N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-21-1999 to 03-29-1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Remarks:

NOTOX B.V., The Netherlands

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 7 weeks old
- Weight at study initiation: ranged between 270 and 292 g.
- Fasting period before study: 18 hours
- Housing: individually housed in Macrolon plastic cages
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Tumhout, Belgium)
- Water (e.g. ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 40-80
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: January 21, 1999 To: April 14, 1999

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared immediately prior to dosing. The required amount of [14C]-test article was weighed into an empty glass container. After addition of the required amount of non-radiolabelled test material, a weighed amount of the vehicle was added to achieve the final concentration. For groups 1M and 2M (50 and 1000 mg/kg bw respectively), the nominal test material concentration was 10 and 200 mg/ml, with a nominal radioactivity concentration of 2.4 MBq/ml. The formulations were stored at ambient temperature on a magnetic stirring device for a maximum of 4 hours.

VEHICLE
- Amount of vehicle (if gavage): Dose volume of 5 mL/kg bw, the nominal radioactivity contained in each dose was approximately 3 MBq.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Immediately after preparation and prior to dose administration, the homogeneity and radioactivity concentration of each formulation were verified by radioanalysis.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (distribution, excretion)
- Tissues and body fluids sampled :
1) Urine samples were collected from all animals over the following time intervals: 0-24, 24-48, 48-72, and 72- 96 hours after administration of [14C]-labeled test substance. The urine was freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation. Faeces were collected on dry ice over the same time intervals as urine. All urine and faeces samples were stored at < -20°C until analysis. At termination the interior of the cages was rinsed with methanol/water (50/50). The cage rinse was weighed and stored at < - 20° until analysis.
2) Volatiles: For all animals, expired CO2 was collected in a 2N NaOH solution, and other volatiles in cellosolve, over the following nominal time intervals: 0-8, 8-24, 24-36, and 36-48 h after the administration of [14C]-test material. Sample weight was determined at the end of each collection period. Samples were refrigerated at approximately 4°C for a maximum of 3 days, within which period the analyses were completed. Following completion of the analysis, the remainder of the study sample was discarded.
3) All animals were anaesthetised using ether at 96 h after administration of [14C]-test article. The thorax and abdomen were opened with a midline incision. By means of aorta punction the maximum possible amount of blood was withdrawn. Care was taken to avoid contamination of other organs with blood. Sampled blood was transferred into preweighed heparinised tubes. A subsample of approximately 1 mL of the heparinised blood was removed for total C analysis. The remaining blood was centrifuged to obtain the plasma which was weighed and stored at < -20°C until analysis. Following the removal of the blood, the following tissues and organs were harvested: liver, kidneys, spleen, abdominal fat, muscle (left hind leg), G.I. tract, lymph nodes (mesenteric lymph nodes and Peyer's patches), gonads (testes, seminal vesicles, and prostate), bone (left femur), thymus, skin, brain and carcass. The weight of each tissue and blood sample was recorded at the time it was harvested. All biological samples were stored at < -20°C until analysis.

Radioactivity measurements:
All samples were subjected to radioactive measurements performed using a Packard scintillation counter (1900TR) with automatic external standard quench correction.

Statistics:

The mean and standard deviation were used to characterise the data, where appropriate (i.e., radioactivity measurement, concentration, etc.). Concentrations of radioactivity in blood, plasma and tissues were calculated as mg equivalents of [14C]-test material /kg of sample. The total amounts of radioactivity in tissue and excreta samples were calculated as a percentage of the administered radioactivity and a 14C mass balance was presented.

Details on distribution in tissues:

- Concentrations of radioactivity in blood, plasma and tissues: For group 1M, the highest mean tissue concentrations were recorded in the spleen and the mesenteric lymph nodes (0.99 and 1.04 mg/kg tissue, respectively). Similar mean tissue levels were recorded in the thymus, G.I tract, Peyer's patches, skin, bone, liver and kidney (0.11-0.55 mg/kg). In the remaining tissues and organs analysed (brain, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle), mean radioactivity concentrations were about an order of magnitude lower (0.012-0.063 mg/kg). For group 2M, the highest mean tissue concentrations were recorded in the spleen and the G.I. tract (26.9 and 18.0 mg/kg, respectively). The mean tissue concentrations in thymus, Peyer's patches, skin, bone, liver and kidney of the animals of group 2M was 3.86-11.2 mg/kg. In the remaining tissues and organs analysed (brain, mesenteric lymph nodes, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle), mean radioactivity concentrations were about an order of magnitude lower (0.11-1.96 mg/kg). The mean concentration in the plasma (0.002 mg/kg for group 1M and <0.16 mg/kg for group 2M) was a factor of at least 12 lower than in the blood (0.056 mg/kg for group 1M and 1.94 mg/kg for group 2M).
- Amounts of radioactivity (percent of dose) in blood, plasma and tissues: The mean amount of radioactivity was highest in the G.I tract (0.14-0.22%), followed by liver (0.057-0.059 %). In all other tissues and organs analysed, the mean amount of radioactivity was <0.007% of the dose. The carcass of the animals of group 1M and 2M contained a mean 1.27 and 0.84% of the dose. The total mean amount of radioactivity recovered from tissues and carcass at 96 h post dose was 1.49 and 1.14% for groups 1M and 2M, respectively.

Details on excretion:

- Volatiles: The amount of expired volatile radioactivity was minimal and the sampling was discontinued after 48 h. For group 1M and 2M, mean recoveries of radioactivity in the CO2 traps were 0.007 and 0.003%, respectively. The amount of expired organic volatiles was <0.001% in anyone sample analysed.
- Faeces: The majority of the radioactive dose was excreted in the faeces, mostly during the first 24 hours post dose (group 1M) or during the first 48 hours post dose (group 2M). Mean total recoveries from the faeces during the 96-h test period were 82 and 97% for groups IM and 2M, respectively (at a mean mass balance of 85 and 98%, respectively) (Table 1).
- Urine: Mean total recoveries from the urine during the 96-h test period were 0.60 and 0.28% for groups 1M and 2M, respectively (Table 1).

Metabolites identified:

not measured

- Each animal received a radioactive dose of [14C]-test material which ranged between 2.9 and 3.0 MBq. The animals of group 1M (nominal dose 50 mg/kg bw) received an actual dose of test material which ranged between 48 and 54 mg/kg bw corresponding with 96 and 108% of the nominal dose respectively. The animals of group 2M (nominal dose 1000 mg/kg bw) received an actual dose of test material which ranged between 1018 and 1065 mg/kg bw corresponding with 102 and 107% of the nominal dose respectively. The administered doses were considered to be sufficiently close to the target levels in order to fulfill the study's objectives.

- No mortalities occurred during the study. On day 3 of the study, animal 5 of group 2 was observed to have a scab near the tail tip. There were no other antemortem observations. During tissue harvest no abnormalities were noted.

Table: Amount (mean ± SD) of total radioactivity found in faeces and urine from male rats dosed with [14C-] test article/kg bw.

Interval (h)	% of dose in faeces		% of dose in urine
	50 mg/kg bw	1000 mg/kg bw	50 mg/kg bw	1000 mg/kg bw
0-24	71.7±18.6	43.9±12.4	0.25±0.38	0.05±0.06
24-48	7.69±5.51	44.8±10.6	0.20±0.17	0.14±0.15
48-72	2.31±2.85	6.6±7.55	0.09±0.08	0.06±0.11
72-96	0.64±0.48	1.24±1.55	0.06±0.06	0.03±0.05
Total	82.3±12.0	96.6±4.46	0.60±0.67	0.28±0.35

Additional information

In a toxicokinetics study according to OECD guideline 417 and in compliance with GLP, the routes and rates of excretion of total radioactivity were determined after single oral doses of 50 and 1000 mg of [14C]-labeled test material per kg body weight to male rats of group 1M and 2M, respectively.

Following the 50 and 1000 mg/kg body weight doses, respectively, only 0.60 and 0.28% of the administered radioactivity was recovered from urine within 96 hours after administration. The majority of the radio label was excreted via the feces (96-99% of recovered radiolabel). Excretion via the feces, however, does not necessarily mean that the compound was not absorbed. Biliary excretion and enterohepatic recirculation, a phenomenon often observed in rats, can also lead to fecal excretion. At the low dose, excretion of radiolabel via the feces occurred predominantly within 24 h post dose. At the high dose however, the fecal excretion of radio label took approximately 48 h, about equal amounts being excreted between 0-24 h and 24-48 h post dose. Excretion of radioactivity via expired air was negligible (< 0.01% of the dose). Cumulative excretion of total radioactivity after oral administration of [14C]-test material was virtually complete within 96 hours after dosing.

The highest mean tissue concentrations were recorded in the spleen and the mesenteric lymph nodes of animals of group 1M (0.99 and 1.04 mg/kg tissue, respectively) and in the spleen and the G.I. tract of animals of group 2M (26.9 and 18.0 mg/kg, respectively). Similar mean tissue levels were recorded in the thymus, G.I. tract, Peyer's patches, skin, bone, liver and kidney of animals of group 1M (0.11-0.55 mg/kg) and in the thymus, Peyer' s patches, skin, bone, liver and kidney of animals of group 2M (3.86-11.2 mg/kg). In the remaining tissues and organs analyzed, mean radioactivity concentrations were about an order of magnitude lower (0.012-0.063 mg/kg in brain, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle of group 1M and 0.11-1.96 mg/kg in brain, mesenteric lymph nodes, blood, testes, prostate, seminal vesicles, abdominal fat and femoral muscle of group 2M). The mean concentration in the plasma (0.002 mg/kg for group 1M and <0.16 mg/kg for group 2M) was a factor of at least 12 lower than in the blood (0.056 mg/kg for group 1M and 1.94 mg/kg for group 2M).

The retention of the administered radiolabel was very limited. Generally, 96 hours after administration the organs and tissues examined contained less than 0.007% of the dose, except the G.I. tract (0.14-0.22%) and the liver (0.057-0.059 %). There were no significant differences in the pattern of distribution between the high and low dose. There was no significant bioconcentration or accumulation in any of the organs and tissues investigated.

Considering the molecular weight of > 2200 g/mol passive uptake via biological membranes and/or enterohepatic circulation is not likely. The findings from the repeated-dose oral toxicity studies indicate that trace amounts are actively taken up via specialized macrophages in the small intestine, because cell proliferation in the mesenteric lymph nodes are the first effects observed. Overall, the systemic exposure is low as indicated by the present data.