Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun 2007 to 20 Sep 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Objective of study:

distribution

excretion

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

Apr 1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Aug 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Version / remarks:

Dec 1994

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nohsan No 8147.

Version / remarks:

Nov 2000

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 7 weeks (high dose), 13 weeks (low dose)
- Weight at study initiation: Low dose: 217 - 224 g (males), 184 - 199 g (females); High dose: 157 - 170 g (females), 126 - 143 g (females)
- Housing: All animals were group housed in stock cages during the initial acclimatisation period. On study animals were housed individually in all glass metabolism cages.
- Diet: ad libitum
- Water: Mains water, supplied by a plastic bottle, ad libitum
- Acclimation period: 7 days (low) 1 day (high)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40 – 70
- Air changes pe hour: 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Jun 2007 To: 20 Sep 2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
- Both dose preparations were formulated as homogenous suspensions and comprised unlabelled and [14C]-test substance in the dose vehicle. For more details see Table 1 in 'Any other information on materials and methods incl. tables'.
- Low dose level: An appropriate amount (14.53 mg) of the [14C]-test substance was accurately weighed into a 5 mL volumetric flask. An appropriate volume (371 μL, equivalent to 4.061 mg) of 10.95 mg/mL test substance stock solution (prepared by dissolving 109.5 mg test substance into 10 mL acetonitrile) was accurately dispensed into the 5 mL volumetric flask. The flask was made up to volume with acetonitrile and gently shaken until the compound was dissolved. Aliquots were removed to determine the radio-diluted specific activity. The solution was transferred with washings of acetonitrile to a mortar board and gently evaporated to dryness under a steady stream of nitrogen. Once dried, the content of the mortar board was lightly wetted with corn oil and ground into a fine paste with a pestle. The paste was then transferred to a pre-weighed glass container. Additional corn oil was added to the mortar board again and ground with the pestle before transferring into the glass container. This process was repeated several times before the contents of the glass jar were then up to the final dose volume to achieve the required target concentration.
- High dose level: The high dose was prepared under another study An appropriate amount (1.818 mg) of test substance was accurately weighed into a 10 mL volumetric flask. An appropriate amount (19.4 mg) of [14C]-test substance was accurately weighed in a glass vial, dissolved in acetonitrile and transferred (with washings), to the 10 mL volumetric flask. The flask was made up to volume with acetonitrile, and gently shaken until the compound was dissolved. Aliquots were removed to determine the radio-diluted specific activity. The high dose formulation was prepared as detailed above using the same method as the low dose formulation.

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg diet

Remarks:

Group 1. Low dose excretion and tissue distribution

Doses / concentrations 2

Dose / conc.:

75 mg/kg diet

Remarks:

Group 2. High dose excretion and tissue distribution

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

- Dose selection rationale: A dose level of 1 mg/kg was selected to represent a no-effect dose following a single oral administration. A dose level of 75 mg/kg was selected, following preliminary dose range finding work, to represent a dose that caused a mild and transient toxicological or pharmacological effect following single oral administration. Each rat was given a single oral dose of nominally 1 mg or 75 mg [14C]- test substance /kg and housed individually in a metabolism cage. Urine, faeces and cage washes were collected at intervals over 7 days after which time the rats were terminated. Selected tissues were removed and together with the residual carcass were analysed for residual radioactivity.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY
- Dose administration: The formulations were administered by gastric gavage at a target dose volume of 5 mL/kg to achieve target dose levels of 1 mg/kg and 75 mg/kg for low and high dose levels, respectively. For both dose levels, the animals received a target radioactive dose of 5 MBq/kg. Each animal was accurately weighed on the day of dosing. The syringes were weighed prior to and following each dosing. The actual dose received by each animal was determined with reference to the radioactive concentration, the weight of dose administered and the calculated specific activity of the dose formulation.
- Tissues and body fluids sampled: Urine and faeces were collected individually and separately by collection over solid carbon dioxide (up to 48 h post dose). Urine and faeces were frozen immediately upon collection. At the end of each faeces collection period, cage wash samples were collected (water).
- Time and frequency of sampling: Urine was collected at intervals of 12, 24, 48, 72, 96, 120, 144 and 168 hours after dosing. Faeces and cage wash were collected at intervals of 24, 48, 72, 96, 120, 144 and 168 hours after dosing.
To investigate pharmacokinetics, terminal blood samples were collected via the vena cava at 168 hours after dosing.
- Other: Animals were terminated by overexposure to anaesthetic vapour followed by cervical dislocation. Each terminal blood sample was divided between two heparinised tubes, one of which was centrifuged to separate plasma. The following tissues were taken for radioactivity analysis: adrenals, bone mineral, brain, renal fat, heart, kidneys, liver, lungs, muscle, ovaries, pancreas, spleen, testes (males), thymus, thyroid, uterus (females), gastrointestinal tract plus contents and residual carcasses. All samples were analysed for radioactivity by liquid scintillation counting either directly or following sample oxidation.

METABOLITE CHARACTERISATION STUDIES
Metabolite characterisation was undertaken in a separate study.

ANALYTICAL METHOD
- Sample oxidation: Duplicate samples (where possible) were oxidised in a Packard Tri-Carb 307 Automatic Sample Oxidiser. The [14C]-carbon dioxide generated was absorbed and mixed with scintillant, prior to analysis by liquid scintillation counting. The efficiency of oxidation of test samples relative to [14C]-standard oxidation efficiencies, which was determined at regular intervals during each series of oxidations. Combustion of standards showed that recovery efficiencies were all greater than 97 %.
- Liquid scintillation counting: All samples prepared in scintillation fluid were subjected to liquid scintillation counting for 5 minutes, together with representative blank samples, using a Packard TR 2100 Liquid Scintillation Analyser with automatic quench correction by an external method. Where possible, samples were analysed in duplicate and allowed to heat and light stabilise prior to analysis. Prior to calculation of each result, a background count rate was determined and subtracted from each sample count rate.

Statistics:

Not applicable.

Results and discussion

Preliminary studies:

Not applicable.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

Seven days after dosing the low dose level rats, the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level rats, radioactive residues were only detectable in the liver (males and females) and kidney (males only). These findings were consistent with the rapid and extensive excretion observed in both sexes. Tissue distribution was similar in both sexes. In mean blood, plasma and other tissues the total radioactivity was below the limit of reliable detection.

Details on excretion:

Excretion of the test substance was fairly rapid, with the majority of the administered radioactivity excreted by 48 hours post dose (89.8 to 102.4% of applied dose). Rapid excretion with main route via faeces (77.3 to 83.0 %) and also (13.3 to 27.1 %) via urine. The routes and rates of excretion were similar for males and females for both low and high dose level animals, although urinary excretion appeared to be slightly greater in females.

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

ANALYSIS OF RADIOLABELLED TEST SUBSTANCE

The HPLC and TLC radiochemical purities of [14C]- test substance as determined prior to the preparation of the dose formulation were 99.7 % and 100.0 % respectively. These purities were considered acceptable and the radiolabelled test substance was used without repurification. The mean radiochemical purities of [14C]-test substance in the low dose level formulation, as determined in pre-dose and post-dose aliquots, were 100.0 %, as determined by both HPLC and TLC. These data indicate that the test material was stable during dose preparation and for the duration of the dosing procedure. The mean radiochemical purities of [14C]-test substance in the high dose level formulation, as determined in pre-dose and post-dose aliquots, was >99.8% and >100.0 %, as determined by HPLC and TLC, respectively. These data indicate that the test material was stable during dose preparation and for the duration of the dosing procedure.

 

ACHIEVED DOSES

The mean achieved concentration of test substance for the low dose formulation, based upon the radioactive analysis, was 0.207 mg/g and 1.013 MBq/g of dose solution. The mean achieved concentration for the high dose formulation was 17.531 mg/g and 1.104 MBq/mg of dose solution. The achieved concentrations for low and high dose levels (taking the specific gravity of corn oil (0.9 g/mL) into account) were within 7 % and 1 % of the test substance target concentrations, respectively. The homogeneity of [C14]-test substance in the low and high dose level preparations was satisfactory. The radiochemical stability of test substance in the dose preparation was also shown to be satisfactory for at least 14 days which covered the period of use in the present study

 

CLINICAL OBSERVATIONS

No clinical observations were observed in either dose group during the study.

RECOVERY OF RADIOACTIVITY

The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 1 mg/kg were 106.0 % for males and 106.4 % for females. The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 75 mg/kg were 95.7 % for males and 100.5 % for females.  

DETAILS ON DISTRIBUTION

- Single low dose: Seven days after dosing, mean blood and plasma concentrations of total radioactivity were below the limit of reliable detection. The highest mean concentration was noted in the liver with values of 0.010 μg equiv/g and 0.005 μg equiv/g in males and females respectively. The kidney also had detectable levels, with mean values of 0.003 μg equiv/g and 0.002 μg equiv/g for males and females respectively. All other tissues had measurements below the limit of reliable detection.

- Single high dose: Seven days after dosing, mean blood and plasma concentrations of total radioactivity were below the limit of detection. The highest mean concentrations were found in the liver with values of 0.586 μg equiv/g and 0.284 μg equiv/g in males and females respectively. In males, the kidney also had detectable levels of total radioactivity with a mean concentration of 0.121μg equiv/g. In females, mean concentrations in the kidney were below the limit of reliable detection. All other concentrations in males and females were below the limit of reliable detection at this terminal time point.

DETAILS ON EXCRETION

- Single low dose: Following a single oral dose of 1 mg [C14]-test substance/kg, the major route of elimination was via the faeces in both males and females, with means of 83.0 and 77.3% of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 19.6 and 27.1 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was rapid with the majority of the administered radioactivity being excreted in the first 48 h post dose (mean of ca 99.1  % and 102.4 % in males and females, respectively). There was no significant radioactivity remaining in the carcass or gastrointestinal tract, indicating excretion was complete by 168 hours post dose.

- Single high dose: Following a single oral dose of 75 mg [C14]-test substance /kg, the major route of elimination was via the faeces in both males and females, with means of 79.4 and 78.5 % of the administered radioactivity recovered by seven days, respectively. Urinary excretion accounted for means of 13.3 and 17.5 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 89.8 and 92.2 % in males and females, respectively). There was no significant radioactivity remaining in the carcass or gastrointestinal tract, indicating excretion was complete by 168 hours post dose.

Table 3 Distribution of radioactivity tissues/organs 168 hours after administration of a single oral dose of [14C]-test substance to rats

Tissue/organ	Group mean tissue residues (µg equivalents of test substance/g )
	Group 1		Group 2
	1 mg/kg		75 mg/kg
	Male (n=4)	Female (n=4)	Male (n=4)	Female (n=4)
Adrenals	<0.002	<0.002	<0.147	<0.079
Bone Mineral	<0.001	<0.001	<0.013	<0.003
Brain	<0.001	<0.001	<0.008	<0.003
Fat- renal	<0.001	0.005	<0.022	<0.218
Heart	<0.001	<0.001	<0.023	<0.00001 -B
Kidneys	0.003	0.002	0.121	<0.057
Liver	0.010	0.005	0.586	0.284
Lungs	<0.001	<0.001	<0.035	<0.005
Muscle	<0.001	<0.001	<0.025	<0.027
Ovaries	NA	<0.002	NA	<0.071
Pancreas	<0.001	<0.001	<0.042	<0.042
Plasma	<0.001	<0.001	<0.007	<0.010
Residual Carcass-A	<0.0001 -B	<0.001	<0.088	<0.053
Spleen	<0.001	<0.001	<0.032	<0.010
Testes	<0.001	NA	<0.012	NA
Thymus	<0.001	<0.001	<0.011	<0.00001 -B
Thyroid	<0.003	<0.002	<0.087	<0.073
Uterus	NA	<0.001	NA	<0.002
Whole Blood	<0.002	<0.001	<0.058	<0.054

A = Value includes residual carcass and blood.

B = The number of significant figures has been raised (to a maximum of 5 decimal places) where appropriate when the value of detection is below the LOD.

Table 4 Recovery of radioactivity in tissues and excreta after administration of a single oral dose of [14C]-test substance to rats

		Group mean excretion data (percentage of radioactive dose recovered)
		Group 1 1mg/kg		Group 2 75 mg/kg
		Male (n=4)	Female (n=4)	Male (n=4)	Female (n=4)
Urine	0 - 12 h 12 - 24 h	14.7 3.2	20.5 4.7	7.5 3.5	7.8 6.8
	24 - 48 h	1.2	1.5	1.7	2.4
	48 - 72 h	0.3	0.3	0.3	0.3
	72 - 96 h	0.1	0.1	0.1	0.1
	96 - 120 h	0.0	0.0	0.1	0.1
	120 - 144 h	0.0	0.0	0.0	0.0
	144 - 168h	0.0	0.0	0.0	0.0
	Subtotal	19.6	27.1	13.3	17.5
Faeces	0 - 24 h	66.8	61.2	58.6	50.4
	24 - 48 h	13.2	14.5	18.4	24.8
	48 - 72 h	2.1	1.3	1.7	2.8
	72 - 96 h	0.7	0.2	0.4	0.3
	96 - 120 h	0.2	0.1	0.2	0.1
	120 - 144 h	0.1	0	0.1	0.1
	144 - 168h	0	0	0.1	0
	Subtotal	83	77.3	79.4	78.5
Cage wash		3.3	1.9	2.9	4.4
GI tract + contents		0.0	0.0	0.0	0.0
Tissues + carcass		<0.1	<0.1	0.1	<0.1
Total Recovery		106.0	106.4	95.7	100.5

Applicant's summary and conclusion

Conclusions:

Following single oral administration of 1 or 75 mg [14C]-test substance to rats, elimination was rapid, with the majority of the administered radioactivity excreted by 48 h post dose. The major route of elimination was via the faeces, with urinary elimination also an important route of excretion. The routes and rates of excretion were similar for males and females for both low and high dose level animals, although urinary excretion appeared to be slightly greater in females. Seven days after dosing the low dose level rats the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level rats radioactive residues were only detectable in the liver (males and females) and kidney (males only). These findings were consistent with the rapid and extensive excretion observed in both sexes. Tissue distribution was similar in both sexes.

Executive summary:

In an OECD TG 417 study in compliance with GLP, eight males and 8 female rats were given a single oral dose of 1 or 75 mg [14C]-test substance/kg to investigate the excretion of radioactivity over seven days. After this period, the rats were killed and residual radioactivity was measured in blood and plasma, selected tissues and the remaining carcasses.

Following a single oral dose of 1 mg [14C]-test substance//kg, the major route of elimination was via the faeces in both males and females, with means of 83.0 and 77.3 % of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 19.6 and 27.1 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 99.2 and 102.4 % in males and females, respectively). The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 1 mg/kg were 106.0 % for males and 106.4 % for females. Following a single oral dose of 75 mg [14C]-test substance//kg, the major route of elimination was via the faeces in both males and females, with means of 79.4 and 78.5 % of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 13.3 and 17.5 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 89.8 and 92.2 % in males and females, respectively). The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 75 mg/kg were 95.7 % for males and 100.5 % for females. Seven days after dosing the low dose level rats, radioactive residues in the majority of tissues were very low and the tissue distribution of radioactivity was similar for both sexes. The liver and kidney were the only two tissues with detectable residues, which were just over the limit of reliable detection.

Seven days after dosing the high dose level rats, radioactive residues were below the limit of reliable detection in all tissues apart from the liver and kidney, and tissue distribution was similar in both sexes. The liver contained mean concentrations of 0.586 and 0.284 μg equiv/g, in males and females respectively. The kidney contained detectable levels in males (0.121 μg equiv/g), but was below the limit of reliable detection in females.

Following single oral administration of 1 mg [14C]-test substance/kg or 75 mg [14C]-test substance/kg to rats, elimination was rapid with the majority of the administered radioactivity excreted by 48 hours post dose. The major route of elimination was via the faeces, with urinary elimination also an important route of excretion. The routes and rates of excretion were similar for males and females and for both the low and high dose level animals, although urinary excretion appeared to be slightly greater in females. Seven days after dosing the low dose level the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level radioactive residues were only detectable in the liver (males and females) and kidney (males only).

Hence, the findings were consistent with the rapid and extensive excretion via faeces and urine observed in both sexes. Tissue distribution was similar in both sexes.