2-methyl-p-phenylenediamine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From Dec. 3, 2003 to Apr. 2, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes

Remarks:

OECD principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methyl-p-phenylenediamine sulphate

Constituent 2

Reference substance name:

2-methyl-p-phenylenediamine

EC Number:

202-442-1

EC Name:

2-methyl-p-phenylenediamine

Cas Number:

95-70-5

Molecular formula:

C7H10N2

IUPAC Name:

2-methylbenzene-1,4-diamine

Constituent 3

Reference substance name:

HAARBRAUN

IUPAC Name:

HAARBRAUN

Details on test material:

UNLABELED TEST SUBSTANCE- Name of test material: Toluene-2,5-diamine sulphate(Haarbraun), Code: A000165 - TSIN: 23005 - Substance type: Pure active substance- Physical state: White-grey powder - Stability: Test material was considered as stable for 5 y if stored dryness and darkness- Stability in solution: The stock solutions of test material in water and water: acetone (4:1) is stable for 8 d measured by HPLC (240 nm) at ambient temperature in presence of light. The stock solutions revealed good stability in water (99.9% and 109%) and water: acetone (4:1) (99.7% and 108%). - Solubility: The solubility in different solvents is as follows: 1 weight%: Water = 9.6 g/L in water < 0.1 weight%: Acetone/water (1:1)RADIOLABELED TEST SUBSTANCE- Name of test material: 2,5-Diamino [ring-U-14C] toluene sulphate - Substance type: Pure active substance- Physical state: Solid- Specific activity: 2.04 GBq/mmol, 55 mCi/mmol (NOTOX substance 133533/B); 1.49 GBq/mmol, 40.37 mCi/mmol; (NOTOX substance 133533/A)- Locations of the label: [Ring-U-14C]- Storage condition of test material: At -20 °C in the absence of moisture, air and light

Radiolabelling:

yes

Remarks:

14C-Toluene-2,5-diamine sulphate

Test animals

Species:

rat

Strain:

other: Wistar Kyoto, WKY/NR Crl BR (inbred)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River, Sulzfeld, Germany - Age at study initiation: 8-9 wk old- Weight at study initiation: All animals were within ± 20% of the sex mean. Body weights ranged from 157-181 g at the time of randomization- Fasting period before study: 18 h- Fasting after test administration: 4 hHousing: Upon receipt from the supplier, animals were individually housed in Macrolon plastic cages containing sterilized sawdust bedding (Type MIII). Following administration of the radioactive dose, rats from Groups 1, 2 were individually housed in stainless steel metabolism cages equipped with a bottom grid and paper bedding and animals of Groups 4, 5 were housed individually in Macrolon plastic cages. Surplus animals remained housed in the Macrolon plastic cages and were used to replace animals that were misdosed or died during dosing- Individual metabolism cages: yes- Diet: Standard pelleted laboratory animal diet (Altromin, code VRF 1, Lage, Germany), ad libitum. The diet was regularly analyzed for nutrients and contaminants.- Water: Tap water, ad libitum- Acclimation period: At least 5 d- Ingredients and/or contaminants of diet, sawdust, nesting material and water were assessed and did not reveal any findings that were considered to have affected study integrity.- Females were non-pregnant and nulliparousENVIRONMENTAL CONDITIONS- Temperature: 21 ± 3°C (actual range)- Humidity: 30-70% (actual range)- Air changes : 15/h- Photoperiod (hrs dark / hrs light): 12 h artificial light and 12 h darkness/day.IN-LIFE DATES: IN-LIFE DATES for different dose groups were as follows:Group 1: From Dec. 4, 2003 to Dec. 8, 2003Group 2: From Dec. 4, 2003 to Dec. 8, 2003Group 4: From March 10, 2004 to March 13, 2004Group 5: From March 10, 2004 to March 13, 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 2M NaOH / water (milli-Q)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Stock solutions of labelled test substance were prepared in milli-Q. Dosing formulations were prepared with the following nominal concentration and specific activity:Group 1: 0.5 mg/mL with a specific activity of 4 MBq/mgGroup 1extra: 0.5 mg/mL with a specific activity of 4 MBq/mgGroup 2: 5 mg/mL with a specific activity of 0.4 MBq/mgGroup 4: 0.5 mg/mL with a specific activity of 4 MBq/mgGroup 5: 5 mg/mL with a specific activity of 0.4 MBq/mgPROCEDURE FOR PREPARATION OF TEST SOLUTION: A measured/weighed amount of labeled and unlabelled test substance was placed into an empty glass container and the formulation was brought to pH 7 with NaOH. Vehicle water (milli-Q) was added to obtain the desired concentration and specific activity. All radiolabelled solutions were prepared immediately prior to dosing (maximum of 4 h). STORAGE OF TEST SOLUTIONS: The vials were covered with aluminum foil before and during the treatment procedure, and the treatment solutions were stored at ambient temperature on a magnetic stirring device.VEHICLE: Water (milli-Q)- Justification for use and choice of vehicle: Based on information from the sponsor on solubility and stability.- Concentration in vehicle: 0.5 and 5 mg/mL - Amount of vehicle (if gavage): 5 mL/kg bw- The radiochemical purity of stock solutions of test material was measured by NOTOX prior to administration and radiochemical stability (before and after treatment) was confirmed by HPLC.HOMOGENEITY AND STABILITY OF TEST MATERIAL: An aliquot of each treatment solution was analyzed using HPLC before and after dosing to confirm the stability of the radiolabelled substance. For HPLC, a flow of 1 mL/min was used with an injection volume of 10 µL, a sample tray temperature of 20° C, a column of nucleosil 100 C18, 250 x 4 mm i.d. (particle size approx. 5 µm) and a column temperature of 40° C. The detection method was UV light absorbance at 240 nm and flow scintillation analysis optimized for 14C. 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 oral treatment

Doses / concentrations

Remarks:

Doses / Concentrations:- Groups 1 and 4(low dose group): 2.5 mg/kg bw (dose volume: 5 mL/kg bw).- Groups 2 and 5 (high dose group) was 25 mg/kg bw (dose volume: 5 mL/kg bw).- Each oral dose of labelled test substance contained approx. 1.5 MBq of radioactivity

No. of animals per sex per dose / concentration:

4 animals/Group 1 and 2; 6 animals/Group 4 and 6

Control animals:

not specified

Details on study design:

- Dose selection rationale: The dose levels were selected based upon repeated dose toxicity studies, oral LD50 was determined to be 100 mg/kg bw, no mortality was expected after a single oral dose of 25 mg/kg bw. The NOAEL in the 90-day study was 2.5 mg/kg bw and the dose levels are a factor 10 apart, which allowed the investigation of differences in the ADME patterns and bioavailability as a function of dose level.- Groups 1 and 2 were used to define the absorption, distribution, metabolism and excretion of 14C Toluene-2,5-diamine sulphate. A total 14C radioactivity mass balance was determined for these groups.- Groups 4 and 5 were used to determine the plasma-kinetics (toxicokinetics) of 14C-Toluene-2,5-diamine sulphate.

Details on dosing and sampling:

MASS BALANCE- Samples collected : Blood, body tissues, urine, and faeces and cage washings from groups 1 and 2- Time of sampling: The urine and faeces were collected at 0-8, 8-24, 24-48, 48-72 and 72-96 h. The blood and body tissues were collected at 96 h (after sacrifice). The cage washings were collected at termination (96 h). Collection and storage of samples: - Blood: Collected by puncturing aorta and transferred into tared heparinized tubes and weighed. Aprox.1 mL of blood was taken for 14C analysis and remaining blood was centrifuged to obtain the plasma which was weighed and stored at ≤ -75 °C.- Body tissues: Following the removal of the blood, the following tissues and organs were harvested: Heart, lung, spleen, gonads (uterus, ovaries: all collected separately), abdominal fat, muscle, adrenals, thymus, thyroid, liver, kidney, brain, bone, GI tract (including contents), residual carcass. The weight of each tissue and blood sample was recorded at the time it was harvested. Tissues and carcasses were stored at ≤ -10 °C prior to analysis.- - Urine and faeces: Samples were freeze-trapped to avoid atmospheric oxidation, evaporation and bacterial degradation.- Cage washings: At termination, the interior of the metabolism cages was rinsed with methanol/water (50/50). The cage rinse was weighed and stored at ≤ -10°C until analyzed.Processing of samples for determination of radioactivity: All radioactive measurements were performed using a Packard scintillation counter (TR-1900)- Faeces: Each sample was homogenized with an equal weight of methanol (if sample size allowed) and triplicates approx. 400 mg of the homogenate (if sample size allowed) were air-dried and combusted (in a Harvey OX500 oxidizer). The combusted samples were trapped in Picosolve C-300 (Packard) present in liquid scintillation vials and the amount of radioactivity was determined.- Urine and cage washings: One, weighed aliquot of 50 µL of urine or 2 mL of the other liquids were transferred to a liquid scintillation counting vial and weighed. The amount of radioactivity was determined using Ultima Gold (Packard) as the scintillation fluid.- Blood: Triplicate aliquots of approx. 200 mg of blood were transferred into combustion cones and combusted following the procedures described for faeces samples.- Tissues: Small tissues or organs (e.g. adrenals, thyroid) were solubilized entirely and unprocessed. Liver, brain and kidneys (without the renal capsule) were homogenized with an equal weight of water using an Ultra-Turrax homogenizer and triplicate aliquots of approx. 400 mg of the homogenate were removed for solubilization and total 14C analysis. For the remaining tissues, except the skin, bone, carcass and GI tract, a single aliquot of approx. 200 mg was solubilized and analyzed for total 14C. Soluene 350 (Packard) was used as the solubilizing agent. Following discoloration by the addition of a H202 solution, the amount of radioactivity in each aliquot was determined using Hionic Fluor (Packard) as the scintillation fluid.- Skin and bone: Skin samples were solubilized in a 6N NaOH and neutralized using HCI, triplicate weighed aliquots of 750 µL of the solubilized sample corresponding with approx. 20 mg of the initial skin sample were transferred to a liquid scintillation counting vial. The bone sample was solubilized in a 6N HCI solution. Following neutralization using a NaOH solution, a weighed aliquot of 750 µL of the solubilized sample corresponding with approx. 75 mg of the initial bone sample was transferred to a liquid scintillation counting vial. Further procedures for solubilization, discoloration and total 14C analysis for skin and bone were identical to those described for tissues.- Carcass + GI: The residual carcass and GI tract were solubilized in successively a 6N NaOH solution and a 12N HCI solution. Following neutralization using a NaOH solution, triplicate weighed aliquots of 750 µL of the solubilized sample were transferred to a liquid scintillation counting vial. Further procedures for solubilization, discoloration and total 14C analysis for the carcass and Gl tract were identical to those described for the tissues. - Plasma: For analysis, one aliquot of approx.100 µL was transferred to a liquid scintillation counting vial and the amount of radioactivity determined. Ultima Gold (Packard) was used as the scintillation fluid.PHARMACOKINETIC STUDY (plasma kinetics)- Tissues and body fluids sampled: Blood (approx. 300 µL was collected alternatively from 3 animals of each group per sampling event)- Time and frequency of sampling: 15 min, 30 min, 1, 2, 4, 8, 24, 48 and 72 h- At approx. half an hour prior to blood sampling the animals of group 4 and 5 were transferred to an incubator set at 40°C where they remained until the time of blood sampling. Sampled blood was transferred into heparinized tubes and centrifuged to obtain the plasma which was stored at ≤-75°C prior to analysis.- Determination of radioactivity in plasma samples: The total amount of 14C radioactivity in plasma samples was determined. Aliquots of approx. 25 µL were transferred to a liquid scintillation counting vial and the amount of radioactivity was determined. Ultima Gold cocktail was used as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter. The level of the parent compound in the plasma samples was determined by HPLC. PHARMACOKINETIC STUDY (excretion)- Tissues and body fluids sampled: Urine, faeces and cage washes- Time and frequency of sampling: 0-8, 8-24 and in additional 24-h intervals after dosing, until 96 h METABOLITE CHARACTERISATION STUDIES- Tissues and body fluids sampled: Urine and faeces - Time and frequency of sampling: 0-8 and 8-24 h (urine); 8-24 h (faeces)- From how many animals: Samples pooled; fractions (50%) of urine and faeces from the animals of groups 1 and 2 were combined and homogenized in order to obtain one urine and faeces sample per group.- Method type(s) for identification: Radio-HPLC (for Metabolite profiles of urine samples and faeces extracts); LC-MS/MS (for Metabolite identification). Detailed description of the analytical methods is provided in the study report.- Storage: The pooled samples of urine and faeces were divided into subsamples which were stored at ≤ -75°C until analysis.Processing of samples: - Extraction of faeces: 4 to 28 g of faeces pool was extracted with 20 mL methanol by vortexing for 2 min and centrifuged for 3 min at (2000 g) and the amount of radioactivity in the supernatant was determined on a 100 µL aliquot. Extraction was repeated until the supernatant contained less than 5% of the total radioactivity. - The methanol extracts were combined (rinsing of each vial for quantitative transfer) and concentrated on a rotavap at 40 °C until approx. 10 mL remained. The activity of the concentrated phase was determined by LSC of a 50 µL aliquot. - The remaining solid residue was extracted consecutively with 20 mL n-hexane, 20 mL methanol pH 2 and 20 ml methanol pH 10 according to the procedure described above. The activity in these phases was determined by LSC of a 100 µL aliquot. - The activity in the remaining pellet was determined by combustion of approx. 400 mg (3 replicates).To increase the extraction recovery, extraction with other solvents was attempted. About 0.4 to 0.5 g of faeces pool was extracted with 5 mL of either tert-butylmethyl ether, dichloromethane, diethyl ether or ethyl acetate, by vortexing for 2 min. - The extract was centrifuged for 5 min at 2000 g and decanted and the amount of radioactivity in the supernatant was determined on a 100 µL aliquot. The extractions were performed three times in total.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

- The mean plasma concentration of Toluene-2,5-diamine sulphate equivalents in the ADME groups 1 and 2 at termination were low (0.003 ± 0.00 and 0.024 ± 0.004 mg/kg of plasma for group 1 and 2 respectively), just above the limit of quantification. - In Group 1 and 2, the average oral absorption was high, 66% (SD: 4) in the low oral dose group and 77% (SD: 2) in high oral dose group, indicating that the majority of the test substance administered orally is absorbed.- The details of individual results are reported in study report.

Details on distribution in tissues:

- The total mean radioactivity (% of dose administered) in blood, tissues and carcass after administration of 14C-Toluene-2,5-diamine sulphate was 0.886 ± 0.162 and 1.351 ± 0.563 for group 1 and 2, respectively.- The total mean concentration of Toluene-2,5-diamine sulphate equivalents in blood, carcass and tissues after a single oral administration was 0.285 ± 0.056 and 7.729 ± 2.253 mg/kg for Group 1 and 2, respectively.- The highest residual concentrations of test substance equivalents were observed in liver, adrenals (high dose only) and thyroid (high dose only). The higher residual concentration in the liver is probably a result of the extensive metabolism of the test substance. - The details of individual results are reported in study report.

Details on excretion:

Urinary Excretion: Excretion via urine was the most important route of elimination for the test substance after oral absorption. Urinary excretion after oral treatment was 62 and 73% for low and high doses, respectively. The rate of excretion was identical for groups 1 and 2 with the bulk of radioactivity excreted during the first 24 h.Faecal excretion: Excretion through faeces was less than compared to excretion via urine. The faeces excretion was 31 and 22% for low and high dose groups, respectively. The excretion of radioactivity in faeces was delayed compared to urine, with significant excretion not until after 8 h post-dose and the highest amount of radioactivity was excreted in the 8-24 h interval. After 24 h, the rate of excretion of radioactivity in the faeces was similar between the low and high oral groups. Total excretion: The total excretion for Group 1 and 2 was 96.927% (SD 2.573) and 97.289% (SD 4.072), respectively. 91-100% of the administered dose was excreted during the study period and in 48 h after treatment excretion was almost.The details of individual results are reported in study report.

Toxicokinetic parameters

Toxicokinetic parameters 1

Toxicokinetic parameters:

Tmax: The mean Tmax for group 4 and 5 was 0.5 and 0.25 h.

Toxicokinetic parameters 2

Toxicokinetic parameters:

Cmax: The mean Cmax for group 4 and 5 was 1.56 and 19.99 mg/kg.

Toxicokinetic parameters 3

Toxicokinetic parameters:

Cmax: Dose normalized mean Cmax for group 4 and 5 were 06. and 0.78 mg/kg/mg x kg

Toxicokinetic parameters 4

Toxicokinetic parameters:

AUC: The mean AUC (last) for group 4 and 5 was 8.93 and 146 mg x h/kg

Toxicokinetic parameters 5

Toxicokinetic parameters:

AUC: The mean AUC(infinity) for group 4 and 5 was 17.59 and 174 h x mg/kg

Toxicokinetic parameters 6

Toxicokinetic parameters:

AUC: The dose normalized mean AUC infinity for group 4 and 5 was 7.10 and 6.79 h x mg/kg/mg x kg.

Toxicokinetic parameters 7

Toxicokinetic parameters:

other: The mean t1/2 for group 4 and 5 was 231 and 28.3 h.

Toxicokinetic parameters 8

Toxicokinetic parameters:

other: The mean elimination rate for group 4 and 5 was 0.0030 and 0.0245 1/h.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

METABOLITE PROFILE STUDY: Urine: With LC-RAD, two large peaks were observed, at 2.40 and 4.50 min, and several smaller peaks and relatively less of the peak at 2.40 min and relatively more of the peak at 4.50 min was present in the high oral dose group. Presence of Totuene-2.5 diamine sulphate in the samples could not be excluded, since the retention time of this compound was close to the retention time of one of the metabolites. With, LC-RAD-MS, three major peaks were observed at approx. 3, 11 and 16 min, with on average 21 %, 38% and 38% of the radioactivity in urine associated with these peaks, respectively. No parent compound was observed in any of the urine samples. Faeces: With LC-RAD, low radioactivity levels were present in the faeces, but up to five metabolic fractions could be distinguished, with a major peak at 4.50 min. With LC-RAD-MS), only a peak was observed in the low oral dose group, at approx. 11 min.METABOLITE IDENTIFICATION STUDY: The largest peak seen (at 11 min) is for a molecule whose mass spectral data are consistent with N,N-diacetyl-14C-toluene-2,5-diamine and the second and third most abundant metabolites have unknown structures but are likely N-acetylated. In the radioactivity chromatograms, the peak of N,N-diacetyl-14C-toluene-2,5-diamine could not be detected in the other groups, but in the MS chromatograms this peak was present in all groups.

Any other information on results incl. tables

ANALYSIS OF FORMULATION AND DOSE VERIFICATION: The radiochemical purity of stock solutions of labeled Toluene-2,5-diamine sulphate in water was on average 96.6% (SD 1.7). The formulations were homogeneous as indicated by relatively small standard deviation of the mean of the top, middle and bottom samples. In addition, there was no relevant difference between the radioactive concentrations measured before and after treatment. The actual concentration of test substance administered was 0.496, 4.963, 0.492 and 4.999 mg/mL in group 1, 2, 4 and 5, respectively. The concentration for extra group 1 was 0.492 mg/mL. The radiopurity of test preparation in Groups 1, 2, 4 and 5 were 97.8, 98.1, 92.9 and 99.2%, respectively. The radiopurity of extra formulation for Group 1 was 98%.

Animals in group 1, 2, 4 and 5 received effective radioactive doses ranging between 1.30 and 2.11 MBq/rat. Animals in low dose groups (1 and 4) received average dose of Toluene-2,5-diamine sulphate was 3.07 (SD 0.07) and 2.48 (SD 0.23) mg/kg bw and animals in high dose groups received 25.96 and 25.63 (SD 0.74) mg/kg bw, respectively.

The details of individual results are reported in study report.

MORTALITY, CLINICAL SIGNS AND MACROPSCOPY

-Animal 22 (high oral dose kinetic group) died approx. 2 h after dosing, of an unknown cause.

- In the low oral dose groups (group 1 and 4), piloerection and a red discharge from the nose were observed from Day 3 onwards. 

- On Day 4, blood in the metabolism cage (due to loss of a nail) and low water consumption were noted for animal 2 (low oral dose group). 

- In the high oral dose groups (groups 2 and 5), piloerection and a red discharge from the nose were observed and in addition in group 5, lethargy, hunched posture, and ptosis were observed. This was probably a result of the combination of test substance ingestion and blood sampling.

- At necropsy, several macroscopic abnormalities were observed: animal 2 pelvic dilation (left), broken nail and animal 5 pelvic dilation (right). These findings are normal for rats of this strain and age and it is not likely that they can be attributed to treatment with the test substance.

- The details of individual results are reported in study report.

BODY WEIGHTS

- Both at the time of randomization and at the day of treatment, body weight of the animals was within 20% of the mean body weight.

RECOVERY OF 14C-TOLUENE-2,5 DIAMINE SULPHATE

- The mean total recovery of radioactivity after a single oral administration of 14C-Toluene-2,5-diamine sulphate was 97.813 ± 2.456 and 98.640 ± 4.173% of administered dose (mean ± SD) for Group 1 and 2, respectively.

- The mean total procedural recovery radioactivity in faeces extracts for Group 1 and 2 was 102.29 and 95.63%, respectively.

TOXICOKINETIC PARAMETERS

- The terminal half-lives and AUC0 were reported as approximations for groups 4 and 5, since either the r<2 was 90%, only two points were used in the calculation or the percentage extrapolated in the AUC0 calculation was > 15%.

- The dose-normalized Cmax value of the high oral dose group was higher than the dose-normalized Cmax of the low oral dose group, but still within the same order of magnitude.

- The level of test substance was determined by radio-HPLC; in group 4 and 5, the test substance was detected at a few time points, but always at a low concentration (maximum of 0.49 mg/kg plasma in group 5). 

- In general, the integration of the different peaks in plasma was difficult, due to the poor separation of the peaks. Between 4 and 5 minutes, several peaks were observed in the chromatogram. 

- Reference standards showed that the retention time of the test substance was around 5.2 - 5.5 min and at a slightly earlier retention time (4.8 - 5.0 min) a large metabolite peak eluted and separation of the parent compound and this metabolite was difficult. This metabolite peak was possibly the same large peak at 4.5 min in urine, observed with LC-RAD method 1. 

- Nevertheless, it can be concluded that the test substance is rapidly metabolized and therefore difficult to detect in plasma.

- The number of points and correlation coefficient for Group 1 and 2 was 3 and 4 and 0.194 and .594 respectively.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: When administered orally, the test substance was absorbed, readily distributed into all organs, extensively metabolized (by N-acetylation metabolism pathway) and excreted via the urine.14C-Toluene-2,5-diamine sulphate when administered orally was extensively absorbed, readily distributed into all organs, extensively metabolized and excreted via the urine and faeces.

Executive summary:

The absorption, distribution, metabolism and excretion of14C-Toluene-2,5-diamine sulphateafter oral administration was determined by following the OECD guideline 417 (Toxicokinetics).

The study was designed to study the fate of 14C-toluene-2,5-diamine sulphate in femaleWistar Kyotorats after oral administration at different dose levels: 2.5 mg/kg bw and 25 mg/kg bw. Four groups of rats were used in the study: Group 1 and 2 (animal number =4) for the mass balance and Group 4 and 5 (animal number =6) for the toxicokinetics.

All test formulations were prepared in water (milli-Q), immediate prior to administration.

The samples of test material formulations were tested for homogeneity and stability by HPLC system with UV detector and radioactivity concentration of each formulation was verified by radioanalysis (liquid scintillation).

 In the mass-balance groups, urine and faeces were collected in 0-8, 8-24, 24-48, 48-72 hr intervals. Animals were euthanized 96 h after dose administration, and several tissues and organs were collected. Total radioactivity in blood, urine, faeces, tissues and organs was determined. Selected urine and faeces samples were pooled per group and the metabolite profile in these pooled samples was investigated. The metabolite profile was analyzed by radio-HPLC and metabolite investigation was performed by LC-MS/MS analysis. The % oral absorption of administered dose was also determined.

In the toxicokinetic groups, blood was sampled alternately from several rats per time point at 0.25, 0.5, 1, 2, 4, 8, 24, 48 and 72 h after dosing. Total radioactivity and toluene-2,5-diamine sulphate equivalent concentrations were determined. The various toxicokinetic parameters were determined. The concentration was determined by HPLC system with UV detector. The radioactivity was determined by 14C liquid scintillation.

In formulation analysis, the actual concentration of test material administered was 0.496, 4.963, 0.492 and 4.999 mg/mL in group 1, 2, 4 and 5, respectively. The concentration for extra group 1 was 0.492 mg/mL. The radioactive purity in Groups 1, 2, 4 and 5 were 97.8, 98.1, 92.9 and 99.2%, respectively. The radiopurity of extra formulation for Group 1 was 98%. Animal in low dose groups (1 and 4) received average dose of Toluene-2,5-diamine sulphate was 3.07 (SD 0.07) and 2.48 (SD 0.23) mg/kg bw and animals in high dose groups received 25.96 and 25.63 (SD 0.74) mg/kg bw, respectively.

Body weight of the animals was within 20% of the mean body weight.

In the low oral dose groups (group 1 and 4), piloerection and a red discharge from the nose were observed from Day 3 onwards. In the high oral dose groups (groups 2 and 5), piloerection and a red discharge from the nose were observed and in addition in group 5, lethargy, hunched posture, and ptosis were observed. This was probably a result of the combination of test substance ingestion and blood sampling. Animal 22 (high oral dose kinetic group) died approx. 2 h after dosing, of an unknown cause.

The mean plasma concentration of Toluene-2,5-diamine sulphate equivalents in Groups 1 and 2 at termination were 0.003 ± 0.00 (66%) and 0.024 ± 0.004 (77%) mg/kg of plasma, indicating that the majority of the test substance administered orally is absorbed.

The total remaining radioactivity in carcass plus tissues was between 0.7 and 7.9% of the administered dose in all groups. The highest residual concentrations of test substance equivalents in the oral routes were observed in liver, adrenals (high dose only) and thyroid (high dose only). The higher residual concentration in the liver is probably a result of the extensive metabolism of the test substance.

In metabolite profile study,two large peaks were observed, at 2.40 and 4.50 min, and several smaller peaks and relatively less of the peak at 2.40 min and relatively more of the peak at 4.50 min was present in the high oral dose group. Three major peaks were observed at approx. 3, 11 and 16 min, with on average 21%, 38% and 38% of the radioactivity in urine associated with these peaks, respectively.

In the metabolite identification study,the largest peak seen (at 11 min) is for a molecule whose mass spectral data are consistent with N,N-diacetyl-14C-toluene-2,5-diamine and the second and third most abundant metabolites have unknown structures but are likely N-acetylated.In the radioactivity chromatograms, theofN,N-diacetyl-14C-toluene-2,5-diaminecould not be detected in the other groups, but in the MS chromatograms this peak was present in all groups.

Reference standards showed that the retention time of the test substance was around 5.2 - 5.5 min and at a slightly earlier retention time (4.8 - 5.0 min) a large metabolite peak eluted and separation of the parent compound and this metabolite was difficult. This metabolite peak was possibly the same large peak at 4.5 min in urine, observed with LC-RAD method 1. 

The test substance is rapidly metabolized and therefore difficult to detect in plasma.

Urinary excretion after oral treatment was 62 and 73% for low and high doses, respectively. Excretion through faeces was less than compared to excretion via urine. The total excretion for Group 1 and 2 was 96.927% (SD 2.573) and 97.289% (SD 4.072), respectively.

The total mean radioactivity (% of dose administered) in blood, tissues and carcass after administration of 14C-Toluene-2,5-diamine sulphate was0.886 ± 0.162 and 1.351 ± 0.563 for group 1 and 2, respectively.

The total mean concentration of Toluene-2,5-diamine sulphate equivalents in blood, carcass and tissues after a single oral administration was 0.285 ± 0.056 and 7.729 ± 2.253 mg/kg for Group 1 and 2, respectively.

The total mean recovery of radioactivity after a single oral administration of 14C-Toluene-2,5diamine sulphate was 97.813 ± 2.456 and 98.640 ± 4.173% of administered dose (mean ± SD) for Group 1 and 2, respectively.

The mean total procedural recovery of the extraction/combustion procedure for Group 1 and 2 was 102.29 and 95.63%, respectively.

The toxicokinetics parameters observed in Groups 4 and 5 were as follows:

The mean Tmax for group 4 and 5 was 0.5 and 0.25 h.

The mean Cmax for group 4 and 5 was 1.56 and 19.99 mg/kg.

Dose normalized mean Cmax for group 4 and 5 were 06. and 0.78 mg/kg/mg x kg

The mean AUClast of group 4 and 5 was 8.93 and 146 mg x h/kg

The mean AUCinfinity for group 4 and 5 was 17.59 and 174 h x mg/kg

The dose normalized mean AUC infinity for group 4 and 5 was 7.10 and 6.79 h x mg/kg/mg x kg.

The mean t1/2 for group 1 and 2 was 231 and 28.3 h.

The mean elimination rate for group 4 and 5 was 0.0030 and 0.0245 1/h.

The absorption, distribution, metabolism and excretion through urine and faeces of 14C-toluene-2,5-diamine sulphate after oral administration to rats was rapid and N-acetylation was the most important metabolic reaction observed for 14C-toluene-2,5-diamine sulphate

This toxicokinetics test is classified as acceptable, and satisfies the guideline requirements of the OECD 417 method.