Registration Dossier

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
other information
Study period:
no data
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Not according to a guideline and there is no data if the study was performed under GLP. Limited reporting.
Objective of study:
distribution
Principles of method if other than guideline:
Method: other
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
No data
Route of administration:
other: oral and i.p.
Vehicle:
other: distilled water
Details on exposure:
no data
Duration and frequency of treatment / exposure:
4 day(s)
Remarks:
Doses / Concentrations:
Males: single oral dose of 100 mg/kg or repeated doses i.p. 5 mg/kg
No. of animals per sex per dose:
no data
Control animals:
no
Details on study design:
The plasma and brain chloramine-T disposition and effects on monoamine  [5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA)] contens  in rat hypothalamus, striatal and cortical tissues were investigated.
Adult male and wistar rats weighing 200-300 g were used. They were  alloted to three groups. Rats in Group 1 were given single oral dose of  100 mg  Chloramine-T/kg. Rats in Groups 2 and 3 were given daily dosage of 5 mg  Chloramine-T/kg or 0.5 ml distilled water i.p. on four consecutive days.  All rats were killed. 
Details on dosing and sampling:
For rats of Group 1, serial blood samples were  collected and brain removed. For rats of Groups 2 and 3 brains were removed and hypothalamus, stratum and frontal  cortex dissected out. Blood and brain samples from Croup 1 rats were  assayed for chloramine-T concentration. Hypothalamus, striatum and  frontal cortex tissues from Groups 2 and 3 rats were assayed for 5-HT and  5-HIAA concentrations
Details on distribution in tissues:
After a single oral dose it was seen that Chloramine-T was rapidly  absorbed and entered the CNS. After 4 repeated i.p. doses The levels of 5-HT were decreased no chances in 5-HIAA were found.
Conclusions:
Interpretation of results: bioaccumulation potential cannot be judged based on study results
After a single oral dose it was seen that Chloramine-T was rapidly absorbed and entered the CNS. After 4 repeated i.p. doses the levels of 5-HT were decreased no chances in 5-HIAA were found.
Executive summary:

Adult male Wistar rats were divided into three group. The plasma and brain  Tosylchloramide sodium disposition and effects on monoamine  [5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA)] contens  in rat hypothalamus, striatal and cortical tissues were investigated. Adult male and wistar rats weighing 200-300 g were used. They were  alloted to three groups. Rats in Group 1 were given single oral dose of  100 mg  Tosylchloramide sodium/kg. Rats in Groups 2 and 3 were given daily dosage of 5 mg Tosylchloramide sodium/kg or 0.5 ml distilled water i.p. on four consecutive days.  All rats were killed. For rats of Group 1, serial blood samples were  collected and brain removed. For rats of Groups 2 and 3 brains were removed and hypothalamus, stratum and frontal  cortex dissected out. Blood and brain samples from Croup 1 rats were  assayed for Tosylchloramide sodium concentration. Hypothalamus, striatum and  frontal cortex tissues from Groups 2 and 3 rats were assayed for 5-HT and  5-HIAA concentrations. After a single oral dose it was seen that Tosylchloramide sodium was rapidly  absorbed and entered the CNS. After 4 repeated i.p. doses the levels of 5-HT were decreased no chances in 5-HIAA were found.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study was not performed according to a standard guideline and pre-GLP. Reporting is too limited to judge the validity of the study.
Objective of study:
metabolism
Principles of method if other than guideline:
No guideline followed.
GLP compliance:
no
Remarks:
pre-GLP
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 200-250g
- Fasting period before study: no
- Housing: Metabowls (Jencons Scientific Apparatus Limited, Hemd hempstead, Herts., U.K.) for the separate collection of urinc and faeces and allowed free access to food and water before and during the experinwnt.
- Individual metabolism cages: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- 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


IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
other: In 1.0 ml 20% ethanol in water or 50% aq. propylene glycol (1 ml)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
no data
Duration and frequency of treatment / exposure:
no data
Remarks:
Doses / Concentrations:
Females: 29 mg/kg 8uCi/rat; in 1.0 ml 20% ethanol in water, 200 mg/kg 15 uCi/rat; in 50% aq. propylene glycol (1ml)
No. of animals per sex per dose:
no data
Control animals:
no
Positive control:
no
Details on study design:
no data
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, expired air
- Time and frequency of sampling: no data

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sample: urine, faeces, expired air
- Time and frequency of sampling: no data
- From how many animals: samples were pooled, how many animals: no data
- Method type(s) for identification: Gas chromatography-mass spectrometry
- Limits of detection and quantification: no data
Statistics:
no data

After oral administration of [methyl-14C]toluene-4 -sulphonamide (Chloramine–T) 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%).

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
After oral administration to rats the label was rapidly eliminated largely in the urine (66 -89% dose) with little in the faeces (2 -8% dose). 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%).
Executive summary:
After oral administration of [methyl-14C]toluene-4 -sulphonamide (Tosylchloramide sodium) 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%).
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
3 May 2006 - 12 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to guideline and under GLP.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: designed for this specific study
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
No details. The stomach content was collected from these non fasted rats. The animals were reserve animals from another study.
Route of administration:
other: not applicable
Vehicle:
water
Details on exposure:
The stomach contents from 2 rats in non-fasted condition was collected. From this sample 7.12 g was homogenized with 4 parts of water giving 32.46 g homogenate with a pH value of 4.79. From the contents including mucosa of the small intestines from the same rats 6.49 g was homogenized with to 4 parts of 0.01 M phosphate buffer pH 6.8, resulting in a homogenate of 35.15 g with a pH of 6.83.
Duration and frequency of treatment / exposure:
one single dose and exposure up to 4 hours
Remarks:
Doses / Concentrations:
For the dose solution, 2.51 mg 14C-halamid was weighed into a vial and 137.8 mg nonlabelled Halamid was added, next 4.35 ml of water was added (total weight: 4.52 g), resulting in a 3.11% Halamid solution containing 1.68 MBq [14C]-Halamid per gram solution. The dose solution was carefully mixed and visually checked for homogeneity before each administration.
No. of animals per sex per dose:
2
Control animals:
other: blanks were also measured
Positive control:
Not applicable
Details on dosing and sampling:
Breakdown of Halamid
For each time point 0.4 ml of the respective homogenate was placed in a eppendorf cup and pre-incubated at 37ºC. for at least 2 minutes. Then 0.1 ml of dose solution was added, mixed and incubation time started. At time points 0, 0.5, 1, 2 and 5 minutes after the start of the incubation, 0.5 ml of ice-cold 0.02 M NaOH in acetonitrile was added to each of the eppendorf cups, mixed and immediately centrifuged for 30 seconds at 13,000 rpm in a eppendorf centrifuge and the supernatant as soon as possible (within 1 minute) injected in the HPLC system. The radioactivity in the supernatant was measured with liquid scintillation counting. Control incubations with demineralised water were used to study the stability during analysis.

VolatiIe metabolites and [14C]-CO2
4 ml of the homogenates were incubated with I mL of the dose solution. The incubations were performed in washing bottles continuously flushed with nitrogen gas (anaerobic conditions) at 37ºC for 4 hours. The effluent was led through washing bottles, placed in serial. The first washing bottle contained 20 % aqueous dimethylsulfoxide to trap possible volatile organic compounds. The second trap contained 1 M KOH to trap [14C]-CO2. Directly after addition of dose solution 2 aliquots of 50 pl were sampled and radioactivity measured to check the concentration of Halamid. At time points 0.5, 1, 2 and 4 h, 2 ml aliquots were sampled and radioactivity measured by liquid scintillation counting.

Determination of radioactivity
Radioactivity was determined by liquid scintillation counting (LSC) using DOT-DPMtm (digital overlay technique using the spectrum library and the external standard spectrum) for quench correction on a Wallac Phamacia model S1414 scintillation counter. Calibration procedures for the instruments are established at the testing facilities. Radioactivity was determined in all collected samples. In brief, samples were counted for radioactivity as follows:
CO2-trap: Aliquots of the CO2 trap samples were added directly to liquid scintiliant (Hionic FluorTM) for LSC.
Volatiles trap: Aliquots of the volatiles trap samples were added directly to liquid scintillant (Ultima GoldTM) and measured with LSC.
Incubation mixture: Samples from the incubation mixture were added directly to liquid scintiliant (Ultima ColdTM) and measured with LSC.

HPLC method capable of separating Chloramine-T trihydrate and the possible metabolite formed p-TSA was developed.

Column: Waters Nova-pak® C18,3.9 x 150 mm cartridge, 4 μm
Mobile phase: 0.01 M K2HP04 buffer, pH 4.0 : acetonitrile = 68 : 32*
Flow rate: 1.0 ml/.min
Detection: UV (λ = 229 nm) RA - liquid scintillation flow cell 0.5 ml, 3 ml/min Ultima Flow™
Injection volume: 1- 2μl
*) Slightly adapted from the study plan (i.e. 60:40) based on results obtained during set up of the analytical method.

Chloramine-T trihydrate was found highly unstable in the homogenates and rapidly to p-TSA. To slow down breakdown of Chloramine-T trihydrate, a 0.02 M NaOH solution in acetontrile was added directly after taking samples.

The linearity and reproducibility of the HPLC-method was determined by injecting two calibration curves consisting of 5, 10, 20, 50 and 100 pg Chloramine-T trihydrate/100 μl water containing 0.01 M NaOH. Calibration curves of the UV-signal of Chloramine-T trihydrate showed good linearity and reproducibility.
The correlation coefficients r2 were > 0.999.
Metabolites identified:
not measured

The breakdown of Chloramine-T trihydrate was very fast. Immediately after addition of Chloramine-T trihydrate to the homogenates ca 90% of the Chloramine-T trihydrate was converted to p-TSA in both homogenates. After 2 and 1 minutes of incubation > 99 % of the Chloramine-T trihydrate present in stomach and small intestinal contents, respectively, was convened to p-TSA. The Chloramine-T trihydrate concentration in the blank incubation showed a small, but not significant decrease.

Sample incubation   Time poiny (minutes)     Peak area based on radio activity     Peak area based on UV 229 nm
     Halamid  p-TSA  Halamid  p-TSA
 Blank  0  88.9 11.1  90.5  9.5 
   5  83.8  16.2  84.0  16.0
 Stomach content  0  7.8 -6.8 92.2 -94.0  10.1 -7.2 89.9 -92.8 
   0.5  4.1 95.9  5.2  94.8 
   1  1.5  98.5  2.0  98.0
   <1.0 >99.0  1.0  99.0 
   5  <1.0 >99.0  0.2   99.8
 Small intestinal contents  0  15.2 -31.1 84.8 -68.9  17.2 -33.0  82.8 -67.0 
   0.5  3.2  96.8 3.6  96.4 
   1  <1.0  >99.0    0.8  99.2
   2  <1.0  >99.0  1.2  98.8
   5  <1.0  >99.0  0.1  99.9

The formation of volatiles and CO2 was almost absent, after 4 h of incubation in the homogenates less than 0.05 and 0.1% of the applied radioactivity.

 Sample incubation  Incubation (hours)  % radioactivity found as   
     Volatiles [14C]-CO2 
 Stomach contents  0.5  0.005  0.006
   1  0.013  0.022
   2  0.017 0.046 
   4 0.042  0.068 
 Small intestinal contents  0.5  0.005  0.020
   1  0.011  0.045
   2  0.021  0.072
   4  0.031  0.103
Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
Chloramine-T trihydrate is not stable in stomach and intestinal contents  of rats
Executive summary:
The study was designed to examine the stability of [14C]-Tosylchloramide sodium, trihydrate in stomach and intestinal contents of rat. To this end, homogenates of stomach and small intestinal contents from male Wistar rats in non-fasted condition were incubated with[14C] Tosylchloramide sodium, trihydrate at a target concentration of 0.6 % (w/w) in the homogenates, assuming a five-fold dilution of the 3% nominal use concentration. At several time points aliquots of the incubation mixture were taken to study the stability of [14C]- Tosylchloramide sodium, trihydrate formation of volatiles. 

The breakdown of Tosylchloramide sodium, trihydrate was very fast. Immediately after addition of Tosylchloramide sodium, trihydrate to the homogenates ca 90% of the Tosylchloramide sodium, trihydrate was converted to p-TSA in both homogenates. After 2 and 1 minutes of incubation > 99 % of the Tosylchloramide sodium, trihydrate present in stomach and small intestinal contents, respectively, was converted to p-TSA. The formation of volatiles and CO2 was almost absent, after 4h of incubation in the homogenates less than 0.05 and 0.1 % of the applied radioactivity was found as volatiles and 14C-CO2, respectively.

Tosylchloramide sodium, trihydrate is not stable in stomach and intestinal contents of rats.

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
3 (not reliable)
Toxicokinetic parameters:
half-life 1st:
Toxicokinetic parameters:
half-life 2nd:
Toxicokinetic parameters:
half-life 3rd:
Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
3 (not reliable)
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2006- July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to guideline and under GLP.
Objective of study:
toxicokinetics
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
ADME and TK groups (1-6) : Rat: Wistar, Crl:WI (outbred, SPF quality).Bile-cannulation group (7) : Rat: Wistar HsdCpb : WU.
- Source: ADME and TK groups (1-6) : Charles River Deutschland, Sulzfeld, Germany. Bile-cannulation group (7) : Harlan, Horst, The Netherlands
- Age at study initiation: Young adult animals of 10-12 weeks old were used.
- Weight at study initiation: body weights ≥ 179 gram
- Fasting period before study: No
- Housing:
ADME and TK groups (1-6):
Upon receipt from the supplier animals were group-housed in Macrolon cages (type MIV, height 18 cm) containing sterilized sawdust bedding (Woody-Clean type 314; Tecnilab-BMI BV, Someren, The Netherlands). Certificates of analysis were examined and then retained in the NOTOX archives. In
addition, paper was provided as nest material (Enviro-dri, Tecnilab-BMI BV, Someren, The Netherlands). Animals of groups 3 and 6 remained housed in these cages during the pre-treatment. Following administration of the radioactive dose and 24 hours prior to dosing, rats from ADME groups (1-3) were individually housed in stainless steel metabolism cages (LxWxH = 18.5x19x20 cm). Animals of TK groups (4-6) were housed individually in
Macrolon cages (LxWxH: 30x1 9x1 9 cm), equipped with a bottom grid and paper bedding. Surplus animals were kept in the Macrolon cages.
Bile-cannulation group (7):
Upon receipt from the IMTC animals were individually housed in Macrolon plastic cages (type Mlll height 15 cm) containing sterilized sawdust bedding (Woody-Clean type 314; Tecnilab-BMI BV, Someren, The Netherlands) and tissues. Certificates of analysis of the bedding were examined for contaminants and archived. After the acclimatization period, animals were individually housed in stainless steel metabolism cages (LxWxH = 18.5x19x20 cm). Two surplus animals were kept in the Macrolon cages.
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum access to pelleted rodent diet (SM RIM-Z from SSNIFFB Spezialdiaten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-22.0
- Humidity (%): 45-90
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES:
Grow 1
Start of administration: 18 July 2006
Termination: 21 July 2006

Group 2
Start of administration: 24 July 2006
Termination: 27 July 2006

Group 3
Start of administration "cold": 21 July 2006
Start of administration C14: 31 July 2006
Termination: 03 August 2006

Group 4
Start of administration: 18 July 2006
Blood sampling for toxicokinetics: 18, 19, 20 and 21 July 2006
Termination: 21 July 2006

Group 5
Start of administration: 24 July 2006
Blood sampling for toxicokinetics: 24, 25, 26 and 27 July
Termination: 27 July 2006

Group 6
Start of administration "cold": 21 July 2006
Start of administration C14: 31 July 2006
Blood sampling for toxicokinetics: 31 July, 01, 02 and 03 August 2006
Termination: 03 August 2006

Group 7
Administration: 28 August 2006
Termination: 30 and 31 August 2006

Experimental completion date: 30 and 31 August 2006

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A weighed amount of unlabeled Chloramine T was placed into an empty glass container. A measured amount of a stock solution of labelled Chloramine T in Milli-Q water and additional Milli-Q water was added to obtain the desired concentration and specific activity. All radiolabelled solutions were prepared immediately prior to dosing and stored at ambient temperature, for a maximum of 4 hours. Before and during the treatment procedure the
treatment solutions were kept on a magnetic stirring device.

The unlabelled Chloramine T formulation used for pre-treatment was prepared immediately prior to dosing and stored at ambient temperature for a maximum of 4 hours.


VEHICLE
Milli-Q water for the labelled formulations.

Milli-U water for the non-labelled Chloramine T formulation (used for the 10 days pre-dosing of group 3 and 6 animals).
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: Stock solutions of [ring-U-14C]Chloramine T were prepared in Milli-Q water. Four dosing formulations were prepared with the following nominal concentration and specific activity:
Group 1 and 4: 4 mg/ml with a specific activity of 0.5 MBq/mg
Group 2 and 5: 40 mg/ml with a specific activity of 0.05 MBq/mg
Group 3 and 6: 4 mg/ml with a specific activity of 0.5 MBq/mg
Group 7: 4 mg/ml with a specific activity of 0.3 MBq/mg
- Amount of vehicle (if gavage): 5ml/Kg
- Lot/batch no. (if required): not applicable
- Purity: not applicable


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Before and after dose administration, the homogeneity and radioactivity concentration of each formulation was verified by radioanalysis. For this purpose, weighed aliquots of 25 µl were taken from the top, middle and bottom of the mixture and analyzed by liquid scintillation counting (LSC) using Ultima Gold (Packard) scintillation cocktail.
Duration and frequency of treatment / exposure:
Single dose for groups 1, 2, 4, 5 and 7. In the case of groups 3 and 6, a single oral dose of labelled Chloramine T was administered following pre-treatment with non-labelled Chloramine T for 10 days.
Remarks:
Doses / Concentrations:
ADME and TK groups:
Groups 1 and 4: low-dose of 20 mg/kg bw.
Groups 2 and 5: high-dose of 200 mg/kg bw.
Groups 3 and 6: low-dose of 20 mg/kg bw.
Each oral dose of [ring-U-14C]Chloramine T contained approximately 10 MBq/kg bw of radioactivity.
Bile-cannulation group 7: a single low-dose of 20 mg/kg bw, containing approximately 6 MBq/kg bw of radioactivity.
No. of animals per sex per dose:
4/sex/dose level (ADME groups: 1-3)
3/sex/dose level (TK groups: 4-6)
4/sex/dose level (bile-cannulation group: 7)
Control animals:
no
Details on study design:
- Dose selection rationale:
Dose levels were chosen based on available acute toxicity data and a 90-d dietary study in rats. The LD50 of a 10% solution of Chloramine T was 935 mg/kg bw. The effect level in the 90-d study was > 150 mg/kg bw/day and the NOAEL was 15 mg/kg bw/day (studies avaialble at that time from 1971). Based on this, the low-dose level in the ADME study was chosen as 20 mg/kg bw and the high-dose level as 200 mg/kg bw. Furthermore, the dose levels were a factor 10 apart, which allowed the investigation of differences in the ADME patterns and bioavailability as a function of dose level.
- Rationale for animal assignment (if not random): random
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled : urine, faeces, blood, plasma, serum, cage washes, bile, bone, Heart, Lung, Spleen, Gonads (testes, seminal vesicles, prostate, uterus, ovaries: all collected separately), Abdominal fat, Muscle, Adrenals, Thymus, Thyroid, Liver, Kidney, Brain, Bone
GI tract (including contents), Skin (shaved), Residual carcass
- Time and frequency of sampling:
For the animals in ADME and bile-cannulation groups (1-3 and 7) bile (group 7 only), urine and faeces were collected over the following time intervals: 0-8, 8-24, 24-48 and 48-72 hours after the dosing with [ring-U-14C]Chloramine T. Urine, bile and faeces were freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation. Urine, bile and faeces samples were stored at ≤ 75°C prior to analysis. At termination, the interior of the metabolism cages was rinsed with methanol/water (50/50). The cage rinse was weighed and stored at 5 -10°C until analyzed. Based on the results of pilot study 462105 (non-GLP), volatiles were not sampled.
For the animals in TK groups (4-6), blood samples from the tail vein of each rat were sampled at the following time-points after dose administration:
15, 30 min, 1 , 2, 4, 8, 24, 48 and 72 hours (max. deviation 1 minute). The amount of sample collected was approximately 300 µl per sampling event. At approximately half an hour prior to blood sampling the animals of groups 4, 5 and 6 were transferred to an incubator set at 40°C where they remained until the time of blood sampling. At the times of blood sampling, the rats were maintained in restrainers. At other times they were housed individually in Macrolon cages and allowed food and water ad libitum. After the final blood sample was obtained, the animals were euthanized by a C02/02 procedure and the carcasses disposed of as radioactive waste. Sampled blood was transferred into tubes containing Liheparin and centrifuged to obtain the plasma which was stored at ≤ -75°C prior to analysis.
At the designated time of euthanasia each animal in ADME groups (1-3) was deeply anaesthetised using isoflurane vapour. The thorax and abdomen were opened with a midline incision. By means of an aorta punction the maximum possible amount of blood was withdrawn, causing the animals to be exsanguinated. Care was taken to avoid contamination of other organs with blood. Sampled blood was transferred into tared tubes containing Li-heparin and weighed. A weighed subsample of approximately 1 ml of the heparinised blood was removed for total 14C analysis. The remaining blood was centrifuged to obtain the plasma which was weighed and stored at ≤ -75°C. The organs listed above were harvested.
Samples from muscle and bone were collected from the left hind leg. The weight of each tissue and blood sample were recorded at the time it was harvested. Tissues and carcass were stored at ≤ -10°C prior to analysis.
At the designated time of euthanasia all animals of bile-cannulation group 7 were euthanized using an oxygen/carbon dioxide procedure. The carcass and GI tract (including contents) were stored at ≤ -10°C prior to analysis.
- Other: None


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling:
Urine:
ADME Groups (1 -3): 0-8 and 8-24 h
Faeces:
ADME Groups (1-3): 8-24 h
- From how many animals: the samples were pooled in order to obtain one urine and faeces sample per group per sex. That is 4 animals per group per sex
- Method type(s) for identification: LC-RAD-PDA-MS and NMR
- Limits of detection and quantification: see: additional information on materials and methods
- Other: None
Statistics:
Mass balance:
The mean and standard deviation was used to characterize the data, where appropriate (i.e. radioactivity measurement, concentration, etc). Concentrations of radioactivity in blood, plasma and tissues were calculated as mg equivalents of Chloramine T /kg of sample, based on the specific activity of the [ring-U-14C]Chloramine T formulation. The total amounts of radioactivity in tissue and excreta samples were calculated as a percentage of the administered radioactivity by the following formula:
Amount excreted (% of administered dose) = (radioactivity matrix (Bq/total matrix)/dose (Bq/animal))* 100%
and a 14C mass balance were presented. A mean recovery of I00 ± 10% of the administered radioactive dose was achieved. For ADME groups (1-3) and the bile-cannulation group (7): Cumulative amounts (expressed as percentage of the dose) excreted in urine, bile and faeces were calculated by summation of the amounts excreted in the individual urine, bile and faeces samples per collection period, respectively.
The oral absorption was calculated by summing the amounts of radioactivity in urine, bile (group 7), cage-wash and tissues and organs.

Kinetic calculations:
Concentrations of radioactivity in plasma were calculated as mg equivalents of Chloramine T /kg of sample, based on the specific activity of the [ring-U-14C]Chloramine T formulation. For each group, all toxicokinetic parameters were calculated from the curves constructed from the mean values at each time point using the WinNonlin 4.0.1 program. Non-compartmental analysis was applied.

Details on absorption:
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. No significant differences were noted in absorption between low and high dosing and single and repeated dosing. The absorption in the bile-cannulation group was lower than in the ADME groups, 75% in both sexes. Urinary excretion of radioactivity in the bile-cannulated animals was about 11 to 23% lower than in the ADME groups and excretion in the faeces was about 7 to 15% higher. The reason for this is unclear: animals in the bile-cannulation group showed more clinical signs and can perhaps be viewed as in worse condition than the other animals, possible influencing absorption. Another explanation can be that the absence of bile influences the absorption of the compound. No sex-differences were noted in oral absorption in any of the groups.
Details on distribution in tissues:
The average total remaining radioactivity in blood, carcass plus tissues in the ADME groups was between 0.6 and 1.4% of the administered dose in all groups. The major part of it was present in carcass, i.e. between 0.4 and 1.1 % of the administered dose in all groups. In the bile cannulation group, the average total remaining radioactivity in carcass and GI tract was less than 1 % of the administered dose.
The residual concentrations of Chloramine T equivalents in group 1 and 3 were in the same
range; concentrations in group 2 (high dose group) were approximately one order of magnitude
higher than in the low dose groups.
The tissue concentration equivalents of Chloramine T were below the concentration observed in blood in all groups. Highest concentrations in tissueslorgans were observed in liver and kidney, which is indicative of the extensive metabolism and excretion of the substance. This indicates that Chloramine T shows no potential for accumulation.
Details on excretion:
Excretion via urine is an important route of elimination for Chloramine T, after oral absorption. 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. The rate of excretion was similar for all ADME groups with the bulk of radioactivity excreted during the first 8h. The excretion rate of radioactivity in the urine in the bile-cannulation group was slightly lower than in the ADME groups in the first time intervals.

Excretion via faeces was a minor route of excretion for Chloramine T equivalents. 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. The excretion of radioactivity in faeces was delayed compared to urine, with significant excretion not until after 8 h post-dose. The highest amount of radioactivity was excreted in the
8-24 h interval.

Excretion via the bile is only a minor route of excretion for for Chloramine T equivalents, after oral absorption. Biliary excretion accounted for 6% of the administered dose in males and 4% in females. Excretion took place mainly in the first interval, from 0-8 hours after dosing.

The results show that 95-101% of the administered dose was excreted during the study period (mean values). Excretion was quick and almost complete within 24h post-dose.
Toxicokinetic parameters:
other: see table below: remarks on results including tables and figures
Metabolites identified:
yes
Details on metabolites:
LC-RAD-PDA-MS metabolite analysis
An overview of the data obtained in the metabolite identification part of this study indicated that a wide variety of compounds have been found that may be metabolites of p-TSA, which is formed from the chemically instable Chloramine T. Most of these compounds could not be identified.
In the urine samples, one major radioactivity peak was observed. Despite analysis in negative and positive modes no identification of potential metabolites was possible. Despite the large numbers of potential metabolites that were found with LC-MS analysis in the faeces extracts of the Wistar rats, only two potential metabolites were identified, i.e. p-sulfonicbenzoic acid and an aromatic methoxy compound. In the radioactivity chromatograms 3 major peaks were observed. None of the identified metabolites did correspond to these peaks.

NMR metabolite analysis
The urine of group 2 males was used for analysis. The unknown metabolite, representing approx. 80% of the radioactivity in the chromatogram, was isolated by prep HPLC. Analysis of 1H and 13C NMR indicated that the isolated metabolite was a para-substituted benzene with COOH and SO2NH2 substituents, and identified as 4-Sulfamoyl benzoic acid. In addition, mass spectrometric analysis was carried out which was consistent with the molecule being 4-Sulfamoyl benzoic acid.

Dose verification: The radioactive dose administered to each animal was calculated from the weight of the dose administered and the mean radioactive concentration of the formulations. Animals in ADME and TK groups (1,-6) received average effective radioactive doses ranging between 10.2 and 11.1 MBq/kg bw. Animals in the bile-cannulation group (7) received an average effective radioactive dose of 6.3 MBq/kg bw. Animals in the low-dose groups received an average dose of Chloramine T between 20.1 and 20.6 mg/kg bw. Animals in the high-dose groups received an average dose of Chloramine T between 190.8 and 205.4 mg/kg bw. All the doses were sufficiently close to the target doses in order to fulfill the study's objectives. Toxicokinetic parameters

        group 4 20 mg/kg bw single oral dose        group 5200 mg/kg bw single oral dose       group 620 mg/kg bw repeated oral dose  
 parameters    m  f  m  f  m  f
 Tmax  hr  0.5 0.5  0.25  0.25 
 Cmax  mg/kg  11.9 11.3  69.6  101  15.7  18.7 
 Dose-norm  Cmax  mg/kg/mg*kg  0.595 0.565  0.347  0.505  0.785  0.935 
 AUClast hr*mg/kg  70.0 53.3  843  680  101  90.8 
Dose-norm AUClast  hr*kg*mg/kg/mg  3.50 2.67  4.22  3.40  5.05  4.54 
AUC∞  hr*mg/kg  97.8* 59.9*   1345*  730*  229*  112*
 Dose-norm  AUC∞  hr*kg*mg/kg/mg  4.89*  3.00* 6.73*  3.65*  11.5*  5.61* 
 % extrapolated  %  28.5 11.0  37.4  6.8  56.1  19.1 
 λz  1/hr  0.012 0.027*  0.008*  0.053*  0.006  0.020 
 t1/2  hr  60.5 26.1*  82.7* 13.2*  120  35.4 
 no. points    3
 Corr coef.  r2  0.9615 0.8746  0.8535  0.6724  0.9795  0.9649 
Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
It can be concluded that Tosylchloramide sodium, trihydrate 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.
Executive summary:

Method

Absorption, distribution, metabolism and excretion of [ring-U-14C]

Tosylchloramide sodium, trihydrate 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 Tosylchloramide sodium 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 Tosylchloramide sodium 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 Tosylchloramide sodium 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 Tosylchloramide sodium 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 Tosylchloramide sodium were below the concentration observed in blood in all groups. 'This indicates that Tosylchloramide sodium 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 Tosylchloramide sodium 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.

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Only a summary of the study.
Species:
other: Horse

A pharmacokinetic study in horses has been conducted to determine  concentrations of p-TSA after topical application 0.5 % Chloramine-T  solution. Six horses with wounds were treated twice a day at intervals of  8 hours over 72 h (6 treatments/per animal in total) by washing (3  animals, 200 ml to 1000 ml) or spraying (3 animals,100 to 250 ml). Blood  samples were collected immediately prior to each treatment, at 2, 4, 6,  and 8 h after the first treatment and at 2, 4, 6, 12, and 24 h after the  last treatment and analyzed with HPLC (LOD 3.2 µg/l) Chloramine-T was not  detected in any of the samples. p-TSA was only detectable in 2 samples  from the 5th treatment. Detected concentrations were 4.4 µg/l and 6.0  µg/l.

Executive summary:

A pharmacokinetic study in horses has been conducted to determine concentrations of p- TSA after topical application 0.5 % Tosylchloramide sodium solution. Six horses with wounds were treated twice a day at intervals of 8 hours over 72 h (6 treatments/per animal in total) by washing (3 animals, 200 ml to 1000 ml) or spraying (3 animals,100 to 250 ml). Blood samples were collected immediately prior to each treatment, at 2, 4, 6, and 8 h after the first treatment and at 2, 4, 6, 12, and 24 h after the last treatment and analyzed with HPLC (LOD 3.2 µg/l) Tosylchloramide sodium was not detected in any of the samples. p-TSA was only detectable in 2 samples from the 5th treatment. Detected concentrations were 4.4 µg/l and 6.0 µg/l.

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:
guideline study
Remarks:
Study performed according to guideline and under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes (incl. certificate)
Radiolabelling:
yes
Species:
human
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
- Other:
sampling time:
Receptor fluid samples were collected from 0-1 h. 1-2 h, followed by 2 2-h intervals until 24 hours after application. 8 h after application, the unabsorbed test substance (dislogeable dose) was removed from the application sit. In the end of the experiment (24 h), the diffusion cell was dismantled and samples were taken form the receptor and donor compartments, each skin membrane was tape stripped 10 times and skin membranes were digested for analysis.
treatment of sample:
Receptor fluid samples were collected in vials for immediate analysis. The unabsorbed test substance (dislogeable dose) was removed from the application site using a 3.5% Teepol solution in water and cotton swabs (skin wash). The cotton swabs were pooled per skin membrane and extracted with 10 ml water containing 0.01 M NaOH for stabilization. An aliquot of the extracted cotton swabs were analyzed. The receptor and donor compartments were washed twice with 1.0 ml water and analyzed. Each skin membrane was tape stripped 10 times at the end of the study using D-squame (Monadem, Monaco). Tape stripping was discontinued in case the epidermis is ruptured. Tape strips were analyzed directly. Skin membranes were digested for analysis in 5ml of a 1.5 M KOH solution containing 20% ethanol. Aliquots of the digested skin membranes were analyzed The mass balance of the test substance was determined using the samples described above (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.
Analysis:
The radioactivity in the samples was determined using a LKB/Wallac S1414 scintillation counter. Ultima GoldTM scintillation liquid (Packard) was added to samples of the receptor fluid (15ml per sample), the diffusion cell washes (15ml per sample); the cotton swab extracts (4ml to a 0.5ml aliquot of each sample), the tape strips (4ml per sample), and to samples of the mock dosing samples (15ml per sample). For the determination of radioactivity in digested skin preparations, 4mL Hionic FluorTM scintillation liquid (Packard) was added to an aliquot (0.5 ml) of each digested skin membrane.
The following HPLC analysis was performed (TNO report V6837/02):
Column: Waters Nova-pak® C18,3.9 x 150 mm cartridge, 4 μm
Mobile phase: 0.01 M K2HP04 buffer, pH 4.0 : acetonitrile = 68 : 32*
Flow rate: 1.0 ml/.min
Detection: UV (λ = 229 nm) RA - liquid scintillation flow cell 0.5 ml, 3 ml/min Ultima Flow™
Injection volume: 1 μl
*) Slightly adapted from the study plan (i.e. 60:40) based on results obtained during set up of the analytical method.
Chloramine-T trihydrate was found highly unstable and rapidly degrading to p-TSA. To slow down breakdown of Chloramine-T trihydrate, a 0.01 M NaOH solution was added to various samples.
Calculations:
- The cumulative penetration of rest substance equivalents was calculated from the receptor fluid samples by the following equation:
Cumulazivr DPMT = DPMT + Σ(DPMT-1, ... DPM1)
DPMT: radioactivity at sampling time T
DPMT-1: radioactivity at the sampling time preceding 1
DPMT1 : radioactivity at the first sampling time
For each receptor fluid sample, background values were subtracted.
- Using the program Microsoft Excel, the 'slope' and 'intercept' of the virtual line through the linear portion (if the penetration curve was calculated. A straight line was mathematically represented by the formula "y = slope*x + intercept", in which y represents the cumulative absorption and x represents time. This formula was used to calculate the maximal flux and the lag time from the mean values of six skin preparations:
Maximal flux =slope
Lag time = - intercept/slope
- The permeability coefficient or Kp value for tritiated water [cm/h] was calculated as follows: Kp = flux constant [μg/cm2/h] / applied concentration [μg/cm3]
- The total absorption is defined as the amount of compound-related radioactivity present in the receptor fluid, the receptor compartment wash, and the skin (excluding tape strips)


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:
Remarks on result:
other:

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   
Conclusions:
The mean total adsorption of a 3% aqueous Tosylchloramide sodium, trihydrate solution on human skin is 9.7%. The mean total adsorption of a 0.5% aqueous Tosylchloramide sodium, trihydrate solution on human skin is 20.0%
Executive summary:

According to OECD 428 and under GLP the percutaneous absorption of  [14C]-Tosylchloramide sodium, 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 Tosylchloramide sodium, 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 Tosylchloramide sodium, 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 Tosylchloramide sodium, trihydrate towards p-TSA was observed in the receptor fluid. Based on UV detection, the relative amount of Tosylchloramide sodium, trihydrate decreased from 71.7 % established a few, minutes after mixing (t=0) to 14.5 % after 48 hours. In the skin wash solution, Tosylchloramide sodium, 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 Tosylchloramide sodium, trihydrate solution on human skin  is 9.7%. The mean total adsorption of a 0.5% aqueous Tosylchloramide sodium, trihydrate solution on human skin is 20.0%

Description of key information

It is shown that [14C] Tosylchloramide sodium is almost instantaneously converted to p-TSA in contact with stomach and intestinal contents of rat. Similarly, also an in-vitro dermal absorption study demonstrated the conversion of Tosylchloramide sodium, resulting to only p-TSA to pass the skin. Rapid and complete oral uptake followed by rapid excretion mainly with urine. Overall dermal absorption about 20%.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
20
Absorption rate - inhalation (%):
100

Additional information

Toxicokinetics, metabolism and distribution TOSYLCHLORAMIDE SODIUM (Tosylchloramide sodium)

Absorption

It was shown in an in vitro study that [14C] Tosylchloramide sodium is rapidly converted to p-TSA in contact with stomach and intestinal contents of rat, after 2 minutes >99%. Therefore systemic exposure will not be to Tosylchloramide sodium but to p-TSA (De Bie, H.J., 2007(Proprietary data).

In a single andrepeated dose toxicokineticsstudy with radiolabelled Tosylchloramide sodium it was shown that radiolabelled material was rapidly absorbed orally, after 0.25-1 hourthe maximum plasma concentration is reached (Tmax). The absorption was high up to 92 and 91 % after a low single dose (males and females, 20 mg/kg bw), 83% after a high single dose (both sexes, 200 mg/kg bw) and 89 and 85% after a low repeated dose (males and females, 20 mg/kg bw). The absorption of the bile canulation group was lower, about 75% after a low single dose (both sexes, 20 mg/kg bw) and biliary excretion accounted for 4-6% of the administered dose (males and females). The urinary excretion of radioactivity was 11-23% lower in the bile-cannulation group compared to the other ADME groups while the faecal excretion was 7-15% higher (Wenker, M.A.M. and Emmen, H.H., 2007 (Proprietary data). The lower absorption may be explained by the absence of bile in the intestine influencing the absorption of the compound. The bile may be required to facilitate absorption from the intestine to the blood and subsequent excretion via the urine. Thus a lower absorption form the intestine leads to a higher excretion in the faeces and a lower excretion in the urine. The difference in results may perhaps also be influenced by the fact that animals in the bile-cannulation group had a cannule and showed more clinical signs.

An older study from literature confirms these results after administration of a single dose of p-TSA to rats (Ball, L.M., et al., 1978).

 

Distribution

At termination of a single andrepeated dose toxicokineticsstudy, 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 major part of it was present in carcass, i.e. between 0.4 and 1.1 % of the administered dose in all groups. In the bile cannulation group, the average total remaining radioactivity in carcass and GI tract was less than 1% of the administered dose (Wenker, M.A.M. and Emmen, H.H., 2007 (Proprietary data ).

 

Anadón, A., et al.(1997)studied the brain disposition of Tosylchloramide sodium. The study suggests that Tosylchloramide sodium is rapidly absorbed and enters the Central Nervous System also indicating a potential storage in the brain. These findings have not been confirmed in any other study with Tosylchloramide sodium or p-TSA and in the study by Wenker (2007) it was shown that the levels in tissues (including the brain) was below the level found in blood, showing that there is no potential for accumulation. In a recently performed 90d study with rats the Functional Observational Battery did not indicate any neurological effects (Van Otterdijk, F.M., 2007 (Proprietary data) nor have such effects been observed in any other study with Tosylchloramide sodium or p-TSA.

 

Excretion

After 4h incubation of [14C]-Tosylchloramide sodium in the stomach and intestinal the formation of gaseous metabolites was negligible, less than 0.05 and 0.1 % of the applied radioactivity was found as volatiles and [14C]-CO2, respectively (De Bie, H.J.,2007(Proprietary data).

After a single (20 or 200 mg/kg bw) or repeated dose (20 mg/kg bw) variable apparent terminal half-lives were observed, ranging from 13.2 to 120 hours. The most important route of excretion is the urine, up to 78 and 87% (males and females, 200 mg/kg bw). The major part of the radioactivity was excreted within the first 8 hours. Faecal excretion was a minor route, up to 13 and 16% (males and females, 200 mg/kg bw). The major part of the radioactivity was excreted within a 8-24 hour period. In the bile canulation group the faecal excretion increased to 25% and the urinary excretion decreased to 66 and 67% (males and females). Biliary excretion accounted for 4 and 6% of the administered dose (males and females). Excretion took place mainly in the first interval, from 0-8 hours after dosing. See section on absorption for explanation of these findings.

These results show that 95-101% of the administered dose was excreted during the study period (mean values). Excretion was quick and almost complete within 24h post-dose(Wenker, M.A.M. and Emmen, H.H., 2007 (Proprietary data). 

These findings of rapid distribution and elimination are supported by a study performed with rats receiving 30 mg/kg bw iv or 100 mg/kg bw orally after which blood samples were collected and plasma concentrations ofTosylchloramide sodiumwere determined.Tosylchloramide sodiumwas characterized by a rapid distribution and elimination phases after i.v. (t1/2α=0.12h, 1/2β=1.41h) and oral dosing (t1/2α=0.42h, 1/2β=1.98h). Plasma clearance was relatively high (0.147 and 0.149 liter/h, respectively) with a volume of distribution at steady state of 0.24 liter (Martinez-Larrañaga, M.R., et al., 1996). An older study from literature also confirms these results after administration of a single dose of p-TSA to rats (Ball, L.M., et al., 1978).

 

Metabolism

For identification of samples from a single andrepeated dose toxicokineticsstudy an LC-PDA-RAD-MS method was developed. The method was based on p-TSA since it has been shown that Tosylchloramide sodium is rapidly converted to p-TSA.Urine and faeces samples were further analyzed for identifying metabolites. In the urine samples, one major radioactivity peak was observed. This peak was identified asbenzoic acid derivative (4-sulfamoylbenozic acid, CAS 138-41-0) (see Fig. 1).

 

See attached document

Figure 1.benzoic acid derivative (4-sulfamoylbenozic acid, CAS 138-41-0)

 

An older study from literature confirms the identity of the major metabolite (Ball, L.M., et al., 1978). Female albino Wistar rats were given a single low and high oral dose (29 mg/kg bw in 1ml 20% ethanol in water and 200 mg/kg bw in 50% aq. propylene glycol (1ml)). This study also identified the small amounts of other metabolites found next to a remaining small amount of parent compound p-TSA (1.5-2.3%). Other identified metabolites were 4-sulphamoylbenzyl alcohol (2.0-3.9%), 4-sulphamoylbenzaldehyde (0-1.5%) and at high dose N-acetyltoluene-4-sulphonamide (2.1-2.3%).

 

Other studies reported in literature on metabolism were largely qualitative and confirm the metabolite4-sulfamoylbenozic acid in urine of dogs and rats(Flaschenträger B., et al., 1934;Minegishi, K. I., et al., 1972).

Despite the large numbers of potential metabolites that were found with LC-MS analysis in the faeces extracts, only two potential metabolites were identified, i.e. p-sulfonicbenzoic acid (Fig. 2a) and an aromatic methoxy compound (Fig. 2b). In the radioactivity chromatograms 3 major peaks were observed. None of the identified metabolites did correspond to these peaks.

 

See attached document

Figure 2a. p-sulfonicbenzoic acid                                                         1b. aromatic methoxy compound

 

Dermal absorption

Thepercutaneousabsorption of [14C]-Tosylchloramide sodium in a 3% and0.5%aqueoussolutionwasevaluated on 6 freshly excised human membranes. A rapid degradation of Tosylchloramide sodium towards p-TSA was observed in the receptor fluid (decrease to 71.7 % minutes after mixing further to 14.5 % after 48h). In the skin wash solution, Tosylchloramide sodium was relatively stable 92% was detected after 48 hours.The 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 mean total adsorption of a 3% aqueous Tosylchloramide sodium solution on human skin is 9.7%. The mean total adsorption of a 0.5% aqueous Tosylchloramide sodium solution on human skin is 20.0% (Maas, Ir. W.J.M., 2007 (Proprietary data).

 

Some additional data for percutaneous absorption is available from cows and horses.

Tosylchloramide sodium is used for teat disinfection and it was investigated if residues were detectable incow blood. Five lactating cows were treated twice daily for eight days with 0.5% Tosylchloramide sodium solution. Blood samples were taken during treatment up to 24h after the last treatment. Samples were analyzed with HLPC (LOD 5 μg/kg) and no p-TSA was detected in any of the samples (EMEA,2001).

A pharmacokinetic study in horses has been conducted to determine concentrations of p-TSA after topical application 0.5 % Tosylchloramide sodium solution. Six horses with wounds were treated twice a day at intervals of 8 hours over 72 h (6 treatments/per animal in total) by washing (3 animals, 200 ml to 1000 ml) or spraying (3 animals,100 to 250 ml). Blood samples were collected immediately prior to each treatment, at 2, 4, 6, and 8 h after the first treatment and at 2, 4, 6, 12, and 24 h after the last treatment and analyzed with HPLC (LOD 3.2 µg/l) Tosylchloramide sodium was not detected in any of the samples. p-TSA was only detectable in 2 samples from the 5thtreatment. Detected concentrations were 4.4 µg/l and 6.0 µg/l (EMEA, 2006).

 

In conclusion, these studies in horses and cows show that after repeated treatment concentrations in serum were generally undetectable or in a few cases very low indicating that dermal absorption is poor and/or rapid metabolism and excretion of the active substance.

 

Accumulation

The tissue concentration ofTosylchloramide sodiumequivalents were below the concentration observed in blood in all groups indicating that Tosylchloramide sodiumequivalents show no potential for accumulation  (Wenker, M.A.M. and Emmen, H.H., 2007 (Proprietary data).

Furthermorea log Pow for Tosylchloramide sodium trihydrate of -1.3 has been determined (IIIA section A3 3.9). Tosylchloramide sodium trihydrate is quickly transformed into p-TSA in contact with organic matter. For p-TSA a logPow value was measured of 0.82 Based on these values no bioaccumulation test is warranted.

The test data show that for p-TSA equivalent residues a worst-case bioconcentration factor of 2.2 was derived using fingerling fish and the p-TSA equivalent concentration. Based on the rapid transformation and low log Pow, Tosylchloramide sodium has a low bioaccumulation potential.

Conclusion

Oral uptake is expected based on information from the available test information (acute oral toxicity) and the range of physico chemical parameters. Dermal absorption would be possible based on physico-chemical information. Based its molecular weight, the distribution is expected. Fast absorption and subsequent elimination is confirmed by the available ADME data from TSA isomers and N-chloro-4-methylbenzenesulfonamide sodium (Tosylchloramide sodium, CAS 127-65-1). The absorption values for hazard assessment would be 100% for the inhalation route, 20% for the oral route and 100% for the dermal route, respectively.

 

References

1.      Anadón, A, et al., 1997, Brain disposition of Tosylchloramide sodium and neurochemical consequences in rats., J Vet Pharmacol Therap, 20 ((Suppl.1)) p:265-266.

2.      Ball, LM, et al., 1978, The fate of saccharine impurities: The excretion and metabolism of (14C)toluene-4-sulphonamide and 4-sulphamoyl (14C)benzoic acid in the rat, Xenobiotica, 8:(183-190).

3.      De Bie, HJ, 2007, Stability of [14C]Halamid in stomach and intestinal contents of rat, TNO Quality of Life, Zeist, The Netherlands, Report No.: 6837/02, May 9, 2007 (Proprietary data).

4.      EMEA, 2001, Committee for Veterinary Medicinal Products. Tosylchloramide Sodium (extension to bovine) Summary Report (2), The European Agency for the Evaluation of Medicinal Products, Report No.: EMEA/MRL/782/01-FINAL, March, 2001.

5.      EMEA, 2006, Committee for Veterinary Medicinal Products. Tosylchloramide Sodium (extension to horses) Summary Report (3), The European Agency for the Evaluation of Medicinal Products, Report No.: EMEA/CVMP/220264/2005-FINAL, February, 2006 .

6.      Flaschenträger B., et al., 1934, Über einen neuartigen Abbau der aliphatischen Kette., Z. Physiol.Chem., 225:p:157-167.

7.      Maas, IWJM, 2007, In vitro percutaneous absorption of [14C]-Halamid® through human skin membranes using flow-through diffusion cells, TNO Quality of Life, The Netherlands, Report No.: V6837/01, January 31, 2007 (Proprietary data).

8.      Martinez-Larrañaga, MR, et al., 1996, Pharmacokinetics of Tosylchloramide sodium in rats., Methods Findings Exp Clin Pharmacol 18:(Suppl. B) p:217.

9.      Van Otterdijk, FM, 2007, 90-Day dietary toxicity study with p-TSA in the rat, Notox, The Netherlands, Report No.: 474042,July 12, 2007 (Proprietary data).

10.   Wenker, MAM and Emmen, HH, 2007, Absorption, distribution, metabolism and excretion of [ring-U-14C]Tosylchloramide sodium in the Wistar rat., NOTOX, 's-Hertogenbosch, The Netherlands, Report No.: 462094, July 12, 2007 (Proprietary data)