Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb 2010 - 20 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

other: Amendment 1

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 3

Reference Type:

other: Amendment 2

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Objective of study:

other: absorption, distribution, metabolism, and excretion characteristics

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF New Test Guidelines for Supporting Registration of Chemical Pesticides 12 Nousan 8147, adopted November 24, 2000, amended June 26, 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

EC Number:

620-056-5

Cas Number:

874195-61-6

Molecular formula:

C14H13F3N4O5S

IUPAC Name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

Radiolabelling:

yes

Remarks:

labelled with 14C in the triazine ring

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, The Netherlands
- Age at study initiation: males: ca. 6 weeks old; females: ca. 8-9 weeks old
- Fasting period before study: The feeding was stopped approx. 16 hours prior to administration and continued approx. 6 hours afterwards.
- Housing: During acclimatisation rats were kept as group of three animals each in Makrolon® cages (type III H or IV) on hemp or softwood litter. During the test rats were kept individually in Makrolon® metabolism cages. With these cages, an almost quantitative and separate collection of urine and faeces was possible.
- Individual metabolism cages: yes
- Diet: The rats were fed with rat/mice maintenance long life diet, supplied by Provimi Kliba AG, Switzerland (approx. 16 g per animal and day).
- Water: Tap water, ad libitum
- Acclimation period: approx. 7 days prior to administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 45-56
- Air changes (per hr): 10-15
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The radiolabelled test compound (KATH 6425) was shipped in solid and dried form. The specific radioactivity amounted to 4.46 MBq/mg (2.676 x 1E8 dpm/mg). For preparation of a stock solution, the compound was dissolved in 25 mL acetonitrile. Subsamples from this solution were taken for radioactivity measurement by liquid scintillation counting (LSC) and identity and purity checks by chromatographic and spectroscopic methods. The stock solution was stored in a freezer at ≤ -18 °C until preparation of the administration suspensions.
The identity of the test compound was confirmed by spectroscopic (LC-MS, 1H-NMR) and chromatographic (HPLC) methods. The radiochemical purity of the test compound in the stock solution was determined by HPLC and amounted to > 99% .
Subsamples from the stock solution were taken for preparation of the administration suspensions for Test 1 (only males) and Test 2 (only females). One day before starting the test, a definite volume of the stock solution with the required amount of test compound was pipetted into a glass flask and concentrated to near dryness under a gentle stream of nitrogen. Afterwards, the residues were reconstituted in 0.5% aqueous Tragacanth® by treatment in an ultrasonic bath. A magnetic stir bar and glass balls were added to the suspensions which were then continuously stirred in a cold room at approx. +5 °C until the administrations. During the individual administrations, the suspensions were stirred at room temperature.
The radiochemical purity and the stability of the test compound in the administration suspensions were determined by HPLC of diluted aliquots after dosing. The purity amounted to > 99% for both tests. Further subsamples of the administration suspensions were taken for radioactivity measurement by LSC and for identity checks by HPLC co-chromatography.
The total amount of radioactivity administered to each animal served as reference value (A0 = 100%) for the percentage calculation of the total radioactivity in the biological samples.

Duration and frequency of treatment / exposure:

single dose
animals were sacrificed 72 h post dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
2 mg/kg bw

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

- Dose selection rationale: 2 mg/kg bw was selected as low dose level

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood (plasma and blood cells), liver, kidney, GIT, heart, brain, testes, ovaries, uterus, adrenal glands, Harderian gland, thyroid gland, spleen, lungs, eyes, skin, femur bone, perirenal fat, leg muscle, carcass
- Time and frequency of sampling:
urine: 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–48 h, 48–72 h
faeces: 0–24 h, 24–48 h, 48–72 h
plasma micro samples: 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 24 h, 28 h, 32 h, 48 h, 52 h, 56 h, 72 h
organs and tissues were sampled at sacrifice

METABOLITE CHARACTERISATION STUDIES
Metabolic profiles in urines and extracts of faeces were measured by reversed phase HPLC with radiometric detection using an acidic water/acetonitrile gradient.

Statistics:

All mean values were calculated as arithmetical mean values and filed with standard deviations.
The coefficient of variation (cv) was calculated from the existing values according to the following formula: v = S / x ̅ × 100 [%]
If the mean values for fraction samples or subsets were calculated from values with < LLQ (= lowest limit of quantification), then the half of the corresponding LOQ value was used in place of the non-determined concentration value.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The test compound was nearly completely absorbed since more than 75% of the administered dose was detected in the urine and the body excluding GIT at sacrifice.

Main ADME results 2

Type:

distribution

Results:

Fast distribution; Cmax was reached within 0.67 and 0.33 h after administration in males and females, respectively. From the maximum, the radioactivity level declined down to ca. 50% of Cmax after 1.5 to 2 h and to < 1% of Cmax within 24 h after dosing.

Main ADME results 3

Type:

metabolism

Results:

The metabolic transformation was based on demethylation, reduction and conjugation and took place at least at 4 different structural positions of Triafamone. Parent compound, 2 major and 9 minor metabolites were identified in urine and faeces samples.

Main ADME results 4

Type:

excretion

Results:

Excretion was fast and occurred mainly by the renal route. Females exhibited a slightly higher renal excretion rate of ca. 85% than males with ca. 77%. The major part of the dose (> 75% males; > 84% females) was excreted within 24 h after treatment.

Main ADME results 5

Type:

other: Radioactive residues in organs and tissues at sacrifice

Results:

Ca. 0.09% of the dose administered was still detected in the body without GIT of males and ca. 0.02% in the ones of females. For all organs and tissues, levels were in a range of

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Recovery
The total recoveries for both tests were nearly quantitative since between ca. 95.5% and 101.9% of the total dose administered were found in the excreta and the bodies of male and females rats at sacrifice. All recovery rates of radioactivity in urine, faeces and organs and tissues at sacrifice are shown in Table 1.
[Triazine-UL-14C]AE 1887196 was nearly completely absorbed in the rats. The absorption rate that was calculated from the sum of the total dose recovered in urine and body excluding GIT was at least 75.5% in males and 89.3% in females (Table 1). The absorption started immediately after dosing as it was observed from the rapid increase of radioactivity in plasma micro samples.

Details on distribution in tissues:

The distribution of the radioactivity within the body was fast and the maximum plasma level (Cmax) was already reached at 0.67 and 0.33 h after administration in males and females, respectively. At these time points, the radioactivity levels in plasma corresponded approximately to ca. 174% the equidistribution concentration for male (Cnorm = 1.74) and to ca. 65% for female rats (Cnorm = 0.65).
The plasma concentrations declined to approx. 50% of Cmax within 1.5 to 2 h and to lower than 1% of the maximum value within 24 h for both sexes. In the time after 24 h, the plasma radioactivity declined further to 0.003 mg/kg for males and below LOQ for females at the time of sacrifice after 72 h. Overall, male rats exhibited about more than twofold higher plasma concentrations than females during the whole experimental period. The mean values of the total radioactivity in plasma were used for a toxicokinetic modelling with TOPFIT software. Reasonable fitting could be achieved with a two compartment model with 1/Y data weighting. The modelling was performed for the time range between 0 and 72 h for males and 0 to 52 h for females. With exception of the mean residence times (MRTtotal, MRTabs, MRTdisp), all other toxicokinetic parameters (tmax, Cmax, t1/2 abs, t1/2 elim and AUC) and the single plasma concentration values were significant higher in male rats. This probably caused by a longer sojourn of test compound related radioactivity in the blood before excretion mainly via urine. The similar MRT-values on the other hand indicated that the absorption and elimination processes were comparable in both sexes.
All important results of the TOPFIT analysis are presented in Table 2.

Details on excretion:

The major route of excretion in the course of the whole experimental period of 72 h was by urine for both sexes. In male rats, ca. 77% of the total dose administered were detected in the urine, from which more than 75% were excreted within 24 h after dosing. Faecal excretion accounted for ca. 25%, from which ca. 24% were excreted within the first day after dosing. A higher renal excretion was observed for female rats. Ca. 85% of the administered dose was detected in the urine (ca. 84% excreted within 24 h) and ca. 10% in faeces (ca. 8% excreted on Day 1). The results are summarised in Table 3 and 4.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Metabolites in urine
In total, ca. 77% of the dose were detected in urine of male rats of which ca. 60% were identified and ca. 17% characterised (identification rate: ca. 77%). In female rats ca. 85% of the dose were detected in urine of which ca. 86% were identified and ca. 4% characterised (identification rate: ca. 82%).
The parent compound and the metabolite AE 1887196-dihydro were detected in the urines of both sexes in very low amounts (< 0.5% of the dose). The main metabolites were AE 1887196-N-desmethyl representing ca. 19–20% of the dose in both sexes, and AE 1887196-dihydro-N-desmethyl with 12.6% in males and 22.3% in females.
Further metabolites with values of more than 5% of the dose were identified as AE 1887196-dihydro-N-desmethyl-hydroxy (6.5% in males), the two rotamers of AE 1887196-dihydro-O-desmethyl (9.2% in males and 13.4% in females), AE 1887196-dihydro-N-desmethyl-hydroxy (6.5% in males), and AE 1887196-Odesmethyl (5.4% in females). The individual values for all other identified metabolites accounted for less than 5% of the dose. The metabolite AE 1887196-dihydro-di-Odesmethyl was excluded from quantification because sufficient amounts were not available for co-chromatographic experiments after isolation from the 0–24 h composite urine sample of male rats (metabolite fraction 4) even though it was identified by LC-MS as a trace compound. Additionally, several metabolites with less than 5% of the dose were investigated by LCMS/MS, but no exact chemical structure could be assigned due to the low quantities.

Extraction efficiency and metabolites in faeces
Composite samples of faeces from both tests were extracted using conventional methods. The resulting acetonitrile/water extracts represented between 95.1% and 99.3% of recovered radioactivity.
In total, ca. 25% of the dose were detected in faeces of male rats of which ca. 20% of the dose were identified and ca. 4.1% characterised (identification rate: ca. 83%). In female rats ca. 9.6% of the dose were detected in faeces of which ca. 7.6% of the dose were identified and ca. 1.9% characterised (identification rate: ca. 80%).
The parent compound was detected only in the faeces of female rats (< 1.4% of the dose). For AE 1887196-dihydro low levels of < 1% of the dose were found in both sexes. In males, AE 1887196-dihydro-N-desmethyl-hydroxy was the only metabolite representing more than 5% of the dose (6.5%). The individual values for all other identified metabolites in the faeces of males and females accounted for less than 3.0% of the dose.

Biotransformation pathway
AE 1887196 was extensively metabolised to approx. 29 metabolites of which 11 were identified by chromatographic and spectroscopic methods. Only trace amounts of the parent compound and AE 1887196-dihydro were found in the excreta. Nearly all metabolites were present in the urines of both sexes. The predominant metabolites were identified as AE 1887196-N-desmethyl and AE 1887196-dihydro-N-desmethyl. All other identified and characterised metabolites represented a minor part of the dose. The metabolic profiles in urines and faeces were very similar and comparable for both sexes. The principal metabolic reactions of [triazine-UL-14C]AE 1887196 in rats were:
- reduction of the keto group of parent compound to a hydroxyl group,
- to a very small extent (< 1% of the dose) conjugation of AE 1887196-dihydro with glucuronic acid,
- N- and O-demethylation reactions, and
- hydroxylation of the phenyl moiety of the main metabolites AE 1887196-N-desmethyl and AE 1887196-dihydro-N-desmethyl.

Please refer to the attachment for the identified metabolites and the proposed biotransformation pathway.

The assignment of parent compound and metabolites after a storage period of approx. one year was generally not affected because of the highly comparable metabolite patterns (fingerprint) of first and later chromatograms. Only the amounts of peaks lower than 5% of the dose might differ slightly.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Radioactive residues in organs and tissues at sacrifice
Approx. 0.09% of the total dose administered was detected in the bodies of male rats at the time of sacrifice 72 hours after dosing and an only very minor amount of 0.01% in the GIT. Residual concentrations of radioactivity in organs and tissues were in a range of

Any other information on results incl. tables

Table 1 Balance of radioactivity in excreta, and organs and tissues

Test number Sex No. of animals Spec. RA [MBq/mg] Dose [mg/kg bw] Test period [h]	1 Male 4 4.46 2 72	2 Female 4 4.46 2 72
Percent of total dose administered
Faeces Urine	24.97 76.80	10.20 85.25
Sum of excreta	101.77	95.45
Renal/faecal ratio	3.08	8.36
Body excluding GIT GIT Body	0.09 0.01 0.11	0.02 0.02 0.04
Balance	101.88	95.49
Percent of total dose recovered
Faeces Urine	24.45 75.44	10.69 89.27
Sum of excreta	99.90	99.96
Renal/faecal ratio	3.08	8.35
Body excluding GIT GIT Body	0.09 0.01 0.10	0.03 0.02 0.04
Balance	100.00	100.00
Norm.-factor	0.98	1.05
Absorption rate*	75.53	89.30

* sum of urine and body excluding GIT

 

Table 2 Toxicokinetic parameter of [triazine-UL-14C]AE 1887196 after oral administration to male and female rats

Test number Sex No. of animals Spec. RA [MBq/mg] Dose [mg/kg bw]	1 Male 4 4.46 2	2 Female 4 4.46 2
tmax[h] measured	0.67	0.33
tmax[h] modelled	0.55	0.35
Cmax[mg/kg] measured	3.60	1.31
Cmax[mg/kg] modelled	3.66	1.31
t1/2 abs[h]	0.11	0.06
t1/2 elim[h]	13.40	9.77
AUC0 -∞[mg/kgxh]	10.20	3.42
MRTtotal[h]	5.93	5.84
MRTabs[h]	1.87	1.65
MRTdisp[h]	4.06	4.19

Remarks:

Exponential analysis was performed with average values of the equivalent plasma concentrations of

four animals each for the time ranges of 0 to 72 (males) and 0 to 52 (females) hours after dosing.

Reasonable fitting was possible with a two compartment model with 1/Y data weighting.

AUC0 -∞Total area under the plasma radioactivity concentration-time curve extrapolated from time 0 to infinity [mg/kg x h for "C"; g/g x h for "Cnorm"]

t1/2 abs Half-life of the absorption phase [h]

t1/2 elim Half-life of the elimination phase [h]

tmax Time at which the maximum radioactivity concentration occurs in plasma following administration of an extravascular dose [h]

Cmax Maximum radioactivity concentration observed in plasma following administration of an extravascular dose [mg/kg for "C"; g/g for "Cnorm"]

MRT total Mean residence time describing the sojourn (elimination and/or metabolism) of the drug (or total radioactivity) in the body [h]

MRT abs Mean residence time of absorption [h]

MRT disp Mean residence time of disposition [h]

 

Table 3 Excretion of radioactivity via urine and faeces

Test number Sex No. of animals Spec. RA [MBq/mg] Dose [mg/kg bw]	1 Male 4 4.46 2	2 Female 4 4.46 2
Excretion of radioactivity via urine and faeces
Sampling period [h]
Faeces
0-24 24-48 48-72	23.22 1.19 0.07	8.34 1.22 0.65
Urine
0-4 4-8 8-12 8-24 24-48 48-72	48.42 16.61 3.84 7.32 0.51 0.10	28.95 30.20 --* 24.65 1.31 0.14
Sum total	101.77	95.45
Excretion in percent of total dose recovered
Faeces
0-24 24-48 48-72	23.22 1.17 0.07	8.74 1.27 0.65
Urine
0-4 4-8 8-12 8-24 24-48 48-72	47.68 16.20 3.78 7.18 0.50 0.10	30.29 31.59 --* 25.88 1.37 0.14
Sum total	99.90	99.96
Norm.-factor	0.98	1.05

* no sample collected

Table 4 Cumulative excretion of radioactivity via urine and faeces

Test number Sex No. of animals Spec. RA [MBq/mg] Dose [mg/kg bw]	1 Male 4 4.46 2	2 Female 4 4.46 2
Cumulative excretion of radioactivity via urine and faeces
Time [h]
Faeces
24 48 72	23.71 24.90 24.97	8.34 9.55 10.20
Urine
4 8 12 24 48 72	48.42 65.03 68.87 76.19 76.69 76.80	28.95 59.15 --* 83.80 85.11 85.25
Sum total	101.77	95.45
Cumulative excretion in percent of total dose recovered
Faeces
24 48 72	23.22 24.38 24.45	8.74 10.02 10.69
Urine
4 8 12 24 48 72	47.68 63.88 67.88 74.84 75.34 75.44	30.29 61.89 --* 87.76 89.13 89.27
Sum total	99.90	99.96
Norm.-factor	0.98	1.05

* no sample collected