diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Objective of study:

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

limited to animal groups B and D (metabolism following a single oral dosage).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

EC Number:

603-923-2

Cas Number:

135590-91-9

Molecular formula:

C16H18Cl2N2O4

IUPAC Name:

diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, SPF-Zucht Kastengrund
- Age at study initiation: 45-53 days, females: 54-80 days
- Weight at study initiation: males: 205-230 g, females: 190-210 g
- Housing: 2 animals per cage
- Individual metabolism cages: metabolism boxes suitable for separate collection of urine and feces
- Diet (e.g. ad libitum): Altromin 1321 (Altrogge, Lage/Lippe, FRG), ad libitum
- Water (e.g. ad libitum): tap water (municipal water supply), ad libitum
- Acclimation period: about 1 day


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 18-26
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The preparations containing the radioactively labelled active substance were dissolved in sesame oil. Separate batches of the application mixture were produced for each of the two dose levels

VEHICLE
- Justification for use and choice of vehicle (if other than water): sesame oil, solubility, handling at dosing
- Concentration in vehicle: about 0.2 (1 mg/kg dose group) and 19.9 mg/g formulation (100 mg/kg dose group); specific activities of radiolabelled substance used for preparation of formulation were 1873.7 MBq/g (1 mg/kg dose group) and 33.59 MBq/g (100 mg/kg dose group), respectively.
- Amount of vehicle (if gavage): 0.90-0.94 g solution/animal


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The solution of radiolabelled test substance in sesame oil was analysed directly and after 24 hours by TLC, UV-VIS and radioactivity determination. The solution was stable during 24 hours at 6°C, a period which was longer than the interval between preparation and the last administration.

Duration and frequency of treatment / exposure:

single gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood (plasma, only high dose group)
- Time and frequency of sampling: urine and faeces over 24 hour-intervals, blood at terminal sacrifice (after 7 d)
- From how many animals: samples from 5 animals per dose pooled
- Method type(s) for identification: HPLC-MS, Liquid scintillation counting
- Limits of detection and quantification: The smallest peak which could be detected unambiguously was about 1 % of the chromatographed radioactivity. If this radioactivity was contained in a 1 % excreta portion, the overall detection limit of this single peak was estimated to be 0.01 % of the dose.

Results and discussion

Preliminary studies:

not performed

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The renal excretion was the preferred excretion route independent from dose level and sex. As a consequence, the gastro-intestinal absorption rate must at least reach the same level (> 65 - 72%). Longer collection intervals would result in higher levels, as was observed in a parallel pharmacokinetics study. Absorption can thus be assumed to be almost complete.

Details on excretion:

The total excretion was fast and already complete within 0-48 hours after oral administration. Thus, 78 - 92% of the dose were excreted within the first 48 hours after dosing. Most of them was excreted with the urine regardless of sex and dose level: 0-24 hours: 58 - 63% of the dose, 24 - 48 hours: 6 - 10% of the dose, 0-48 hours: 65 - 72% of the dose. The faeces of the male animals contained more radioactivity than those of the females regardless of the dose level: 0-48 hours: 24 - 26% of the dose in the males, 13 - 14% of the dose in the females.

In view of the short collection period (0 - 48 hours after dosage), the summed radioactivity in urine and feces resulted in a good balance. Thus the recovered radioactivity amounted to 90 - 92% of the dose in the males and 78 - 86% in the females, regardless of the dose level. Since the main objection of this study was the identification of the residues, excreta of later periods were not investigated. For total balance a pharmacokinetic study was referred to.

High excretion rates were observed within a relatively short interval regardless of dose level and sex. This indicates that the test compound and its metabolites are not retained for long in the animal organism and, consequently, that the enterohepatic circulation would not play a significant role. This conclusion is confirmed by the relatively low portions excreted with the feces.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

HPLC separations of native urine samples showed four different peaks. Peak identification could be performed by thermospray HPLC-MS in the positive mode. This combination showed the [M + H]+ and the [M + NH4]+ions in each case. For additional confirmation of the attributions, the retention times of the HPLC peaks were compared with those of synthesized reference compounds. The following components were observed in the urine: Hoe 113225 [1-(2,4-dichlorophenyl)-5-ethoxycarbonyl-5-methyl-2-pyrazoline-3-carboxylic acid], 109453 [1-(2,4-dichlorophenyl)-5-methyl-2-pyrazoline-3,5-dicarboxylic acid], 094270 [1-(2,4-dichlorophenyl)-5-methyl-pyrazole-3-carboxylic acid]. A difference between the sexes can be evidently observed: The females excreted a significant portion of Hoe 113225 whereas the males increased the proportion of Hoe 109453 in the urine at the same amount. The unchanged parent compound Hoe 107892 could not be detected in the urine.

Whereas the parent Hoe 107892 was the main portion in the feces of the males (0-24 hours: 8% of the dose), the dicarboxylic acid Hoe 109453 predominated in the feces of the females (0-24 hours: 4 - 6%) regardless of the dose level. Hoe 094270 and Hoe 113225 could also be detected in each faeces fraction at lower levels (0.1 - 6% of the dose). The non-extractable portion amounted to 1%, the polar portion which remained in the aqueous phase amounted to 2% of the dose.

Due to a high radioactivity level observed in the plasma during a parallel pharmacokinetic study, plasma samples of the highly dosed rats (male and female) were analysed. In order to reduce the volume carefully and precipitate the proteins, these samples were freezedried and re-dissolved in methanol. HPLC analysis of the methanol solution resulted in a single peak with the same retention at both sexes. The peak obtained from the female rats was identified as Hoe 109453 by HPLC-MS.

Any other information on results incl. tables

Percentage of metabolites in urine:

	Males		Females
Metabolite	0 - 24 h	24 - 48 h	0 - 24 h	24 - 48 h
Hoe 113225	0.2 - 2	< 0.1	13 - 26	0.4 - 1.2
Hoe 109453	54 - 56	6 - 9	34 - 43	5 - 8
Hoe 094270	2 - 3	0.3	2	0.2 - 0.3

In the urine the dicarboxylic acid Hoe 109453 was the main component in each animal group. The monoester Hoe 113225 was renally excreted in significant portions within the first 24 hours only by the female animals, whereas the Hoe 109453 portion was 2-fold as high as these levels in the urine of the male rats. The pyrazol Hoe 094240 was observed in the 0-24 hours urine of each animal group at the same low level. As expected, the parent compound was not excreted with the urine. Whereas the parent compound Hoe 107892 was the main component in the feces of the male rats, the dicarboxylic acid Hoe 109453 predominated in the feces of the females. Since the fecally excreted radioactivity was higher in males than in females, these findings indicate either a somewhat lower intestinal absorption or a higher biliary excretion of Hoe 107892 in male animals.

Based on the findings in urine and feces the metabolism of Hoe 107892 in the rat is assumed to involve the following steps: the consecutive hydrolysis (saponification) of the two carboxylic acid ester groups and a decarboxylation of one of the carboxylic groups, resulting in an aromatization of the pyrazoline ring. The radioactivity in the blood plasma could be attributed exclusively to the relatively polar dicarboxylic acid Hoe 109453, the main residue component in the urine. No other residue component could be detected.