N-ethyl-o(or p)-toluenesulphonamide

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2006 - 01 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed accrding to guideline and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tosylchloramide sodium

EC Number:

204-854-7

EC Name:

Tosylchloramide sodium

Cas Number:

7080-50-4

Molecular formula:

C7H13ClNNaO5S

IUPAC Name:

Sodium p-toluenesulfonchloramide, trihydrate

Details on test material:

Non-radiolabeled
Test material name: Halamid
Other name: Chloramine T trihydrate
Batch number: 50210
Appearance: white powder
Chemical purity: 99.7%
Supplier: Sigma-Aldrich
Storage conditions: ambient temperature

radiolabeled
Test material name: [14C]-Halamid
Other name: [ring-U-14C]-Chloramine T (trihydrate)
Batch number: CFQ14688
Specific activity: 23 mCi.mmol
Radiochemical purity: 97.2% (HPLC, on 29 March 2006)
Supplier: GE Healthcare
Storage conditions: ≤ 18 ºC

Radiolabelling:

yes

Test animals

Species:

human

Administration / exposure

Type of coverage:

open

Vehicle:

water

Duration of exposure:

8 hours

Doses:

300 or 50 µg/cm2, 3% or 0.5% solutions in water.

No. of animals per group:

6 skin membranes were used per dose and 2 skin membranes were prepared from each donor for each test group (A and B)

Control animals:

no

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human skin membranes were prepared from freshly excised skin obtained from three donors directly after abdominal surgery.
Donor 1: TNA 18/06, born in 1964, arrival at TNO on 26 June 2006
Donor 2: TNA 21/06, born in 1969, arrival at TNO on 28 August 2006
Donor 3: TNA 22/06, born in 1951, arrival at TNO on 30 August 2006
- Ethical approval if human skin: informed consent was provided by all donors.
- Type of skin: abdominal
- Preparative technique:
Human skin was dermatomed using a Dermatome 29 mm (Nouvag GmbH, Germany) to a recorded thickness of approximately 400μm. The exact thickness of all skin membranes was measured with a digimatic micrometer (Mirutoyo Corporation. Japan) and recorded.
- Thickness of skin (in mm): see: any other information on material and methods incl. tables
- Membrane integrity check: Not performed in order to start the experiment as soon as possible. An integrity test requires additional culturing time af the tissue wich may result in loss of viability.
- Storage conditions: The transportation of the skin to the laboratory was carried out as soon as possible after dissection (ca. 2 hours after receipt; skin from donor 1 was stored for about 4 hours in the refrigerator at the hospital until transport), while the skin was kept in a container placed on ice. After arrival at the laboratory, for all three donors, subcutaneous fat was removed and the skin dics were kept at 2-10 ºC during the night.
- Justification of species, anatomical site and preparative technique: The data is used for human risk assessment.

PRINCIPLES OF ASSAY
- Diffusion cell: The skin membranes were placed in 9 mm flow-through automated ditfusion cells (PenneGear Inc., Riegelsvilie, PA. USA).
- Receptor fluid: mixture of Dulbecco's Minimum Eaglc Medium (DMEM) and Ham F12 culture medium (3:l) supplemented with Epidermal Growth Factor (EGF, 10μg/L ), hydrocortisone (400 μg/L), gentamicin (50 mg/L) and Foetal Calf Serum (FCS, 10 %. v/v).
- Solubility of test substance in receptor fluid: The assumption was made that 10% of a 300 µg/cm2 ( = ca 192 µg a.i./skin membrane) dose reaches the recpetor fluid in 24 h. In this case the required solubility of Hlamid is 19.2 µg in 38.4 ml (flow rate ca 1.6 ml/h), i.e. approximately 0.5 µg/ml. The solubility of Halamid in water is 142 µg/ml and thus exceeds the required solubility by a factor 284. The solubilty of the degradation product p-TSA in water is 3 µg/ml and exceeds the required solubility by a factor 6. Therefore the solubility in the receptor fluid is considered to be sufficient.
- Static system: not applicable
- Flow-through system: The receptor fluid was pumped at a speed of cu 1.6 mL/h
- Test temperature: The mean skin surface tempenture was 32 ± 1 ºC
- Humidity: ambient humidity
- Occlusion: not applicable
- Reference substance(s): none

Results and discussion

Absorption in different matrices:

see: remarks on results including tables and figures

Total recovery:

- Total recovery: The mean recovery was 95.4% (high dose) and 94.0% (low dose)
- Recovery of applied dose acceptable: yes
- Results adjusted for incomplete recovery of the applied dose: no
- Limit of detection (LOD):
- Quantification of values below LOD or LOQ:

Percutaneous absorption

Remarks on result:

other:

Any other information on results incl. tables

Stability:

In the receptor fluid, a rapid degradation of Chloramine-T trihydrate towards p-TSA was observed. A few minutes after preparing a 30mg/l solution 71.7% Chloramine-T trihydrate was left. The concentration further decreased from 48.9%, 29.2% to 14.5% after 7, 24 and 48 hours.

Directly after dosing, the dose solutions were analyzed by radio-HPLC. The relative amount of Chloramine-T trihydrate in the both dose solutions was 94 70 based on W detection. Based on radio-activity, this percentage was lower (ca  72%) due to additional peaks in the beginning of the radio-chromatogram  most probable related to a breakdown of the radio-label. Degradation will most likely take place to p-TSA (as is observed in the stability test using receptor fluid). It is not expected that only the radio-activity associated with the unknown peaks will be absorbed through the skin. A rapid a degradation of Chloramine-T trihydrate to p-TSA on the skin surface was confirmed by analyzing the cotton swab extracts. After 8 hours contact time, only ca 23 and ca 2.3 % Chloramine-T trihydrate was left in the coton swab extracts of the high and low dose group, respectively. It is therefore reasonable to assume that the main compound reaching the receptor fluid will be p-TSA. In the skin wash solution, Chloramine-T trihydrate was relatively stable deceted in time at 92% until 48 hours.

* The exact concetration is described in the method section.

**Total absorption is defined as the amount in the receptor fluid, the receptor compartment wash and the skin membrane, excluding the amount in the tape strips.

The in vitro percutaneous penetration of Halamid

Halamid	A		B
concentration measured (g/L)	30.0*		5.0*
Dose (µg/cm2)	300*		50 *
n	6		6
penetration into the receptor fluid after 24 h	% of dose	µg/cm2	% of dose	µg/cm2
	4.01	12.0	11.49	5.74
maximal flux (µg/cm2/h)	0.652		0.367
Lag time (h)	2.7		4.0
Total absorption (%)**	9.7		20.0

Applicant's summary and conclusion

Conclusions:

The mean total adsorption of a 3% aqueous Chloramine-T trihydrate solution on human skin is 9.7%. The mean total adsorption of a 0.5% aqueous Chloramine-T trihydrate solution on human skin is 20.0%. These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide.

Executive summary:

According to OECD 428 and under GLP the percutaneous absorption of  [14C]-Chloramine-T trihydrate in a 3% and 0.5% aqueous solution was evaluated on 6 freshly excised human membranes (2 from one donor). The chemical stability of Chloramine-T trihydrate was determined prior to the conduct of the study aiming at a later analysis of receptor fluid and skin wash fractions obtained in the main study. Skin membranes from three different human donors were used. The exposure time was 8 hours, after which the test compound was washed from the skin and the post-exposure time was 16 hours. Samples were taken from receptor fluid samples, skin wash. Receptor compartment wash, donor compartment wash, tape strips, and digested skin. All collected samples were analyzed with liquid scintillation counting (LSC).To determine the extent of (metabolic) degradation of Chloramine-T trihydrate into p-TSA, samples of the skin wash were analyzed by radio- LC. The total absorption is defined as the amount in the receptor fluid, the receptor compartment wash and the skin membrance, excluding the amount in the tape strips.

A rapid degradation of Chloramine-T trihydrate towards p-TSA was observed in the receptor fluid. Based on UV detection, the relative amount of Chloramine-T trihydrate decreased from 71.7 % established a few, minutes after mixing (t=0) to 14.5 % after 48 hours. In the skin wash solution, Chloramine-T trihydrate was relatively stable detected in time at 92% until 48 hours. For the high dose, the mean penetration of test compound-related radioactivity into the receptor fluid after 24 hours was 12.0 μg/cm2 which was 4.01% of the dose applied. The mean maximal flux was 0.652 μg/cm2/h  the lag time was 2.7 h. The mean total absorption, was 9.7%. For the low dose, the mean penetration of test compound-related radioactivity into the receptor fluid after 24 hours was 5.74 μg/cm2 which was 11.49 % of the dose applied The mean maximal flux was 0.367 μg/cm2/h and the lag time was 4.0 h. Slight differences between donors were observed. The mean total absorption was 20.0 %. The mean recovery was 95.4% (high dose) and 94.0 % (low dose). The mean total adsorption of a 3% aqueous Chloramine-T trihydrate solution on human skin  is 9.7%. The mean total adsorption of a 0.5% aqueous Chloramine T trihydrate solution on human skin is 20.0%. METHOD

Absorption, distribution, metabolism and excretion of [ring-U-14C]Chloramine T in the Wistar rat. The study was conducted according to the following guidelines: OECD Guideline no. 417: Toxicokinetics. Seven groups of rats were included in the study: Three (n=4 males and 4 females) for the massbalance, three (n=3 males and 3 females) for the toxicokinetics and one (n= 4 males and 4 females) for bile collection. Rats were dosed with a single oral dose of 20 mg/kg b.w. or 200 mg/kg b.w., or dosed repeatedly with 20 mg/kg b.w.for 10 days with unlabelled test substance

prior to a labeled dose. In the mass-balance groups, urine, faeces and bile (bile-cannulation group only) were collected in 0-8, 8-24, 24-48 and 48-72 hr intervals. Animals were euthanized 72 hours after dose administration, and several tissues and organs were collected. Total radioactivity in urine, faeces, bile, tissues and organs was determined. Selected urine and faeces samples were pooled per group and the metabolite profile in these pooled samples was investigated. In the toxicokinetic groups, blood was sampled from each rat at the following time points: 0.25, 0.5, 1 , 2, 4, 8, 24, 48 and 72 hour after dosing. Total radioactivity and Chloramine T equivalent concentrations were determined.

 

RESULTS

No mortality was observed in the study. One animal in the bile-cannulation group was killed in extremis, due to problems with the bile cannula. This animal displayed piloerection and hunched posture.

Clinical signs were observed mainly in the high dose ADME group and the bile cannulation group and consisted of piloerection.

From the plasma data it was evident that oral absorption proceeded fast, with Tmax values of 0.25 to 1 hour. Plasma concentration versus time curves of Chloramine T equivalents after oral dosing were similar, with similar Tmax values, and dose-normalised Cmax and AUC, in the same

order of magnitude. This suggests linear kinetics over the investigated dose range. Variable apparent terminal half-lives were observed, ranging from 13.2 to 120 hours. In general, it appeared that females had a shorter half-life of radioactivity than the males, but this was not reflected in the mass-balance data. The most important route of excretion of Chloramine T equivalents was urine. Urinary excretion accounted for 90% for males and 88% for females after low dosing, 81 % for males and 79% for females after high dosing and 87% for males and 78% for females after repeated dosing. In the bile-cannulation group urinary excretion accounted for 67% for the males and 66% for the females after oral dosing.

Faecal excretion was only a minor route of excretion. Faecal excretion accounted for 10% for the males and females after low dosing, 13% for the males and females after high dosing and 13% for the males and 16% for the females after repeated dosing, in the ADME groups. In the bile-cannulation group faecal excretion accounted for 25% for the males and 23% for the females. Biliary excretion accounted for 6% of the administered dose in males and 4% in females, in the bile-cannulation group. The absorption of Chloramine T equivalents was high, 92% and 91 % for the males and females in group 1 respectively, 83% in the males and females of group 2, and 89% and 85% of the males and females in group 3 respectively. The absorption in the bile-cannulation group was lower than in the ADME groups, 75% in both sexes. At termination of the study, the average total remaining radioactivity in blood, carcass plus tissues was between 0.6 and 1.4% of the administered dose in all groups..

The tissue concentration equivalents of Chloramine T were below the concentration observed in blood in all groups. 'This indicates that Chloramine T shows no potential for accumulation. The average total recovery of radioactivity in groups 1 to 3 and 7 was between 96 and 102% of

the administered dose. In the urine samples, one major radioactivity peak was observed, which accounted for 80-92% of the radioactivity in the radio-chromatogram and 65-81 % of the applied dose. This metabolite was isolated by prep HPLC from the urine pool of group 2 males and then identified as being 4. Sulfamoyl benzoic acid by NMR and Mass Spectrometry. Based on the radioactivity data (same major peak present in all groups and sexes at a similar retention time) it was concluded that the major radioactivity peak represented 4-Sulfamoyl benzoic acid in all groups.

In the radioactivity chromatograms of the faeces extracts 3 major peaks were observed, but these peaks could not be identified with MS. Two potential minor metabolites were identified i.e. p-sulfonic-benzoic acid and an aromatic methoxy compound.

 

CONCLUSIONS

It can be concluded that Chloramine T administered orally was highly absorbed and excreted mainly via the urine. The data indicated that there were no sex differences in urinary, faecal or biliary excretion of radioactivity and no sex differences in total absorption. In addition, no differences were observed in urinary, faecal, biliary excretion or total absorption between the low and high dose or between single and repeated administrations. Linear plasma kinetics were observed. One major metabolite was observed in urine of all groups and identified as 4-Sulfamoyl benzoic acid. After oral administration of [methyl-14C]toluene-4 -sulphonamide (p-TSA) to rats the label was rapidly eliminated largely in the urine (66 -89% dose) with little in the faeces (2 -8% dose). The 14C in the faeces was 4 -sulphamoylbenzoic acid, which probably originated in the tissues since the gut flora was unable to effect this biotransformation.The urine of rats given Chloramine–T contained 4– sulphamoylbenzoic acid as the major metabolite (93 % of the urinary 14C) together with small amounts of unchanged compound (1.5–2.3% of urinary 14C) 4–sulphamoylbenzyl alcohol (2.0–3.9%), 4 -sulphamoylbenzaldehyde (0–1.5 %) and at higher doses N–acetyltoluene–4–sulphonamide (2.1–2.3%). These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide.