N-(cyclohexylthio)phthalimide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

Toxicokinetic

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(cyclohexylthio)phthalimide

EC Number:

241-774-1

EC Name:

N-(cyclohexylthio)phthalimide

Cas Number:

17796-82-6

Molecular formula:

C14H15NO2S

IUPAC Name:

N-(cyclohexylthio)phthalimide

Details on test material:

Certificate of Analysis: No 060707/I3
Test batch No.: 041/07
Active substance: 99,42 % weight
Weight loss: 0,08 % weight
Toluene insolubles: 0,05 % weight
Ash: 0,008 % weight
Melting point: first liquid 92,3°C
complete liquid 92,5 °C
Sieve retention: 2,0 mm 0 %
0,5 mm 0 %

Radiolabelling:

yes

Remarks:

14C-radiolabeled test article

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Species: rats, Wistar strain
Source: Velaz Praha, Czech Republic
Number and Sex: 140 males
Body weight: Young rats (initial body weight 160-180 g, Animal delivery protocols No. 24/2007, 4/2008 and 5/2008) were quarantined for 1 week before dosing. At the beginning of experiments initiation mean of body weight was 184,9±20,3 g for absorption, distribution, elimination (urine, faeces) or metabolism. In bile elimination experiment an average of body weight was higher (256±17,6) g for better cannulationof bile duct or duodenum.
Housing: Rats were housed in the groups of up to five per cage, in a wooden grating floor polyethylene cages. The care of animals in the Experimental Animal Quarters, hameln rds, a.s., is directed by the SOP 003/53204/07.
Acclimation: Rats were acclimatised for one week according SOP 001/53204/07 - Quarantine of animals. The humidity of air and temperature was routinely monitored, automatically recorded and controlled (SOP 012/53204/07). Rats were kept in cages under a 12 h light/ dark cycle maintained at 22,2±0,5°C temperature and relative humidity were in the accordance with OECD guidline (range 22±3°C and 30-70%).
Animals were allowed to drink water and feed rat diet (Top Dovo, Slovak Republic) ad libitum expect overnight prior to the dose, when food was withheld until 4 h postdose.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

Olive Oleum Raffinatum

Details on exposure:

Dose preparation
I. part: 14C-Duslin was admistered together with unlabelled compound. Duslin after weighing (10 mg Duslin at low dose and 30 mg at high dose per ml olive oil)was transferred into a frictional dish. There was added small volume of dichlormethane and solution of 14C-Duslin. DCM was evaporated under nitrogen (SOP 042/211/03). Olive oil was slowly added. The test article was stirred by pestle infrictional dish until the test article was completely suspended. The stock solutions were prepared for both the doses, to allow dose volumes of 1 ml per 200 g body weight (5 ml/kg). The nominal radioactive quantity of 14C-Duslin was 50 µCi/kg, which approximated to 10 µCi per 200 mg of body weight.
II. part: The same amount of unlabelled Duslin was suspended in appropriate volume of olive oil in frictional dish.
Suspensions were stored at room temperature maximally 6 days. The stability of administerd form was confirmed by HPLC.

Duration and frequency of treatment / exposure:

0 - 6 h
6 - 24 h
24 - 48 h
48 - 72 h

Doses / concentrations

Remarks:

Doses / Concentrations:
50mg/kg, 150 mg/kg

No. of animals per sex per dose / concentration:

I. part
Animals for biological samples of absorption, distribution and elimination
Rats (n=120) in experiment were divided into six groups. The groups No. 1 and No. 4 consist of fifty rats with five animals/time interval of blood or organs and tissues collecting.
Group No. 1: dose 50 mg/kg (n=50) - colection of plasma, organs and tissues
Group No. 2: dose 50 mg/kg (n=5) - collection of urine and faeces
Group No. 3: dose 50 mg/kg (n=5) - collection of bile
Group No. 4: dose 150 mg/kg (n=50) - collection of plasma, organs and tissues
Group No. 5: dose 150 mg/kg (n=5) - collection of urine and faeces
Group No. 6: dose 150 mg/kg (n=5) - collection of bile
II. part
Animals for biological samples of metabolism
These animals were administered only non-labelled test article for LC/UV analysis of biological samples in the cause of contamination of column by isotope. The rats (n=20) were divided into four groups, each consisting of five animals.
Group No. 1: - collection of native plasma, bile, urine and faecs
Group No. 2: dose 150 mg/kg - collection of plasma
Group No. 3: dose 150 mg/kg - collection of urine and faeces
Group No. 4: dose 150 mg/kg - collection of bile

Control animals:

no

Details on study design:

- Dose selection rationale: A toxicokinetic study was carried after administration of the dooses at 50 and 150 mg/kg body weight. These doses used were derived from NOAEL value 50 mg/kg/day and LOAEL 150 mg/kg/day for Sprague Dawley Albino rats in 24 months chronic toxicity of Duslin.

Details on dosing and sampling:

Sample colection:
Absorption
The rats (5 animals per time period per dose group) were slightly anesthetized with diethyl ether. The same rats were used for two outlying time intervals - first is blood sampling form eye and secon from the heart. Whole blood was collected from the retroorbital venoplex or by cardiac puncture before sacrifice of animals at 10, 20, 40 min. and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48 and 72 h postdose. As anticoagulant was used heparin.
The blood was taken over from the syringe or caplillary into the marked centrifugation test tube and centrifuged for 15 min. at 4000 rpm and 5 °C . The plasma separated by an automatic pipette was frozen at -15°C until the radioanalysis.
Distribution
From the animals were removed selecting organs and tissues (heart, brain, lung, liver, kidney, testes, thymus, stomach, intestine, colon, a strip of skin from spine, skeletal muscle from hind leg, spleen and abdominal fat) at six time points. The organs and tissues were collected at 2, 8, 16 ,24, 48 and 72 H after the dose, flushed with saline, and divided at two parts, which were weighted. The first part was immediately sampled and second part of them stored frozen at -15°C until analyzed.
Elimination
Urine and faeces: Rats(5 per dose group) were exposed once via p.o. by 14C-Duslin and were placed in a metabolism cage. Excreta (urine and feaces) were collected in portion of 0 to 6 h, 6 to 24 h, and subsequently in 24 h intervals up to 72 h after dosing. After each collection, metabolism cages were rinsed with 0.25 % Neodisher PM5. The supernantan was decanted and the liquid and solid debris retained separately for total14C measurements. Urine and cage washing volumes and faeces weight were measured. Rinse volumes were collected and all samples of elimination were stored at -15°C before analysis.
Bile: Rats(5per dose) were all time of experiment under i.p. urethane anaesthesia (1mg/kg). Cannula was surgically implanted into the bile duct and bile flow was stabilised minimally for 20 min. Duslin was administered via the duuodenum, each rat received a slow-push of dose. Consequently, bile was collected from the supplier with a cannula on calibrated tubes at time intervals 0-2, 2-4 and 4-6 h postdose.
Carcas: Whole rats ( including all organs and tissues) at the end of elimination expreriment were used for thej mass balance study. They were digested for several days in ethanolic KOH ( 1 l/200 g bw).
Metabolism: The same procedures mwere used for collection of the biological samples after administration of unlabelled Duslin as is described upper . Blood samples were collected at 40 min. and 5 h post exposure from the retroorbital sinus or by puncture from heart at 24 h. Rat urine was collected for 0-24, 24-48, 48-72 h after dosing in metabolic cages. The samples of urine were aliquot, filtered to remove any particulates, and transferred to a plastic tube. Whole bile was collected for 6h. The native samples were taken from animals with were administrered placebo-olive oil. The samples of biological samples were stored at -20°C until analysis.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Main ADME results 2

Type:

distribution

Main ADME results 3

Type:

metabolism

Main ADME results 4

Type:

excretion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The rats (5 animals per time period per dose group) were slightly anesthetized with diethyl ether. The same rats were used for two outlying time intervals - first is blood sampling form eye and secon from the heart. Whole blood was collected from the retroorbital venoplex or by cardiac puncture before sacrifice of animals at 10, 20, 40 min. and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48 and 72 h postdose. As anticoagulant was used heparin.
The blood was taken over from the syringe or caplillary into the marked centrifugation test tube and centrifuged for 15 min. at 4000 rpm and 5 °C . The plasma separated by an automatic pipette was frozen at -15°C until the radioanalysis.

Details on distribution in tissues:

From the animals were removed selecting organs and tissues (heart, brain, lung, liver, kidney, testes, thymus, stomach, intestine, colon, a strip of skin from spine, skeletal muscle from hind leg, spleen and abdominal fat) at six time points. The organs and tissues were collected at 2, 8, 16 ,24, 48 and 72 H after the dose, flushed with saline, and divided at two parts, which were weighted. The first part was immediately sampled and second part of them stored frozen at -15°C until analyzed.

Transfer into organs

Transfer into organs 1

Test no.:

#4

Transfer type:

other: blood or organs

Observation:

distinct transfer

Transfer into organs 2

Test no.:

#6

Transfer type:

other: blood or organs

Observation:

distinct transfer

Details on excretion:

Urine and faeces
Rats (five per dose group) were exposed once via p.o. by 14C- Duslin and were placed in a metabolism cage. Excreta (urine and faeces) were collected in portion of 0 to 6 h, 6 to 24 h, and subsequently in 24 h intervals up to 72 h after dosing. After each collection, metabolism cages were rinsed with 0,25% Neodisher PM5. The supernatant was decanted and the liquid and solid debris retained separately for total 14C measurements. Urine and cage washing volumes and faeces weight were measured. Rinse volumes were collected and all samples of elimination were stored at -15°C before analysis.
Bile
Rats (five per dose) were all time of experiment under i.p. urethane anaesthesia (1 mg/kg). Cannula was surgically implanted into the bile duct and bile flow was stabilised minimally for 20 min. Duslin was administerd via the duodendum, each rat received a slow-push of dose. Consequently, bile was collected from the supplier with a cannula on calibrated tubes at time intervals 0-2, 2-4 and 4-6 h postdose.
Carcass
Whole rats (including all organs and tissues) at the end of elimination experiment were used for the mass balance study. They were digested for several days in ethanolic KOH ( 1 liter/200 g bw.)

Toxicokinetic parameters

Toxicokinetic parameters 1

Test no.:

#5

Toxicokinetic parameters:

AUC: 56,99 µg.h/ml, Dose: 50 mg/kg

Toxicokinetic parameters 2

Toxicokinetic parameters:

AUC: 98,29 µg.h/ml, Dose: 150 mg/kg

Toxicokinetic parameters 3

Test no.:

#5

Toxicokinetic parameters:

Cmax: 15,75 µg/ml, Dose: 50 mg/kg

Toxicokinetic parameters 4

Toxicokinetic parameters:

Cmax: 38,07 µg/ml, Dose: 150 mg/kg

Toxicokinetic parameters 5

Test no.:

#5

Toxicokinetic parameters:

Tmax: 0,6 h, Dose: 50 mg/kg

Toxicokinetic parameters 6

Toxicokinetic parameters:

Tmax: 0,4 h, Dose: 150 mg/kg

Toxicokinetic parameters 7

Toxicokinetic parameters:

other: MRT = 35,24 h, Dose: 50 mg/kg

Toxicokinetic parameters 8

Toxicokinetic parameters:

other: MRT = 14,64 h, Dose: 150 mg/kg

Toxicokinetic parameters 9

Toxicokinetic parameters:

other: t1/2 el. = 28,13 h, Dose: 50 mg/kg

Toxicokinetic parameters 10

Toxicokinetic parameters:

other: t1/2 el. = 13,73 h, Dose: 150 mg/kg

Toxicokinetic parameters 11

Toxicokinetic parameters:

other: CL = 154,84 ml/h, Dose: 50 mg/kg

Toxicokinetic parameters 12

Toxicokinetic parameters:

other: CL = 267,45 ml/h, Dose: 150 mg/kg

Toxicokinetic parameters 13

Toxicokinetic parameters:

other: Vel = 6283,75 ml, Dose: 50 mg/kg

Toxicokinetic parameters 14

Toxicokinetic parameters:

other: Vel. = 5296,43 ml, Dose: 150mg/kg

Toxicokinetic parameters 15

Toxicokinetic parameters:

other: Vss = 5452,88 ml, Dose: 50 mg/kg

Toxicokinetic parameters 16

Toxicokinetic parameters:

other: Vss = 3916,10 ml

Toxicokinetic parameters 17

Toxicokinetic parameters:

other: kel. = 0,02 (1/h), Dose: 50 mg/kg

Toxicokinetic parameters 18

Toxicokinetic parameters:

other: kel. = 0,05 (1/h), Dose: 150 mg/kg

Metabolite characterisation studies

Metabolites identified:

not specified

Details on metabolites:

Only the samples in the 150 mg/kg dose were profiled for metabolism because samples in the 50 mg/kg dose would have to a little amount for analysis.
Plasma: After p.o. administration to rats of Duslin at high dose, the plasma metabolite profiles at 40 min. were investigated. Applying the same criteria as those used for defining all metabolite peaks, only small peak was detected in the UV profile of all animal plasma extracts associated with the same retention time for Duslin. There were not observed differences among chromatographic profiles of plasma samples collected at 5 hours after administration.
Urine: Urine samples collected at 24 consecutive hours intervals of treatment with high dose were analysed. Some varations in the metabolite profile were seen over these periods. There were observed significant differences among chromatographic profiles of blank urine and samples collected 24 and 48 hours after administration. Low trace of a concentration close to the retention time for parent compound was present in the urine samples at 24 h. 48 hours after high dose administration, urinary content no consistent of peak associated with this retention time. However, the presence of its peak was documented in urine after 72 h, whereas LC/UV/MS analysis did not confirm presence of Duslin in the samples.
Bile: There were not observed significant differences between chromatographic profiles of native bile (blank) and samples of bile collected during 6 hours after intraduodenal administration of Duslin.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no data
CONCLUSION

OECD Guideline No. 417 - Toxicokinetics (Performed by the Biological Department hameln rds, a.s., 19th May, 2008)
Toxicokinetic study using Wistar rats (140 male rats) orally exposed to 14C radiolabelled substance ( phenyl ring-U-14C), and using olive oil as vehicle, provided information about ADME (absorption, distribution, elimination, metabolism). The animals for evaluation of metabolism had administered unlabelled Duslin.
The amount and rate of absorption ( a time-dependent concentration in plasma and pharmacokinetic parameters), pattern of distribution (organ and tissue levels), route and rate of elimination ( excretion by urine, faeces, bile ) and metabolism were evaluated.

Absorption
Following two different single oral doses (50 or 150 mg/kg ) of 14C-labelled Duslin, agent-related material was rapidly absorbed and detected in plasma reaching a low maximum concentration. When given low dose of 50 mg/kg, the maximum plasmatic concentration (Cmax) was ~0.15% of dose per ml (15.8 ug/mL) and achieved at Tmax 0.6 h(36 min). At high dose of 150 mg/kg, Cmax was 38.1 ug/mL and reached faster (Tmax 0.4h= 24min), if compared to low dose. The 3-fold increase in dose resulted in approximately 2-fold increase of Cmax and AUC 0-∞. The terminal elimination half-time was twice longer (28h) for low dose than for high dose (14h). Duslin had high elimination rate that manifested significant distribution into organ and tissues.

Distribution
The organs and tissues were collected at 6 time points: 2, 6, 16, 24, 48, 72 hours post dose. The amount of Duslin in stomach decreased quickly, maximal radioactivity was observed after 2h. It declined to half level after 8h, indicating rapid transit from the GI tract. The distribution into organs and tissues was biphasic, first time during 2-8h and second time during 16-24h. High content expressed as percent of administered activity per g tissue (%AA/g) was found in majority of tested organs: liver, kidney, lung, spleen, fat, skin, and particularly in heart; the lowest was detected in brain and testes. At both doses, spleen, fat and skin contained the highest concentrations at the end of experiment. Abdominal fat and skin accumulated large amounts of radioactivity.
The observations at 2h following a single low oral dose of (14C)-Duslin showed high concentrations in liver (0.101%AA/g), lung (0.114%AA/g) and kidney (0.133%AA/g). Concentration in kidney decreased quickly; and it completely disappeared after first 8h. At 2h the high concentrations were determined also in heart (0.111%AA/g), spleen (0.096%AA/g), abdominal fat (0.086%AA/g), thymus (0.082%AA/g) and skin (0.061%AA/g). At 8h post dose, further decline in (14C)-Duslin-derived radioactivity was found in the majority of tissues but in some of the organs (liver, kidney, spleen, colon, fat and thymus), activity increased again at 16-24h.
The different observations were found at high dose of 150 mg/kg. Compared to low dose, at the beginning (2 and 8h), the elimination organs (liver, kidney and lung) had 2-4 fold lower percent of administered dose per gram tissue. Excluding the GI tract (stomach, intestine and colon), only heart (0.08%AA/g), spleen (0.05-0.116%AA/g), abdominal fat (0.04-0.07%AA/g) and skin (0.05-0.08%AA/g) were measured organs that reached maximal concentrations at 2-8 and 16-24 h. No further decline was observed after 48h. Three days after dosing, the residues of Duslin ( >=0.05%AA/g) were found in skin and abdominal fat with large amounts found also in spleen.

Elimination
Renal excretion was a major elimination route for Duslin. Elimination by urine in rat peaked in first 6 hours; accounting for 50-60 % of the administered dose. Elimination of compound-derived radioactivity was complete at the final sampling time 72h. The cumulative percent in urine collected at time intervals 0-6, 6-24, 24-48 and 48-72h was 60, 79, 80 and 81%AA at low dose 50, 77, 79, and 80%AA at high dose,respectively. Cumulative urinary elimination was 80-81%.
Faecal excretion was a minor elimination route for Duslin, representing less than 5% of dose. The rate of excretion through faeces was minimal after 6h (0.1-0.3%AA) and peaked between 24 to 72h. The cumulative percent dose eliminated in the faeces was 4.7%AA for low dose and 2.1%AA for high dose, respectively.
The amount of dose eliminated by bile was only 0.21-0.33 % within 6 hours. That indicated that biliary elimination of Duslin after intraduodenal administration was negligible.
The clinical signs of toxicity were not observed during experiments; however, strong odour was presented after administration of Duslin, suggesting the possibility of the elimination of volatile compounds by air also.

Metabolism
Duslin standard of 10ug/ml was used in the LC/UV chromatographic report. Retention time of Duslin was between 5.5-6.5 min. The limit of quantification was 0.01 ug/mL. Only the samples of the 150 mg/kg dose were used for the assessment of Duslin metabolism.
The plasma metabolites at 40 min.after dosing were investigated. Only small peak was detected in the UV profile of all animal plasma extracts associated with the same retention time for Duslin. There were no observed differences among chromatographic profiles of plasma samples collected 5h after administration.
Urine samples collected at 24 consecutive hour intervals of treatment were analyzed. Some variations in the matabolite profiles were seen over these periods. There were observed significant differences among chromatographic profiles of blank urine and samples collected 24 and 48h after administration. Low trace of a concentration close to the retention time for parent compound was in the urine sample at 24h, 48 hours after dose administration, there was no peak in urinary content associated with this retention time. however, the presence of the peak was documented in urine after 72h again, whereas LC/UV/MS analysis did not confirm presence of Duslin in the samples.
Only one undefined metabolite was observed in plasma and urine. There was no free parent compound, also any unchanged Duslin or ots derivative no could be detected in bile. There were no significant differences between chromatographic profile of native bile(blank) and samples of bile collected during 6h after intraduodenal administration of Duslin.