Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-11-07 to 1990-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 140 to 150 g, except of three males weighed 157, 190 and 209g
- Fasting period before study: no data
- Housing: 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): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
other: gavage and topical
Vehicle:
water
Details on exposure:
PART 1: FATE AND DISPOSITION OF [14C]TB AFTER ADMINISTRATION BY GAVAGE AND ANALYSIS BY WOLE BODY AUTORADIOGRAPHY

- The sample of C12 AAPB (N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner)
carboxylate) isotopically labelled with 14C in the carboxymethyl ammonium moiety ([14C]TB) was prepared in house. The sample had a specific activity of 3.0 µCi/mg and was assigned the ESL sample number S18320T01.

- Each rat was dosed by gavage with 1.0 ml of aqueous [14C]TB solution containing 29.115 x 10exp6 dpm of 14C (4,372 µg, 13.11 µCi).
PART 2: SKIN ABSORPTION OF [14C]TB

- [14C]TB, ESL sample number 518320T01 was used in this experiment as in study part 1.
- Each rat was treated topically with 100µl of 3% aqueous [14C]TB containing 19.942 x 10exp6 dpm of 14C (2,990µg, 8.98µCi).

PART 3: FATE AND DISPOSITION OF [14C]TB AFTER ADMINISTRATION BY GAVAGE

- [14C]TB, ESL sample number 518320T01 was used in this experiment as in study part 1.
- Each rat was dosed by gavage with 1.0 ml of aqueous [14C]TB solution containing 29.540 x 10exp6 dpm of 14C (4,435 µg, 13.31 µCi).

PART 4: FATE AND DISPOSITON OF [1-14C]TB AFTER ADMINISTRATION BY GAVAGE OR TOPICAL APPLICATION
- The sample N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner) carboxylate, isotopically labelled with 14C in the 1 carbon of the lauroyl moiety ([1-14C]TB) was synthesised in house. The sample had a specific radioactivity of 7.83µCi/mg, was stored as an aqueous solution at 730µCi/ml and was assigned the ESL sample number S18320T02.

- Two aqueous solutions of [1-14C]TB were prepared at concentrations of approximately 10µCi/ml for gavage and 10µCi/0.1ml for topical application.
Gavaged rats received a test solution containing 27.177 x 10exp6 dpm of 14C (1,563µg, 12.24 µCi) and topically treated rats received 25.390 x 10exp6 dpm of 14C (1,461µg, 11.44µCi).
Duration and frequency of treatment / exposure:
- single gavage and up to 48 h observation after dosing
- single topical application under occlusion for 48 hours
- single topical application and rinsing of the skin after 10 minutes contact, covering of the application sites and subsequent observation for 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
[14C]TB gavage approximately 30mg/kg (87.3µCi/kg)
[14C]TB topical approximately 20mg/kg (60µCi/kg; approximately 0.3 mg/cm²)
[1-14C]TB gavage approximately 10mg/kg (81.5µCi/kg)
[1-14C]TB topical approximately 10mg/kg (81.5 µCi/kg; approximately 0.15 mg/cm²)
No. of animals per sex per dose:
[14C]TB gavage: 5 females and 5 males (first experiment in which only 2 rats were permitted to remain in test for 48 hours) and additional 3 females and 3 males in a second experiment in which all rats were permitted to remain in test for 48 hours.
[14C]TB topical: six females and 6 males
[1-14C]TB gavage: three females and three males
[1-14C]TB topical: three females and three males
Control animals:
no
Details on dosing and sampling:
PART 1: FATE AND DISPOSITION OF [14C]AAPB AFTER ADMINISTRATION BY GAVAGE AND ANALYSIS BY WOLE BODY AUTORADIOGRAPHY

- Treatment of animals: Colworth Wistar rats weighing 140g to 150g were used in this experiment. The rats were housed in metabolism cages specifically designed for the collection of urine, faeces and expired air for 14C analysis. The experiment was conducted between November 7th - 9th 1989.
Each of 5 female and 5 male rats were dosed by gavage with 1.0 ml of test solution. After treatment the rats were placed in individual metabolism cages.

- Sampling: At 2, 4, 8, 24 and 48 hours after dosing, two rats (one of each sex) were killed by CO2 asphyxiation and quickly frozen in a petroleum spirit/solid CO2 mixture in preparation for analysis by whole body autoradiography. All the cages were fitted with absorption towers containing ethanolamine: 2-methoxyethanol (1:2 v/v) for the collection of expired 14C02 and where produced, urine and faeces were also collected for the assay of 14C levels. At the end of the experiment the cages were washed down with distilled water to collect any 14C remaining.

- Analysis of C14: Throughout this experiment, 14C was determined by liquid scintillation counting. All aqueous samples were counted in a Beckman LS 3801 spectrometer (Beckman-RIlC Ltd., High Wycombe, England.) using Liquiscint (National Diagnostics, Aylesbury, England). The counting efficiency of each sample was estimated by the Beckman H number system.
The urine samples, made up to 25 ml with distilled water, and the test solution were counted directly in liquid scintillator; 0.5 ml of solution was normally prepared in 10 ml of scintillator. The cage washings were made up to 25 ml with distilled water and then counted as the urine.
The 14C content of the faeces was assayed by first freeze drying and then combusting weighed amounts in a Packard 306 sample oxidiser using Carbamate-1 CO2 absorbent and Oxosol 306 scintillator (both National Diagnostics Ltd).
Aliquots of CO2 absorber (1ml) (comprising 2-methoxyethanol:ethanolamine, 2:1 by volume) were counted in 10ml 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.

- Whole body autoradiography (WBA): The rats were killed and immediately frozen. During the whole of the procedure the rats were kept in a frozen state. Rats were trimmed with a previously cooled saw and embedded in a block of 1% cooled 'Solvite' wallpaper paste. The embedding mould was surrounded with 40 - 60° petroleum ether and solid carbon dioxide. Each rat in its block was then fixed on to the microtome stage in the cryostat (Bright Cryostat, Model OT/SEL) by adding some cooled solvite to the edges of the block. The block was levelled prior to sectioning.
When the required levels were reached, 20 µm sections were taken on to tape strips (50 mm wide, 3M 810 tape). Sections were taken to ensure that all
organs and glands were shown in at least one of the sections.
Sections were dried overnight in the cryostat, the tape strips were dusted with fine talc and stuck on to X-ray film (Hyperfilm beta max) in a dark room. The films were then put under vacuum in black envelopes and left at 4°C for the required exposure times, which were determined from control sections treated in the same way.
The X-ray films were developed with Kodak LX 24 Developer and Ilford Hypam fixer. After drying the films were examined for areas of radioactivity.
A further section at each level was stained with Haematoxylin and Eosin to identify the tissues if necessary.

- Thin layer chromatography (TLC) of faecal extracts: Two samples of powdered faeces from one female rat and one male rat, collected during the first 24 hours, was extracted with 3 x 100ml of Karl Fischer grade methanol. At each extraction stage the methanol was collected, the volume measured and 0.5ml aliquots removed for radioactivity assay. The three extracts from each sample were pooled and the volumes reduced by rotary evaporation to approximately 10mI. The supernatant was removed from the sediment and the sediment dissolved in 4ml distilled water. Aliquots of extract and dissolved sediment were assayed for radioactivity.
Sufficient methanol (10µl) was spotted onto Machery-Nagel silica gel G thin layer chromatography (TLC) plates (supplied by Camlab, Cambridge) to give at least 10,000 dpm per track. [14C]TB marker was run alongside the extracts. The plates were then developed in 2 solvent systems:
a) Chloroform:methanol (1:1)
b) Methanol:water (70:30)
They were allowed to develop at least 15cm up the plate.
The developed plates were removed from the tank and the solvent front marked. After drying in air the plates were scanned on a Ray test Rita-3200
Radio-TLC-analyser (Ray test, Lablogic, Sheffield) and plots obtained. The data obtained by radio TLC was supplemented with autoradiograms of the developed plate using DEF 2 X-ray film (Eastman Kodak Company).

PART 2: SKIN ABSORPTION OF [14C]TB

- Treatment of animals: Colworth Wistar rats weighing approximately 150g were used in this experiment. The rats were housed in metabolism cages specifically designed for the collection of urine, faeces and expired air for 14C analysis. The experiment was conducted between 21st - 23rd November 1989.
Six female and six male rats were clipped on the dorsal surface 24 hours prior to topical application. Each rat was then placed in its individually labelled metabolism cage 24 hours before treatment. During this time control samples of urine and faeces were collected and immediately before treatment control samples of CO2 absorber were also collected.
Immediately prior to treatment the rats were anaesthetised with cyclopropane and the 10cm² treatment site marked with a felt tipped pen on the dorsal surface.
100µl of test solution was then applied to the marked area on two groups of rats, 3 female and 3 male, and the site protected with an occlusive patch. The rats were then returned to their own individual metabolism cages.
Two other groups of rats, 3 female and 3 male, were similarly treated and the test solution allowed to remain in contact with the skin for 10 minutes.
Unbound [14C]TB was then rinsed off with distilled water into a 250ml volumetric flask fitted with a large funnel. The treatment site was protected
with a non-occlusive patch and the rats returned to their own individually labelled metabolism cage.
All the cages were fitted with absorption towers containing ethanolamine:2-methoxyethanol (1:2 v/v) for the collection of expired 14C02. Urine and
faeces were also collected for the assay of 14C levels at 24 and 48 hours. At 48 hours the rats were anaesthetised with cyclopropane and heart blood
withdrawn for assay of radioactivity. The rats were then killed by cervical dislocation, the patches removed and the treated area of skin plus a border of up to 2 cm excised from the carcass. The cages were then washed down with distilled water and all samples plus the carcass were assayed for
radioactivity.

- Analysis of 14C: The 14C content of samples was assayed by liquid scintillation counting in a Beckman LS3801 counter.
Liquiscint (National Diagnostics Ltd.) was the scintillator cocktail used throughout this study for aqueous and ethanolic solutions.
The urine samples, made up to 25 ml with distilled water, and the test solution were counted directly in liquid scintillator; 0.5 ml of solution was normally prepared in 10 ml of scintillator. The cage washings were made up to 50 ml with distilled water and then counted as for the urine.
Aliquots of CO2 absorber (1ml) (comprising 2-methoxyethanol:ethanolamine, 2:1 by volume) were counted in 10ml 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.
14C levels in faeces were determined after combusting in a Packard 306 sample oxidiser. Each sample for combustion was freeze dried and weighed and triplicate sample weights of up to 300mg pelleted and combusted.
Blood was assayed by counting duplicate 0.5ml samples which had been sequestered with LiEDTA.
Excised skins were solubilised in 25ml Soluene-350 (Canberra-Packard) and replicate 0.5ml samples were assayed for radioactivity in Liquiscint after the addition of 3 drops of glacial acetic acid to each sample.
Patches were extracted twice, once with 50ml of water then again with 50ml of 1% CTAB solution and replicate 0.5ml samples from each extraction assayed for radioactivj.ty.
Carcasses were digested overnight in 250ml of 10% sodium hydroxide solution. 50ml of concentrated hydrochloric acid was added to the digest and made up to 1 litre with 250ml ethanol and then water. 1ml replicate samples were assayed for radioactivity.
During the course of treatment a 100µl aliquot of the dosing solution was made up to 250ml with water. Replicate 0.5ml samples were then assayed for radioactivity to determine the amount of radioactive test material applied to each rat.

PART 3: FATE AND DISPOSITION OF [14C]TB AFTER ADMINISTRATION BY GAVAGE

This further experiment was conducted in which rats dosed by gavage were under test for 48 hours. These results will be compared with those obtained previously in experiment part 1 when only 2 rats were permitted to remain in test for 48 hours. The labelled material was administered to rats by gavage and its fate investigated by whole body autoradiography (WBA) as reported in study Part 1.

- Treatment of animals: Olac Wistar rats weighing 140g - 150g were used in this experiment. The rats were houses in metabolism cages specifically designed for the collection of urine, faeces and expired air for 14C analysis. Each rat was placed in its individually labelled metabolism cage 24 hours before treatment. During this time control samples of urine and faeces were collected, and immediately before treatment control samples of CO2 absorber were also collected. The experiment was conducted between January 16th - 18th 1990.
Each of 3 female and 3 male rats were dosed by gavage with 1.0ml of test solution. After treatment the rats were placed in individual metabolism cages
fitted with absorption towers containing ethanolamine:2-methoxyethanol (1:2 v/v) for the collection of expired 14C02. Urine and faeces were also collected for the assay of 14C levels. At the end of the experiment the cages were washed down with distilled water to collect any 14C remaining. Carcasses were also assayed for radioactivity content.

- Analysis of samples for 14C: The 14C content of samples was assayed by liquid scintillation counting in a Beckman LS3800 counter.
Liquiscint (National Diagnostic Ltd.) was the scintillation cocktail used throughout this study for aqueous and ethanolic solutions. Urine samples were measured and the volume noted and 0.5ml aliquots were counted directly. Cage washings were similarly assayed.
Aliquots of CO2 absorber (1ml) (comprising a mixture of 2-methoxyethanol and ethanolamine, 2:1 by volume) were counted in 10ml of cocktail prepared by dissolving 12.5g of 2,5-diphenyloxazole (PPO) and O.75g of p-bis-(2-(5-phenyloxazolyl))-benzene (POPOP) in 1420ml of toluene and 1071ml of methanol.
14C levels in faeces were determined after combusting in a Packard 306 sample oxidiser. Each sample for combustion was freezed dried and weighed, and triplicate sample weights of up to 300mg pelleted and combusted.
Carcasses were digested overnight in 250ml of 10% sodium hydroxide solution. 50ml of concentrated hydrochloric acid was added to the digest and made up to 1 litre with 250ml ethanol and then water. One replicate samples were assayed for radioactivity.
During the course of treatment, dose equivalents were made up to 250ml with distilled water. Replicate 0.5ml samples were then assayed for radioactivity to determine the amount of radioactive test material applied to each rat.

- Thin layer chromatography of urine: Urine from female rats collected at 6-24 hours were pooled and aliquots removed for thin layer chromatographic separation (TLC). Urine (6-24h) from male rats was similarly processed. Sufficient urine sample (100µl) was spotted onto Machery-Nagel silica gel G plates (supplied by Camlab, Cambridge) to give approximately 10,000 dpm per track. [14C]TB was run alongside the urine samples.
The plates were developed in 2 solvent systems:
(a) Chloroform:methanol (1:1 by volume)
(b) Methanol:water (7:3 by volume)
The solvent front was allowed to develop to the top of the plate (20cm). The developed plates were removed from the tank and the solvent front marked. After drying in air the plates were scanned on a Ray test Rita-3200 Radio-TLC-analyser (Ray test, Lablogic, Sheffield) and plots obtained. The data
obtained by radio TLC was supplemented with autoradiograms of the developed plates using DEF2 X-ray film (Eastman Kodak Company).

PART 4: FATE AND DISPOSITON OF [1-14C]TB AFTER ADMINISTRATION BY GAVAGE OR TOPICAL APPLICATION

- Treatment of animals: Twelve Colworth Wistar rats mainly weighing approximately 140g were used in this experiment. The exception was the 3 male rats treated by gavage. These weighed 157, 190 and 209g. The rats were housed in metabolism cages specifically designed for the collection of urine, faeces and expired air for 14C analysis. The experiment was conducted between 12th-14th December 1989. Three male and three female rats had the hair from their backs removed by clipping 24 hours prior to topical application. Three male and female rats for treatment by gavage were not clipped. Each rat was then placed in its individually labelled metabolism cage 24 hours before treatment. During this time control samples of urine and faeces were collected and immediately before treatment control samples of CO2 absorber were also collected.
-- Intubation of [l-14C]TB: Each of 3 male and 3 female rats were dosed by gavage with 1.0ml of test solution and returned to their labelled metabolism cage. All cages were fitted with absorption towers containing ethanolamine:2-methoxyethanol (1:2 v/v) for the collection of expired 14CO2. Samples of trapped expired air were assayed for radioactivity content at hourly intervals for 6 hours, then at 24 and 48 hours. Urine and faeces were collected at 6, 24 and 48 hours.
At 48 hours the rats were killed by cervical dislocation and the carcasses assayed for radioactivity. The cages were then washed down with distilled
water to collect any 14C remaining.
-- Topical application of [1-14C)TB: Immediately prior to treatment the 3 male and 3 female rats, previously clipped, were anaesthetised with cyclopropane and the 10cm² treatment site marked with felt-tipped pen on the clipped dorsal surface. 0.1ml of test solution was applied to the marked area and the site protected with an occlusive patch. The rats were returned to their labelled metabolism cage and samples collected as after intubation. After killing, the patches were removed for extraction and assay of radioactivity and the treated areas of skin plus a border of up to 2cm excised for assay of 14C content.

- Analysis of samples for 14C: The 14C content of samples were assayed by liquid scintillation counting in a Beckman LS3800 counter.
Liquiscint (National Diagnostics Ltd.) was the scintillation cocktail used throughout this study for aqueous and ethanolic solutions.
Urine samples were measured and the volume noted and 0.5ml aliquots were counted directly. Cage washings were similarly assayed.
Aliquots of CO2 absorber (1ml) (comprising 2-methoxyethanol and ethanolamine, 2:1 by volume) were counted in 10ml 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.
14C levels in faeces were determined after combusting in a Packard 306 sample oxidiser. Each sample for combustion was freezed dried and weighed and· triplicate sample weights of up to 300mg pelleted and combusted.
Excised skins were solubilised in 25ml Soluene 350 (Canberra-Packard) and replicate 0.5ml samples were assayed for radioactivity in Liquiscint after the addition of 3 drops of glacial acetic acid to each sample.
Patches were extracted twice with 50ml of 50% aqueous ethanol and replicate 0.5ml samples from each extraction assayed for radioactivity.
Carcasses were digested overnight in 250ml of 10% sodium hydroxide solution. Fifty ml of concentrated hydrochloric acid was added to the digest and made up to 1 litre with 250ml ethanol and then water. One ml replicate samples were assayed for radioactivity.
During the course of treatment, dose equivalents were made up to 250ml with distilled water. Replicate 0.5ml samples were then assayed for radioactivity to determine the amount of radioactive test material applied to each rat.

- Thin layer chromatography of urine and faeces: Two grams of powdered freeze dried urine collected during the 6-24 hours period
from one orally dosed male rat and one female rat, was extracted with 3 x 100ml of Karl Fischer grade methanol. At each stage the extract was withdrawn, the volume measured and aliquots removed for radioactivity assay. The three extracts from each faecal sample were pooled and the volume reduced by rotary evaporation. The supernatant was removed from the sediment and the sediment dissolved in 4ml distilled water. Aliquots of extract and residue was assayed for radioactivity.
Sufficient methanol extract (10µl) was spotted onto Machery-Nagel silica gel G thin layer chromatography (TLC) plates (supplied by Camlab, Cambridge) to give at least 10,000 dpm per track. [1-14C]TB marker has run alongside the extracts.
The plates were then developed in 2 solvent systems:
a) chloroform:methanol (1:1)
b) methanol:water (70:30)
The solvent front was allowed to develop at least 15cm up each plate.
The developed plates were removed from the tank and the solvent front marked. After drying in air the plates were scanned on a Ray test Rita-3200 Radio-TLCanalyser (Ray test, Lablogic, Sheffield) and plots obtained. The data obtained by RTLC was supplemented with autoradiograms of the developed plate using DEF2 X-ray film (Eastman Kodak Company).
Faecal samples collected from male rats (29-31) and female rats (32-34) between 6 and 24 hours after dosing and remaining after assays for 14C content were pooled and 2.0g from each group extracted and examined for 14C metabolites as described above.
Samples of pooled urine (50µl) from male rats 23-25, collected at 0-6 hours, was separated alongside a marker of [1-14C]TB in system (b) and proceeded as described above. Similarly 50µl samples of pooled urine from male rats 23-25 and 3 female rats 26-28, collected at 6-24 hours were examined as above using systems (a) and (b).
In order to examine the 14C metabolites the 6-24 hour samples of urine from male rats 29-31 and female rats 32-34 were pooled and the two samples shell frozen and freeze dried. The residue was then extracted with 2ml methanol and 50µl aliquots developed in systems (a) and (b) alongside a [1-14C]TB marker and one of the urine extracts to which [1-14C]TB marker had been added. The developed plates were examined as described above.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
[14C]TB gavage: Whole body autography indicated poor absorption from the intestine.
[14C]TB topical: After topical application of the 3% [14C]TB solution and leaving under occlusion for 48 hours, penetration through the skin was poor as shown by the low levels of 14C in the urine, expired air and faeces. Rinsing the skin after 10 minutes contact time removed 96% of the applied dose. Approximately 2.5% of the dose remained on the skin and excreta contained approximately 0.1% of the applied dose.
[1-14C]TB gavage: Whole body autography indicated poor absorption from the intestine as after application of [14-C]TB
[1-14C]TB topical: After application of the [1-14C]TB solution under occlusion and without rinsing, skin absorption was similarly low as after topical application of [14C]TB
Details on distribution in tissues:
[14C]TB gavage: The liver, kidney and urinary bladder were the only tissues showing significant levels of 14C in the body by whole body autoradiography. Traces of 14C remained in the liver at 48 hours after dosing.
[14C]TB topical: No relevant tissue distribution detected by whole body autoradiography.
[1-14C]TB gavage: After gavage administration of the [1-14C]TB similar results as after gavage administration of [14-C]TB were obtained.
[1-14C]TB topical: No relevant tissue distribution detected by whole body autoradiography.
Details on excretion:
[14C]TB gavage: After gavage administration of [14C]TB most of the 14C dose was rapidly recovered in the faeces, only 5% was excreted in the urine and 0.8% in the expired air.
[14C]TB topical: After topical application of the 3% [14C]TB solution and leaving under occlusion for 48 hours, low levels of 14C in the urine (2.0%), expired air (0.28%) and faeces (0.5%) were found. When the skin after 10 minutes contact time was rinsed the excreta contained only 0.1% of the applied dose.
[1-14C]TB gavage: After gavage administration of the [1-14C]TB similar results as after gavage administration of [14-C]TB were obtained.
[1-14C]TB topical: After topical application of the [1-14C]TB solution and leaving under occlusion for 48 hours without rinsing low levels as after application of [14C]TB were found, in the expired air (0.45%), urine (1.2%) and faeces (0.46%)

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
At least 90 % of applied test item was excreted in the faeces as unchanged parent material (as was confirmed by thin layer chromatography (TLC) analysis in the case of labelling at the carboxymethylammonium moiety).
Up to approximately 2% was analysed in expired air as CO2.
Structure elucidation of urinary and faecal metabolites was not subject of the study.

Any other information on results incl. tables

PART 1: FATE AND DISPOSITION OF [14C]TB AFTER ADMINISTRATION BY GAVAGE AND ANALYSIS BY WOLE BODY AUTORADIOGRAPHY - DETAILED RESULTS

- Fate of [14C]TB administered by gavage to rats as an aqueous solution: The 14C from [14C]TB was rapidly excreted by the rat mainly in the faeces. Two rats sacrificed at 24 hours had excreted some 76% (female) and 96% (male) in the faeces. Over the 48 hours after dosing a steadily increasing small amount of 14C was detected in urine. The 2 hour urine sample contained some 0.6% of the dose and this increased to approximately 5.5% at 48 hours. Only small amounts of 14C were recovered in the expired air. There was an increase in 14CO2 output from 2 hours when some 0.6% had been expired to 48 hours when this had increased to approximately 0.8%.

- Whole body autoradiography: At 2 hours after dosing, there was radioactivity in the liver, kidney cortex and bladder of both male and female rats. There were also large amounts of 14C present in the stomach and intestine contents. The blood and other organs in the body were not visualised.

At 4 hours after dosing, the kidney cortex was clearly visible and the levels in the liver were increased. Some 14C was still present in the stomach but most of the 14C was still in the intestinal contents.

At 8 hours, both sexes had 14C in the kidney cortex but the male levels were apparently lower. Both sexes had 14C in the liver but no 14C was left in the stomach contents at this time reflecting the ingestion of non-radioactive diet. In the female the radioactivity was mainly in the intestine and faecal pellets in the rectum. The male, however, shoved most of the radioactivity in the faecal pellets with only a small amount detectable in the intestine.

The 24 hour by the female rat showed a large haemorrhagic mass in the intestine but no radioactivity was detectable in this mass. Most radioactivity in both male and female rat was in the faecal pellets in the rectum with smaller amounts in the liver and intestine.

At 48 hours, only a slight trace of radioactivity is detected in the liver, intestine and faecal pellets.

Throughout the experiment the levels of 14C in the autoradiograms fell as 14C was expelled from the body in the faeces and urine. The only tissue where significant levels of 14C were seen to persist for slightly longer times was the liver.

- (TLC) analysis: Triplicate methanol extractions of the 0-24 hour faeces samples from rats 4 and 9 removed 89.5 and 94.3% of the radioactivity respectively. Aliquots of these extracts were separated by TLC. Autoradiograms of these TLC separations of the extracts and the [14C]TB marker indicated that the [14C]TB was excreted unchanged by the rat. The small amount of impurity present in the [14C]TB test material was also present in the faecal extracts. No attempt was made to identify this compound.

- CONCLUSIONS: This result of this experiment indicates that after administration of the [14C]TB by gavage to rats at a dose of approximately 30mg/kgbw, the TB is only poorly absorbed from the intestinal tract. The amount absorbed amounted to <10% of the dose as shown by the recoveries of 14C in the excreta. Most of the 14C dose was recovered in the faeces as unchanged TB within 48 hours of dosing. The urinary 14C accounted for only 5.5% of the dose and no attempt was made to identify the nature of the labelled material. The distribution of the 14C in the body, as shown by WBA was limited to the liver, kidney cortex and urinary bladder and 14C levels in other tissues were not detectable by this technique. The fact that 14CO2 levels were low would suggest that the [14C]carboxymethy ammonium moiety is not extensively metabolised since cleavage of the C-N bond would liberate a two carbon metabolite (probably ethanol) which would be metabolised extensively to CO2. The simple TLC examination of the faecal 14C indicated that the [14C]TB was eliminated in the faeces unchanged.

PART 2: SKIN ABSORPTION OF [14C]TB - DETAILED RESULTS

- The penetration of [14C]TB through rat skin under occlusion: Under the occlusive conditions of application approximately 6% (female rats) and 2% (male rats) of the radioactivity applied to the skin was recovered in the excreta and the carcass at 48 hours after application. The urine was the main route of excretion in both sexes but the female rat urine contained approximately 3% of the dose and the male rat urine 1%. The expired air and faeces contained much lower levels of 14C.

At the end of the experiment much of the applied dose 51% in female rats and 74% in mal, rats remained on the skin. The occlusive patch extracts contained 19% (female rats) and 7% (male rats) of the applied dose but the extraction technique employed did not result in a quantitative extraction of the 14C. This was reflected in the relatively poor overall recoveries of 14C from the occlusive patched animals.

- Penetration of [14C]TB through rat skin after rinsing and under non occlusive conditions: Under simulated user conditions of "apply and rinse off" without occlusion of the treated skin, approximately 96% of the 14C dose was recovered in the skin rinsings and approximately 4% remained bound to the treated skin and associated patch. At 48 hours the urine contained 0.01% (female) and 0.06% (male) of the applied 14C. Carcasses contained 0.04% (female) and 0.07% (male) of the applied radioactivity.

No detectable radioactivity was found in any samples of expired air or in faeces from female rats. Male rat faeces and expired air contained 0.02% of the dose.

- CONCLUSIONS: In the previous experiment (Part 1) [14C]TB administered by gavage was excreted rapidly and mainly in the faeces by female and male rats.

Little (approximately 1%) radioactivity was expired as 14CO2 during 48 hours, and the urine contained approximately 5% of the dose. This indicated that absorption from the intestinal tract was poor.

Under conditions of occlusion penetration of [14C]TB through clipped rat skin, as shown by the levels of 14C excreted during the 48 hours of skin contact, was small. Female rats absorbed approximately 6% of the applied 14C and male rats approximately 2%. The rats excreted most of the 14C in the urine, with the faeces and expired air as minor routes of elimination.

This pattern of excretion was also observed following penetration of 14C from the topically applied [14C]TB which remained on the skin after rinsing. Skin rinsing removed approximately 96% of the applied 14C. Approximately 4% of the dose remained on the skin and penetration was equivalent to approximately 0.1% of the dose applied which was 300µg/cm.

PART 3: FATE AND DISPOSITION OF [14C]TB AFTER ADMINISTRATION BY GAVAGE - DETAILED RESULTS

- Fate of [14C]TB administered by gavage to rats as an aqueous solution: Following gavage of [14C]TB the radioactivity was rapidly excreted by the rat and mainly in the faeces. At 24 hours the rats had excreted some 76% (female) and 84% (male) of the radioactivity in the faeces. These recoveries in the faeces had increased to approximately 86% and 90% respectively by 48 hours.

A small amount of 14C was detected in the urine, being about 3.9% (female) and 3.3% (male) at 48 hours. There was little 14CO2 in expired air. At 2 hours some 0.5% of the 14C had been expired and at 48 hours had increased to 1.4% (female) and 1.0% (male). Approximately 1% of the administered radioactivity was measured in the carcass. The relatively rapid rate of excretion was reflected in the much greater amounts of 14C being recovered during the first day than during the second day.

- TLC analysis of urine: A comparatively large volume of urine sample (100µl) had to be applied to each track in order to obtained detectable amounts of radioactivity. The quality of the autoradiogram from a plate developed in system (b) is not good. When developed in system (a) the radioactivity present in the urine barely migrated from the origin whilst the [14C]TB marker had an Rf = 0.16. The 14C component in urine and the [14C]TB marker travelled much further in system (b), [14C]TB marker with Rf = 0.65 migrated farther then the radioactive urine component.

- CONCLUSIONS: The 14C from [14C]TB is rapidly excreted mainly in the faeces. Approximately 3.5% of the 14C dose was excreted in the urine and approximately 1% was expired as 14CO2.

TLC examination of the urine was attempted, and although the quality of separation was not ideal due to the high loading on the plates, no parent [14C]TB was seen in the urine. The urinary metabolites were more polar than the parent [14C]TB.

This experiment confirmed the findings of the experiment reported in part 1 of this study.

PART 4: FATE AND DISPOSITON OF [1-14C]TB AFTER ADMINISTRATION BY GAVAGE OR TOPICAL APPLICATION - DETAILED RESULTS

- Fate of [1-14C] TB administered by gavage to rats as an aqueous solution: The 14C from [1-14C]TB was rapidly excreted by the rat mainly in the faeces. During the first 24 hour period the faeces contained some 80% of the dose. Only small amounts of 14C was detected in the expired air and at 48 hours less than 2% of the dose had been expired by any of the 6 rats. Less than 5% was excreted in the urine during the first 24 hours. Some 1.0-1.8% remained in the carcass at 48 hours.

- Fate of topically applied aqueous [1-14C] TB in the rat: During the 48 hour test period some 90-98% of the applied dose was associated with the treated skin and the protective occlusive patch.

The results from rats 33 and 34 which were seen to have gnawed their patch during the second day and hence probably ingested some [1-14C]TB, were not included in the following results. Approximately 0.5% of the dose was recovered in the faeces. Most of this 14C was measured in the faeces collected during the first 24 hours. Some 0.3 -0.6% of the dose was expired as 14CO2 , and most of this was recovered during the first 24 hours. Excretion of 14C in the urine followed the same pattern and during the 48 hours of the experiment approximately 1.2% of the dose was excreted. The carcass 14C levels were somewhat variable but approximately 1.2% of the applied dose remained at 48 hours. Thus overall skin penetration was low with a total mean penetration of approximately 3.5% of the dose over the 48 hours of occlusive application.

The 14C excreted by the two female rats which gnawed at their occlusive patch on day 2 had increased amounts of 14C in expired air and urine on day 2 compared with the other rats. Also comparatively large increases of 14C (5 to 10 times) was measured in the faeces compared with day 1.

- TLC analysis: The urine samples collected from rats gavaged with [1-14C]TB were separated into at least 3 radioactive components (system b) none of which had an Rf value corresponding to [1-14C]TB.

The methanol extracts of the faecal samples from gavaged rats, calculated to have extracted in excess of 85% of the radioactivity, were found by TLC separations to contain one main region of radioactivity. The region had an Rf value compatible with that of [1-14C]TB.

Low levels of 14C were detected in samples of urine and methanol extracts of faeces from the topically treated rats. Hence comparatively large masses of urine and the faecal extracts had to be applied to the TLC plates. The quality of the resultant TLC separations were not good. However, the indications were that the urine samples contained 14C metabolites only. In system (a) one major and 3 or 4 minor radioactive components were present, none of which corresponded with the [1-14C]TB marker.

The results obtained for the separations of faecal extracts from topically treated rats suggest that in addition to a 14C component corresponding to the [1-14C]TB marker, at least one 14C metabolite may be excreted in the faeces.

- CONCLUSIONS: The distribution of 14C in excreta and expired air from rats dosed by gavage with [14C]TB was similar to that found from rats gavaged with [1 -14C]TB. Most of the 14C was rapidly excreted in the faeces as unchanged test material. Some 1% was expired as 14C02 and approximately 5% excreted in the urine.

The distribution patterns of 14C in excreta and expired air from rats topically treated with [1-14C]TB and with [14C]TB was very similar. The predominant route of excretion following topical application however was different from an orally administered dose, with greater amounts of 14C being measured in urine (1 to 3%) than in expired 14CO2 (0.3 to 0.5%) and faeces (0.2 to 0.8%). An approximately tenfold decrease in these values was also observed when the [14C]TB dose was rinsed off following a 10 minute contact.

The TLC separation of methanol extracts from faeces produced by rats treated orally with either [1-14C] TB or [14C]TB were found to contain only the parent material. In some instances the faecal extracts contained traces of the impurities present in the starting material. When run in system (b) the impurity in [1-14C]TB was slower than TB whilst that in [14C]TB was faster.

The low levels of 14C in the urine was not conclusive to good quality separations. However, unchanged TB was observed in TLC separations of urine collected from rats following oral or topical treatment with either [14C]TB or [1-14C]TB. TLC separations of urine from rats treated orally with [1-14C]TB contained one major and several other minor 14C metabolites on system (b). Under similar conditions, urine from orally administered [14C]TB produced just one detectable 14C metabolite. No tests were carried out to establish the nature and possible similarity of these two major metabolites. TLC separations were not carried out on urine from rats topically treated with [14C]TB and comparisons were not possible.

TB is poorly absorbed through the intestinal tract or skin and although metabolism of the absorbed material is extensive the metabolism does not completely degrade the molecule. The results indicate that the dodecyl moiety is not extensively removed from the TB during metabolism. Complete liberation of the 1-14C dodecyl chain by the action of peptidase activity would lead to much larger proportion of the 14C being liberated as 14CO2 . It is likely however that some degradation of the alkyl chain by omega and beta oxidations does occur and a small proportion of the 1-14C is completely oxidised. This is similar to many other anionic surfactants (Black and Howes 1980, Absorption, metabolism and excretion of anionic surfactants. Anionic surfactants - Biochemistry, Toxicology and Dermatology. Editor Gloxhuber. C., Marcel Dekker, Inc. New York.).

Table 1: Recoveries (%) of 14C from rats treated orally or dermally with radiolabelled C12 AAPB

Exposure

(Contact time/follow-up duration)

Sex

CO2 total

Urine total

Faeces total

Rinsing

Patch extracts

Skin

Cage washing

Carcass

Resorbed

total*

Overall total

Gavage 30 mg [14C]TB/kg (48h) study 1

F

0.78

5.62

117.99

-

-

-

0.01

-

-a)

124.40

Gavage 30 mg [14C]TB/kg (48h) study 3

F

0.19

-

43.44

-

-

-

0.25

39.30

-b)

82.99

Gavage 30 mg [14C]TB/kg (48h) study 3

F

1.32

4.00

86.68

-

-

-

0.04

1.38

6.7

93.42

Gavage 30 mg [14C]TB/kg (48h) study 3

F

1.39

3.89

86.30

-

-

-

0.05

1.26

6.54

92.89

Gavage 30 mg [14C]TB/kg (24h) study 1

M

0.75

6.48

96.17

-

-

-

0.02

-

-a)

103.42

Gavage 30 mg [14C]TB/kg (48h) study 1

M

0.79

5.40

13.04

-

-

-

0.02

-

-a)

19.25

Gavage 30 mg [14C]TB/kg (48h) study 3

M

1. 03

4.01

92.49

-

-

-

0.05

1.09

6.13

98.67

Gavage 30 mg [14C]TB/kg (48h) study 3

M

1.02

3.55

88.00

-

-

-

0.06

1.08

5.65

93.71

Gavage 30 mg [14C]TB/kg (48h) study 3

M

0.97

2.43

87.91

-

-

-

0.05

0.85

4.25

92.21

Gavage 10 mg [1-14C]TB/kg (48h) study 4

F

1.50

3.96

86.60

-

-

-

0.10

1.03

6.49

93.19

Gavage 10 mg [1-14C]TB/kg (48h) study 4

F

1.88

4.89

79.11

-

-

-

0.04

1.75

8.52

87.67

Gavage 10 mg [1-14C]TB/kg (48h) study 4

F

1.43

3.74

81.93

-

-

-

0.18

1.18

6.35

88.46

Gavage 10 mg [1-14C]TB/kg (48h) study 4

M

1.21

4.11

86.61

-

-

-

0.05

1.28

6.6

93.26

Gavage 10 mg [1-14C]TB/kg (48h) study 4

M

1.04

4.47

90.33

-

-

-

0.05

1.07

6.58

96.96

Gavage 10 mg [1-14C]TB/kg (48h) study 4

M

1.30

4.84

84.02

-

-

-

0.10

1.24

7.38

91.50

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

F

0.311

2.683

0.602

-

16.710

50.948

0.090

1.133

4.729

72.477

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

F

0.236

1.740

0.481

-

18.764

53.646

0.065

1.850

4.307

76.782

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

F

0.406

3.691

1.294

-

20.840

47.242

0.055

3.806

9.197

77.334

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

M

0.241

1.234

0.231

-

9.783

67.375

0.020

0.371

2.077

79.255

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

M

0.286

1.183

0.201

-

4.510

81.943

0.060

0.266

1.936

88.449

Dermal occl. 20 mg [14C]TB/kg (48h/48h) study 2

M

0.191

1.359

0.241

-

7.973

72.229

0.015

0.316

2.107

82.343

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

F

0.58

1.15

0.53

-

19.02

77.66

0.02

1.30

3.56

100.26

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

F

<2B

1.67

3.23

-

13.72

70.23

0.20

9.41

-c)

98.46

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

F

1.53

2.54

4.85

-

10.85

79.96

0.11

6.22

-c)

106.06

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

M

0.31

1.07

0.36

-

12.11

88.87

0.02

0.40

2.14

103.14

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

M

0.41

1.50

0.54

-

12.30

82.50

0.04

0.90

3.35

98.19

Dermal occl. 10 mg [1-14C]TB/kg (48h/48h) study 4

M

0.46

1.01

0.39

-

15.15

81.83

0.01

1.72

3.58

100.57

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

F

0.005

0.025

0.005

94.487

0.617

2.467

<BLK

0.070

0.105

98.676

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

F

0.005

0.015

0.005

98.927

0.737

2.648

<0.005

0.030

0.055

102.372

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

F

<BLK

0.010

0.005

91.881

0.737

2.542

<BLK

0.030

0.045

95.205

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

M

0.035

0.100

0.025

100.231

0.913

3.485

0.005

0.095

0.255

104.889

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

M

0.015

0.035

0.015

92.022

1.249

3.004

<BLK

0.095

0.160

96.435

Dermal open 20 mg [14C]TB/kg (10min/48h) study 2

M

0.010

0.050

0.015

100.998

1.234

3.921

0.005

0.025

0.100

106.258

- = not measured, B = Background, BKL = Blank

* Resorbed = CO2 + Urine + Carcass in gavage studies and CO2 + Urine + Faeces + Carcass in dermal studies

a) Total = excreta + cage washings could not be calculated, because carcass 14C levels could not be calculated because they were analysed by whole body autoradiography (WBA).

b) Obviously rogue results for carcass and faeces, therefore not included in the evaluation.

c) Protective patches damaged during second day. Animals have gnawed their patch during the second day and hence probably ingested some [1 -14C]TB, they had increased amounts of 14C in expired air and urine on day 2 compared with the other rats. and also comparatively large increases of 14C (5 to 10 times) was measured in the faeces compared with day 1. Therefore, the results of these two female rats were not included in the evaluation.

 

study 1: 14C from rats dosed by gavage with [14C]TB for whole body autoradiography. Each rat was dosed by gavage with 1.0 ml of an aqueous test solution of [14C]TB containing 4,372µg (29.115 x 10exp6 dpm 14C).

study 2: 14C from rats dosed topically with [14C] TB. All rats were treated topically with 100 µl of a 3% solution of [14C]TB (containing 2.990µg. 19.942 x 10exp6 dpm 14C) in water spread over 10 cm of clipped skin. For one group of female and male rats the treated area was covered with an occlusive patch and the excreta collected separately for 48 hours, when the rats were killed. A further group of female and male rats were treated as above with the same test solution except that the test solution was left in contact with the skin for 10 minutes before rinsing off with distilled water and the site protected with a non occlusive protective patch for 48h.

study 3:14C from rats dosed by gavage with [14C]TB. Each rat was 6 dosed by gavage with 1.0ml of aqueous [14C]TB containing 4,435µg

(29.54 x 10exp6 dpm of l4C).

study 4: 14C from rats dosed orally or dermally with [1-14C]TB: The rats were dosed with 1.0 ml of an aqueous solution of [1-14C]TB (1,563 µg, 27.177 x 10exp6 dpm 14C or the rats were topically dosed with 1.0m1 of aqueous [1-14C]TB containing 1,563 µg (27.177 x 10exp6 dpm of 14C). The treated area was covered with an occlusive patch and the excreta collected separately for 48 hours.

 

[14C]TB =:N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner) carboxylate) isotopically labelled with 14C in the carboxymethyl ammonium moiety

[1-14C]TB = N(lauroylaminopropyl) N,N dimethyl-N carboxymethyl ammonium (inner) carboxylate, isotopically labelled with 14C in the 1 carbon of the lauroyl moiety

Applicant's summary and conclusion

Executive summary:
Due to an IT bug, text and tabels in this section can not be formated. Therefore, executive summary is given in section "overall remarks"