Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 March 2008 to 07 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidelines in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
UNLABELLED TEST ARTICLEIdentification: DUSANTOX 86Certificate of Analysis No 040707/A9Lot number: 005/07Purity: The certificate of analysis No. 040707/A9Storage conditions and Stability: The test substance was maintained at laboratory temperature 20-26°C protected against sunlight. The storage conditions were monitored. In these conditions of storage test article is stable for 24 months. LABELLED TEST ARTICLEIsotope: 14C-Dusantox 86 [diphenylamine ring – U – 14C]Synthesis: Institute of Isotopes Co., Ltd., Budapest, HungaryCertificate of analysis: No. NP 125865Code: CC – 458Commod. Code: 2844403000Batch No.: XVII/3Description: solidRadiochemical purity: 100%Specific activity: 0.097 mCi/mg (3.58 MBq/mg)Molar activity: 39.39 mCi/mmol (1457.43 MBq/mmol)Activity: 1.48 mCi (54.8 MBq)Amount: 15.3 mgStorage: 4 -8°CSolvent: [14C]-Dusantox 86 was diluted in 1 ml dichloromethane
Radiolabelling:
yes
Remarks:
C14

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Male albino rats of the Wistar strain (N=140, initial bodyweight 150-180g), were obtained from Velaz, Praga, Czech Republic. The average weight at the start of the experiment was 199 ± 45g. For elimination by bile animals with high bodyweight (300 ± 12g) were used for better cannulation of gall-bladder.Rats were quarantined for 5 days before dosing. The animals were housed in sawdust-lined polypropylene holding cages in groups of up to five per cage.The animals were kept in a clean air-conditioned room with controlling of temperature and relative humidity under a 12h light/dark cycle (light 6-18h and dark 18-6h). The roomer temperature was 21.3 ± 0.6°C and relative humidity of 49.2 ± 2.7%.The animals had free access to water and were fed laboratory chow diet (Top Dovo, PD Horné Dubové) except when they were fasted overnight before peroral dosing to 4h after dosing.Before experiments the rats was designated by color on a tail and cage with data card (number of study, time interval, dose level and name of study director).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
VEHICLE Identification: Olive oil (Olivae oleum raffinatum)
Producer: G. Heess
Lot number: L709175
Expiration: 09/2008
DOSE LEVELS: Dusantox 86 was single administered to the rats at two doses. The dose levels were 10 mg/kg and 80 mg/kg. The doses were derived from 28-day toxicity study of compound in Wistar rats.
DOSE PREPARATION. part of study: Dusantox 86 (2mg at low dose and 16mg at high dose per 1ml olive oil) after weighing was transferred into a frictional dish. There was added a small volume of dichloromethane (1-2ml) and a solution of [14C]-Dusantox 86 in DCM with appropriate radioactivity. DCM was evaporated under nitrogen and olive oil was added after this. The test article was stirred by pestle in frictional dish until it was completely suspended in vehicle. The stock emulsions were prepared for both the doses by the same procedure, to allow dose volume of 1ml per 200g bodyweight (5ml/kg). The nominal radioactive quantity of 14C-Dusantox 86 was 50 μCi/Kg, which approximated to 10 μCi per animal. Suspensions were stored at room temperature maximally 5 days. II. part of study: The amount of Dusantox 86 at high dose was suspended in appropriate volume of olive oil in frictional dish by the same procedure without adding of isotope 14C-Dusantox 86.
DOSE ADMINISTRATION: Rats directly before starting of application were weighed and administered volume of dose suspension was calculated on an actual bodyweight. Single dose suspension of Dusantox 86 was administered orally by gavage with metal probe. In the case of elimination by bile test article was applied intraduodenaly using polyethylene cannula.
Duration and frequency of treatment / exposure:
Dusantox 86 was single administered to the rats at two doses.
Doses / concentrations
Remarks:
Doses / Concentrations:The dose levels were 10 mg/kg and 80 mg/kg.
No. of animals per sex per dose:
I. part of study – Total number of 120 rats in experiment was divided into six groups with 3 groups belonging to each dose:
Group No. 1: dose 10mg/kg (n=50) collection of plasma, organs and tissues
Group No. 2: dose 10mg/kg (n=5) collection of urine and faeces
Group No. 3: dose 10mg/kg (n=5) collection of bile
Group No. 4: dose 80mg/kg (n=50) collection of plasma, organs and tissues
Group No. 5: dose 80mg/kg (n=5) collection of urine and faeces
Group No. 6: dose 80mg/kg (n=5) collection of bileII.
part of study – Animals (n=20) are divided into four groups which contained 5 animals:Group No. 1: olive oil – collection of plasma, bile, urine and faecesGroup No. 2: dose 80mg/kg – collection of plasmaGroup No. 3: dose 80mg/kg – collection of urine and faecesGroup No. 4: dose 80mg/kg – collection of bile
Control animals:
yes, concurrent vehicle
Positive control:
Postive control not used in this study.
Details on study design:
The doses were derived from 28-day toxicity study of compound in Wistar rats. ANALYTICAL METHODSRadiochemical Analysis: The radioactivity was determined by liquid scintillation counting techniques using a scintillation spectrometer Packard TriCarb 2000CA.The aliquots of biological samples after preparation in the scintillation vessels were mixed with 15ml of scintillation liquid. Then all samples after cooling were measured for the radioactivity. The samples were routinely counted for 5 min. Counting efficiency was corrected by external standard procedure with appropriate quench curves. The instrument was calibrated before counting of samples. There was used standard etalon for 14-carbon. The quantification limit of radioactivity was set approximately 100 dpm after subtraction of twice background (about 50 dpm). All radioactivity measurements were done once. LC-MS AnalysisChromatographic conditions:Chromatographic system: Agilent 1100 Series LC/MSDColumn: Zorbax ECLIPSE XDB C8 100 x 3, 3.5μm, (Agilent, USA) S.N. USU 001316Mobile phase: A – 0.1% formic acid in deionized water, B – acetonitrile, A/B (20/80)Flow rate: 0.5ml/min, Column temperature: 35°CInjection volume: 5μlDetection MS, Ionization mode: API-ES, Polarity: positive, Monitored ions: SCAN 50 – 1000Fragmentor: 120 V, VWD, 290nm, Gain: 1, Drying gas temp.: 350°CDrying gas flow: 6 1/min, Nebulizer pressure: 30 psgi, Capillary voltage: 3000 VSample preparation:1. 0.5ml of biological material was mixed with acetonitrile and vortex for 20 seconds.2. Centrifuge for 10 minutes at 350rpm and collect clean supernatant to autosampler vials.
Details on dosing and sampling:
BIOLOGICAL MATERIAL
Collection, sampling and preparation of biological material are made according to SOP 047/211/03.Adsorption: Blood samples were collected first time from retroorbital plex in heparinised glass cailares and second time from heart in vacutainers containing lithium heparin and anticoagulant at 16 time points (10, 20 and 40 min and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48 and 72h). The blood samples were immediately centrifuged (4000g for 15min at 4°C). The plasma was harvested and remained frozen for radioactivity. Plasma (0.2ml) was taken into the scintillation vessels and dissolved by the addition of an ethanolic solution of KOH (0.16ml) at the room temperature for 24h. Then it was neutralised by the addition of 1 N HCI (0.8ml) and deionised water (0.16ml). After mixing it was discoloured by a few (2-3) drops of hydrogen peroxide. Distribution: The rats (5 rats per time point) were sacrificed by diethyl ether overdose at 2, 8, 16, 24, 48 and 72h. The organs and tissues, brain, stomach, heart, kidney, liver, lung, skeletal muscle, skin, spleen, stomach, intestines, colon, testes and abdominal fat, respectively were collected. The tissues were immediately excised after collection of blood, cleaned rinsing with saline, blotted dry and divided to two parts which were weighed. First parts were placed into a calibrated glass test tube and treated to a four-fold volume of ethanolic KOH. Second parts were frozen and stored in Petri dishes at -15°C and used only the repeat analysis of radioactivity was necessary measured.
Elimination
Urine and faeces: The animals (five per dose) after dosing were placed in metabolism cages. Urine and faeces were collected over four time periods: 0 to 24h, 24 to 47h, 48 to 72h and 72 to 96h. The metabolism cages were thoroughly washed with Neodisher PM5 (2.5% w/v), and the washings and debris were collected into a calibrated container. The volume or urine and case washing was measured and faeces weighed. All samples were transferred to a freezer as soon as possible after collection and stored until analyzed. There were faecal pellets to produce a homogenate; the amount of 1g was homogenized in 10ml of water. Aliquot of faeces homogenate was taken off and processed in the same way as organ or tissue samples. The volumes of homogenate and digestive samples in KOH solution were measured.Bile: Five rats for each dose were anesthetized by intraperitoneal injection or urethane (1g/kg). The bile ducts were surgically cannulated with polyethylene tubing, which were exteriorized at the calibrated tube. Bile flow was stabilised minimally for 20 min. Further the rats were intraduodenally dosed. The samples of bile were collected at time intervals 0 to 2h, 2 to 4h and 4 to 6h post dosing. Preparation of bile samples was processing in the same way as plasma.The mass balance study: At the end of elimination, the rats of each dose were sacrificed by an overdose of diethylether, and the carcasses were retained for mass balance examination. Carcasses were individually dissolved, over several days, in approximately 0.5 litre of 4M potassium hydroxide in ethanol and the total volume of the digest recorded. The samples were retained at laboratory temperature. From the volume resulted by the dissolution of the whole sacrificed animals separately in ethanolic KOH was taken sample and processed in the same way as plasma. Metabolism: Collection of native sample of biological materials was made after application of vehicle olive oil. Other rats have received a single high dose of 80 mg/kg of unlabelled Dusantox 86. The samples of plasma were collected at 6 and 25h post-dosing. Bile was collected at 2-hour intervals after intraduodenal application. Urine and faeces were collected during 24h and 48h. all biological samples were stored frozen at approximately -20°C before LC-MS analysis. The faeces before analysis were freshly homogenised in 10ml CAN by the same procedure as is described in the first part of the study.
Statistics:
CALCULATIONSEvaluation of concentration: Evaluation of concentrations of Dusantox 86 in biological samples were calculated using Excel program from measured radioactivity. The concentration was expressed as the percent of applied activity (AA) after recalculation to a constant volume of plasma (%AA/ml) or constant amount of organ and tissue mass (%AA/g wet tissue), respectively. Particularly the levels were expressed at μg of Dusantox 86 which were calculated from %AA in all biological samples. The contents in the samples of bile, urine and faeces or carcass were expressed on the total amount of dose (%AA). The concentrations in urine and cage washing samples were cumulated. Dose proportions recovered in individual urinal and faecal samples were calculated to establish the mass balance over 96h. Finally, total recovery of radioactivity was calculated from values of concentration of faeces, urine, cage washing and carcass, respectively. The concentration in all biological samples were calculated on the assumption that all a radioactivity was in the form an unchanged compound.The concentration data are expressed as individual values or as mean ± SEM (n=5).Pharmacokinetic Analysis: Plasma concentration profiles were fitted to a non-compartment model using the licensed software KineticaTM ThermoFisher. These pharmacokinetic parameters were calculated based on concentration expressed at μg of Dusantox 86 per ml plasma: the maximal concentration (Cmax), the time to reach peak concentration (Tmax), terminal elimination rate constant (kel), mean residence time (MRT), the apparent volume of distribution during the terminal elimination phase (Vel) and at steady-state (Vss), area under the plasma concentration-time curve up to infinitive (AUC0-oo), total clearance (CL) and the elimination half-life (t1/2el). No statistical analysis was performed on the pharmacokinetic parameters.

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Type:
distribution
Type:
metabolism
Type:
excretion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The typical radioactivity profile in plasma increased progressively peaking at the same time (Tmax = 6.8h) for both doses with a maximal plasma concentration Cmax of 1.59 μg/ml at low and 10.81 μg/ml at high dose. The concentrations in plasma time-dependent declined in an exponential manner resulting in an AUC0-oo of 11.52 μg/h/ml at low dose and which approximately 10-fold increased at high dose to value 122.13 μg/h/ml. The terminal elimination half-life (t1/2el) of Dusantox 86 was 12.87h at low dose. It was eliminated from plasma with constant of elimination kel of 0.054 h-1. Mean residence time (MRT) was 16h. The total body clearance (CL) was 154ml/h. The apparent volume of distribution for elimination (Vel) and at steady state (Vss) was very similar (2.9L and 2.5L). The plasmatic levels of Dusantox 86 expressed in μg per ml plasma demonstrated marked dose-dependent differences. However, 14C-Dusantox 86 derived radioactivity (%AA/ml) was without significant changes. Following a single oral administration of high dose, the elimination half-life was approximately two-fold longer t1/2el = 20.61 h; the same as mean residence time MRT = 28.62h. Total body clearance was CL = 116ml/h and constant of elimination kel = 0.034h-1. Compared to low dose the volumes of distribution Vel and Vss were logically higher and analogically similar (3.3 – 3.5L). The concentration of the radioactivity after oral administration demonstrated a clear dose-dependence on the time-period of determination, but the difference of plasmatic levels gradually decreased after 24h.
Details on distribution in tissues:
At low dose, the radioactivity in most gastrointestinal tissues, such as stomach and intestine, was highest at the first and second sampling point of 2h (0.8 – 0.9%AA/g) and 8h (app. 0.6%AA/g). It declined as a function of time during 48h (from 0.2 to 0.05-0.1%AA/g). Significant amount of radioactivity was sequestered in the liver between 8 – 24h, when the radioactivity declines with time from 0.2 to 0.1%AA/g. The significant amount of radioactivity (0.35%AA/g) was observed in the spleen at 8h. The organs and tissues such as heart, skin, colon and lung, respectively maximally contained ~0.05-0.08% of the administered dose per g tissue during the first 24h. The radioactivity levels in other tested organs and tissues, including kidney, muscle and adipose fat were lower than 0.05%AA/g. It was noted that brain and testes did not accumulate radioactivity during all time of the experiment; there were observed very low concentrations 0.01-0.02%AA/g.There appeared to be no gross difference in the tissue levels of radioactivity between doses; however, the concentrations of Dusantox 86 expressed in μg of compound at dose of 80mg/kg are approximately 8-fold higher. At the beginning of the experiment (2h post dosing), the GIT tissues (stomach and intestine) analysis showed the high radioactivity (0.5 - 0.9%AA/g). Liver contents also high radioactivity (0.3 – 0.4% AA/g) at the same time intervals than at low dose (between 8 – 24h). Also the high radioactivity was observed in spleen at 8h (0.25%AA/g). Similarly than at low dose, heart, skin, colon and lung, contented approximately 0.05 – 0.13%AA/g, whereas in kidney, muscle and adipose fat, respectively, was detected maximally 0.06%AA/g. Analogically, low concentrations at high dose were observed in brain and testes (0.01 – 0.02%AA/g) too.The radioactivity in the organs and tissues, did not peak until as late as 48h post-dosing. No retention of radioactivity in tissues other than liver, spleen, stomach and intestine was noted.
Details on excretion:
Excretion of 14C-Dusantox 86 in the urine was low; only 0.4% of the dose was recovered in the urine as total radioactivity after oral administration of a 10 ng per kg dose of [14C]-Dusantox 86 to rats. Mean recoveries of Dusantox 86 at high dose of 80 mg/kg accounted for 0.3%AA.Administration of 14C-Dusantox 86 to rats revealed that the compound is predominantly eliminated via the faecal route, accounting for 79-97% of the administered radioactive dose. The maximal amount of radioactivity 86% of the low administered dose was eliminated in the first 24 hours. Faecal elimination of total radioactivity in rats was 96.8 ± 11.2%AA during 4 days. The time-dependent excretion by the faeces at low dose over 96h was similar to that at high dose. Of this, the bulk was eliminated in the faeces at high dose, and the majority (~69%AA) of this was within the first 24h; and summary 79.3 ± 9.4% of dose after four days.Intraduodenal dosing of [14C]-Dusantox 86 to bile-duct cannulated rats showed no extensive biliary secretion of radioactive material, with approximately 0.04% of the dose eliminated by this route at low dose and the same amount excreted at high dose during 6h post-dosing. The radioactivity remained in the organism in carcass on day 4 was about 2.5 – 2.7% of administered dose at both cases.Total recovery of administered radioactivity calculated from urine, cage washing, faeces and carcass was 99.6 ± 11.3%AA at low dose and only 82.3 ± 9.6%AA at high dose during 96h.

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
Plasma: Two major peaks were observed in LC/UV chromatograms of plasma extract 6 hours after administration of Dusantox 86 at 80mg/kg. Identity of Dusantox 86 was confirmed by MS Scan detection. Retention time (tR) for Dusantox 86 observed on the base of its standard is ~ 5.9 min. Molecular weight of Dusantox 86 is 405 g/mol (m/Z – 406 for [M+H]+). Molecular weight of unknown metabolite is 421 g/mol (m/z – 422 for [M+H]+) with retention time about 2.7 minute. Relative abundance of metabolic with molecular weight of 421g/mol in relation to Dusantox 86 is about 5% (calculated from peak area). Faeces: Three main peaks were observed in LC/UV chromatograms of faeces extract 24 hours after administration of Dusantox 86. The major peak with retention time about 6.0 minute was identified as unchanged compound. Identity of Dusantox 86 was confirmed by MS Scan detection. Molecular weight of Dusantox 86 is 405 g/mol (m/z – 406 for [M+H]+). Molecular weight of first unknown metabolite is 421 g/mol (m/z – 422 for [M+H]+) and with retention time about 2.7 minute corresponds to plasmatic metabolite of Dusantox 86. Its relative abundance is the same as in plasma (5%). Molecular weight of second unknown metabolite was 437 g/ml (m/z – 438 for [M+H]+) and its retention time was about 1.5 minute. Relative abundance of this metabolite in relation to Dusantox 86 is about 25% (calculated from peak area). Urine: Comparison of UV chromatographic profile of blank urine and urine collected 24 hours after administration of Dusantox 86 at dose of 80mg/kg was undertaken. Parent compound or its metabolites were not detected. Bile: The LC-MS analysis of the bile confirmed neither the identification of Dusantox 86 nor its metabolites in samples. It seems that Dusantox 86 is not available metabolized after intraduodenal application or the levels of compounds are under detection level.Although there are no indications of the presence others of unknown derivatives of compound in the tested biological samples, it cannot be excluded that Dusantox 86 is in blood and excreted into faeces in the form of as yet unknown metabolites, which were detected by the analytic technique used. For extract identification of the metabolites reference standards are necessary.

Any other information on results incl. tables

Table 3 Dusantox 86. Toxicokinetic – Pharmacokinetic parameters of14C-Dusantox 86 in plasma after p.o. administration

Parameter

Dose

10mg/kg

80mg/kg

Cmax[μg/ml]

1.59

10.81

Tmax[h]

6.8

6.8

AUC0-oo[μg.h/ml]

11.52

122.13

kel.[1/h]

0.054

0.034

MRT [h]

16.08

28.62

t1/2 el.[h]

12.87

20.61

CL [ml/h]

154

116

Vel.[ml]

2859.8

3456.4

Vss[ml]

2476.4

3327.7

 

Table 4 Dusantox 86. Toxicokinetic – Mean organ and tissue concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 10 mg/kg

Organ/Tissue

% AA/g

Time (h)

2

8

16

24

48

72

Brain

0.0206±0.0030

0.0164±0.0023

0.0149±0.0019

0.0106±0.0025

0.0099±0.0019

0.0089±0.0008

Heart

0.0462±0.0069

0.0832±0.0100

0.0452±0.0046

0.0335±0.0067

0.0296±0.0043

0.0314±0.0070

Lung

0.0376±0.0020

0.0774±0.0120

0.0429±0.0051

0.0256±0.0068

0.0242±0.0028

0.0263±0.0042

Kidney

0.0287±0.0039

0.0394±0.0061

0.0381±0.0028

0.0288±0.0049

0.0299±0.0092

0.0217±0.0026

Liver

0.0428±0.0036

0.2050±0.0368

0.0994±0.0202

0.1013±0.0292

0.0504±0.0163

0.0467±0.0069

Spleen

0.0581±0.0055

0.3514±0.1098

0.0535±0.0024

0.0351±0.0061

0.0485±0.0081

0.0330±0.0018

Stomach

0.8825±0.2485

0.5602±0.1010

0.2026±0.0448

0.1768±0.0664

0.0549±0.0286

0.0403±0.0079

Colon

0.0500±0.0023

0.0557±0.0084

0.0540±0.0066

0.0442±0.0160

0.0249±0.0021

0.0253±0.0025

Intestine

0.7986±0.2421

0.05643±0.1356

0.2108±0.0546

0.3198±0.2217

0.1014±0.0647

0.0558±0.0103

Testes

0.0123±0.0005

0.0111±0.0014

0.0142±0.0009

0.0112±0.0021

0.0119±0.0019

0.0100±0.0007

Fat

0.0341±0.0060

0.0355±0.0064

0.0681±0.0101

0.0432±0.0119

0.0356±0.0045

0.0250±0.0057

Muscle

0.0239±0.0032

0.0289±0.0041

0.0286±0.0020

0.0170±0.0032

0.0155±0.0018

0.0127±0.0017

Skin

0.0522±0.0051

0.0570±0.0058

0.0608±0.0059

0.0506±0.0066

0.0606±0.0143

0.0597±0.0057

 

Table 5 Dusantox 86. Toxicokinetic – Mean organ and tissue concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 80 mg/kg

Organ/Tissue

% AA/g

Time (h)

2

8

16

24

48

72

Brain

0.0123±0.0011

0.0178±0.0021

0.0292±0.0123

0.0214±0.0056

0.0181±0.0044

0.0122±0.0010

Heart

0.0471±0.0049

0.0996±0.0241

0.1256±0.0763

0.0496±0.0155

0.0420±0.0085

0.0294±0.0031

Lung

0.0343±0.0020

0.0827±0.0239

0.1253±0.0794

0.0454±0.0185

0.0444±0.0165

0.0335±0.0026

Kidney

0.0177±0.0018

0.0350±0.0086

0.0794±0.0435

0.0328±0.0127

0.0263±0.0065

0.0171±0.0005

Liver

0.0359±0.0053

0.3208±0.0671

0.3853±0.2843

0.1142±0.0560

0.0677±0.0262

0.0416±0.0067

Spleen

0.0835±0.0182

0.2429±0.1054

0.0782±0.0368

0.03302±0.0052

0.0337±0.0040

0.0295±0.0009

Stomach

0.4802±0.0338

0.3973±0.0697

0.2653±0.0439

0.0864±0.0257

0.0698±0.0191

0.0402±0.0088

Colon

0.0338±0.0054

0.0492±0.0110

0.0932±0.0432

0.0524±0.0173

0.0352±0.0077

0.0293±0.0029

Intestine

0.8762±0.4551

0.3882±0.1190

0.4834±0.2487

0.0909±0.0341

0.0591±0.0111

0.0613±0.0112

Testes

0.0109±0.0015

0.0110±0.0010

0.0180±0.0061

0.0109±0.0021

0.0120±0.0017

0.0108±0.0008

Fat

0.0480±0.0071

0.0375±0.0052

0.0605±0.0265

0.0327±0.0024

0.0429±0.0049

0.0364±0.0068

Muscle

0.0368±0.0047

0.0311±0.0053

0.0503±0.0242

0.0248±0.0072

0.0227±0.0053

0.0238±0.0027

Skin

0.0286±0.0029

0.0482±0.0045

0.0701±0.0213

0.0422±0.0082

0.0561±0.0124

0.0342±0.0047

Table 6 Dusantox 86. Toxicokinetic – Mean urine concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 10 mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 24

0.1750

0.1690

0.1935

0.1141

0.1958

0.1695±0.033

24 – 48

0.0695

0.1133

0.097

0.0681

0.1203

0.0936±0.024

48 – 72

0.0758

0.1125

0.0516

0.0446

0.0493

0.0668±0.028

72 – 96

0.0475

0.064

0.0596

0.041

0.0555

0.0535±0.009

Σ

0.3678

0.4588

0.4017

0.2678

0.4209

0.383±0.073

 

Table 7 Dusantox 86. Toxicokinetic – Mean urine concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 80 mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 24

0.1221

0.1402

0.1792

0.1068

0.1489

0.1394±0.028

24 – 48

0.0685

0.0581

0.0734

0.0654

0.0949

0.0721±0.014

48 – 72

0.0539

0.1431

0.0459

0.0590

0.0211

0.0646±0.046

72 – 96

0.0254

0.0407

0.0446

0.0443

0.0494

0.0409±0.009

Σ

0.2699

0.3821

0.3431

0.2755

0.3143

0.317±0.047

Table 8 Dusantox 86. Toxicokinetic – Mean faeces concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 10 mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 24

89.4

73.7

97.3

76.9

92.6

86.0±4.6

24 – 48

9.3

8.5

10.3

6.7

6.5

8.3±0.7

48 – 72

0.95

1.41

2.11

1.07

1.99

1.5±0.2

72 – 96

1.08

1.18

1.03

1.11

0.85

1.1±0.1

Σ

100.7

84.8

110.7

85.8

101.9

96.8±11.2

 

Table 9 Dusantox 86. Toxicokinetic – Mean faeces concentration-time profile [14C]-Dusantox 86 in rats after p.o. administration at 80 mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 24

54.0

72.0

74.6

76.6

68.2

69.1±4.0

24 – 48

9.4

12.3

3.0

6.4

4.9

7.2±1.7

48 – 72

0.53

3.71

0.87

0.99

1.70

1.6±0.6

72 – 96

1.17

1.09

1.15

1.98

0.96

1.3±0.2

Σ

65.1

89.1

80.5

86.0

75.8

79.3±9.4

Table 10 Dusantox 86. Toxicokinetic – Mean bile concentration-time profile of14C – Dusnatox 86 after intraduodenal administration at 10mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 2

0.0016

0.0031

0.0029

0.0120

0.0303

0.01±0.005

2 – 4

0.0024

0.0129

0.0034

0.0215

0.0647

0.021±0.012

4 – 6

0.0045

0.0120

0.0044

0.0158

0.0304

0.013±0.005

Σ

0.0085

0.028

0.0107

0.0493

0.1254

0.044±0.048

 

Table 11 Dusantox 86. Toxicokinetic – Mean bile concentration-time profile of14C – Dusnatox 86 after intraduodenal administration at 80mg/kg

Time interval (h)

% AA

Order of animals

Mean±SEM

1

2

3

4

5

0 – 2

0.0139

0.0010

0.0064

0.0062

0.0055

0.007±0.002

2 – 4

0.0235

0.0043

0.0203

0.0108

0.0178

0.015±0.008

4 – 6

0.0212

0.0061

0.0185

0.0220

0.0197

0.018±0.003

Σ

0.0586

0.0114

0.0452

0.0390

0.0425

0.039±0.017

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study resultsThe aim of the present study was to assess Toxicokinetic behaviour of Dusantox 86 and recoveries from absorption, distribution, elimination and metabolism following single oral administration in rats at 10 and 80 mg/kg, i.e. 2 and 16 mg per 200g bodyweight. Absorption: The time point at which the plasma concentrations of Dusantox 6 were maximal is the same for both doses (Tmax = 6.8h). The maximum plasmatic concentration was low and increased with high dose level from 1.6 to 10.8μg/ml. In addition, the area under the plasma curve was proportionally 10-fold smaller at low dose (11.5 vs. 122 μg/h/ml).Elimination constant (kel) is rate of elimination of Dusantox 86 in organism (its value 0.1 responses to elimination half-life ~ 7h). Elimination constant is 0.05h-1 at low dose and elimination half-life 12h. Elimination constant at high dose was lower (0.03h-1) and the terminal elimination half-life was longer (20.6h). About five of elimination half-times are sufficient for a fully elimination of a compound for organism (i.e. 65-100h).Also MRT, which accuses mean of time when Dusantox 86 is in organism as unchanged form, was approximately 2-fold longer at high dose (28.6 vs. 16h). The total clearance was higher for low dose (154 vs. 116 ml/h). Vel and Vss values range from 2.5 to 3.5L for both doses. High apparent volumes of distribution mean a low concentration of compound in plasma, thus also suggesting that alterations in the processes of degradation or distribution do contribute to increase circulating compound levels at high dose.This data indicated no significant association of Dusantox 86 to rat plasma. Distribution: [14C]-Dusantox 86 is not extensively distributed into tissues of rats after an oral dose. It is not absorbed very good and rapidly from the gastrointestinal tract. The levels in organs and tissues following oral administration at low dose are the highest only in GU tract (stomach and intestine) and in liver and spleen (0.2% of administered dose/g tissue) 8h after dosing. Approximately 0.1 – 0.3% of the total radioactivity remained in the other organs and tissues on first days, which decreased with time to 0.1%AA/g on second day respectively. After 72h, concentrations in all tissues had decreased by ≤0.03% of the dose per g, only liver, stomach, intestine and skin obtained high content (0.04 – 0.06%AA/g).Organ and tissue distribution of radioactivity was stated to be similar following oral administration of both doses. However, the highest concentrations were observed at high dose. For example, the same of radioactivity at high dose in liver at 8h (0.2 – 0.3%AA/g) corresponding with 10-fold high amount of test article in this organ. At the 8h time point, there were observed also higher concentration of total radioactivity in spleen (0.25 – 0.35%AA/g). The following tissues, such as lung, kidney, adipose fat, muscle and skin had high concentration (0.05 – 0.07%AA/g). At the end of experiment (3day), levels in most organs were reduced maximally to 0.06% of the administered dose per g tissue. The gastrointestinal tissues and organs of metabolism (liver or spleen) contained the highest radioactivity among all tissues. Liver is primary tissues of distribution of Dusantox 86 in rats. Liver tissue levels were higher than those of other tested organs and tissues; this was due to the relatively rapid elimination be faeces. Elimination: Dusantox 86 and its metabolites were mainly excreted in faeces with more than 80% of a radioactive dose recovered within 24h at low dose or 70% at high dose. By day 4 987% of the administered radioactivity at low dose and 79% at high dose was recovered in faeces and only 0.3 – 0.4% in urine, respectively. Despite no extensive biliary excretion, the majority of the radioactivity was rapidly excreted in faeces. Bile samples examined after 6 hours post-treatment were shown to 0.04 % of dose. Significant amounts (~2.5%) of radioactive dose were still associated with the carcass at the end of experiment (after 4 days). The recovery of total radioactivity was 99.6% at low dose and only 82.2%AA at high dose after 96h.Metabolism: The LC/UV/MS analysis of rat plasma samples indicated that Dusantox 86 was presented as unchanged compound and one minor metabolite (5%). The major peak with retention time about 6.0 minute was identified as unchanged compound (M.w. 405g/mol). Metabolite in plasma with its retention time 2.7 min and molecular weight of 421g/mol corresponds probably to hydroxyderivate of Dusantox 86.In the faeces was observed predominantly unchanged Dusantox 86 and it two metabolites. The retention time (tR 2.7 min) and molecular weight (421g/mol) of first metabolite was the same as for metabolite detected in plasma. Second metabolite had tR 1.5 min and M.w. 437g/mol. On the base of molecular weight of both metabolites can be confirmed that likely these metabolites are hydroxyl- and dihydroxyderivate of Dusantox 86. UV chromatographic reports of blank material were compared to urine and bile samples after Dusnatox 86 administration. There were no observed differences between extracts of blank and samples collected after administration of Dusantox 86 in case of urine and bile. LC-MS profiles of rat plasma, faeces, urine and bile suggest that Dusantox 86 is not extensively metabolized in vivo. There were detected only one metabolite in plasma and two metabolites in faeces, respectively.
Executive summary:

The Toxicokinetic of Dusantox 86 was studied in male Wistar rats at two oral dose levels 10 and 80 mg/kg.

Maximum plasmatic levels of Dusantox 86 generally occurred to 7h after dosing, consistent with no high or rapid adsorption. Pharmacokinetic parameters for the elimination of test article from the plasma of rats show that its terminal elimination half-life is approximately 2-fold at high dose. The high apparent elimination and the steady-state volume of distribution indicated low concentration of Dusantox 86 in plasma and pronounced tissue accumulation.

The results from the distribution do not suggest a potential for accumulation in any tested organs and tissues, apart from liver. Spleen and liver content a maximum level around 0.2-0.35% of dose per g tissue at 8h and approximated to 0.05% AA/g 72h post-dosing. The GIT tissues, such as stomach and intestine had the highest concentrations at the beginning of experiment (0.8-0.9% AA/g).

Elimination of Dusantox 86 is predominantly via the faeces (≥80% AA) and small extent via the urine (0.3-0.4% AA), suggesting a significant non-renal pathway. The relative contributions of these routes were dose dependent. The excretion by faeces at low dose was higher and faster than at high dose (97 vs. 79% AA). In all cases, approximately 2.5-2.7% of the dose remained in the carcass of rats at 96h. The total recovery of radioactivity accounted for 82% of the high dose and more than 99% of the low dose. Elimination via the bile ranged from less than 0.05% AA at the both intraduodenal doses.

The LC/UV/MS profile of the plasma extracts showed a single major peak and one minor peak. Major peak was identified as unchanged compound on the basis of standard Dusantox 86 and eluted at approximately tR6 min. There was detected also one metabolite with tR2.7 min and molecular weight of 421g/mol. On the basis of molecular weight it is probably a hydroxyderivate of Dusantox 86. Analyses identified either parent compound or metabolites in urine or bile. In summary, Dusantox 86 is excreted by the faeces mostly as unchanged compound and its two probably hydroxyl- and dihydroxy- metabolites, first with the same retention time and molecular weight as metabolite in mplasma and second metabolite with tR1.5 ming and M.w. 437g/mol. It can be concluded that Dusantox 86 is not rapidly and mightily metabolized in rats.