Bis(N,N',N''-trimethyl-1,4,7-triazacyclononane)-trioxo-dimanganese (IV) di(hexafluorophosphate)monohydrate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP and similar to OECD method 417.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

The [14C]Mn Catalyst was synthesised in the Radiochemical Section at URL Vlaardingen, Netherlands, a non-GLP compliant laboratory but a competent radiochemical laboratory. The radiochemical purity of the sample had not been determined but a specific activity of 9.lµCi/mg was quoted (URLV data).

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Olac Wister

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- six male and six female Olac Yistar rats weighing 160 ± 7g and 156 ± 4g respectively
- Housing: individually labelled metabolism cages specifically designed for the collection of urine, faeces and expired air
- Individual metabolism cages: yes (All the cages were fitted with traps containing ethanolamine: 2-methoxyethanol (1:2 v/v) for the collection of expired air).
- Diet (e.g. ad libitum):PCD diet ad libitum.
- Water (e.g. ad libitum): water ad libitum.
- Acclimation period: approximately 24 hours prior to dosing

Prior to treatment control urine, faeces and C02 absorber were collected and prepared for 14C analysis to give a pre-dose background 14C figure for each rat

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

16.2mg (147µCi) sample of [14C]Mn Catalyst was dispensed at a concentration in the vehicle of (:nominally) a lOµCi/ml (l.08mg/ml) solution for dosing the rats.

- Amount of vehicle (if gavage): 15ml of distilled water

The radiochemical purity of the sample had not been determined but a specific activity of 9.lµCi/mg was quoted (URLV data). The material was given the ESL sample number S1926001.

Duration and frequency of treatment / exposure:

4, 8, 24, 48 and 96 hours one rat of each sex was sacrificed and prepared for whole body autoradiography (WBA). . Af ter 96 hours two rats were sacrificed and the carcasses prepared for 14C analysis.

Doses / concentrations

Remarks:

Doses / Concentrations:
Dosing by gavage with 1 ml of the [14C]Mn Catalyst test solution in water containing 1.08 mg (10.SµCi of 14C).

No. of animals per sex per dose / concentration:

Six male and six female rats

Control animals:

other: pre-dose background 14C figure for each rat.

Details on study design:

Analysis of 14C

Throughout the study 14C activity was determined by liquid scintillation counting. Samples were counted in a Beckman LS3800 spectrometer using Liquiscint (National Diagnostics, Aylesbury, England) as the scintillation cocktail except for the C02 absorber and faeces samples.

Triplicate 1 ml aliquots of C02 absorber were counted in l0ml of cocktail prepared by dissolving 12.5g of 2,5-diphenyloxazole (PPO) and 0.75g of p-bis (2-(5-phenyloxazolyl))-benzene (POPOP) in 1420ml of toluene and 1071ml of methanol. The urine samples were made up to a suitable volume wi th distilled water and triplicate aliquots of 0.5ml were counted in l0ml of Liquiscint.

The 14C content of the faeces was determined by freeze drying and then weighing, pelleting and combusting duplicate samples of about 300mg in a sample oxidiser using C02 absorber and scintillator.

Carcass was digested in 250ml of 10% aqueous sodium hydroxide at 60°C. Fifty ml of concentrated hydrochloric acid and 250ml of ethanol were added and the total digest made up to a litre wi th water. Triplicate 1 ml aliquots were taken and counted in 15 ml of Liquiscint.

The 14C content in the cage washings was analysed by making the cage wash volume up to 50ml in distilled water and counting triplicate 0.5ml aliquots in lOml of Liquiscint.

During the course of the treatments dose equivalents were made up to 250ml with ethanol.Five aliquots of 0.5ml were counted in l0 ml of Liquiscint to determine the amount of radioactive test material dosed to each rat.

TLC was carried out on the [14CJ Mn Catalyst test solution, the 0-8 hour urine samples from one representative male and female rat and pooled 8-24 hour faeces.

The UV absorption of the samples was measured in matched quartz lcm pathlength cuvettes over a wavelength range of 350nm to 220nm.Each sample was scanned against an appropriate solvent blank . A time course experiment using absorption spectroscopy to moni tor the rate of degradation of Mn Catalyst in the presence of filtered rat urine was carried out.A 0.004% aqueous solution of Mn Catalyst (5ml) was spiked wi th 0.Sml of ei ther water or filtered rat urine. Absorption spectroscopy was carried out on the samples immediately after preparation and at various times over 48 hours . The samples were stored in the dark at room temperature for the 48 hours.

Whole body autoradiography: The rats were killed by C02 asphyxiation and immediately frozen in 60-80° petroleum ether saturated with solid C02. Each rat was fixed onto the microtome stage in the cryostat and longitudinal sections cut from tail to head until the required organs were exposed. When the required levels were reached 20µm sections were taken and mountedon X-ray film. After 2, 4 and 8 weeks exposure the X-ray films were developed and then examined for areas of radioactivity.

Details on dosing and sampling:

Thin layer chromatography (TLC} was carried out on the test solution to demonstrate stability of the [14C]Mn Catalyst over the dosing period.

After dosing, the cages were sealed and air drawn through the cage and C02 absorber at 1 L/minute. The C02 absorber was sampled for 14C analysis at 2, 4, 8, 24 and 48 hours.

Urine and faeces were collected at 8, 24, 48, 72 and 96 hours and prepared for 14C analysis.

At 4, 8, 24, 48 and 96 hours one male and one female rat was sacrificed by C02 asphyxiation and prepared for whole body autoradiography.
The two remaining rats (one male and one female) were sacrificed at 96 hours and the carcasses prepared for 14C analysis .
After eac h rat had been sacrificed the cages and excreta separators were rinsed down wi th distilled water and the rinsings prepared for 14C analysis .

Results and discussion

Main ADME results

Main ADME results 1

Type:

excretion

Results:

urinary: 70% male and 79% female @ 96hrs

Main ADME results 2

Type:

excretion

Results:

faecal route 24% males and 18% females at 96 hrs

Main ADME results 3

Type:

excretion

Results:

expiration as 14CO2 accounted for 9.3% males and 2% females

Main ADME results 4

Type:

absorption

Results:

2% remained in carcasses after 96 hrs of both male and female

Toxicokinetic / pharmacokinetic studies

Details on absorption:

After 96 hours there was still approximately 2% of the 14C dose remaining in the carcasses of the single male and female rats analysed.

The [14C]Mn Catalyst was readily absorbed from the gastro-intestinal tract and excreted rapidly mainly in the urine.

Details on distribution in tissues:

Whole body autoradiography indicated widespread tissue distribution of 14C with liver, kidney, salivary glands and thyroid as principle organs of 14C accumulation. This 14C was seen to be quickly removed from these organs .

WBA results reflected those of the turnover study, showing the rapid and almost complete elimination of the [14C]Mn Catalyst from the body within 24 hours of dosing from all tissues, wi th the exception of the thyroid gland, gastro-intestinal tract, preputial gland and a very low level remaining in the liver and salivary glands. The main areas of deposition in both males and females was in the contents of the gastro-intestinal tract, reflecting the method of dosing and in the kidney medulla and the bladder, being the main routes of excretion.

At 4 and 8 hours there are significant levels of 14C deposition in tissues other than those of the intestinal tract, the highest concentrations being seen in the liver,submaxillary gland, Harderian gland, thyroid, pituitary, incisor tooth root and prepuptial glands. Low levels were seen in many other tissues including the lateral nasal gland (Steno's gland), intra-orbital lacrymal gland, major sublingual gland, lungs, pancreas, spleen, kidney, adrenal glands, testes and bone marrow.

By 24 hours only very small amounts were seen remaining in the intestinal tract but still some in the thyroid.
By 48 hrs traces of deposi tion were still persisting in the thyroid at 48 hours and at a very low level in the liver.
At 96 hrs no deposi tion was seen remaining in the autoradiograms.

Details on excretion:

The recoveries of 14C from the male and female rats demonstrated that excretion of [14C]Mn Catalyst from the body was mainly in the urine and that excretion was virtually complete by 24 hours after dosing. There was no significant difference in disposition between the sexes. Other lesser, though significant, routes of excretion were via the faeces and as 14C02 expiration.

In both sexes excretion of the [14C]Mn Catalyst was rapid with more than 96% of the dose being excreted during the first 24 hours .

Urinary excretion of the 14C labelled material was very rapid in the female rat taken for WBA at 4 hours with over 50% of the dose having been excreted.

The main route of excretion was via the urine with just under 70% and 79% being excreted in the male and female urine respectively up to 96 hours .

Faecal excretion was the secondary route of excretion wi th 24% (males) and 18% (females) passing out of the body by this route.

Expiration as 14C02 accounted for 9.3% of the dose in the male rat and 5.4% in the female rat.

There appears to be no bilary excretion of the [14C]Mn Catalyst because the bile ducts within the liver contain no 14C.

By 96 hours virtually all traces of 14C have been eliminated from the body, with only the male preputial gland holding on to any significant levels of 14C activity.

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

Thin layer chromatography: TLC appeared to showed that Mn Catalyst spiked with rat urine underwent a chemical reaction which resulted in complete breakdown of the Catalyst.
The 14C labelled urinary metabolites were shown to be common to both sexes.

The major point of note from the chromatography was that the [14C]Mn Catalyst broke down completely in the presence of rat urine.

The major breakdown product in control urine appeared to be the same material as the major urinary metabolite and impurity in the test solution.

The 14C labelled urinary metabolite in the TLC appeared as a single spot which coincided with the Rf value of the impurity in the [14C]Mn Catalyst test solution thus demonstrating that the materials may be the same. There did not appear to be any [14C]Mn Catalyst in the urine indicating rapid breakdown in urine.

TLC separation of the 8-24 hour faecal extracts in methanol; there were five 14C labelled metabolites common to both male and female faeces. In the faecal extracts there were small levels of parent material and metabolites all of which were present in the [14C]Mn Catalyst test solution. The faecal metabolite did not appear to be present in the test solution.

UV absorption spectroscopy of Mn Catalyst in the presence of rat urine:

The absorption spectrum of Mn Catalyst in water alone does not changed significantly over a period of 48 hours. In contrast the absorption spectrum of Mn Catalyst spiked with rat urine changes significantly over the same period. Immediately after the addition of rat urine the absorbance below 260nm had reduced significantly. After 24 hours the absorption peak at about 280nm had disappeared leaving absorption peaks at about 315nm and 265nm.By 48 hours the overall level of UV absorbance below 340nm had become considerably reduced with a single peak at about 255nm.

The Mn Catalyst is the dinuclear Mn (IV) species of the compound. In the presence of reducing agents this will be broken down to the free ligand
(N,N ',N''-trimethyl-triazocyclononane [MeTACN] ) and Mn (II) salts (Dr. T. Swarthoff, private communication). The observed rapid breakdown of the Mn Catalyst in urine monitored by TLC and UV absorption spectroscopy probably reflects such a reducing reaction.

Any other information on results incl. tables

Clinical observations: None of the rats showed any signs of ill health during the course of the experiment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results remained in carcasses after 96 hrs of both male and female
The 14C from the labelled Mn Catalyst was extensively absorbed from the intestinal tract of both male and female rats and both excreted the 14C rapidly mainly via the urine . The expired air contained approximately 9% of the 14C in male rats and 5% in female rats. This slight sex difference in absorption and metabolism was reflected in the urine and faeces recoveries.The rate of excretion was rapid but traces (2%)of 14C remained in the carcass at 96 hours after dosing.

WBA indicated widespread tissue distribution of 14C with liver, kidney, salivary glands and thyroid as principle organs of 14C accumulation. This 14C was seen to be quickly removed from these organs .

TLC examination of urinary 14C indicated breakdown of the Catalyst probably to the ligand which was excreted extensively in the urine. Instability of Mn Catalyst in the presence of rat urine was demonstrated by UV absorption spectroscopy.The 14C02 produced indicated a slight degree of N-demethylation of the ligand.