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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standars, well documented and acceptable for assessment

Data source

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
The purpose of the metabolism study was to determine if high and low doses of SD 32899 were metabolised in different ways and also to find a suitable metabolite for monitoring exposure of plant workers to the compound.
GLP compliance:
no

Test material

Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall)
- Age at study initiation:
- Weight at study initiation: 226 - 275 g
- Fasting period before study: Starved from midnight prior to dosing the following morning.
- Housing: Individually housed in plastic cages.
- Individual metabolism cages: yes (after third dose)
- Diet: Given food 1 hour after dosing.
- Water: ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
No data

Administration / exposure

Route of administration:
other: oral: blunt-ended needle
Vehicle:
DMSO
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Doses were prepared immediately before use. They were assayed quantitatively by liquid scintillation counting and qualitatively by high pressure liquid chromatography immediately after dosing was completed. The 14C-labelled 32899 was used within 5 days of preparation.
The doses of 14C-32899-oxide mixed with "cold" 32899 were assayed by liquid scintillation counting and spectroscopic analysis.

DIET PREPARATION
Not applicable

VEHICLE
All doses were administered in 1 mL of DSMO/kg of body weight.
Duration and frequency of treatment / exposure:
Three exposures at 48 hour intervals.
Doses / concentrations
Remarks:
Doses / Concentrations:
Low dose, high specific activity 14C-32899 (2 mg/kg):
Dose 1: 1.88 mg/kg, dose 1: 2.11 mg/kg and dose 3: 2.53 mg/kg

High dose, low specific activity 14C-32899 (350 mg/kg):
Dose 1: 350 mg/kg, dose 2: 349 mg/kg and dose 3 338 mg/kg

Low dose 2 mg/kg (1.6 mg/kg 32899 and 0.4 mg/kg 14C-32899-oxide:
Dose 1: oxide: 0.47; 32899: 1.58
Dose 2: oxide: 0.40; 32899: 1.41
Dose 3: oxide: 0.40; 32899 1.50
No. of animals per sex per dose:
Sixteen male animals in total, each exposed to three doses as follows:

Five animals used for low dose 14C-32899
Five animals used for high dose 14C-32899
Three animals used for low dose 14C-32899-oxide
Three animals used for high dose 14C-32899-oxide
Control animals:
no
Details on study design:
HPLC analysis
Column: Hichrom spherisorb S5-ODS 4.9 x 250 mm.
Solvents: A) Methanol:Water 80:20
B) Methanol
(both calculated 1% acetic acid)
Gradient: 10% B for 2 min then linear gradient to 75% B in 6 min
Flow: 1 mL/min
Detection: U.V. Pye 4020 spectrophotometer, wavelength 274 nm. Radioflow. Berthold Lb 503 HPLC Radioactivity monitor.
Chart recorder: Linseis series 2000
Injection: Waters U6K injection valve.

Spectrophotometric analysis
Analysis carried out using a Varian Cary 210 spectrophotometer. 32899 was measured at 305 nm (where the oxide gave no interfering absorption). Standards were prepared in DSMO and diluted with chloroform.

Liquid scintillation counting (LSC)
LSC was carried out using a Packard Tricarb 460CD liquid scintillation counter using ES 299 (Packard) scintillation cocktail. Tissue cimpbustions (Packard 306 B) were counted in Permafluor V (Packard) scintillation cocktail.

Thin Layer Chromatography
All analytical TLC was carried out using Merck 0.25 mm thickness F254 silica gel plates. The following solvent systems were used:
System A: Acetone/hexane (1/1, v/v)
B: Ethyl acetate/formic acid/water (70/4/4)
C: Butanol/acetic acid/water (6/1/1) (BAW)
D: Toluene/ethyl acetate/acetic acid (75/25/1)
Analysis of plates was by Numelec TLC analyser, Berthold TLC scanner or by autoradiography.

Dose assay
A) Qualitative analysis: The dose solution was diluted 1 in 10 with dichloromethane or chloroform. This solution was then used for HPLC analysis. Column eluate (2 mL fractions) were collected and assayed by scintillation counting. 32899 and 32899-oxide standards were prepared in either dichloromethane or methanol.
B) Quantitative analysis: Duplicates of the dose solution (0.15 mL) were diluted in volumetric flask to 50 mL with toluene. 3 x 0.1 mL aliquots were assayed by scintillation counting.
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
Blood samples (approx. 350 µL) were taken by tail vein bleeding at 1, 3, 5, 7, 9, 24 and 48 hours after dosing. After the 48 hour bleed the animals were redosed and the schedule repeated. Thirty minutes after the second 48 hour bleed the animals were dosed for a third time, then placed in all-glass metabolism cages for 48 hours.
The blood samples were stored at 4°C in heparinised vessels.
Urine and faeces were collected at 24 and 48 hours.
Forty eight hours after the third dose the rats were sacrificed by exsanguination using direct heart puncture under anaethesia. The following tissues were also removed; posterior tibial nerves (left and right), brain, and fat (peri-renal). These tissues and the remaining carcass were stored at -20°C until analysed.

Tissue analysis
A) Blood (50 µL duplicated samples) was assayed by combustion using a Packard 306 B tissue oxidiser (24 hour urine samples were used for efficiency calculations).
B) Brain and fat samples were homogenised by stirring with a broad spatula. 50-100 mg duplicate samples were assayed by combustion.
Tissue extractions
Brain and fat composite samples were prepared for each compound and dose level; these consisted of 50% of each individual sample. These tissue composites were then extracted with 2 x 10 vol (w/v) of methanol using an Ultra Turrax homogeniser (0.5-L min). All the remaining nerve tissues at each dose level were combined and extracted similarly with 2 x 80 vol (w/v) of methanol. The extracts were assayed by LSC. and the residues were assayed by combustion. The extracts were also analysed by TLC and HPLC if sufficient activity was present.

Urine and faecal analysis
Individual urine samples were assayed by LSC. Composite samples were then prepared at each dose level by mixing 2 mL of each individual sample together. These compositesamples were analysed by TLC and HPLC.
The faecal samples were homogenised using 2-3 volumes of distilled water (v/w) and an Ultra Turrax homogeniser (1-2 min, half speed). Duplicates (approx. 100 mg) were then assayed by combustion in a Packard Tissue Oxidiser.
Composite faecal sample (20% of each individual sample) at each dose level were prepared and extracted with 5 volumes of methanol (v/w) by stirring for 45 minutes. The metabolic extract was assayed (2 x 0.5 mL) by LSC and then taken down to a small volume by rotary evaporation and analysed by TLC and HPLC.
The residues were assayed by combustion.

Isolation and identification of major urinary metabolite
The major urinary metabolite of 14C-32899 was isolated using the 24 h, high dose urine composite. Preparative TLC was carried out on Kontes Quantagram (1 mm thickness) TLC plates, developed in solvent system B. The position of the major metabolite was determined by viewing under U.V. light and radio-chemically by using a Numeloc TLC analyser. The appropriate area was then scraped and eluted with methanol and reduced to dryness by rotary evaporation. The residue was taken up in 2 mLs of chloroform and assayed (2 x 100 µL) by scintillation counting. The solution (which contained 60% of the applied material) was analysed by mass spectrometry and TLC.
The urinary metabolites at the 32899 low dose level and after dosing with 32899-oxide were analysed by co-chromatography in three systems.

Results and discussion

Main ADME resultsopen allclose all
Type:
other: µg equivalents 32899/mL of blood
Results:
2 µg/kg-1: After final dose: 0.0135 +/- 0.0068
Type:
other: µg equivalents 32899/mL of blood
Results:
350 µg/kg-1: After final dose: 3.70 +/- 2.02
Type:
other: µg equivalents 32899 oxide/mL of blood
Results:
0.4 µg/kg-1: After final dose: 0.0028 +/- 0.0025
Type:
other: µg equivalents 32899 oxide/mL of blood
Results:
12 µg/kg-1: After final dose: 0.0330 +/- 0.012
Type:
other: µg equivalents per g of tissue - posterior tibial nerve
Results:
Low dose 32899: 0.0112 +/- 0.0043
Type:
other: µg equivalents per g of tissue - posterior tibial nerve
Results:
Low dose 32899 - oxide: Below level of sensitivity - 0.012 µg equivalents 32899 - oxide/g tissue
Type:
other: µg equivalents per g of tissue - posterior tibial nerve
Results:
High dose 32899: 3.7 +/- 2.0
Type:
other: µg equivalents per g of tissue - posterior tibial nerve
Results:
High dose 32899 - oxide: 0.046 +/- 0.023
Type:
other: µg equivalents per g of tissue - brain
Results:
Low dose 32899: Below level of sensitivity - 0.002 µg equivalents 32899/g tissue
Type:
other: µg equivalents per g of tissue - brain
Results:
Low dose 32899 – oxide: Below level of sensitivity - 0.003 µg equivalents 32899 - oxide/g tissue
Type:
other: µg equivalents per g of tissue - brain
Results:
High dose 32899: 0.20 +/- 0.12
Type:
other: µg equivalents per g of tissue - brain
Results:
High dose 32899 – oxide: Below level of sensitivity - 0.003 µg equivalents 32899 - oxide/g tissue
Type:
other: µg equivalents per g of tissue - fat
Results:
Low dose 32899: 0.0031 +/- 0.0017
Type:
other: µg equivalents per g of tissue - fat
Results:
Low dose 32899 – oxide: Below level of sensitivity - 0.003 µg equivalents 32899 - oxide/g tissue
Type:
other: µg equivalents per g of tissue - fat
Results:
High dose 32899: 2.2 +/- 0.90
Type:
other: µg equivalents per g of tissue - fat
Results:
High dose 32899 - oxide: 0.012 +/- 0.005

Toxicokinetic / pharmacokinetic studies

Details on absorption:
14C-32899: Blood kinetic studies
Low dose (2 mg/kg): Peak levels for both doses occurred one hour after dosing and were 0.14 (dose 1) and 0.21 (dose 2) µg equivalents of 32899/mL blood. The profile shows a typical biphasic elimination, a rapid fall in blood levels during the first 5 hours after dosing followed slower rate of decrease over the next 48 hours.
High dose (350 mg/kg): Following the first dose, a peak value of 17.4 µg equivalents 32899/mL blood was observed after nine hours. After the second dose an initial peak, at 3 hours, of 19.8 and second peak, at 9 hours, of 18.4 µg equivalents 32899/mL blood were seen. This double peak is an artifact. None of the mean concentrations of radioactivity in the blood between 3 h and 9 h are statistically significantly different from each other (at the 0.05 level).

32899-oxide blood kinetic studies
Low dose (0.4 mg/kg 14C-32899 and 1.6 g mg/kg-1 unlabelled 32899): The peak values observed were 0.029 (1 hour after dose 1) and 0.020 (3 hours after dose 2) µg equivalents 32899-oxide/mL blood. Elimination of activity from the blood was rapid and levels fell below the limit of sensitivity (ca. 0.002 µg equivalents per mL of blood) after 24 hours.
High dose (13 mg/kg 14C-32899-oxide and 317 mg/kg unlabelled 32899): The peak values observed were 0.52 (1 hour after dose 1) and 0.45 (3 hours after dose 2) µg equivalents 32899-oxide/mL blood.

Summary of the kinetics of [14C]-32899 in blood
From the data it is clear that low doses of 32899 rapidly produce a peak in the blood concentrations, whilst high doses give a later maximum. This could be due either to enterohepatic circulation or to saturation of the absorption process of the compound from the gastro intestinal tract.
The residue levels in the blood do not drop to zero between the doses at either the high or the low dose levels.
The data show that 48 h after each of the three doses the concentrations in the blood steadily increase. Although there is some scatter of the values they are statistically sigificantly different from each other at the P=0.05 level (high dose) and at the P=0.01 level (low dose). This indicates that neither a high (350 mg/kg) or a low (2 mg/kg) dose is completely cleared from the body 48 h after the exposure has occurred.
The blood profiles due to the 14C-32899-oxide are similar to those for the 14C-32899 and indicate that the levels of oxide in the doses of 14C-32899 do not significantly alter the results obtained. The concentrations of oxide dropped below the limits of detection (ca. 0.002 µg equivalents per mL of blood) 48 h after each of the first two low doses (about 0.4 mg of kg of body weight). Hence it is difficult to ascertain if there was accumulation in the animals, but the fact that after the third dose the residues were above the level of detection implies that there was.
Analysis of the data from the high dose (about 12 mg per kg of body weight) of 14C-32899-oxide indicate that there are definately signs of accumulation in the blood on repeated dosing at 48 h intervals. The mean levels at 48 h are statistically significantly different from each other at the P = 0.01 level.
Details on distribution in tissues:
Level of reactivity in tissues
For both dose levels of 14C-32899-oxide and for low doses of 14C032899 the levels of activity are close to or below the respective limits of detection. After high doses of 14C-32899 the posterior tibial nerve tissue showed the highest level of activity (3.7 µg equivalents of 32899 per g of tissue) of the three tissues assayed. This indicates some selectivity in the uptake of the compound and/or its metabolites into the peripheral nervous system in comparison to the central nervous system or other fatty tissues.
The levels of radioactivity found after dosing with 14C-32899-oxide were low and clearly show that the results from the experiments carried out using 14C-32899 contaminated with small amounts of 14C-32899-oxide have not been significantly affected by the presence of the oxide.
The residues of radioactivity in the posterior tibial nerve and fat show that 32899 is taken up more efficiently by these tissues than is 32899-oxide. The ratios of concentrations in the tissues are much higher (78 for the nerves, 183 for the fat) than was the ratio of the dose given (27).

Tissue extractions
In general about 90% of the labelled material was extracted, although it is of particular interest to note that after low dosages only 60-70% of the radioactivity was extracted from the nerve tissue.
The methanol extracts of posterior tibial nerve tissue from animals given high doses of 14C--32899 was examined by HPLC and TLC.
The HPLC analysis showed a major peak, which had a retention time corresponding to authentic 32899 oxide, and contained about 74% of the extracted radioactivity. The minor HPLC peak, which was due to less polar materials, contained about 23% of the extracted radioactivity.
The TLC analysis was carried out using two solvent systems. In system B (ethyl acetate: formic acid:water) the major peak (about 63%) co-chromatographed with authentic 32899-oxide whilst most of the rest of the material ran as a brosd peak near the solvent front indicating that it was less polar in nature. In system A (acetone:hexane) the polar 32899-oxide (about 16% of the radioactivity) remained on the origin whilst the less polar metabolites were split into four main components (about 80% of the radioactivity together). None of the four peaks corresponded to 32899.
It was impossible to identify any of these metabolites, or to determine why the proprtion of radioactivity chromatographing as 32899-oxide varied, due to a shortage of material.
Details on excretion:
Recovery of administered radioactivity
After low dosages, the recoveries in urine and faeces, were 83% for 14C-32899 and 82% for 14C-32899 -oxide over 48 hours. Following dosing at high levels, the recoveries were considerably lower, being only 48% for 14C-32899 and 39% for 14C-32899 -oxide over 48 hours. To account for the shortfall in recovery the intestines in the high dose animal carcasses were removed, homogenised, and assayed by combustion. A further 48% was found in the 14C-32899 dosed animals, givign a total recovery of 97%. The recovery from the intestines of the animals receiving 14C-32899 -oxide was 37%, giving a total recovery of 76%.
These results indicate a difference between the way a high and a low dose of 14C-32899 is handled in the animal. The high dose taking longer to be excreted. It is likely that not all of the high dose was absorbed and that the absorption that occurred did so throughout the passage time through the intestines.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Urinary metabolites
Composite urinary samples were analysed by TLC. The major metabolite in each case co-chromatographed in four solvent systems with 32899-oxide. Urines from animals given high doses of 14C-32899 and 14C-32899-oxide were analysed also by HPLC. The major metabolites again co-chromatographed with 32899-oxide. the major metabolite from animals given high doses of 14C-32899 was confirmed as 32899-oxide by isolation (TLC) followed by mass spectral analysis. The spectrum corresponded with that obtained from authentic 32899-oxide.
Some minor metabolites, both more and less polar than 32899-oxide were detected in the urines of animals given nigh doses of 14C-32899 but they occurred in such low concentrations that it was impossible to identify them.

Faecal metabolite profile
HPLC and TLC analyses were carried out on the methanolic extracts of faecal composites. Over 80% of the radioactivity (82%, for 14C-32899 and 89% for 14C-32899-oxide, both high and low doses, 24 and 48 hour samples results averaged) was extracted.
The major metabolites in each case co-chromatographed on TLC in three solvent systems with 32899-oxide. After high and low doses of 14C-32899 and high doses of 14C-32899-oxide there was some evidence that one of the minor metabolites could have been 32899. (32899 in animals dosed with 32899-oxide could be due to oxygen exchange or reductive metabolism of 32899-oxide). All other metabolites were present at too low a concentration to be identified.

Any other information on results incl. tables

The test compound was found to be very unstable. this was probably due to one or both of two factors: oxidation of the very small quantities which were being used, and the 14C-label in the compound inducing radio-chemical decomposition. It was found to be impossible to obtain a sample of 32899 completely free of 32899 -oxide. This meant that all studies on 32899 were carried out on a mixture of 32899 and 32899 -oxide. In order to determine any effects of the 32899 -oxide on the studies a second series of studies was carried out using non-radio-labelled 323899 mixed with 14C-32899 -oxide in similar proportions to those used in the main studies.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
The results suggest that it is not impossible that repeated doses as low as 2 mg per kg of body weight will, if given over a sufficient period at two day intervals, result in significant concentrations of the compound in tissues.
Executive summary:

The metabolism of low (2 mg per kg) and high (350 mg/kg) oral doses of the SHOP catalyst ligand 32899 in male rats has been investigated. Residues in blood after two successive doses and metabolites after a third dose have been monitored. Blood residues indicate that 48 h is not sufficient time for rats to completely eliminate either a high or a low dose. The results suggest that it is not impossible that repeated doses as low as 2 mg per kg of body weight will, if given over a sufficient period at two day intervals, result in significant concentrations of the compound in tissues.

At both dose levels, 32899 -oxide is the major excreted metabolite; although this is excreted principally in the faeces of rats, it is likely to be a suitable metabolite for monitoring exposure of humans to 32899 by urine analysis.