1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

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:

28 Nov 2012 - 06 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted in 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted in 1998

Qualifier:

according to guideline

Guideline:

other: Japan/FAMIC-ACIS Annex 2.3 1 to Notification 12 Nousan 8147: Studies of Metabolic Fate in Animals

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Specific details on test material used for the study:

RADIOLABELLING INFORMATION
- Radiochemical purity: > 98% for all aliquots of the test compound
- Specific activity: 3.92 MBq/mg (105.93 μCi/mg) for test 1 to 6 and test 9, 3.81 MBq/mg (103.07 μCi/mg) for test 7 and 8
- Locations of the label: pyrazole-carboxamide moiety

Radiolabelling:

yes

Remarks:

pyrazole-carboxamide-14C

Species:

rat

Strain:

other: Wistar Unilever HsdCpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland
- Age at study initiation: 6 - 7 weeks (males), 8 - 9 weeks (females)
- Weight at study initiation: approximately 200 g
- Housing: individual in Makrolon metabolism cages
- Diet: rat/mice maintenance long life diet (V1574-000 Ered I or V1534-000, R/M-H, 10 mm, ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 22.3
- Humidity (%): 22.3 - 61.1
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Six aliquots of the radiolabelled test compounds were dissolved in acetonitrile plus 0.1% acetic acid and stored in a freezer at min. -18°C. For each administration suspension, an adequate aliquot of the corresponding stock solution was concentrated to near dryness under a gentle stream of nitrogen. For the high-dose experiments (test 3, 4, 7 and 8) the radiolabelled test compound was radiodiluted with the non-radiolabelled test compound (ratio of 1:200 or 1:1). The individual portions of test compound were formulated with 0.5% aqueous Tragacanth and the pH was adjusted to pH 4 with acetic acid. Afterwards the individual administration suspensions were stirred overnight at approx. 4°C and at room temperature during the administration process.

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

2 other: mg/kg bw (nominal)

Remarks:

Actual dose: 1.94 - 2.01 and 1.90 - 2.11 mg/kg bw in males and females, respectively
see table 1

Dose / conc.:

20 other: mg/kg bw (nominal)

Remarks:

Actual dose: 23.63 and 20.88 mg/kg bw in males and females, respectively
see table 1

Dose / conc.:

200 other: mg/kg bw (nominal)

Remarks:

Actual dose: 191.72 and 197.97 mg/kg bw in males and females, respectively
see table 1

No. of animals per sex per dose / concentration:

3 (test 6 and 9)
4 (test 1 - 5 and test 7, 8)

Control animals:

no

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the toxicological properties of the test substance. The low dose level was fixed at 2 mg/kg bw, which is in the range of the NOEL of the subchronic toxicological studies. Dose levels of 20 and 200 mg/kg bw were selected for the high dose experiments, which are sufficiently below the LD50 value.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, plasma, bile, organs and tissues
- Time and frequency of sampling: see table 2
Method type(s) for identification: Liquid scintillation counting

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, bile
- Time and frequency of sampling: see table 2
- From how many animals: samples pooled see table 3
- Method type(s) for identification: HPLC-UV, ESI-MS/MS, NMR

- Limits of detection and quantification:
HPLC analysis: All peaks corresponding to a radioactivity signal approximately 2.5 x background noise were integrated.
Liquid scintillation counting: The limit of detection (LOD) was predefined on the base of the background radioactivity-counting rate, which was in the range between 10 – 36 cpm, depending on the instrument used. For the calculations, a reference point of 20 cpm was set as average background radioactivity. The LOD was established as twice the average background radioactivity. Single background subtraction and a quench and counting efficiency correction for transformation of gross counts (cpm) into net counts (dpm) were automatically performed by the instruments. Samples with individually measured values below 20 dpm (after correction for the background radioactivity) were not quantified and labelled as n.q. (= LOQ = (average background activity x freeze drying factor / representative sample weight) x (1 / specific radioactivity of the test compound)

Statistics:

The values of the radioactivity measurements were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outliers were marked and not used for the calculations of arithmetic means, standard deviations [SD] or correlation coefficients (CV). Also, the limit of quantitation (LOQ) was determined for the radioactivity measurements. The decision, if a value was below the LOQ was ascertained with the 1/3-2/3 rule: if more than 1/3 of the
single values which were used for the calculation of the mean value were below the LOQ, the calculated mean value was also set below the LOQ and was not considered in arithmetic means.

Type:

absorption

Results:

41.2 and 29.3% for males and females, respectively

Type:

distribution

Results:

< 0.4% (low and high dose rats) and approximately 1% (bile-duct cannulated rats) of the administered dose was recovered in the bodies (excluding gastrointestinal tract); higher organ concentrations in females compared to males

Type:

metabolism

Results:

Parent compound (PC) was the main compound in the excreta (51.32 - 66.42% and 88.84 - 107.98% of the recovered dose in low and high dose tests, respectively). PC-deshydrochloro-dihydrate, PC-dihydroxy and PC-hydroxy-N-methyl represent major metabolites.

Type:

excretion

Results:

> 99% of the recovered dose excreted via urine and faeces within 72 h after administration with the exception of bile duct-cannulated rats (94% and 99% for males and females, respectively)

Details on absorption:

The absorption of the test substance started immediately after administration as shown by the concentration of radioactivity in the plasma of the low dose tests (2 mg/kg bw). For all low dose tests and the high dose test at 20 mg/kg bw with male rats, the maximum plasma concentration (Cmax) was reached within 1 h (tmax) after the administration. The highest plasma concentration was measured at 7 h for females of the high dose test with 20 mg/kg bw. Female rats showed a higher absorption rate for plasma compared to male rats. There was no radioactivity detected in all plasma samples of the high dose tests with 200 mg/kg bw and in samples at 48 h and 72 h of the tests with 20 mg/kg bw. Low dose tests with bile-duct cannulated males and females showed that about 35% for males and 24% for females of the recovered dose was distributed via the enterohepatic circulation. Absorption rates were calculated by summation of the recovered radioactivity in urine, bile and body without gastrointestinal tract and amounted to 41.2% for males and 29.3% for females.

Details on distribution in tissues:

In males and females of the low dose group (2 mg/kg bw) the maximum of the plasma concentration of the radioactivity was measured approx. 1 h after dosage (tmax). In the high dose tests with 20 mg/kg bw, tmax was reached at 1 h for males and delayed to approx. 7 h for females after dosing. In all low dose tests the maximum of the dosed normalised concentration ranged from 0.131 to 0.199 (Cmax). In tests with 20 mg/kg bw Cmax amounted to ≤ 0.008 only, due to a restricted absorption behaviour of the test compound. For tests with 200 mg/kg bw there was no radioactivity detected in the plasma samples. For all low dose tests the plasma concentration declined to < 2% of Cmax within 72 h post administration indicating no retention of compound related residues in the body of the animals. The calculated AUC0 - ∞-value for low dose females was approx. 2-times higher compared to males after oral administration. High dose rats at 20 mg/kg bw showed an approx. 20-times lower exposure compared to low dose rats, due to the restricted absorption at higher dose levels.

There were very low residues in organs and tissues of all low and high dose tests. Female rats showed higher organ concentrations compared to male rats.
At sacrifice < 0.4% of the administered dose (mean values) was found in the bodies (excluding GIT) of the low and high dose rats and approx. 1% in bodies of the bile duct-cannulated. Negligible amounts of radioactivity were detected in the GITs (0.002 to 0.178% of dose), excluding GITs from bile-duct cannulated rats, showing that the elimination of the radioactivity was nearly completed at sacrifice. For bile duct-cannulated approx. 4.597% and 0.066% of the recovered dose was detected in the GIT of males and females, respectively. The highest equivalent concentrations were detected in the liver of all low and high dose tests. They ranged from 0.0526 mg/kg bw to 0.4247 mg/kg bw. Concentrations in plasma and blood cells were very low and amounted to ≤ 0.0062 mg/kg bw, only. Noticeable was the amount of radioactivity in perirenal fat of low dose females (e.g. 0.0261 mg/kg bw), because there was no radioactivity detected in fat of male rats.The concentrations in the other organs and tissues ranged from 0.0007 to 0.0115 mg/kg bw. From the renal and faecal excretion and from the elimination kinetics of total radioactivity from plasma it was concluded that small amounts of residual radioactivity in organs and tissues are subject for further elimination.

Details on excretion:

Generally, in the low and high dose tests the excretion was almost completed 72 h after administration. At this time more than 99% of the recovered dose had been excreted via urine and faeces. As an exception, the excretion rate for bile duct-cannulated rats amounted approx. 94% for males and 99% for females of the recovered dose. In all low and high dose tests the main portion of radioactivity was excreted latest after 48 h. In all tests the excretion was mainly faecal and ranged from 92 to 99% of the recovered radioactivity, with an exception of the tests with bile duct-cannulated rats, in which approx. 54% for males and 70% for females of the recovered dose were detected in faeces and approx. 35% for males and 24% for females of the recovered dose in bile. For all low dose tests (2 mg/kg bw) the urinal excretion rate ranged from 4.51% to 6.58 % of the recovered dose. Negligible urinal excretion rates were measured for the high dose tests at 20 and 200 mg/kg bw and ranged from 0.08% to 0.57% of the recovered dose. These low urinal excretion rates gave a clear hint, that there is a reduced absorption of the test substance at higher dose rates.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 0.72

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 0.40

Key result

Test no.:

#5

Toxicokinetic parameters:

half-life 1st: 0.20

Key result

Test no.:

#7

Toxicokinetic parameters:

half-life 1st: 0.49

Key result

Test no.:

#8

Toxicokinetic parameters:

half-life 1st: 0.18

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 27.9

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 2nd: 18.0

Key result

Test no.:

#5

Toxicokinetic parameters:

half-life 2nd: 30.1

Key result

Test no.:

#7

Toxicokinetic parameters:

half-life 2nd: 14.3

Key result

Test no.:

#8

Toxicokinetic parameters:

half-life 2nd: 4.1

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 1.21

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 2.36

Key result

Test no.:

#5

Toxicokinetic parameters:

AUC: 1.27

Key result

Test no.:

#7

Toxicokinetic parameters:

AUC: 0.06

Key result

Test no.:

#8

Toxicokinetic parameters:

AUC: 0.12

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 0.138

Remarks:

measured value

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 0.199

Remarks:

measured value

Key result

Test no.:

#5

Toxicokinetic parameters:

Cmax: 0.131

Remarks:

measured value

Key result

Test no.:

#7

Toxicokinetic parameters:

Cmax: 0.00665

Remarks:

measured value

Key result

Test no.:

#8

Toxicokinetic parameters:

Cmax: 0.00812

Remarks:

measured value

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 0.151

Remarks:

calculated value

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 0.214

Remarks:

calculated value

Key result

Test no.:

#5

Toxicokinetic parameters:

Cmax: 0.131

Remarks:

calculated value

Key result

Test no.:

#7

Toxicokinetic parameters:

Cmax: 0.00695

Remarks:

calculated value

Key result

Test no.:

#8

Toxicokinetic parameters:

Cmax: 0.00940

Remarks:

calculated value

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 1

Remarks:

measured value

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 1

Remarks:

measured value

Key result

Test no.:

#5

Toxicokinetic parameters:

Tmax: 1

Remarks:

measured value

Key result

Test no.:

#7

Toxicokinetic parameters:

Tmax: 1

Remarks:

measured value

Key result

Test no.:

#8

Toxicokinetic parameters:

Tmax: 7

Remarks:

measured value

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 1.59

Remarks:

calculated value

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 1.60

Remarks:

calculated value

Key result

Test no.:

#5

Toxicokinetic parameters:

Tmax: 1.02

Remarks:

calculated value

Key result

Test no.:

#7

Toxicokinetic parameters:

Tmax: 1.35

Remarks:

calculated value

Key result

Test no.:

#8

Toxicokinetic parameters:

Tmax: 3.97

Remarks:

calculated value

Metabolites identified:

yes

Details on metabolites:

There were no sex-specific metabolites detected. Slight differences of the metabolite concentrations between males and females were observed within the dose groups. Tentatively, male rats showed more hydroxylation compared to female rats. The identification rate of parent compound and metabolites was high and amounted to between 89.59 and 108.29% of the administered dose parent compound was the main compound in the excreta for all tests. Parent compound was detected between 51.32 and 66.42% of the dose in all low dose tests (2 mg/kg bw) including pre-treatment and bile-duct cannulation. Parent compound was nearly completely excreted via faeces in the high dose tests with 20 and 200 mg/kg bw and amounted between 88.84% and 107.98% of the recovered dose. In general, there were no different metabolites found for males and females. The quantity of the individual metabolites was not significantly sex related. Major metabolites were parent compound-deshydrochloro-dihydrate, parent compound-dihydroxy and parent compound-hydroxy-N-methyl and amounted up to 8.7% of the dose.
Further prominent metabolites were, parent compound-benzylalcohol-Gluc, parent compound-hydroxypyridyl-Gluc, parent compound-deschloro-desmethyl-amide-dihydroxy, parent compound-despyridyl, parent compound-benzylalcohol, parent compound-pyridinyl-pyrazole-5-carboxylic acid and parent compound-5-hydroxypyridine. These prominent metabolites were detected and amounted up to 4.3% of dose. The majority of metabolites amounted to < 2.5% of dose and were identified as parent compound-pyrazole-5-N-methyl-amide-hydroxy, parent compound-pyrazole-5-amide, parent compound-pyrazole-5-N-methyl-amide, parent compound-pyrazole-5-carboxylic acid, parent compound-deschloro-GSH-thio-conjugate, parent compound-despyridyl-benzylalcohol, parent compound-despyridyl-N-methyl-quinazolinone-pyrazole-3-carboxylic acid, parent compound-hydroxy-N-methyl-hydroxypyridyl-Gluc, parent compound-N-methylquinazolinone-hydroxy-Gluc, parent compound-N-methyl-quinazolinone-hydroxypyridyl-Gluc, parent compound-deschloro-desmethyl-amide, parent compound-phenylhydroxy, parent compound-desmethyl-amide, parent compound-desmethyl-amide-hydroxy, parent compound-N-methyl-quinazolinone-benzylalcohol, parent compound-deschloro-hydroxy, parent compound-hydroxy, parent compound-despyridyl-N-methyl-quinazolinone, parent compound-Nmethyl-quinazolinone-hydroxypyridyl, parent compound-quinazolinone and parent compound-N-methyl-quinazolinone.
The most important metabolic reaction was the hydroxylation in the pyridinyl moiety, the N-methyl moiety and the methyl group of phenyl moiety leading to mono- and/or dihydroxy compounds. By trend, male rats showed slightly more hydroxylation compared to female rats. Hydroxylation in other positions of the test substance was also detected, but was not exactly located by structure elucidation. Conjugation with glucuronic acid was observed as a follow-up reaction after hydroxylation. Another important reaction was the intra-molecular condensation (cyclisation) of parent compound and was leading to quinazolinone compounds. Cleavage was detected for the phenyl moiety and was leading to an amide followed by oxidation to carboxylic acid or by methylation to an N-methyl amide. Other cleavage reactions were detected for the pyridine and tetrazole ring. In case of the tetrazole ring the cleavage was followed by an oxidation leading to a carboxylic acid. Minor metabolic reactions were the demethylation of the N-methyl group and the deschlorodination in the pyridine ring. Another observed reaction was the conjugation of the deschlorodinated parent compound with two times glutathione followed by degradation of one of the glutathione groups to mercapto alcohol. An overview of the principal metabolic reactions of pyrazole-carboxamide-14C - radiolabelled test substance in rats is presented below:
- hydroxylation in the pyridinyl moiety, the N-methyl moiety and the methyl group of phenyl moiety leading to mono- and/or dihydroxy compounds. Hydroxylation in other positions was also detected, but was not exactly located by structure elucidation
- conjugation with glucuronic acid after hydroxylation
- intra-molecular condensation (cyclisation) of parent compound leading to quinazolinone compounds
- cleavage of the phenyl moiety leading to an amide followed by oxidation leading to a carboxylic acid or by methylation leading to an N-methyl-amide
- cleavage of the pyridine ring
- cleavage of the tetrazole ring followed by oxidation leading to a carboxylic acid
- demethylation of the N-methyl group
- deschlorodination in the pyridine ring
- conjugation two times with glutathione after deschlorodination in the pyridine ring followed by degradation of one of the glutathione groups to mercapto alcohol

Table 4 Recovery as percent of total radioactive dose administered (mean values)

Test ID	1	2	3	4	5	6	9	7	8
Urine Bile Faeces	4.66 - 98.58	6.69 - 94.47	0.09 - 108.86	0.53 - 93.69	4.95 - 103.09	5.66 38.92 59.64	4.45 24.71 71.55	0.34 - 107.02	0.41 - 109.75
Total	103.24	101.16	108.95	94.22	108.05	104.22	100.71	107.35	110.16
Body excluding GIT	0.189	0.396	n.c.	0.011	0.204	1.009	0.459	0.012	0.013
GIT	0.029	0.151	n.c.	0.178	0.073	4.870	0.066	0.002	0.003
Total in body	0.218	0.547	n.c.	0.189	0.278	5.879	0.525	0.014	0.017
Balance	103.46	101.71	108.95	94.40	108.32	110.10	101.24	107.37	110.18

GIT = gastrointestinal tract, n.c. = not calculated (due to value below lower limit of quantification), - = no sample collected

Table 5 Recovery as percent of total radioactive dose administered (mean values)

Test ID	1	2	3	4	5	6	9	7	8
Urine Bile Faeces	4.51 - 95.28	6.58 - 92.88	0.08 - 99.92	0.57 - 99.23	4.68 - 95.06	5.17 35.12 54.18	4.41 24.46 70.61	0.31 - 99.67	0.37 - 99.61
Total	99.79	99.46	100.00	99.80	99.74	94.48	99.48	99.99	99.99
Body excluding GIT	0.183	0.390	n.c.	0.012	0.193	0.925	0.453	0.011	0.012
GIT	0.028	0.148	n.c.	0.192	0.068	4.597	0.066	0.002	0.003
Total in body	0.211	0.538	n.c.	0.203	0.261	5.522	0.519	0.013	0.015
Norm. - factor	0.967	0.982	0.918	1.060	0.951	0.910	0.988	0.932	0.908
Absorption rate [%]						41.2	29.3

GIT = gastrointestinal tract, n.c. = not calculated (due to value below lower limit of quantification), - = no sample collected

 

Table 6 Residues in organs and tissues at sacrifice – equivalent concentration [mg active substance equivalent/ kg sample] (mean values)

Test ID	1	2	3	4	5	7	8
Blood cells	0.0020	0.0035			0.0017
Plasma	0.0035	0.0057	n.c.	n.c.	0.0062	0.0022	0.0030
Carcass	0.0008	0.0042	n.c.	n.c.	n.c.	n.c.	n.c.
Heart	0.0011	0.0029	n.c.	n.c.	0.0011*	n.c.	n.c.
Brain	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.
Kidneys	0.0052	0.0115	n.c.	n.c.	0.0046	0.0038	0.0060
Liver	0.0593	0.1109	n.c.	0.4247	0.0658	0.0526	0.0636
Testes	0.0007	-	n.c.	-	0.0011	n.c.	-
Ovaries	-	0.0078	-	n.c.	-	-	n.c.
Uterus	-	0.0054	-	n.c.	-	-	n.c.
Adrenal gland	n.c.	0.0086	n.c.	n.c.	n.c.	n.c.	n.c.
Thyroid gland	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.
Spleen	n.c.	0.0029	n.c.	n.c.	n.c.	n.c.	n.c.
Lung	0.0018	0.0045	n.c.	n.c.	0.0026	n.c.	n.c.
Skin	0.0015	0.0048	n.c.	n.c.	0.0018	n.c.	n.c.
Bone femur	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.
Perirenal fat	n.c.	0.0261	n.c.	n.c.	n.c.	n.c.	0.0075*
Muscle leg	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.	n.c.

n.c. = not calculated, - = no sample collected, * = mean value calculated with half of lower limit of quantification (LLQ) for values < LLQ

 

Table 7 Balance of the test substance and metabolites excreted with urine, bile and faeces (low dose; % of dose administered)

Name: Parent compound-	Test ID
	1	2	5	6	9
pyrazole-5-N-methyl-amide-hydroxy	0.50	0.72	0.66	0.31	0.20
pyrazole-5-amide	0.21	0.16	0.14	0.14	0.85
pyrazole-5-N-methyl-amide	1.56	0.51	1.04	1.72	1.08
pyrazole-5-carboxylic acid	2.12	1.68	1.79	2.40	1.63
deschloro-GSH-thio-conjugate	0.80	0.38	0.37	3.72	1.72
despyridyl-benzylalcohol	1.24	0.65	1.43	1.30	0.70
deshydrochloro-dihydrate	4.44	5.36	4.76	0.93	1.01
despyridyl-N-methyl-quinazolinonepyrazole- 3-carboxylic acid	0.38	0.80	0.50	0.27	0.10
hydroxy-N-methyl-hydroxypyridyl-Gluc	0.72	0.43	0.92	1.96	0.88
benzylalcohol-Gluc	1.44	1.58	3.16	2.18	1.72
dihydroxy	3.92	2.05	4.79	0.47	0.10
hydroxypyridyl-Gluc	-	-	-	4.45	2.63
deschloro-desmethyl-amide-dihydroxy	1.23	2.35	2.61	1.52	1.22
despyridyl	3.00	3.41	2.71	2.01	1.46
N-methyl-quinazolinone-hydroxy-Gluc	1.04	0.77	1.87	1.97	1.36
benzylalcohol	3.24	2.48	3.25	0.98	1.15
pyridinyl-pyrazole-5-carboxylic acid	3.21	3.37	3.34	1.95	0.86
N-methyl-quinazolinonehydroxypyridyl- Gluc	-	-	-	1.37	0.88
deschloro-desmethyl-amide	1.14	0.80	1.26	-	-
hydroxy-N-methyl	6.16	3.93	8.71	3.91	2.88
phenylhydroxy	1.80	2.54	2.09	1.51	0.90
5-hydroxypyridine	2.87	4.28	3.12	1.57	1.52
desmethyl-amide	0.96	0.54	1.85	0.19	0.10
desmethyl-amide-hydroxy	-	-	-	0.32	0.39
parentcompound	54.34	53.58	21.32	56.93	66.42
N-methyl-quinazolinone-benzylalcohol	1.00	1.55	1.06	1.00	0.29
deschloro-hydroxy	-	-	-	0.23	0.14
hydroxy	0.40	0.91	0.47	0.42	0.21
despyridyl-N-methyl-quinazolinone	1.00	1.30	0.86	2.68	1.88
N-methyl-quinazolinone-hydroxypyridyl	0.06	0.28	-	0.21	0.16
quinazolinone	0.32	0.04	0.04	0.39	0.14
N-methyl-quinazolinone	0.48	0.95	0.35	0.87	0.65
Total identified	99.59	97.38	104.46	100.34	95.21
Total characterised	2.01	1.42	1.94	3.81	2.59
Number of characterised metabolites	7	5	6	11	9
Maximum value of a characterised metabolite	0.72	0.85	0.61	0.94	0.77
Solids of faeces	1.14	1.21	0.85	0.07	1.40
Urine not analysed	0.23	0.09	0.03	-	-
Faeces not analysed	0.27	1.06	0.76	-	1.50
Total	103.24	101.16	108.05	104.22	100.71

- = not detected

Table 8 Balance of the test substance and metabolites excreted with urine, bile and faeces (high dose; % of dose administered)

Name: Parent compound-	Test ID
	3	4	7	8
pyrazole-5-N-methyl-amide-hydroxy	-	-	-	-
pyrazole-5-amide	-	-	-	-
pyrazole-5-N-methyl-amide	-	-	0.35	0.21
pyrazole-5-carboxylic acid	-	-	0.31	0.16
deschloro-GSH-thio-conjugate	-	-	-	-
despyridyl-benzylalcohol	-	-	0.14	-
deshydrochloro-dihydrate	-	0.15	0.66	0.63
despyridyl-N-methyl-quinazolinonepyrazole- 3-carboxylic acid	-	-	-	-
hydroxy-N-methyl-hydroxypyridyl-Gluc	-	-	0.13	0.07
benzylalcohol-Gluc	-	-	0.27	0.14
dihydroxy	-	-	0.58	0.28
hydroxypyridyl-Gluc	-	-	-	-
deschloro-desmethyl-amide-dihydroxy			0.31	0.12
despyridyl	-	-	0.53	0.32
N-methyl-quinazolinone-hydroxy-Gluc	-	-	0.11	0.10
benzylalcohol	-	-	0.49	0.29
pyridinyl-pyrazole-5-carboxylic acid	-	-	0.41	0.30
N-methyl-quinazolinonehydroxypyridyl- Gluc	-	-	-	-
deschloro-desmethyl-amide	-	-	0.26	0.12
hydroxy-N-methyl	0.24	0.15	1.21	0.80
phenylhydroxy	-	-	0.20	0.18
5-hydroxypyridine	-	0.12	0.22	0.32
desmethyl-amide	-	-	0.27	0.19
desmethyl-amide-hydroxy	-	-	-	-
parentcompound	107.98	88.84	98.55	103.19
N-methyl-quinazolinone-benzylalcohol	-	0.21	0.40	0.35
deschloro-hydroxy	-	-	-	-
hydroxy	-	-	-	-
despyridyl-N-methyl-quinazolinone	-	-	-	0.9
N-methyl-quinazolinone-hydroxypyridyl	-	-	-	-
quinazolinone	-	-	-	-
N-methyl-quinazolinone	0.07	0.12	-	0.15
Total identified	108.29	89.59	105.41	108.01
Total characterised	0.23	0.16	0.85	0.71
Number of characterised metabolites	1	1	3	2
Maximum value of a characterised metabolite	0.23	0.16	0.47	0.55
Solids of faeces	0.26	0.28	0.71	0.80
Fraction of faeces not analysed (distillate)	-	0.03	-	-
Urine not analysed	0.09	0.53	0.34	0.41
Faeces not analysed	0.08	3.63	0.05	0.23
Total	108.95	94.22	107.35	110.16

- = not detected

Description of key information

Absorption, distribution, excretion and metabolism, oral (OECD 417):

absorption rates: to 41.2% for males and 29.3% for females

distribution: tmax = 1 h males, 1 - 7 h females, no retention of compound related residues in the body of the animals

excretion: completed after 72 h, mainly fecal

metabolism: no sex-specific metabolites, mainly parent compound in excreta, see metabolic pathway in the attached background material in the robust study summary

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

41.2

Additional information

A reliable study regarding absorption, distribution, excretion and metabolism of the test substance is available.

The toxicokinetic behavior (absorption, distribution, excretion) and metabolism of the test substance was investigated in the Wistar Unilever HsdCpb:WU rat according to OECD Guideline 417 and in compliance with GLP (M-549940-01-1). The test compound was radiolabeled with 14C in the pyrazole-carboxamide moiety of the molecule. Three groups of 4 male and 4 female rats received the test substance at single doses of 2, 20 and 200 mg/kg bw orally via gavage as a suspension in water and Tragacanth at pH 4. Two additional groups of 3 males and 3 females received the test substance at a single dose of 2 mg/kg bw following bile-duct cannulation. A further group of 4 males was pre-treated with the non-radiolabeled test substance as a suspension in water and tragacanth at pH 4 daily via oral gavage for 14 days followed by a single dose of the radiolabeled test substance at 2 mg/kg bw. The animals were sacrificed at the latest three days after dosing and in case of bile-duct cannulation after two days. The total radioactivity that included the test compound and the metabolites was determined in plasma samples, urine, bile, feces, organs and tissues at sacrifice. The metabolism was investigated in urines, bile and extracts of feces.

Between 94.4% and 110.2% of the administered dose were recovered from measurement of the total radioactivity in plasma samples, urines and feces, as well as in organs and tissues at sacrifice.

Absorption:

The absorption of the test substance started immediately after administration as shown by the concentration of radioactivity in the plasma of the low dose tests (2 mg/kg bw). For all low dose tests including the test with the pre-treatment and the intermediate dose test at 20 mg/kg bw with male rats, the maximum plasma concentration (Cmax) was reached within 1 h (tmax) after the administration. The highest plasma concentration was measured at 7 h for females of the high dose test with 20 mg/kg. Female rats showed a higher absorption rate for plasma compared to male rats. There was no radioactivity detected in all plasma samples of the high dose tests with 200 mg/kg bw and in samples at 48 and 72 h of the tests with 20 mg/kg bw. Low dose tests (2 mg/kg bw) with bile-duct cannulated males and females showed that about 35% for males and 24% for females of the recovered dose was distributed via the enterohepatic circulation. Absorption rates were calculated by summation of the recovered radioactivity in urine, bile and body without gastrointestinal tract and amounted to 41.2% for males and 29.3% for females.

Distribution:

The distribution of the test substance from the central compartment to the different organs and tissues was observed by measuring the concentration of the total radioactivity in plasma. After a single oral administration of 2 mg/kg bw to male and to female rats (low dose) the maximum of the plasma concentration of the radioactivity was measured approx. 1 h after dosage (tmax). In the high dose tests with 20 mg/kg bw, the measured maximum of the plasma concentration was reached at 1 h for males and delayed to approx. 7 h for females after dosing. In all low dose tests the maximum of the dosed normalized concentration ranged from 0.131 to 0.199. In tests with 20 mg/kg bw the highest dose normalized concentration amounted to ≤ 0.008 only, due to restricted absorption behaviour of the test compound. For tests with 200 mg/kg bw there was no radioactivity detected in the plasma samples. For all low dose tests the plasma concentration declined to < 2% of the maximum concentration within 72 h post administration. This indicates no retention of compound related residues in the body of the animals. Plasma dose normalized concentrations of the low dose tests as well as the tests at 20 mg/kg bw were calculated with a two-compartment model by TOPFIT. The weighting of 1/y or 1 were used, due to a very fast elimination phase after reaching the plasma maximum followed by a slower elimination phase after approx. 24 h.

Excretion:

Generally, in all tests the excretion was almost completed 72 h after administration. At this time more than 99% of the recovered dose had been excreted via urine and feces. As an exception, the excretion rate for bile duct-cannulated rats amounted approx. 94% for males and 99% for females of the recovered dose. In all low and high dose tests the main portion of radioactivity was excreted latest after 48 h. In all tests the excretion was mainly fecal and ranged from 92 to 99% of the recovered radioactivity, with an exception of the tests with bile duct-cannulated rats, in which approx. 54% for males and 70% for females of the recovered dose were detected in feces and approx. 35% for males and 24% for females of the recovered dose in bile. For all low dose tests (2 mg/kg bw) the urinal excretion rate ranged from 4.51% to 6.58 % of the recovered dose. Negligible urinal excretion rates were measured for the high dose tests at 20 and 200 mg/kg bw and ranged from 0.08% to 0.57% of the recovered dose. These low urinal excretion rates gave a clear hint, that there is a reduced absorption of the test substance at higher dose rates.

Residues in organs and tissues at sacrifice:

Generally, there were very low residues in organs and tissues of all low and high dose tests. Female rats showed higher organ concentrations compared to male rats. At sacrifice < 0.4% of the administered dose (mean values) was found in the bodies (excluding gastrointestinal tract) of the low and high dose rats and approx. 1% in bodies of the bile duct-cannulated. Negligible amounts of radioactivity were detected in the gastrointestinal tracts (0.002 to 0.178 % of dose), showing that the elimination of the radioactivity was nearly completed at sacrifice. For bile duct-cannulated approx. 4.597% and 0.066% of the recovered dose was detected in the gastrointestinal tract of males and females, respectively. The highest equivalent concentrations were detected in the liver of all low and high dose tests. They ranged from 0.0526 mg/kg to 0.4247 mg/kg. Concentrations in plasma and blood cells were very low and amounted to ≤ 0.0062 mg/kg, only. Noticeable was the amount of radioactivity in perirenal fat of low dose females (e.g. 0.0261 mg/kg), because there was no radioactivity detected in fat of male rats. The concentrations in the other organs and tissues ranged from 0.0007 to 0.0115 mg/kg. From the renal and fecal excretion and from the elimination kinetics of total radioactivity from plasma it was concluded that small amounts of residual radioactivity in organs and tissues are subject for further elimination.

Metabolism:

For investigation of the metabolism, urine, bile and feces were sampled at different time points in the individual tests. Faeces were conventionally extracted with a mixture of acetonitrile/water (8/2, v/v) plus formic acid followed by an exhaustive extraction with acetonitrile/water (1/1; v/v) and acetonitrile/water (1/1; v/v) plus formic acid using microwave assistance. The conventional extraction rate ranged from 85.7 to 99.8% of the radioactivity in faeces. After exhaustive extraction, only between 0.07 and 1.21% of the recovered dose were detected in the post extraction solids. There were no losses during sample preparation. Parent compound and metabolites were analyzed and quantified in the urines, bile and the extracts of feces by HPLC based on reversed phase chromatography with an acidic water/acetonitrile gradient. There were no sex-specific metabolites detected. Slight differences of the metabolite concentrations between males and females were observed within the dose groups. Tentatively, male rats showed more hydroxylation compared to female rats. The identification rate of parent compound and metabolites was high and amounted to between 89.59 and 108.29% of the administered dose. Parent compound was the main compound in the excreta for all tests. Parent compound was detected between 51.32 and 66.42% of the dose in all low dose tests (2 mg/kg bw) including pre-treatment and bile-duct cannulation. Parent compound was nearly completely excreted via feces in the tests with 20 and 200 mg/kg bw and amounted between 88.84% and 107.98% of the recovered dose. In general, there were no different metabolites found for males and females. The quantity of the individual metabolites was not significantly sex related. Major metabolites were parent compound-deshydrochloro-dihydrate, parent compound-dihydroxy and parent compound-hydroxy-N-methyl and amounted up to 8.7% of the dose. Further prominent metabolites were parent compound-benzylalcohol-Gluc, parent compound-hydroxypyridyl-Gluc, parent compound-deschloro-desmethyl-amide-dihydroxy, parent compound-despyridyl, parent compound-benzylalcohol, parent compound-pyridinyl-pyrazole-5-carboxylic acid and parent compound-5-hydroxypyridine. These prominent metabolites were detected and amounted up to 4.3% of dose. The majority of metabolites amounted to < 2.5% of dose and were identified as parent compound-pyrazole-5-N-methyl-amide-hydroxy, parent compound-pyrazole-5-amide, parent compound-pyrazole-5-N-methyl-amide, parent compound-pyrazole-5-carboxylic acid, parent compound-deschloro-GSH-thio-conjugate, parent compound-despyridyl-benzylalcohol, parent compound-despyridyl-N-methyl-quinazolinone-pyrazole-3-carboxylic acid, parent compound-hydroxy-N-methyl-hydroxypyridyl-Gluc, parent compound-N-methyl-quinazolinone-hydroxy-Gluc, parent compound-N-methyl-quinazolinone-hydroxypyridyl-Gluc, parent compound-deschloro-desmethyl-amide, parent compound-phenylhydroxy, parent compound-desmethyl-amide, parent compound-desmethyl-amide-hydroxy, parent compound-N-methyl-quinazolinone-benzylalcohol, parent compound-deschloro-hydroxy, parent compound-hydroxy, parent compound-despyridyl-N-methyl-quinazolinone, parent compound-N-methyl-quinazolinone-hydroxypyridyl, parent compound-quinazolinone and parent compound-N-methyl-quinazolinone. Parent compound and metabolites were isolated from urine, bile and extracts of feces of the low dose tests by HPLC and identified by spectroscopic methods. They were assigned in the other profiles of the urines and feces extracts based on their retention times and the comparison of the metabolite pattern shown in the profiles recorded with the profiling method. Unidentified metabolites were characterized by extraction and chromatographic behavior. But probably, more metabolites may be present as indicated by broad non-resolved zones in the chromatogram of bile.

The most important metabolic reaction was the hydroxylation in the pyridinyl moiety, the N-methyl moiety and the methyl group of phenyl moiety leading to mono- and/or dihydroxy compounds. By trend, male rats showed slightly more hydroxylation compared to female rats. Hydroxylation in other positions of the test substance was also detected, but was not exactly located by structure elucidation. Conjugation with glucuronic acid was observed as a follow-up reaction after hydroxylation. Another important reaction was the intra-molecular condensation (cyclisation) of parent compound and was leading to quinazolinone compounds. Cleavage was detected for the phenyl moiety and was leading to an amide followed by oxidation to carboxylic acid or by methylation to an N-methyl amide. Other cleavage reactions were detected for the pyridine and tetrazole ring. In case of the tetrazole ring the cleavage was followed by an oxidation leading to a carboxylic acid. Minor metabolic reactions were the demethylation of the N-methyl group and the deschlorodination in the pyridine ring. Another observed reaction was the conjugation of the deschlorodinated parent compound with two times glutathione followed by degradation of one of the glutathione groups to mercapto alcohol. An overview of the principal metabolic reactions of the radiolabeled test substance in rats is presented below:

•            hydroxylation in the pyridinyl moiety, the N-methyl moiety and the methyl group of phenyl moiety leading to mono- and/or dihydroxy compounds; hydroxylation in other positions was also detected, but was not exactly located by structure elucidation

•            conjugation with glucuronic acid after hydroxylation

•            intra-molecular condensation (cyclisation) of parent compound leading to quinazolinone compounds

•            cleavage of the phenyl moiety leading to an amide followed by oxidation leading to a carboxylic acid or by methylation leading to an N-methyl-amide

•            cleavage of the pyridine ring

•            cleavage of the tetrazole ring followed by oxidation leading to a carboxylic acid

•            demethylation of the N-methyl group

•            deschlorodination in the pyridine ring

•            conjugation two times with glutathione after deschlorodination in the pyridine ring followed by degradation of one of the glutathione groups to mercapto alcohol