a NaOH solution, triplicate weighed aliquots of 750 μL of the solubilized sample were transferred to a liquid scintillation counting vial.
Solvable was used as the solubilizing agent. Following discoloration by the addition of a H2O2 solution, the amount of radioactivity in each aliquot was determined using Hionic Fluor as the scintillation fluid.

Carcass + GI tract: The residual carcass and GI tract were solubilized in successively a 6N NaOH solution and a 12N HCl solution. Following neutralization using a NaOH solution, triplicate weighed aliquots of 750 μL of the solubilized sample was transferred to a liquid scintillation counting vial. Solvable was added as a solubilizing agent. Following discoloration by the addition of a H2O2 solution, the amount of radioactivity was determined using Hionic Fluor as the scintillation fluid.

Remaining Tissues: For the remaining tissues, except the bone, carcass and treated skin, a single aliquot of approximately 200 mg was solubilized and analysed for total 14C. Small tissues or organs (e.g. adrenals, thyroid) were solubilized entirely and unprocessed. Liver, brain and kidneys (without the renal capsule) were homogenized with an equal weight of water using an Ultra-Turrax homogeniser and triplicate aliquots of approximately 400 mg of the homogenate were removed for solubilisation and total 14C analysis.
Further procedures for solubilisation, discoloration and total 14C analysis are identical to those described for bone.

Storage of remaining samples: The remainder of each sample (urine, faeces, plasma and blood) was stored at ≤ -15°C for possible future analysis, except metabolite samples which were kept at ≤ -75°C. Samples will be discarded 8 weeks after issuing the draft report, after obtaining the sponsors consent.

Toxicokinetic plasma analysis
The total amount of radioactivity in the plasma of Groups 4 and 5 were measured by LSC using Ultima Gold as scintillation fluid.

Metabolite investigation
Pooling of samples and sample treatment
Constant fractions of urine and faeces from the animals of Group 1-3 were combined and homogenized in order to obtain one urine and one faeces sample per gender per group. Based upon the pilot study (505497) the following intervals were pooled:
Urine: Group 1-3: 6-24 and 24-48 h
Faeces: Group 1-2: 6-24 and 24-48 h; Group 3: was pooled per time interval (6-24 and 24-48 separately) in order to obtain two faeces samples per gender.
Pre dose samples of urine and faeces were also be pooled, in order to obtain one pre dose urine and faeces sample per group.
Total 14C was determined in the pooled samples by LSC (except pre dose samples). The pooled samples of urine and faeces were divided into small sub-samples which were immediately frozen and stored at ≤ -75°C until analysis.

Extraction of faeces
Different solvents were investigated for their extraction efficiency (results not reported, described in raw data). Based on this experiment, the extraction procedure as described below was performed:
Approximately 10 g of faeces pool was extracted with 20 mL methanol by vortexing for 2 minutes. The extract was separated from the solids by means of centrifugation for 5 min at 2000 g and decanted. This extraction was performed three times and combined to one extract. The amount of radioactivity was determined by LSC of a 100 μL aliquot.
The activity in the remaining pellet was determined by combustion of approximately 200 mg (3 replicates) on a on a PerkinElmer Oxidiser.

Statistics:

None specified in the study report.

Results and discussion

Preliminary studies:

Detailed under study number 505497 (non-GLP).

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Oral absorption of DVS005 (Weston 705T) was found to be low (range 4.01 to 7.75%) although this was higher after repeated administration compared with a single dose.

Main ADME results 2

Type:

distribution

Results:

The average total remaining radioactivity in blood, carcass plus tissues was between 0.397 and 0.844% of the administered dose after oral dosing.

Main ADME results 3

Type:

metabolism

Results:

All radioactive peaks in the urine samples were below 5% of the administered dose.

Main ADME results 4

Type:

excretion

Results:

Urinary excretion accounted for approximately 3% after a single oral dose and approximately 6% after repeated administration after 48 hours. Faecal elimination accounted for 81 to 92% in both males and females after 48 hours.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Oral Absorption: Oral absorption was calculated by summing the amount of radioactivity in urine, cage wash and tissues and carcass, and ranged between 4.01 to 7.75%, with a higher oral absorption after repeated administration compared with a single dose.

Details on distribution in tissues:

The half-lives could be accurately determined for males. For the females the half-lives were reported as approximations, since the span of time points used for its calculation was less than twice its calculated value and the correlation coefficient was below 0.9. As a consequence, AUC∞ was also reported as an approximation.
At a dose of 100 and 1000 mg/kg DVS005 (Weston 705T) administered orally, absorption was slow with a tmax ranging between 4 and 6 hours after dosing. Tlast ranged between 10 to 48 after dosing and was later in males compared with females. In order to compensate for the observed later tlast values between the males and females and different dose levels, partial areas up to 10 hours after administration were calculated for all groups.
After oral administration of 100 mg/kg DVS005 (Weston 705T) the average peak concentration in plasma was 1.96 and 0.835 mg/kg for respectively males and females. The average exposure, expressed as AUC0-10, was 16.9/6.81 h*mg/kg in respectively males/females.
After oral administration of 1000 mg/kg DVS005 (Weston 705T) the average peak concentration in plasma was 30.1 and 13.7 mg/kg for respectively males and females. The average exposure, expressed as AUC0-10, was 213/96.5 h*mg/kg in respectively males/females.
After absorption DVS005 (Weston 705T) was eliminated with an apparent terminal half-life ranging between 6.11 and 11.5 hours, where accurate determination was possible.
A more or less dose proportional increase was noted from 100 to 1000 mg/kg DVS005 (Weston 705T) in both genders in plasma. Higher exposures in terms of Cmax and AUC were noted in males compared with females at both dose levels.

Details on excretion:

Urinary excretion
Excretion via urine was only a minor route of elimination for DVS005 (Weston 705T) after oral administration. After a single oral dose of 100 mg/kg DVS005 (Weston 705T) urinary excretion was 3.29% in males and 3.26% in females of the administered dose after 48 hours. After a single oral dose of 1000 mg/kg DVS005 (Weston 705T) this was 3.66% in males and 3.16% in females of the administered dose after 48 hours. After repeated oral administration of 100 mg/kg DVS005 (Weston 705T) urinary excretion was 6.51% in males and 6.38% in females of the administered dose after 48 hours. The excretion rate was slightly higher in the repeated dose group compared with the single oral dose group resulting in a slightly higher amount of radioactivity excreted in the urine.

Faecal elimination
After oral administration DVS005 (Weston 705T) was mainly elimination via the faeces. Faecal elimination represented 91.4% and 86.2% after 48 hours of the administered oral dose of 100 mg/kg DVS005 (Weston 705T) in males and in females respectively. In the high dose group (1000 mg/kg) this was respectively 92.1% and 88.4% in males and females of the administered dose after 48 hours.
After repeated oral administration of 100 mg/kg DVS005 (Weston 705T) faecal elimination was 81.5% in males and 90.5% in females of the administered dose after 48 hours.

Elimination of total radioactivity
After a single oral administration of 100 mg/kg DVS005 (Weston 705T) 95.3% and 90.1% of the administered dose was eliminated during the study period for respectively males and females. After a single oral administration of 1000 mg/kg DVS005 (Weston 705T) this was respectively 96.3% and 92.8% for males and females. After 14 days repeated oral administration of 100 mg/kg DVS005 (Weston 705T) this was respectively 88.9% and 98.0% for males and females. Elimination was rapid and almost complete within 48 hours post-dose, most of the radioactivity was eliminated within the 6 to 24 hours time interval.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Recovery after faeces extraction
Recovery and distribution of the radioactivity in the faeces pool is shown in Table 16. The procedural recovery of the extraction/combustion procedure ranged between approximately 98 to 122%.
Approximately 90% of the radioactivity in the pools was extracted with methanol and 10% of the faeces associated radioactivity remained unextractable. In one sample, males Group 3 (6-24 hr) a very high recovery of 161% was noted. This is considered an accidental finding probably caused by not homogenous sample.

Metabolite identification – LC-MS/MS
In the radioactivity chromatogram of the urine sample obtained after oral administration of DVS005 (Weston 705T), nine radioactivity peaks were observed. All radioactive peaks in the urine samples were below 5% of the administered dose (maximal 2.8% of the dose was observed for M7 in urine sample Ur3F). As only metabolites > 5% of the administered dose should be identified, metabolite identification was not necessary in urine samples.
All peaks observed in the radioactivity chromatogram of the faeces samples obtained after oral administration of DVS005 (Weston 705T) were also observed in the stock solution of [phenol-U-14C] DVS005. Based on these data it was concluded that no metabolites were detected in faeces and therefore metabolite identification in faeces was not necessary.

Any other information on results incl. tables

Urinary excretion

Recovery of radioactivity in urine after a single or repeated oral does of [phenol-U-14C]DVS005 (Weston 705T)

GROUP 1 (100 MG/KG B.W., Single dose, ADME)

Animal no.	Percentage of administered dose in urine sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 1 2 3 4	0.458 0.337 0.234 0.302	2.16 2.25 1.91 2.63	0.359 0.709 0.860 0.963	2.98 3.29 3.00 3.90
Mean SD	0.333 0.094	2.24 0.299	0.723 0.264	3.29 0.427
Females 21 22 23 24	0.189 0.449 0.259 0.256	2.59 2.42 2.56 1.75	0.708 0.544 0.542 0.780	3.49 3.41 3.36 2.79
Mean SD	0.288 0.112	2.33 0.391	0.643 0.120	3.26 0.318

GROUP 2 (1000 MG/KG B.W., Single dose, ADME)

Animal no.	Percentage of administered dose in urine sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 5 6 7 8	0.288 0.556 0.261 0.243	2.09 3.11 2.26 1.92	1.42 0.939 0.790 0.793	3.81 4.61 3.31 2.93
Mean SD	0.337 0.147	2.35 0.528	0.979 0.307	3.66 0.724
Females 25 26 27 28	0.402 0.450 0.225 0.562	2.43 1.55 1.25 1.83	0.730 0.968 1.28 0.962	3.56 2.97 2.75 3.36
Mean SD	0.410 0.140	1.76 0.502	0.985 0.225	3.16 0.366

GROUP 3 (100 MG/KG B.W., Repeated dose, ADME)

Animal no.	Percentage of administered dose in urine sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 9 10 11 12	0.652 0.024 0.737 0.708	4.05 5.67 4.55 4.69	1.69 0.971 1.50 0.788	6.39 6.67 6.79 6.19
Mean SD	0.530 0.339	4.74 0.680	1.24 0.428	6.51 0.270
Females 29 30 31 32	0.982 0.643 0.171 0.867	4.51 3.88 5.47 3.64	1.26 1.50 1.37 1.20	6.76 6.03 7.01 5.71
Mean SD	0.666 0.359	4.38 0.818	1.33 0.133	6.38 0.610

 

Faecal elimination

Recovery of radioactivity in faeces after a single or repeated oral doses of [phenol-U-14C]DVS005 (Weston 705T).

 

GROUP 1 (100 MG/KG B.W., Single dose, ADME)

Animal no.	Percentage of administered dose in faeces sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 1 2 3 4	n/s n/s n/s n/s	99.5 75.6 83.7 87.8	1.80 6.82 3.60 6.87	101.3 82.4 87.3 94.7
Mean SD		86.7 9.96	4.77 2.50	91.4 8.30
Females 21 22 23 24	n/s n/s n/s n/s	86.0 74.9 86.2 50.1	2.65 8.28 3.90 32.7	88.6 83.1 90.1 82.8
Mean SD	n/a n/a	74.3 17.0	11.9 14.1	86.2 3.73

n/s: no sample obtained

n/a: not applicable

 

GROUP 2 (1000 MG/KG B.W., Single dose, ADME)

Animal no.	Percentage of administered dose in faeces sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 5 6 7 8	0.005 n/s n/s n/s	81.4 71.4 82.2 88.6	7.7 18.0 11.7 7.4	89.1 89.5 93.9 96.0
Mean SD		80.9 7.09	11.2 4.95	92.1 3.39
Females 25 26 27 28	n/s n/s 0.001 0.002	80.8 86.3 82.9 85.4	2.86 5.31 6.92 3.07	83.7 91.6 89.8 88.5
Mean SD	0.001 0.001	83.9 2.49	4.54 1.93	88.4 3.39

n/s: no sample obtained

 

GROUP 3 (100 MG/KG B.W., Repeated dose, ADME)

Animal no.	Percentage of administered dose in faeces sample during time intervals post-dose [h]
	0-6	6-24	24-48	Total
Males 9 10 11 12	n/s n/s n/s n/s	69.7 69.0 73.4 75.2	7.97 14.0 10.7 6.18	77.7 83.0 84.1 81.4
Mean SD		71.8 2.96	9.70 3.40	81.5 2.81
Females 29 30 31 32	n/s n/s n/s n/s	99.6 69.2 87.1 78.5	5.03 14.0 3.36 5.11	104.6 83.2 90.4 83.6
Mean SD	n/a n/a	83.6 12.9	6.87 4.81	90.5 10.0

n/s: no sample obtained

n/a: not applicable

 

Elimination of total radioactivity

Total elimination of radioactivity after a single or repeated oral doses [phenol-U-14C]DVS005 (Weston 705T).

 

GROUP 1 (100 MG/KG B.W., Single dose, ADME)

Animal No.	Percentage of administered dose eliminated during time interval post-dose [h]
	0-6	6-24	24-48	Cage wash	Total
Males 1 2 3 4	0.458 0.337 0.234 0.302	101.7 77.9 85.6 90.5	2.15 7.53 4.46 7.83	0.520 0.556 0.408 0.584	104.8 86.3 90.7 99.2
Mean SD	0.333 0.094	88.9 9.97	5.49 2.70	0.517 0.077	95.3 8.33
Females 21 22 23 24	0.189 0.449 0.259 0.256	88.5 77.3 88.7 51.8	3.36 8.82 4.44 33.5	0.550 0.445 0.708 1.00	92.6 87.0 94.1 86.6
Mean SD	0.288 0.112	76.6 17.4	12.5 14.2	0.676 0.242	90.1 3.86

 

GROUP 2 (1000 MG/KG B.W., Single dose, ADME)

Animal No.	Percentage of administered dose eliminated during time interval post-dose [h]
	0-6	6-24	24-48	Cage wash	Total
Males 5 6 7 8	0.293 0.556 0.261 0.243	83.5 74.5 84.4 90.5	9.14 19.0 12.5 8.18	0.659 0.391 0.394 0.785	93.6 94.5 97.6 99.7
Mean SD	0.338 0.147	83.2 6.58	12.2 4.88	0.557 0.197	96.3 2.85
Females 25 26 27 28	0.402 0.450 0.226 0.564	83.3 87.9 84.1 87.3	3.59 3.83 8.28 7.88	0.628 1.04 1.06 0.547	87.9 93.2 93.7 96.3
Mean SD	0.410 0.141	85.6 2.28	5.90 2.53	0.820 0.270	92.8 3.52

 

GROUP 3 (100 MG/KG B.W., Repeated dose, ADME)

Animal No.	Percentage of administered dose eliminated during time interval post-dose [h]
	0-6	6-24	24-48	Cage wash	Total
Males 9 10 11 12	0.652 0.024 0.737 0.708	73.7 74.7 77.9 79.9	9.66 14.9 12.2 6.97	1.21 0.278 0.850 0.973	85.2 90.0 91.7 88.5
Mean SD	0.530 0.339	76.6 2.86	10.9 3.41	0.827 0.394	88.9 2.74
Females 29 30 31 32	0.982 0.643 0.171 0.867	104.1 73.1 92.6 82.2	6.29 15.5 4.73 6.31	0.867 1.59 0.812 1.25	112.2 90.8 98.3 90.6
Mean SD	0.666 0.359	88.0 13.4	8.21 4.92	1.13 0.364	98.0 10.2

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Average total recovery of radioactivity in all dose groups was between 90 and 99% of the applied dose.

Executive summary:

Absorption, Distribution, Metabolism and Excretion of [phenol-U-14C]DVS005 (Weston 705T) in the Wistar rat after a single and repeated oral dose.

The study was conducted according to the following guidelines:

• ICH M3 (R2): Note for guidance on non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, December 2009.

• OECD Guideline no. 417: Toxicokinetics (adopted April 4, 1984)

 

Five groups of rats were included in the study: 3 groups of 4 males and 4 females for the mass balance (ADME) and 2 groups of 6 males and 6 females for toxicokinetic evaluation (TK).

Rats were dosed by oral gavage with a single dose of 100 or 1000 mg/kg DVS005 (Weston 705T) or a repeated dose (14 days) of 100 mg/kg DVS005 (Weston 705T).

In the mass-balance groups, urine and faeces were collected in pre-dose (-24-0 h), 0-6, 6-24 and 24 48 hr intervals. Animals were euthanized 48 hours after dose administration, and a range of tissues and organs were collected. Total radioactivity in urine, faeces, tissues and organs was determined. Selected urine and faeces samples were pooled per group and the metabolite profile in these pooled samples was investigated.

In the toxicokinetic groups, blood was sampled alternately from three rats per time point at 1, 2, 4, 6, 8, 10, 24 and 48 hour after dosing. Total radioactivity and DVS005 (Weston 705T) equivalent concentrations were determined.

 

RESULTS and CONCLUSIONS

No mortality was observed in the study.

After a single and repeated oral dose of 100 mg/kg DVS005 (Weston 705T) no clinical signs were noted. At a single oral dose of 1000 mg/kg piloerection, salivation and ptosis were noted in 2 male animals, in the females no clinical signs were noted at this dose level.

At necropsy, in 2 female animals (single and repeated oral dose of 100 mg/kg) liquid was noted in the uterus. In one male animal (1000 mg/kg) the testicle and epididymis on the left side were reduced in size. At the incidence observed, these were considered signs of no toxicological significance.

At a dose of 100 and 1000 mg/kg DVS005 (Weston 705T) administered orally, absorption was slow with a tmax ranging between 4 and 6 hours after dosing. Tlast ranged between 10 to 48 after dosing and was later in males compared with females.

A more or less dose proportional increase was noted from 100 to 1000 mg/kg DVS005 (Weston 705T) in both genders in plasma. Higher exposures in terms of Cmax and AUC were noted in males compared with females at both dose levels.

Oral absorption of DVS005 (Weston 705T) was found to be low (range 4.01 to 7.75%) although this was higher after repeated administration compared with a single dose. Of this, urinary excretion represented the major component (range 3 to 6%). Altogether 9 metabolites, all below 5% of the administered dose, were found in the urine.

Faecal elimination accounted for 81 to 92% on the radioactivity in both males and females after 48 hours, for respectively the low and high dose group. In the repeated dose group (100 mg/kg) the faecal elimination was slightly lower compared with the single dose groups. Since no metabolites were found in the faeces it was considered that this material represented unabsorbed material.

At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was between 0.397 and 0.844% of the administered dose after oral dosing. The mean percentage of radioactivity in the carcass was 0.122-0.157%.

Average total recovery of radioactivity in all dose groups was between 90 and 99% of the applied dose.