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

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

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: In accordance with GLP and similar to OECD protocol 427.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

411-760-1

EC Name:

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

Cas Number:

116633-53-5

Molecular formula:

C18 H42 Mn2 N6 O3 . 2 F6 P

IUPAC Name:

dimanganese(4+) bis(1,4,7-trimethyl-1,4,7-triazonane) bis(hexafluoro-λ⁵-phosphanuide) hydrate trioxidandiide

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 Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 166 ± 6g (male) and 148 ± 7g (female)
- Fasting period before study: unknown
- Housing: rats were housed in individually-labelled metabolism cages specifically designed for the collection of urine, faeces and expired air for 14C analysis .
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Dorsal hair was removed with clippers from all rats at 24 hours prior to treatment.

IN-LIFE DATES: Between 23rd July and 25th July 1991.

Each rat was then placed in its individual metabolism cage and control (background) urine and faeces samples were collected and, immediately before treatment, control samples of C02 absorber were also collected.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: 50% aqueous ethanol

Duration of exposure:

48 hours

Doses:

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

The topically applied dose was 95µg/cm 2.

Topically treated male and female rats received 0.95mg (8.7µCi) of [14C]Mn Catalyst over 9.62 cm2 of clipped skin with occlusion.

No. of animals per group:

4 male amd 4 female rats

Control animals:

no

Details on study design:

Topical application of [ 14C]Mn Catalyst: Immediately prior to dosing, the rats were anaesthetised with cyclopropane and a rubber 0-ring of 35mm internal diameter was attached to the dorsal skin with Permabond superglue. Each rat was then treated topically with 100µ1 of test solution which was spread over the 9.62 cm2. The treated area was then dried using a hair dryer and a rubber disc of 38mm diameter mounted onto the 0-ring with the superglue to provide an occlusive cover.

All cages were fitted with absorption towers containing ethanolamine: 2-methoxyethanol (1:2 by volume) for the collection of expired 14C02. Samples of trapped expired air were assayed for radioactivi ty content at intervals of 4, 8, 24 and 48 hours. Urine was collected at 8, 24 and 48 hours and faeces at 24 and 48 hours. At 48 hours the rats were killed by gaseous anesthetic when the protective device was removed and cut into small pieces ready for solvent extraction.The rats were then reweighed.

The treated skin site with a 1 cm border was excised and rinsed with approximately 20ml of 50% aqueous ethanol . The skin and the rinsings were both analysed for 14C.The carcass remains were prepared for 14C analysis.The cages and excreta separators were rinsed down with distilled water and the rinsings analysed for 14C.

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

The urine samples were made up to a suitable volume with distilled water (e.g. l0ml or 25ml). 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.

The protective devices were extracted twice at room temperature in 50ml of 50% aqueous ethanol and triplicate 0.S ml aliquots assayed for radioactivity in l0ml of Liquiscint.

The skin rinsings were weighed and triplicate 0.5ml aliquots counted in l0 ml of Liquiscint.

Excised skins were dissolved overnight at 60°C in 25ml of Soluene-350 tissue solubiliser and triplicate 0.5ml aliquots assayed for radioactivity in l0 ml of liquiscint following the addition of three drops of glacial acetic acid to each sample.

Carcasses were solubilised in 250ml of 10% sodium hydroxide overnight at 60°C. To the digest was added 50ml of concentrated hydrochloric acid and the total volume was adjusted to l000 ml with 250ml of ethanol and water. Triplicate 1.0ml aliquots were then assayed for radioactivity in 15ml 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.

Thin layer chromatographic (TLC) analysis of the test solution, urine, test site washings, and extract of a protective device. Two TLC systems used were :

1. Polygram silica gel G plates developed in chloroform: methanol:water (65:25:4 by volume).( known as system 1)

2. KC18 reverse phase plates developed in 0.29M ammonium dihydrogen orthophosphate: acetonitrile: methanol (60:28:12 by volume) - HPLC (due to the fact that this system was recommended for high performance liquid chromatographic separation of Manganese Catalyst).(known as system 2).

Results and discussion

Signs and symptoms of toxicity:

no effects

Remarks:

(male) and from 148 ± 7g to 136 ± llg (female)

Dermal irritation:

no effects

Absorption in different matrices:

When [14C]Mn Catalyst was applied to male and female rat skin in an aqueous ethanolic solution and left in contact under occlusive conditions for 48 hours, a proportion of the 14C dose penetrated the skin as shown by the presence of 14C in excreta and tissues of the rat.The main route of excretion was in the urine although approximately 1% of the applied dose was in the carcasses at 48 hours.

The topically applied dose was 95µg/cm 2 and during 48 hours penetration through male rat skin was 2.3% to 17.5% and 0.6% to 4.9% through female rat skin.There was a significant difference between the sexes in the fate of [14C]Mn Catalyst. Significant amounts (between 5 and 35% of the 14C dose) were transferred from skin to the protective device during 48 hours which may have reduced the amount of 14C available for percutaneous absorption.

The predominant route of excretion was in the urine. TLC of the rinsings showed that {14C]Mn Catalyst was unstable on the skin


- Skin wash: Some 14% (male) and 27% (female) of the dose was rinsed from the skin at 48 hours
- Skin test site: Some 51% (male) and 41% (female) was retained on the skin.
- Carcass: 48 hours - 1.0% (male) and 0.7% (female)
- Urine: 6.5% (male) and 1.3% (female) from mean absorption values of some 8.7% and 2.7% respectively.
- Cage wash + cage wipe: inclusion of 14C recoveries from cage rinsings, possibly originating from urinary contamination, increased these urine values to some 6.6% and 1.7% respectively.
-:Protective device: Carbon-14 extracted from the protective device amounted to some 12% (male) and 22% (female) of the dose.

The wide variation between the groups of rats in the levels of percutaneously absorbed 14C may possibly be due to several parameters. The test solution used in these experiments was without added perborate which is included in the commercial product to stabilise the Mn Catalyst. Also the the variable conditions of pH which occur on skin and the presence of the adhesive on the protective device was thought to support the degradation of Mn Catalyst to the free ligand.

Total recovery:

Thin layer chromatography

In system 1 the bulk of the urinary radioactivity was confined to a single region with three other minor radioactive regions separated from the urine. Also in system 1, the major impurity in the [14C]Mn Catalyst marker in control urine now had an Rf value of 0.22.

In system 2 the urine sample contained one major region of radioactivity. This region coincided with the major impurity in the [14C]Mn Catalyst in control urinE . In control urine the [14C]Mn Catalyst had an Rf value of 0.59.

Autoradiograms of TLC separations of an aliquot of skin washings which were developed in systems 1 and 2 and two major regions of radio activity were separated by both systems which are probably the Mn Catalyst and the free ligand.This indicates that reduction of the [14C]Mn Catalyst has probably occurred on the skin.

Percutaneous absorption

Percutaneous absorption 1

Dose:

95µg/cm2

Parameter:

percentage

Absorption:

ca. 0.6 - ca. 4.9 %

Remarks on result:

other: 48 hrs

Remarks:

2.64 ± 1.98 µg/cm2 through female rat skin

Percutaneous absorption 2

Dose:

95µg/cm2

Parameter:

percentage

Absorption:

ca. 2.3 - ca. 17.5 %

Remarks on result:

other: 48 hrs

Remarks:

8.66 ± 6.23 µg/cm through male rat skin

Applicant's summary and conclusion

Conclusions:

After topical application of [14C]Mn Catalyst to rat skin under conditions of occulusion a considerable proportion of the 14C dose was absorbed through skin.