Pyrasulfotole

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr 2003 - 18 Feb 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Aug 1995

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC, USA)
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: for oral experiments: 188 - 225 g, for intravenous experiments: 193 - 250 g
- Housing: following dosing, rats were housed individually in Nalgene rodent metabolism cages which allowed collection of urine, faeces, and respired 14CO2 and volatile metabolites
- Diet: Certified Rodent Diet 5002 Meal (PMI Nutrition International, St. Louis, USA), ad libitum
- Water: not further specified, ad libitum
- Acclimation period: 6 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50 - 58
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

other: oral or intravenous

Vehicle:

other: oral formulation: water; intravenous formulation: 0.9% sodium chloride

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

[Phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone Oral Dose Experiment (O-LDE-M)
A 0.2-mL aliquot of the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone stock solution was transferred into a 5-mL volumetric flask, and the aliquot was diluted to 5 mL with acetonitrile (ACN). The solution was radioassayed and concentrated slightly using a stream of nitrogen gas. An additional aliquot (0.03 mL) of the [phenyl-UL-14C] AE 0317309 stock solution was transferred into the 5-mL volumetric flask, and the solution was diluted to 5 mL with ACN. The solution was radioassayed and transferred to a 50-mL pear-shaped flask. A nonradioactive test substance stock solution was prepared by transferring 65.5 mg of nonradioactive test substance (K-1196) into a 10-mL volumetric flask and diluting the contents to mark with ACN. A 1.77-mL aliquot of the nonradioactive test substance stock solution was transferred to the 50-mL pear-shaped flask. The sample was concentrated to dryness at 30 to 35°C using a rotary evaporator (Büchi; Westbury, NY). The sample was transferred to a 10-mL, graduated mixing cylinder using multiple 1-mL rinses with H2O, and the sample was diluted to 7.0 mL with H2O. The dose solution was mixed, radioassayed (0.056 mCi/mL; 8.54 mCi/mmol; 2.37 mg/mL), and aliquots were analyzed by HPLC and mass spectrometry.

[Pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone Oral Dose Experiment (O-LDE-M)
A 0.2-mL aliquot of the [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone stock solution was transferred into a 10-mL volumetric flask, and the aliquot was diluted to 10 mL with ACN. The solution was radioassayed and transferred to a 50-mL pear-shaped flask. A nonradioactive test substance stock solution was prepared by transferring 72.4 mg of nonradioactive test substance (K-1196) into a 10-mL volumetric flask and diluting the contents to mark with ACN. A 1.87-mL aliquot of the nonradioactive test substance stock solution was transferred to the 50-mL pear-shaped flask. The sample was concentrated to dryness at 30 to 35°C using a rotary evaporator. The sample was transferred to a 10-mL, Reacti-Vial reaction vial (Pierce; Rockford, IL) containing a magnetic stir bar, using multiple 1-mL rinses with H2O, and the sample was diluted to 7.0 mL with H2O. The dose solution was stirred, radioassayed (0.054 mCi/mL; 8.72 mCi/mmol; 2.26 mg/mL), and aliquots were analyzed by HPLC and mass spectrometry.

[Phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone Intravenous Experiment (IV-LDE-M)
A 3.6-mL aliquot of the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone stock solution (C-938B) was transferred into a 10-mL volumetric flask, and the aliquot was diluted to 10 mL with ACN. The solution was radioassayed and concentrated slightly using a stream of nitrogen gas. An additional aliquot (1.0-mL) of the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone stock solution was transferred into the 10-mL volumetric flask, and the solution was diluted to 10 mL with ACN. The solution was radioassayed and transferred to a 50-mL pear-shaped flask. A nonradioactive test substance stock solution was prepared by transferring 65.0 mg of nonradioactive test substance (K-1267) into a 10-mL volumetric flask and diluting the contents to mark with ACN. A 1.90-mL aliquot of the nonradioactive test substance stock solution was transferred to the 50-mL pear-shaped flask. The sample was concentrated to dryness at 30 to 35°C using a rotary evaporator. The sample was transferred to a 10-mL, Reacti-Vial reaction vial containing a magnetic stir bar, using multiple 1-mL rinses with saline solution, and the sample was diluted to 8.3 mL with saline solution. The dose solution was stirred, radioassayed, and filtered using a syringe filter (0.22 μm). The filtered dose solution was mixed and radioassayed (0.063 mCi/mL; 10.0 mCi/mmol; 2.29 mg/mL), and aliquots were analyzed by HPLC and mass spectrometry.

[Pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone Intravenous Experiment (IV-LDE-M)
A 3.0-mL aliquot of the [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone stock solution (C-1024A) was transferred into a 10-mL volumetric flask, and the aliquot was diluted to 10 mL with ACN. The solution was radioassayed and transferred to a 50-mL pear-shaped flask. A 12.8 mg aliquot of nonradioactive test substance (K-1267) was transferred to the 50-mL pear-shaped flask. The sample was mixed and concentrated to dryness at 30 to 35°C using a rotary evaporator. The sample was transferred to a 10-mL, Reacti-Vial reaction vial containing a magnetic stir bar, using multiple 1-mL rinses with saline solution, and the sample was diluted to 7.1 mL with saline solution. The dose solution was stirred, radioassayed and filtered using a syringe filter. The filtered dose solution was stirred and radioassayed (0.062 mCi/mL; 9.97 mCi/mmol; 2.24 mg/mL), and aliquots were analyzed by HPLC and mass spectrometry.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

10 mg/kg bw/day

Remarks:

oral dose of [phenyl-UL-14C] AE 0317309

Dose / conc.:

9.81 mg/kg bw/day

Remarks:

intravenous dose of [phenyl-UL-14C] AE 0317309

Dose / conc.:

9.88 mg/kg bw/day

Remarks:

oral dose of [pyrazole-3-14C] AE 0317309

Dose / conc.:

9.6 mg/kg bw/day

Remarks:

intravenous dose of [pyrazole-3-14C] AE 0317309

No. of animals per sex per dose / concentration:

5 males per radiolabel (oral administration)
4 - 5 males per radiolabel (intravenous administration)

Control animals:

no

Details on study design:

- Rationale for animal assignment: Each rat was assigned a unique consecutive number at the time of arrival from Charles River Laboratories. The rats were chosen for inclusion in the experimental group based on overall healthy appearance, gradual weight gain over the acclimation period, and similar weights.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, tissues (bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, testes, and thyroid), cage washes
- Time and frequency of sampling:
urine: 6, 12, 24 and 48 hours post-treatment (in the [phenyl-UL-14C] AE 0317309 oral experiment also after 52 hours)
faeces: at 24-hour intervals until sacrifice (in the [phenyl-UL-14C] AE 0317309 oral experiment also after 52 hours)
blood and tissues: at sacrifice (in general 48 hours post-treatment; 52 hours in the [phenyl-UL-14C] AE 0317309 oral experiment)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling:
urine: 6, 12, 24 and 48 hours post-treatment (in the [phenyl-UL-14C] AE 0317309 oral experiment also after 52 hours)
faeces: at 24-hour intervals until sacrifice (in the [phenyl-UL-14C] AE 0317309 oral experiment also after 52 hours)
- From how many animals: 4-5 (pooled samples)
- Method type(s) for identification: radioassay and HPLC-MS-MS

Statistics:

All calculations were performed using Microsoft Excel 2002 (10.5815.4219; SP-2).

Preliminary studies:

A preliminary experiment was conducted in which male and female rats were administered a single oral dose of [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone at a rate of 100 mg/kg bw. In this preliminary experiment, the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone was well absorbed, with absorption of approximately 91% of the administered dose for the females and 88% for the males. The elimination of the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was very rapid, with the majority of the administered dose being excreted via urine during the first 24 hours following dosing for both male and female rats.
Radioactivity in the tissues was below 0.5% of the administered dose for both sexes at 72 hours after the administration. 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was observed as the major radioactive component in the urine and faeces, representing approximately 82% and 89% of the total excreted radioactivity in males and females, respectively.

Type:

absorption

Results:

The test substance was readily absorbed with 57 - 62% of the dose recovered in urine within 6 hours and 73 and 75% within 52 and 48 hours, respectively.

Type:

distribution

Results:

≤ 2% of the administered dose remained in the carcass and tissues at sacrifice. Residue levels were highest in liver and kidney.

Type:

metabolism

Results:

87 - 95% was excreted unchanged; three minor metabolites were identified in urine and faeces. The major metabolic pathway occurred via N-demethylation of the test substance.

Type:

excretion

Results:

Total excretion was rapid, > 96% was excreted within 24 hours (approx. 90% in the urine, 10% in the faeces)

Details on absorption:

[Phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone:
The [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was readily absorbed following oral administration, with 62% of the dose recovered in the urine within 6 hours and a total of 73% of the dose recovered in the urine at the time of sacrifice (52 hours). The intravenous experiment showed that 10% of the dose was found in the faeces at the time of sacrifice (48 hours).

[Pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone:
The [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was readily absorbed following oral administration with 57% of the dose recovered in the urine within 6 hours and a total of 75% of the dose recovered in the urine at the time of sacrifice (48 hours). The intravenous experiment showed that 8% of the dose was found in the faeces at the time of sacrifice (48 hours).

Details on distribution in tissues:

In all experiments, ≤ 2% of the administered dose remained in the carcass and tissues at sacrifice. In all experiments, the highest residues were found in the liver and kidney (see Table 1, attached background material).

Details on excretion:

[Phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone - oral experiment
The distribution of [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone residues among urine, faeces, cage wash, and tissues for each rat in the [phenyl-UL-14C] AE 0317309 O-LDE-M experiment is shown in Table 2 (attached background material). Following oral dosing, [phenyl-UL-14C] AE 0317309 residues were rapidly excreted (102% of the administered dose was excreted within 24 hours). The total radioactive recovery was 108% of the administered dose. The majority of the radioactivity was excreted in the urine (73%), with less being excreted in the faeces (31%). Much of the administered dose was rapidly excreted in the urine within 6 hours of dosing (62%).

[Pyrazole-3-14C] AE 0317309 - oral experiment
The distribution of [pyrazole-3-14C] AE 0317309 residues among urine, faeces, cage wash, and tissues for each rat in the [pyrazole-3-14C] AE 0317309 O-LDE-M experiment is shown in Table 2 (attached background material). Following oral dosing, [pyrazole-3-14C] AE 0317309 residues were rapidly excreted (106% of the administered dose was excreted within 24 hours). The total radioactive recovery was 111% of the administered dose. The majority of the radioactivity was excreted in the urine (75%), with less being excreted in the faeces (32%). Much of the administered dose was rapidly excreted in the urine within 6 hours of dosing (57%).

[Phenyl-UL-14C] AE 0317309 - intravenous experiment
The distribution of [phenyl-UL-14C] AE 0317309 residues among urine, faeces, cage wash, and tissues for each rat in the [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone IV-LDE-M experiment is shown in Table 2 (attached background material). Following intravenous dosing, [phenyl-UL-14C]5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone residues were rapidly excreted (96% of the administered dose was excreted within 24 hours). The total radioactive recovery was 100% of the administered dose. The majority of the radioactivity was excreted in the urine (87%), with less being excreted in the faeces (10%). Much of the administered dose was rapidly excreted in the urine within 6 hours of dosing (83%).

[Pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone - intravenous experiment
The distribution of [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone residues among urine, faeces, cage wash, and tissues for each rat in the [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone IV-LDE-M experiment is shown in Table 2 (attached background material). Following intravenous dosing, [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone residues were rapidly excreted (97% of the administered dose was excreted within 24 hours). The total radioactive recovery was 106% of the administered dose. The majority of the radioactivity was excreted in the urine (91%), with less being excreted in the faeces (8%). Much of the administered dose was rapidly excreted in the urine within 6 hours of dosing (84%).

Metabolites identified:

yes

Remarks:

, 3 minor metabolites were identified

Details on metabolites:

All individual residues which accounted for ≥ 5% of the administered dose were identified; additionally, some components that were present in smaller quantities were also identified. Identification of the residues was accomplished by comparison of the mass spectral data to that of authentic reference standards when available. Some residues were tentatively identified by comparison of their HPLC retention times with those of authentic reference standards or components which had been identified in other matrices.

The total amount of hydroxymethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone identified in the urine and faeces from both oral experiments was 2% of the administered dose. The total amount of hydroxymethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone identified in the urine and faeces from both intravenous experiments was 1 - 2% of the administered dose.

The total amount of desmethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone identified in the urine and faeces from both oral experiments was 8% of the administered dose. The total amount of desmethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone identified in the urine and faeces from both intravenous experiments was 6% of the administered dose.

The total amount of AE B197555 identified in the urine and faeces from the [phenyl-UL-14C] oral and intravenous experiment was 1% of the administered dose.

From all experiments, 87 to 95% of the administered dose was excreted unchanged as (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone. Hydroxymethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone, desmethyl 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone, and AE B197555 were observed as minor metabolites in the urine and faeces. Greater than 96% of the administered dose in each experiment was identified. The major metabolic pathway occurred via N-demethylation of the test substance.

See Table 3 for the metabolite profile in excreta and Figure 1 for structure, chemical name and common name of standards and metabolites (attached background material).

Conclusions:

The toxicokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics). The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. With the use of radioactive-labelled test material, the present study demonstrated that (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone was readily absorbed after oral administration and excretion was rapid. Greater than 96% of the administered dose was excreted within 24 hours following oral administration. In all experiments, the majority of the radioactivity was excreted in the urine. Faecal excretion represented 8 to 10% of the administered dose during the intravenous experiments.
No volatile residues were detected, and no mineralization was observed. Residue levels in all tissues were highest in liver and kidney.
The test substance was not greatly metabolised, as 87 to 95% of the administered dose was excreted unchanged. Three metabolites were identified in urine and faeces. The major metabolic pathway occurred via N-demethylation of the test substance to yield desmethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone.

Description of key information

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) No. 1907/2006 and ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), an assessment of the toxicokinetic behaviour of the target substance is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical properties and studies in which the toxicokinetic behaviour of (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone was investigated.

Based on a toxicokinetics study in rats (M-258714-01-1, 2005), it was observed that (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone was readily absorbed after oral administration and excretion of the test substance was rapid. In the preliminary phase of the study, absorption was approximately 88% and 91% of the orally administered dose for males and females, respectively. In the main phase of the study, >96% of the administered dose was excreted within 24 hours following oral administration. In all experiments, the majority of the radioactivity was excreted in the urine. Faecal excretion represented 8 to 10% of the administered dose during the intravenous experiments.

No volatile residues were detected, and no mineralization was observed. Residue levels in all tissues were highest in liver and kidney.

The test substance was not greatly metabolised, as 87 to 95% of the administered dose was excreted unchanged. Three metabolites were identified in urine and faeces. The major metabolic pathway occurred via N-demethylation of the test substance to yield desmethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone.

Based on the data from the toxicokinetic study, it is assumed that (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone is readily absorbed via the oral route. No dermal absorption data is available, however, systemic toxicity was observed in a sub-acute dermal study (M-263216-01-2, 2005), demonstrating (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone is dermally absorbed. Data relating to inhalation absorption is limited to an acute study (M-121743-01-2, 2004), in which some minor clinical findings were noted, implying some level of absorption may have taken place. A worst-case value of 100% inhalation absorption is used for the chemical safety assessment. For the purposes of DNEL route-to-route extrapolation (oral to inhalation) calculations, a value of 100% absorption is assumed for both routes (i.e. the rate or oral absorption in rat is considered to be equivalent to the rate of inhalation absorption in man), which is considered to be appropriate due to the high levels of oral absorption observed in the toxicokinetic study. Furthermore, based on the rapid excretion of the test material and its metabolites, it is concluded that there is no bioaccumulation potential.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - inhalation (%):

100

Additional information

A reliable study on the absorption, distribution, metabolism and excretion of 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone after single oral and intravenous doses in rats is available.

In the available toxicokinetic study (M-258714-01-1, 2005) that was conducted according to OPPTS 870.7485 and GLP, radiolabelled test substance was administered to male Wistar rats. Two different radiolabels were used: [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone and [pyrazole-3-14C] of 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone, with the radiolabel located in the phenyl ring and at position 3 of the pyrazol ring, respectively. Single oral doses of each radiolabel of the test substance were administered to five rats at dose rates of 10.0 and 9.88 mg/kg body weight, respectively. In two separate experiments, male Wistar rats with surgically implanted jugular cannulae were dosed intravenously with [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone (four rats) or [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone (five rats) at dose rates of 9.81 and 9.60 mg/kg body weight to investigate the absorption of the test substance.

In all experiments, urine and faeces were collected in separate containers cooled with dry ice. The weight of urine at each interval was determined, and aliquots were radio-assayed. Each faeces sample was weighed, homogenized and aliquots of the faecal homogenates were oxidized and radio-assayed. All urine and faeces samples were stored frozen in a freezer (<-18°C) when not in use. Following completion of each experiment, each metabolism cage was washed with methanol/water (Me0H/H2O; 1:1), and the washes from each cage were separately radio-assayed. The urine and faeces collection containers were also washed with Me0H/H2O (1:1), and the combined washes from the urine and faeces containers for each rat were radio-assayed.

At the conclusion of each experiment individual rats were anesthetized with halothane and each rat was exsanguinated through cardiac puncture using a 10-mL disposable syringe pre-treated with heparin. Triplicate blood samples were pipetted into combustion pads for oxidation and radio-assay. In all of the experiments, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, testes, and thyroid were collected and weighed; aliquots of each tissue were oxidized and radio-assayed. The gastrointestinal tract (GIT) of each rat was collected and weighed, and the entire sample was homogenized, oxidized and radio-assayed. The remaining carcass was dissolved in 3 N ethanolic potassium hydroxide (KOH) solution and aliquots of the KOH solution were radio-assayed. In an attempt to detect and collect respired 14C02 and all other volatile compounds in the [phenyl-UL-14C] and [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone oral dose experiments, the rats were housed in metabolism cages equipped with a flow-through system which allowed for the separation and collection of respired gases. Respired gas collection was discontinued 24 hours post-treatment. Respired gases were not collected during the [phenyl-UL-14C] and [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone intravenous (i.v.) experiments. Identification and quantitation of the metabolites in the urine samples and faeces extracts were accomplished by using reverse phase HPLC and MS/MS.

Preliminary Experiment

A preliminary experiment was conducted in which male and female rats were administered a single oral dose of [phenyl-UL-14C]5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl)methanone at a rate of 100 mg/kg body weight. [phenyl-UL-14C]5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was well absorbed, with absorption of approximately 91% of the administered dose for the females and 88% for the males. The elimination of the [phenyl-UL-14C]5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was very rapid, with the majority of the administered dose being excreted via urine during the first 24 hours following dosing for both male and female rats. Radioactivity in the tissues was below 0.5% of the administered dose for both sexes at 72 hours after the administration. [phenyl-UL-14C]5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was observed as the major radioactive component in the urine and faeces, representing approximately 82% and 89% of the total excreted radioactivity in males and females, respectively.

Absorption

[Phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone:

The [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was readily absorbed during the oral experiment, with 62% of the dose recovered in the urine within 6 hours and a total of 73% of the dose recovered in the urine at the time of sacrifice (52 hours). The [phenyl-UL-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone i.v. experiment showed that 10% of the dose was found in the faeces at the time of sacrifice (48 hours).

[Pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone:

The [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone was readily absorbed during the oral experiment, with 57% of the dose recovered in the urine within 6 hours and a total of 75% of the dose recovered in the urine at the time of sacrifice (48 hours). The [pyrazole-3-14C] 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone i.v. experiment showed that 8% of the dose was found in the faeces at the time of sacrifice (48 hours).

Distribution

In all experiments, <2% of the administered dose remained in the carcass and tissues at sacrifice. In all experiments, the highest residues were found in the liver and kidney.

Excretion

Following dosing, test substance residues were rapidly excreted (96 - 106% of the administered dose was excreted within 24 hours). The total radioactive recoveries ranged from 100 - 111% of the administered dose. The majority of the radioactivity was excreted in the urine (73 to 75% and 83 to 91% in the oral and intravenous experiments, respectively), with less being excreted in the faeces (8 to 10% and 31 to 32% in the intravenous and oral experiments, respectively). Much of the administered dose was rapidly excreted in the urine within 6 hours of dosing (56.8 - 84.1%).

Metabolite Identification and Characterization

All individual residues which accounted for > 5% of the administered dose were identified; additionally, some components that were present in smaller quantities were also identified. Identification of the residues was accomplished by comparison of the mass spectral data to that of authentic reference standards when available. Some residues were tentatively identified by comparison of their HPLC retention times with those of authentic reference standards or components which had been identified in other matrices.

Proposed Metabolic Pathway

From all experiments, 87% to 95% of the administered dose was excreted as unchanged test substance.

Hydroxymethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone, desmethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone and AE B197555 were observed as minor metabolites in the urine and faeces. Greater than 96% of the administered dose in each experiment was identified. The major metabolic pathway occurred via N-demethylation of the test substance.

Conclusions

The excretion of the test substance was rapid. Following oral administration, greater than 96% of the administered dose was excreted within 24 hours. In all experiments, the majority of the radioactivity was excreted in the urine. Faecal excretion represented 8 to 10% of the administered dose during the intravenous experiments. No volatile residues were detected, and no mineralization was observed. Residue levels in all tissues were highest in liver and kidney. The excretion profile indicates that the test substance was rapidly absorbed after gavage administration.

Once absorbed, the test substance was not greatly metabolised, as 87 to 95% of the administered dose was excreted unchanged. Hydroxymethyl (5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone, desmethyl 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone and AE B197555 were observed as minor metabolites in the urine and faeces. Greater than 96% of the administered dose in each experiment was identified. The major metabolic pathway occurred via N-demethylation of the test substance to yield desmethyl 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone.

 

Supporting information on the toxicokinetic behaviour:

In accordance with Regulation (EC) No. 1907/2006, Annex VIII, Column 1, Item 8.8 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), supporting evidence on the toxicokinetic behaviour was obtained based on the available substance-specific data on physico-chemical and toxicological properties. The test substance is a solid beige coloured powder with a water solubility of 69.1 g/L at 20 °C, a molecular weight of 362.3 g/mol and a vapour pressure 6.8E-9 hPa at 25 °C. The octanol/water partition coefficient (log Pow) was determined to be -1.362 at 23 °C, pH 7.

Absorption

In general, absorption of a substance depends on the potential to cross biological membranes, which is determined by the molecular weight, the log Pow and water solubility. Mostly, substances cross the membranes by passive diffusion, which requires sufficient solubility in water and lipids, a capability, which is described by the log Pow. In general, log Pow values between -1 and 4 are favourable for absorption whereas ionic substances are thought not to readily diffuse across biological membranes. Chemicals that do not offer these properties may be absorbed via active processes including facilitated diffusion, active transport or pinocytosis (ECHA, 2017).

Oral:

In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (>1 mg/L). As the test substance has a molecular weight of 362.3 g/mol, a water solubility of 69.1 g/L (at 20 °C) and a log Pow value of -1.362 (at 23 °C and pH 7), systemic exposure by oral absorption is anticipated. The toxicokinetic study in rats (M-258714-01-1, 2005) showed that the test compound was readily absorbed from the intestinal lumen. Therefore, (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone is considered to be absorbed along the gastrointestinal tract. This is also supported by systemic toxicity, which occurred after oral administration of the test substance in various repeated dose toxicity studies.

Dermal:

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low if the water solubility is < 1 mg/L; low to moderate if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. However, if water solubility is above 10,000 mg/L and the Log Pow is below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption (ECHA, 2017).

The log Pow is predicted to fall in the range of > - 1.362 < 1 at the pH of the skin (4.5 - 5.5). The log Pow, water solubility and molecular weight of (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone indicate that the substance is likely to be absorbed via the skin.

In a 28-day repeated dose dermal toxicity study, the NOAEL was considered to be 10 mg/kg bw/day, based on the treatment-related systemic findings in animals administered 100 and 1000 mg/kg bw/day (M-263216-01-2, 2005). The observed adverse effects show that the substance is absorbed through the skin, although it is not possible to quantify the rate of absorption.

Inhalation

(5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone is a solid with very low vapour pressure (6.8E-9 hPa at 25 °C), and therefore low volatility. Under normal use and handling conditions, inhalation exposure and availability for respiratory absorption of the substance in the form of vapours, gases, or mists is considered to be negligible (ECHA, 2017). However, the substance may be available for inhalatory absorption after inhalation of aerosols, if the substance is sprayed (e.g. as a formulated product). In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed. The particle size distribution of (5-hydroxy-1,3-dimethylpyrazol-4-yl)(2-mesyl-4-trifluoromethylphenyl) methanone shows that the average of the median particle size D50 was 11.5 µm while the D10 was 1.7 µm. 90% of the particle volume or particle mass had a lower particle diameter than 38.4 µm (D90). This indicates that the majority of the substance has the potential to be inhaled, ≥90% has the ability to reach the thoracic region and ≥50% has the ability to penetrate into the alveolar region following exposure via the inhalation route.

Minimal treatment-related effects were observed in the acute inhalation toxicity study (M-121743-01-2, 2004), these were limited to ungroomed hair / coat, piloerection and mild hypothermia noted during the clinical observations, which were transient and resolved by Day 4 post-exposure This indicates some level of absorption may have occurred, however, it is not possible to quantify this. Due to the limited information available, a worst-case approach is taken and absorption via the inhalation route is assumed to be 100%.

For the purposes of long-term systemic DNEL route-to-route extrapolation (oral to inhalation) calculations, a value of 100% absorption is assumed for both routes (i.e. the rate or oral absorption in rat is considered to be equivalent to the rate of inhalation absorption in man), which is considered to be appropriate due to the high levels of oral absorption observed in the toxicokinetic study. Furthermore, based on the rapid excretion of the test material and its metabolites, it is concluded that there is no bioaccumulation potential.

The distribution, metabolism and excretion properties for 5-hydroxy-1,3-dimethylpyrazol-4-yl)(α,α,α-trifluoro-2-mesyl-p-tolyl) methanone are discussed above under study M-258714-01-1 (2005).

Reference list:

ECHA (2017). Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance. Version 3.0, November 2017. European Chemicals Agency, Finland.

ECHA (2012). Guidance on information requirements and chemical safety assessment, Chapter R.8: Characterisation of dose [concentration]-response for human health. Version 2.1, November 2012. European Chemicals Agency, Finland.