cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jun 2001 to 07 Aug 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

April 1984

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Test Data for Registration of Agricultural Chemicals, Nohsan No 8147, Agricultural Production Bureau

Version / remarks:

November 24, 2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Directive 94/79/EC Annex 1, Toxicological and Metabolism Studies No. L 354/18, 51

Version / remarks:

December 21, 1994

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Cas Number:

94361-06-5

Molecular formula:

C15H18ClN3O

IUPAC Name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanBrl: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Female rats were nulliparous and non-pregnant
- Age at study initiation: 9 weeks
- Weight at study initiation: about 180 g
- Housing: Open Plexiglas metabolism cages
- Diet: certified standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 42-74
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Jun 2001 To: 11 Jul 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: a mixture of polyethylene glycol 200/ethanol/water 2/2/1 (v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in a mixture of polyethylene glycol 200/ethanol/water 2/2/1 (v/v) at a concentration of 0.2 mg/mL.
Each animal received 0.5 mL of the administration solution by stomach tube.

DOSE FORMULATION STABILITY
The radioactivity of the test substance in the administration solution was checked at the first time of dosing, after the eighth dosing, and after the last dosing.

Duration and frequency of treatment / exposure:

Daily, for 14 consecutive days

Doses / concentrations

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

Measured concentrations (mg/kg bw/day): group T1: 0.58 - 0.62; group T2: 0.59 - 0.61; group T3: 0.56 - 0.60; group T4: 0.58 - 0.60
Measured radioactive concentration: 280 kBq (7.6 µCi)

No. of animals per sex per dose / concentration:

16 female rats, which were assigned to 4 subgroups, i.e. T1, T2, T3, and T4

Control animals:

no

Details on study design:

- Dose selection rationale: The dose level was selected with the intention to have a most authentic test system, i.e. not essentially inducing enzyme activity and not running into saturation effects of transport and metabolism processes.
- Animals from the different subgroups were sacrifed at different time points after start of administration. T1 at day 1, T2 at day 7, T3 at day 14 and T4 at day 20.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: excreta, urine, faeces, serial blood, plasma, serum, cage washes, adrenals, bone, brain, fat (abdominal), heart, kidneys, liver, lungs, muscle (skeletal), ovaries, pancreas, spleen, thymus, thyroid, uterus, whole blood
- Time and frequency of sampling:
Excreta: only collected from the animals of Subgroup T4 in daily intervals from day 0 to day 20;
Serial blood: specimens were taken daily only from the animals of Subgroup T4. During the dosing period the sampling was performed prior to the daily dosing;
At defined time points, i.e. 1 day (Subgroup T1), 7 days (Subgroup T2), 14 days (Subgroup T3), and 20 days (Subgroup T4) after the first administration 4 animals each were sacrificed and the remaining tissues were collected. The later two time points corresponded to 1 and 7 days after the last dose, respectively.
At the end of the collection period each cage of Subgroup T4 was rinsed thoroughly with water/ethanol 1/1 (v/v).

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: daily
- From how many animals: 10% of the total urine volume and 20% of the total faeces weight from the individual animals, were pooled for the time interval day 0-1, day 6-7, and day 13-14 from the animals of Subgroup T4.
- Method types for identification: Liquid scintillation counting, TLC

ANALYTICAL METHOD
Radioactivity was measured by Liquid Scintillation Counting (LSC) on Packard Tri-Carb scintillation counters, model 2000CA9 and 2500TR9, equipped for computing quench-corrected disintegrations per minute (dpm). Background values were measured with each specimen sequence using the respective scintillation mixture without any specimens. Recovery tests of the sample oxidizer were performed by combusting standards of the blend of n-amyl alcohol, 1,3-butandiol, and 14C-stearic acid) 9 at the beginning of each day and with each combustion sequence (consisting of about 20 specimens). Combustion and trapping efficiencies were always above 95% except for bone (Subgroup T1, 80-85 %) and serial blood (Subgroup T4 day 1 through day 3, 77-96%). However, all reported data are therefore uncorrected.
The pattern of radioactivity on thin layer plates was detected by using a Packard Instant Imager The TLC plates were exposed for an appropriate time interval. Further processing of the images including quantification of the radioactive zones was performed on the Instant Imager software. Alternatively, a Bio-Imaging Analyzer, model BAS 500010. The TLC plates were exposed on phosphor imaging plates in a lead shielding box. Thereafter, the plates were scanned and visualized on the image reader BAS 5000. Further processing of the images including quantification of the radioactive zones was performed on the TINA software.

TLC was performed on precoated plates of silica gel SI 60 F254 and reversed phase RP 18 F254, 200 x 200 mm 0.25 mm thick. The plates were developed without chamber saturation.
For the determination of the stability of [Phenyl-U-14C] test substance in the administration vehicle the following systems were used:
Plate: Silica gel SI 60 F254 (without chamber saturation)
CRM1 toluene / acetic acid (50/50 v/v);
Plate: Reversed phase RP18F254 (without chamber saturation);
CRM2 acetonitrile / water / tetrahydrofurane (60/40/0.1 v/v/v);

Urine metabolite pattern analysis (two-dimensional TLC):
Plate: Silica gel SI 60 F254
1. Dimension SS5: acetic acid / isopropanol / formic acid / water (65/25/5/5 v/v/) without chamber saturation
2. Dimension SS6: chloroform / methanol / formic acid / water (65/25/5/5 v/v) with chamber saturation

Feaces metabolite pattern analysis (two-dimensional TLC):
Plate: Silica gel SI 60 F254
1. Dimension SS1: ethyl ether /ethanol / formic acid (95/5/0.5 v/v) developed two times without chamber saturation
2. Dimension SS: methylene chloride / methanol (95/5 v/v) developed two times with chamber saturation

Non-radioactive components were located as dark quenching spots against the fluorescent background when dried plates were viewed under UV light (254 nm).

Statistics:

Mean values and standard deviation were calculated according to the general equation.
The calculated absorption, excretion, total recovery, tissue residues in [ppm] and [% of daily dose, % of total dose] are based on duplicate measurements, except for the adrenals and thyroids, and the results of the blood kinetics which were performed on a single aliquot measurement. All radioactivity counting of specimens were corrected for background by subtraction of appropriate blank values from specimen count rates to give net "dpm" per specimen.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The orally administered test substance was rapidly absorbed from the gastro-intestinal tract into the systemic circulation.

Details on distribution in tissues:

The residues in all tissues reached maximum levels 7 days after start of dosing. Highest levels were determined in liver (1.4 ppm) and adrenals (0.9 ppm), lungs (0.6 ppm) and fat (0.5 ppm). All other tissues reached maximum levels below 0.3 ppm.

Details on excretion:

Four days after start of dosing the amount of daily excretion remained nearly constant during the dosing period. The major part of the daily administered dose was excreted with the faeces (55 %) and a somewhat smaller part was excreted via kidneys (40 %).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The chromatography of urine revealed a metabolite pattern, consisting of at least 21 metabolite fractions. The pattern was qualitatively and quantitatively not influenced by the multiple dosing. TLC analysis of the faeces extracts revealed at least 13 metabolite fractions. The extractability and the metabolite pattern of the faeces were essentially identical for all three selected sampling intervals with slight quantitative variations mainly between the first time interval (0-1 day) and the later selected time intervals. These quantitative variations are based on the time shift of faecal excretion for the first time interval after start of dosing. Unchanged parent (Fr.9 and Fr. 10) was excreted via faeces at a rate of about 13 % of the daily dose. Besides unchanged parent the metabolite M1 could be assigned to the faecal metabolite pattern by chromatography, accounting for about 6 % of the daily dose.

Any other information on results incl. tables

Animal observation

The animals were checked for appearance and behaviour at arrival and at each sampling time during the experiment. No unusual behaviour of the animals was observed.

Stability of the Test substance in the Application solution

The formulated test substance was found to be stable during the application period as checked by TLC. The analysis after the first, eighth and after the last of 14 consecutive daily doses revealed that the test substance represented more than 96% of the radioactivity of the application solution.

Absorption

The orally administered test substance was rapidly absorbed from the gastro-intestinal tract into the systemic circulation.

Distribution

During the dosing period the residues of all selected tissues and organs reached plateau residue level seven days after start of dosing. The highest residue values were found in liver and adrenals accounting for 1.4 ppm and 0.9 ppm test substance equivalents, respectively. Besides liver and adrenals relatively high levels were also found in lungs (0.6 ppm) and fat (0.5 ppm). All other tissues and organs reached maximum levels below 0.3 ppm test substance equivalents. The calculated half life (t./2) for the depuration of the residual radioactivity from tissues and organs, assuming a mono phasic first order kinetics, were for most of the selected tissues in the range of 1 to 3 days. The fastest depletion rate was calculated for brain, pancreas and thymus (t./a = 25-26 hours). The higher persistence of the residues in whole blood as compared to plasma led to a whole blood/plasma residue ratio of 5:1 seven days after the last dose, indicating a partial association of the residues to blood cells. The total amount of residues in tissues and organs 7 days after the last of 14 consecutive daily doses accounted only for 0.09 % of the total administered dose.

Excretion

During the dosing period, i.e. 14 consecutive daily doses, a steady state in terms of excretion was reached 4 days after the first administration. Thereafter the amount of daily excretion remained nearly constant until the end of dosing, accounting for about 40 % and 55 % of the daily dose for urine and faeces, respectively. Three days after the last dose the administered test substance was almost completely excreted. The major part of the administered test substance was excreted with the faeces accounting for 56 % of the total dose. A somewhat lower part, i.e. 40 % of the total dose, was excreted with the urine.

Table 1. Percentage recoveries of administered radioactivity in urine and faeces during and following 14 daily oral doses.

	Excretion after multiple administrations
	% of total administered dose			% of daily administered dose
Sampling Interval	Urine	Faeces	Total	Urine	Faeces	Total
0-1d	1.48	1.81	3.29	20.82	25.55	46.37
1-2d	1.97	3.29	5.26	27.70	46.55	74.05
2-3d	2.29	3.33	5.62	31.96	46.38	78.34
3-4d	2.58	3.67	6.25	36.31	51.66	87.97
4-5d	2.55	3.63	6.18	35.89	50.96	86.85
5-6d	2.69	4.47	7.16	37.88	63.04	100.92
6-7d	2.85	3.80	6.65	39.81	53.10	92.91
7-8d	2.98	3.61	6.59	41.38	50.17	91.55
8-9d	2.84	4.25	7.09	39.70	59.47	99.17
9-10d	2.62	4.09	6.71	36.66	57.30	93.96
10-11d	3.02	4.34	7.36	42.18	60.75	102.93
11-12d	2.96	3.62	6.58	41.19	50.30	91.49
12-13d	3.04	4.58	7.62	42.79	64.50	107.29
13-14d	2.96	4.07	7.03	41.02	56.33	97.35
14-15d	1.58	1.94	3.52	21.84	26.85	48.96
15-16d	0.95	0.76	1.71	13.14	10.47	23.61
16-17d	0.42	0.53	0.95	5.76	7.41	13.17
17-18d	0.22	0.28	0.5	3.28	3.88	6.96
18-19d	0.10	0.13	0.23	1.40	1.83	2.23
19-20d	0.08	0.08	0.16	1.15	1.13	2.28
Total	40.17	56.29	96.46

 

Table 2. Distribution of metabolic fractions in urine and faeces after single and
multiple oral dosing with [14C]-test substance

	Urinary Metabolite Profiles			Faecal metabolite profiles
Sample	Urine 0-1d	Urine 6-7d	Urine 13-14d	Sample	Faecal 0-1d	Faecal 6-7d	Faecal 13-14d
Metabolite fraction Fr1 Fr2 Fr3 Fr4 Fr5 Fr6 Fr7 Fr8 Fr9 Fr10 Fr11 Fr12 Fr13 Fr14 Fr15 Fr16 Fr17 Fr18 Fr19 Fr20 Fr21	0.9 0.8 2.4 1.2 1.2 0.2 0.9 1.2 0.2 0.2 0.1 2.0 2.3 4.5 0.3 11.3 0.2 0.4 0.1 0.1 0.2	1.8 1.8 4.3 3.3 1.9 0.4 2.1 3.6 0.4 0.7 0.2 4.3 3.7 6.0 1.2 1.8 0.5 0.5 0.2 0.2 0.7	1.3 1.3 3.1 3.0 1.4 0.4 1.7 2.4 0.4 0.6 0.1 4.0 2.9 6.0 4.8 2.9 3.3 0.6 0.2 0.2 0.4	Metabolite Fraction Fr1 Fr2 Fr3 Fr4 Fr5 Fr6 (M1) Fr7 Fr8 Fr9 (parent) Fr10 (parent) Fr11 Fr12 Fr13 Non-extractable	2.3 0.6 0.1 0.1 3.1 2.2 2.2 0.3 1.1 8.4 2.4 0.5 0.4 1.9	5.7 1.1 0.8 0.8 7.0 6.2 3.3 1.1 1.1 12.8 7.0 1.3 1.2 3.6	6.8 1.2 1.4 0.7 7.1 6.9 3.4 1.3 1.1 12.3 7.5 1.4 1.3 3.8
Total	20.8	39.8	41.0	Total	25.6	53.1	56.3

 

Table 3. Tissue distribution of test substance following multiple oral dosing

Group	T1	T2	T3	T4	Half Life (hours)
Dose (mg/kg bw)	0.60	0.60	0.58	0.59
Days after initiation	1	7	14	20
Days after last dose	1	1	1	7
Adrenals Blood Bone Brain Fat Heart Kidneys Liver Lungs Muscle Ovaries Pancreas Plasma Spleen Thymus Thyroid Uterus	0.6461 0.0288 0.0113 0.0544 0.3298 0.0687 0.1403 0.6591 0.3475 0.0366 0.0959 0.1093 0.0371 0.0544 0.0363 0.0626 0.0434	0.9347 0.0631 0.0250 0.0846 0.4895 0.1137 0.2546 1.3698 0.5618 0.0584 0.1567 0.2239 0.0658 0.0928 0.0673 0.1134 0.0710	0.8639 0.0687 0.0244 0.0864 0.5078 0.1161 0.2615 1.2526 0.5537 0.0599 0.1531 0.1901 0.0669 0.1068 0.0631 0.1057 0.0772	0.0314 0.0152 0.0021 0.0016 0.0122 0.0066 0.0327 0.0883 0.0185 0.0018 0.0060 0.0040 0.0028 0.0076 0.0014 <LQ 0.0031	30 66 41 25 27 35 48 38 29 28 31 26 31 38 26 n/a 31

 

Applicant's summary and conclusion

Conclusions:

The routes and rates of excretion did not change and the tissue distribution pattern of residues did not change following single or multiple dose administration. [14C]-test substance was rapidly absorbed into the systemic circulation following administration of multiple oral doses of 0.5 mg/kg to female rats. The absorbed [14C]-test substance was rapidly and almost completely excreted mainly via the faeces and to a lesser extent through the urine. The metabolite profiles of [14C]-test substance revealed that the compound was extensively metabolised and analysis showed complex metabolic patterns, which were qualitatively similar in the urines with the exception of Fr15 and Fr17, which disproportionably rose in comparison to other metabolites after 14 days or multiple dosing. The metabolite profile in the faeces showed slight quantitative differences between day 0 -1 and day 14 but these were not considered to be significant. Radioactive residues reached a plateau after 7 days and remained steady until cessation of dosing. Upon cessation levels were depleted rapidly from tissues with half-lives in the range of 1-3 days, with the blood>kidneys>liver=spleen all showing the longest retention times.

Executive summary:

In a GLP compliant study, performed in accordance with OECD TG 417, the disposition of the test substance, was investigated in the rat after multiple oral administrations. [Phenyl-U-14C] labelled test substance was orally administered to 16 female rats. The dosing was performed by 14 consecutive daily doses at a nominal dose level of 0.5 mg/kg body weight. Four animals each were assigned to subgroups (T1-T4) which were sacrificed at 4 different time points after start of administration and the test substance related residues were determined in tissues and organs. The excreted radioactivity was determined in urine and faeces of subgroup T4 at daily intervals.

The test substance was rapidly absorbed from the gastrointestinal tract into the systemic circulation and the totally administered dose was almost completely excreted, i.e. within 3 days after termination of dosing. After start of dosing a steady state in terms of excretion was reached within 4 days. The major part of the daily administered dose was excreted with the faeces (about 55 %) and a somewhat smaller part was excreted via kidneys (about 40 %). In sum 56 % and 40 % of the totally administered dose was excreted with the faeces and the urine, respectively. Seven days after the last dose only 0.09 % of the total administered dose remained in tissues and organs. The blood kinetics showed increasing residue values with ongoing administrations up to 8 days after of start dosing. Thereafter the residues in blood kept almost constant at a plateau level of about 0.075 ppm test substance equivalents during the further dosing period. After the last of 14 consecutive daily doses the residues in whole blood decreased to about half the maximum concentration within 3 days.

The tissue residues determined at 4 different time points during and after the dosing period showed a similar profile as observed in blood kinetics. The residues in all selected tissues and organs reached their plateau residue levels 7 days after start dosing. Highest levels were determined in liver (1.4 ppm) and adrenals (0.9 ppm), followed by lungs (0.6 ppm) and fat (0.5 ppm). All other tissues and organs revealed low maximum levels not exceeding 0.3 ppm test substance equivalents. After reaching the maximum the residue levels kept almost constant (plateau) during the further dosing period. Thereafter the tissue residues declined rapidly reaching values lower than 10 % of the maximum concentration within seven days after the last dose. The calculated half- lives (t1/2) for the depuration ranged from 1 to 3 days. The metabolite pattern of urine and faeces, investigated at three different time intervals during the dosing period, i.e. day 0 - 1, 6 - 7, and 13 - 14, were not influenced by multiple dosing. About 13 % of the daily dose were excreted as unchanged parent via faeces.

In summary the test substance was rapidly absorbed and almost completely excreted after multiple administrations. The residues in tissues and organs reached a plateau within 7 days after start of dosing. There is no evidence for an accumulation of the test substance and/or related residues after multiple dosing.