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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

April 1984

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

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

Radiolabelling:

yes

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

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

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.

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 %).

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.

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

 

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 1985 to Sep 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

1984

Deviations:

yes

Remarks:

Radioactive residues in tissues determined for a reduced number of animals (3 males and 3 females). This deviation is considered not to compromise the scientific validity of the study.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Remarks:

Kfm:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 190 - 305 g
- Housing: individual metabolic cages allowing sampling of blood from the tail vein and collection of bile, urine and faeces quantitatively and separately at preset time points

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 42 ± 1
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Jan 1985 To: Sep 1985

Route of administration:

other: gastric intubations or by injection into the femoral vein

Vehicle:

DMSO

Details on exposure:

Dose levels were 10 and 130 mg/kg bw of the test substance dissolved in DMSO. Animals were administered the test substance (0.3 to 0.5 mL) either by gastric intubations or by injection into the femoral vein. The radioactive dose ranged from 153-257 mCi/kg bw except for bile-cannulated animals (25-32 mCi/kg bw) pre-treatment in the multidose experiment was with unlabelled compound.

Duration and frequency of treatment / exposure:

- Experiment 1: single intravenous dose
- Experiment 2, 3 and: single oral dose
- Experiment 4: 14 daily oral doses

Dose / conc.:

10 mg/kg bw (total dose)

Dose / conc.:

130 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

- Experiment 1, 2, 3 and 5: 5 animals/sex
- Experiment 4: 6 animals/sex

Control animals:

no

Details on study design:

Administration outline:
- Experiment 1: intravenous administration of a single dose of [14C]-test substance, 10 mg/kg (5 animals/sex)
- Experiment 2: Oral administration of a single dose of [14C]-test substance, 10 mg/kg (5 animals/sex)
- Experiment 3: Oral administration of a single dose of [14C]-test substance, 10 mg/kg, in bile duct cannulated rats (5 animals/sex)
- Experiment 4: Oral administration of multiple doses: 14 daily doses (10mg/kg) of unlabelled test substance followed by a single dose of [14C]-test substance 10 mg/kg (6 animals/sex).
- Experiment 5: Oral administration of a single dose of [14C]-test substance 130 mg/kg (5 animals/sex).

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, serum, bile, carcass
- Time and frequency of sampling for the bile duct cannulated rats the urine and bile were collected at intervals of 0-24, 24-48, 48-72 and 72-144 hours. Faeces was collected at 0-144 hours. Radioactivity in carcass was determined after 144 hours.
- Time and frequency of samplien other animals: sampling of urine was after 4, 7 and 24 hours and at daily intervals thereafter. Faeces were collected at daily intervals.
- About 50 mg of blood were taken from the rat tail vein, at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 24, 48, 72, 96, 120, 144 and 168 hours.
- Distribution studies of residual radioactivity: All intacts rats were sacrificed by ether overnarcosis at 168 hrs post-dose. The distribution of residual radioactivity at this time point was determined either by liquid scintillation counting (LSC) on selected dissected organs (in 3 rats/sex/study) or by whole-body autoradiography (WBAR) (in 2 rats/sex/study). The latter method was used in addition to LSC since its high optical resolution and sensitivity allow to detect radioactivity in small organs (which cannot be dissected and/or processed by LSC, e.g. membranes) and in the different tissues of some organs. For WBAR the rats were deep-frozen immediately after sacrifice in a mixture of n-hexane and dry ice at -75 °C, shaved and embedded in a 2% semi-liquid gel of Na-CMC (carboxymethylcellulose). Thin sagittal slices were obtained according to the method of Ullberg [3] at -23 oC in a cryomicrotome where they were dried for 48 hrs prior to exposure at -23 °C to RPl films for 3 weeks. The films were processed manually for 5 min at 20 °C in a developer.

ANALYTICAL METHOD
The amount of radioactivity in the administered solution, urine, bile, blood, faeces, tissues and carcass was measured by liquid scintillation counting. The samples were either dissolved in an appropriate scintillation cocktail or processed by combustion or digestion. Quench correction was performed by the external standard ratio (ESR) method. The radioactivity of the standard counting solution used to find the quench correction curve was determined using radiolabelled hexadecane as internal standard. The obtained "ESR vs counting efficiency" curves were fitted by polynomial equations of second degree. Conversion of accumulated counts in dpm/mg or dpm/mL was done by an on-line connected computer. All instruments used to generate "quench" correction curves for the unknown samples were initially calibrated with quench tritium standards. If the experimental values were within the range of 100 ± 3% of their true values, the parameters of all curves of the respective instrument were regarded to be valid.
Under the conditions of this study, the detection limit in all samples proessed by liquid scintillation counting was F=0.0006, corresponding to 6 and 78 ng equiv./g for the low and high doses, respectively. For the whole body radiography, the limit of detection was F=0.008.

Details on absorption:

Summing up the fraction of radioactivity excreted via the bile and urine by the cannulated rats it appears the minimum absorption was 85.1 ± 5.1 % and 86.4 ± 1.8 % of the dose in males and females, respectively.

Details on distribution in tissues:

At study termination (168 h) were highest in the liver and adrenals (cortex). The next highest residues were found in the renal fat, kidney, spleen (after i.v application only). In almost all cases, radioactive levels were higher in females than in males.

Details on excretion:

Excretion predominately occurred through the bile accounting for 75.5% and 59.6% in males and females, respectively. Excretion via the urine accounted for 9.5 and 26.8 % in males and females, respectively.

Metabolites identified:

not measured

Details on results

Absorption

Summing up the fraction of radioactivity excreted via the bile and urine by the cannulated rats it appears the minimum absorption was 85.1 ± 5.1 % and 86.4 ± 1.8 % of the dose in males and females, respectively. Unabsorbed materials were represented by the fraction of the dose in the faeces of these animals (4.5 ± 1.9% for males and 4.8 ± 0.9% for females). The extent of absorption was well confirmed by the urinary quotients p.o./i.v. after low- and high doses as well as after pretreatment, especially in the male rats (84-86%). In the case of females the quotients were slightly higher (92-100 %). The absorption occurred more slowly after a high dose, as demonstrated by the delayed maximal blood levels and the much lower extent of the urinary excretion - within the first day after administration. Half-lives of absorption were about 2-3 hrs after low - and 6-9 hrs after high doses. Comparison of the urinary values between intact and bile duct cannulated rats enabled estimation of the reabsorption of radioactivity from the GIT to be about 15%.

Distribution

By comparing the distribution patterns of both sexes in all the experiments it appeared that the tissue levels in females were slightly higher than in males. The highest ones were those of liver, adrenals, renal fat and kidney (p.o. and i.v.) and spleen (i.v.). The other levels were low. After a single oral low dose to male rats the mean levels at 168 hrs p.d. in liver, adrenals, renal fat and kidney were 590, 320, 190 and 150 ng equ. parent drug/g, respectively. In females, the corresponding concentrations were 520, 540, 260 and 140 ng/g of tissue. After a single intravenous dose, most levels were higher than in the oral study. They amounted to 920, 640, 330 and 230 ng/g by males and 1220, 960, 510 and 270 ng/g by females in liver, adrenals, renal fat and kidney. Concentrations up to 550 ng/g were found in the spleen. After an acute high oral dose, most tissue levels in both sexes were quite similar to those in the low dose study, except in muscles and skin by females (higher levels). After pretreatment the radioactivity tissue levels were slightly lower than the corresponding ones of the acute oral study. The highest concentrations amounted in the liver, adrenals/renal fat and kidney to nearly 350, 190 and 90 ng equiv. parent drug/g of tissue, respectively.

Whole-body autoradiograms confirmed well these results. In particular WBAR demonstrated that the highest levels of radioactivity were those of the adrenal cortex whereas only traces were present in the adrenal medulla. Moreover high radioactivity amounts were still present in the intestinal content and confirmed thus that the excretion of compound related materials was not completed at this time point.

Excretion

As demonstrated by the amount of radioactivity recovered in the bile of the operated animals (acute low oral dose) the drug and/or its metabolites were mainly excreted via this route 75.5±10.9 and 59.6±3.3 % of the dose by males and females, respectively, within 144 hrs. Biliary excretion was thus higher for male- than for female rats. At least 90% of the biliary excretion occurred within the first day following the administrations, demonstrating a rather rapid hepatic clearance. The extent of the urinary excretion in intact rats was similar in all the experiments and somewhat higher by the female- than the male rats. It amounted to nearly 30 and 40% of the dose in males and females, respectively. Interestingly the mean rate of the urinary excretion was only 0.1% during the first day following the high dose whereas in all other experiments it was up to 6 times higher. The excretion of radioactive materials was not completely terminated at 144 hrs p.d. since up to 1% of the dose was recovered in the excreta in the time interval 144-168 hrs in all experiments. At the end of the investigation, the total excretion amounted to 84.2-100% of the dose.

Table 1. Cumulative excretion of radioactivity in rat urine, faeces, and bile (% of applied dose)

	10 mg/kg p.o		130 mg/kg p.o		10 mg/kg p.o bile cannulated		10 mg/kg p.o multiple dose		10 mg/kg i.v
	m	f	m	f	m	f	m	f	m	f
Urine
0-24h	9.0	14.5	2.4	2.2	3.4	5.5	13.3	15.3	13.7	12.1
0-48h	20.6	33.6	9.0	13.5	9.2	25.3	21.7	29.0	25.9	25.8
0-72h	24.4	37.3	20.4	30.2	9.4	26.2	25.6	32.7	29.3	34.0
0-144h	27.7	40.7	28.0	41.4	9.5	26.8	27.6	34.7	32.6	38.2
0-168h	27.8	41.0	28.5	41.7			27.8	34.9	32.9	38.4
Faeces
0-24h	12.4	18.7	9.3	1.6			31.6	17.8	16.6	6.7
0-48h	36.4	38.3	24.5	9.5			48.7	36.5	49.1	25.8
0-72h	46.3	47.4	42.4	29.1			55.1	44.6	57.8	40.7
0-144h	57.8	53.4	59.8	42.5	4.5	4.8	59.6	48.8	67.6	50.5
0-168h	58.6	53.9	60.3	43.0			60.1	49.2	68.5	50.8
Bile
0-24h					73.9	53.8
0-48h					74.7	57.4
0-72h					75.3	59.0
0-144h					75.5	59.6
Carcass					0.6	1.0
Total	86.5	94.9	88.8	84.8	90.0	92.1	88.0	84.1	101.3	89.2

Table 2. Blood level parameters in the rat

	10 mg/kg p.o		130 mg/kg p.o		10 mg/kg p.o multiple dose		10 mg/kg i.v
	m	f	m	f	m	f	m	f
Cmax[ug/ml]	2.2	2.5	13.4	23.6	1.7	1.2	4.1	2.9
TCmax[h]	3.0	9.0	43	36	5.0	5.0	n.a	n.a
AUC*	6.0	8.5	5.7	9.6	3.7	4.0	8.6	11.1
T½ elimination [h]	29	31	25	30	30	33	26	29

 

Table 3. Residues in the rat 168 hours after dosing, in mg/kg equivalents of the test substance.

	10 mg/kg p.o		130 mg/kg p.o		10 mg/kg p.o multiple dose		10 mg/kg i.v
	m	f	m	f	m	f	m	f
Blood	0.05	0.06	0.03	0.04	0.04	0.03	0.07	0.10
Plasma	0.04	0.04	0.03	0.05	0.02	0.02	0.05	0.07
Brain	0.01	0.02	0.01	0.02	0.01	0.01	0.02	0.04
Testicles	0.02		0.01		0.01		0.01
Epididymis	0.02		0.02		0.01		0.02
Uterus		0.04		0.07		0.03		0.07
Ovaries		0.10		0.05		0.05		0.46
Muscle	0.01	0.02	0.01	0.08	0.01	0.01	0.03	0.04
Skin	0.05	0.06	0.06	0.35	0.03	0.08	0.05	0.12
Renal Fat	0.19	0.26	0.21	0.34	0.07	0.10	0.33	0.51
Heart	0.02	0.04	0.02	0.04	0.02	0.02	0.04	0.09
Thymus	0.01	0.02	0.01	0.02	0.02	0.01	0.05	0.10
Pancreas	0.03	0.04	0.02	0.04	0.03	0.03	0.06	0.13
Thyroid	0.02	0.08	0.02	0.07	0.02	0.02	0.05	0.10
Bone marrow	0.02	0.02	0.01	0.03	0.01	0.02	0.15	0.13
Lymph nodes	0.02	0.03	0.02	0.04	0.02	0.03	0.05	0.08
Salivary Glands	0.02	0.04	0.02	0.03	0.02	0.02	0.04	0.07
Spleen	0.02	0.04	0.02	0.04	0.02	0.02	0.55	0.50
Adrenal	0.32	0.54	0.25	0.41	0.17	0.21	0.64	0.96
Kidney	0.15	0.14	0.14	0.14	0.10	0.07	0.23	0.27
Lung	0.05	0.05	0.04	0.05	0.03	0.05	0.17	0.32
Liver	0.59	0.52	0.42	0.50	0.36	0.32	0.92	1.22

Conclusions:

The test substance was well absorbed in the rat, had a high bioavailability and was almost completely excreted. Excretion predominately occurred through the bile accounting for 75.5% and 59.6% in males and females respectively. Excretion via the urine accounted for 9.5 and 26.8 % in males and females, respectively. Total excretion between males and females was almost identical but the pattern differed with slightly more radioactivity being excreted via the bile by males than females and the converse being the case for excretion via the urine. After seven days the compound was almost completely eliminated from the body, residues in tissues and organs were very low, however at the high dose and in animals administered by i.v there was an apparent increase in residue retention in the tissues of females in comparison to the males. Resides were predominately associated with organ involved in elimination, including the kidney, renal fat and liver as well as the adrenal glands.

Executive summary:

In a GLP compliant study, equivalent to OECD TG 417, the absorption and disposition characteristics of radiolabelled test substance and its metabolites were investigated in male and female rats after acute low (10 mg/kg) oral and intravenous-, acute high (130 mg/kg) oral- and multiple low (10 mg/kg) oral doses. The blood levels of radioactivity, excretion of drug related materials and distribution of residues in the body were determined by liquid scintillation counting and whole-body autoradiography. The absorption was almost complete (at least 86%) independently from the dose level and regimen. It was delayed after high doses but remained linear in the investigated dose range. The dose normalized maximal blood levels did not differ significantly from one experiment to the other except in the high dose study (males) due to delayed absorption. They amounted to about 2.2 µg/mL for a 10 mg/kg dose. The average AUC were however slightly higher in the female compared male rats. The drug and its metabolites were slowly eliminated from blood with mean elimination half-lives of nearly 30 h which did not differ significantly from one experiment to the other. The excretion of the compound derived materials occurred mainly via the bile and was almost completed at the end of the investigation, although radioactive residues were found in most tissues at this time. The highest levels were observed in the adrenal cortex and liver after i.v. dosing (1.2 µg/g) followed by renal fat, kidney, spleen. The tissue concentrations were the slightly higher in the females than in males in all experiments. However there was no unusual retention of drug derived materials in the rats and multiple dosing did not influence the distribution patterns. In conclusion, the test substance was well absorbed in the rat, had a high bioavailability and was almost completely excreted. Excretion predominately occurred through the bile accounting for 75.5% and 59.6% in males and females respectively. Excretion via the urine accounted for 9.5 and 26.8 % in males and females, respectively. Total excretion between males and females was almost identical but the pattern differed with slightly more radioactivity being excreted via the bile by males than females and the converse being the case for excretion via the urine. After seven days the compound was almost completely eliminated from the body, residues in tissues and organs were very low, however at the high dose and in animals administered by i.v there was an apparent increase in residue retention in the tissues of females in comparison to the males. Resides were predominately associated with organ involved in elimination, including the kidney, renal fat and liver as well as the adrenal glands.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr 1993 to 29 Sep 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR VAF/Plus

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 50 - 61 days
- Weight at study initiation: 225 - 250 g
- Fasting period before study: yes
- Housing: Individually in Nalgene metabolism cages
- Individual metabolism cages: yes
- Diet: rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Apr 1993 To: 29 Sep 1993

Type of coverage:

open

Vehicle:

other: Sentinel® 40 WG

Remarks:

water used as a dose vehicle in spray dilutions

Duration of exposure:

0.5, 1, 2, 4, 10 and 24 hours

Doses:

- Nominal doses: 0.15, 0.015 and 0.0025 mg a.i./cm2 skin
- Actual doses: 0.1670, 0.0182 and 0.0025 mg a.i./cm2 skin
- Actual doses calculated as follows: group means of the actual dose received for each rat at each dose level
- Dose volume: 9-10 µL/cm2 skin
- Rationale for dose selection: The doses were selected to give a range of concentrations in order to determine the effect of concentration on the absorption and elimination of the test substance in male rats following a single dermal dose

No. of animals per group:

24 rats per dose level, plus a control group of 6 rats

Control animals:

yes

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: Dose suspensions were prepared by dissolving the required amount of a mixture of unlabelled test substance and [14C]-test substance with an appropriate volume of the formulation base and water

APPLICATION OF DOSE: Using a syringe, 90-100 µL of the dose preparation was applied and spread evenly across the surface of the defined skin site (10 cm2). The syringe was weighed before and after dosing to determine the amount of dose given. After the dose had dried, a template was attached to the back of each animal and fitted with a non-occlusive cover to prevent loss of test compound from the application site. Control rats were given a dermal application of an aqueous suspension of the blank formulation

TEST SITE
- Preparation of test site: hair clipping 24 hours prior to dosing (an area of 40 cm2)
- Area of exposure: 10 cm2
- Type of cover / wrap if used: non-occlusive patch, 6.0 x 8.0 cm sheet made of cellulose and pectin based material

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Sub-groups of 4 treated rats were skin washed and terminated at 0.5, 1, 2, 4, 10 and 24 hours after dosing. Animals were anaesthetised for the skin washing procedure. The application site washed 5 times with a mild soap solution (5% in water) using cotton-wool balls, and rinsed with water after each washing step.

SAMPLE COLLECTION
Sub-groups of 4 treated rats were anaesthetised and terminated by exsanguination at 0.5, 1, 2, 4, 10 and 24 hours after dosing. One control rat was terminated at each time point. The terminal blood sample, treated skin, template, cover and carcass was retained for analysis. Urine, faeces and an ethanol:water (1:1 v/v) cage wash were collected at the termination of each experiment. Urine and faeces were frozen upon excretion by collection over solid carbon dioxide.

SAMPLE PREPARATION
- Storage procedure: Urine, faeces and carcass samples were stored at approximately -10°C prior to analysis. Blood, covers, cotton wool balls from skin washes, skin and cage wash samples were refrigerated prior to analysis.
- Preparation details: see "Any other information on materials and methods incl. tables"

ANALYSIS
- Method type(s) for identification: liquid scintillation counting and HPLC for concentration and stability determination
- Liquid scintillation counting results (cpm) converted to dpm as follows: disintegration per minute (dpm) values were calculated using the appropriate instrument-stored quench correction data.
- Limits of detection and quantification: typical limit of detection (LOD) values are reported for each sample type as percent of dose.

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Absorption in different matrices:

- Non-occlusive cover: less than 4 % AD
- Skin wash: 74 - 97 % AD
- Skin test site: 2.08 ± 0.8 % AD and 1.22 ± 0.65 % AD for all mid and high dose animals, respectively
- Blood: Small amounts of radioactivity were found in the blood. The blood concentrations did not appear to increase with exposure time.
- Carcass: at high dose: 0.75 ± 0.32 % AD; at mid dose: from 0.59 % AD after 0.5 hour to 2.00 % AD after 10 hours of exposure, between 10 and 24 hours, residues in carcasses increased to 10.1 % AD
- Urine: less than 1 % AD
- Cage wash + cage wipe: less than 0.1 % AD; amounts in excess of 0.25 % AD were found occasionally (4/72 cage washes)
- Faeces: Very little radiolabel was excreted in the faeces by the end of 24 h. An average of less than 1% of the dose was found in feces at any exposure time and at any dose level. Faeces from one animal exposed to the low dose for 24 hours contained 2.15 %AD compared to an average of 0.08 % AD for the other three 24 hour animals

Total recovery:

- Total recovery: 85 - 114 %AD
- Recovery of applied dose acceptable: yes, mean recovery of radiolabel from each group of animals was greater than 92 %AD

Key result

Time point:

10 h

Dose:

0.0025 mg/cm2

Parameter:

percentage

Absorption:

16.01 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Time point:

24 h

Dose:

0.0182 mg/cm2

Parameter:

percentage

Absorption:

14.31 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Time point:

24 h

Dose:

0.167 mg/cm2

Parameter:

percentage

Absorption:

2.94 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Table 1. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.1670 mg/cm².

	Recovery (% of applied dose)
	High dose level: 0.1670 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.00	0.00	0.04
Faeces	0.00	0.00	0.00	0.00	0.01	0.01
Cage wash	0.00	0.00	0.00	0.00	0.00	0.02
Carcass + blood	0.92	0.53	0.73	0.85	0.66	0.82
Absorbed dose	0.92	0.53	0.73	0.85	0.67	0.89
Treated skin	0.97	2.10	1.12	0.90	0.67	2.05
Skin wash	96.99	95.70	94.46	93.34	93.74	92.57
Template	1.67	0.08	1.23	0.87	0.52	1.73
Cover	0.00	0.00	0.00	0.00	0.01	0.03
Unabsorbed dose	98.67	95.78	95.69	94.21	94.27	94.32
Total recovered	100.55	98.41	97.54	95.95	95.61	97.27

 

Table 2. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.0182 mg/cm².

	Recovery (% of applied dose)
	Mid dose level: 0.0182 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.01	0.12	1.19
Faeces	0.00	0.00	0.00	0.00	0.01	0.37
Cage wash	0.00	0.00	0.01	0.01	0.03	0.24
Carcass + blood	0.59	0.82	1.04	1.42	2.00	10.08
Absorbed dose	0.59	0.82	1.05	1.43	2.16	11.88
Treated skin	2.19	2.08	2.85	1.28	1.66	2.43
Skin wash	97.02	87.90	94.40	91.82	88.14	80.08
Template	1.00	2.30	0.83	1.97	1.46	1.58
Cover	0.00	0.00	0.00	0.07	0.04	0.48
Unabsorbed dose	98.02	90.21	95.23	93.85	89.64	82.14
Total recovered	100.80	93.11	99.12	96.56	93.45	96.45

Table 3. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.0025 mg/cm².

	Recovery (% of applied dose)
	Low dose level: 0.0025 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.20	0.81	0.92
Faeces	0.00	0.00	0.00	0.01	0.02	0.60
Cage wash	0.01	0.01	0.01	0.41	0.09	0.23
Carcass + blood	0.86	2.74	4.48	10.30	9.90	5.95
Absorbed dose	0.87	2.75	4.50	10.92	10.81	7.69
Treated skin	3.28	3.98	6.06	4.91	5.20	8.91
Skin wash	84.99	90.81	82.16	76.47	76.05	74.48
Template	1.02	0.63	1.18	1.64	1.90	2.02
Cover	2.09	1.34	0.17	0.05	0.15	0.39
Unabsorbed dose	88.11	92.79	83.51	78.16	78.10	76.90
Total recovered	92.25	99.52	94.07	93.98	94.11	93.50

Conclusions:

Absorption at the high dose level of 0.167 mg/cm2 was minimal, amounting to 2.94 % including the dose absorbed and the dose retained in skin after 24 hours. Absorption at the mid dose level of 0.0182 mg/cm2 was low, amounting to 14.31 % including the dose absorbed and the dose retained in skin after 24 hours. At the low dose level of 0.0025 mg/cm2, absorption increased from 4.15 % including the dose absorbed and the dose retained in skin after 0.5 hours to a plateau level of approximately 16 % including the dose retained in skin after 10 hours. At all three dose levels investigated, the majority of the applied test substance was readily washed from the application site skin after the exposure period. The absorbed dose was excreted mainly in urine and to a lesser extent in faeces.

Executive summary:

In a GLP compliant study the test substance in Sentinel 40WG formulation base was administered dermally to male Sprague Dawley rats at dose levels of approximately 0.0025,  0.015 and 0.15 mg/cm2, in dosing volumes of about 0.1 mL, applied to 10 cm2  of dorsal skin. The test material was left on the skin for 0.5, 1, 2, 4, 10 and 24 hours. Four rats from each dose group were used for each exposure period. The application site was protected by a template and covered by a non-occlusive patch. For all animals, the application site was washed with a 5 % soap/water solution at the end of the exposure period prior to termination. Non-occlusive patches, skin washes, templates, skin, cage washes, urine, feces, blood and carcasses were analyzed for radioactivity.   The test material was absorbed to some extent via the dermal route of exposure. Absorption was not proportional to dose level except for an exposure time of 0.5 hour when the average absorption at all dose levels was approximately 0.79 % of the doses. At a dose level of 0.0025 mg/cm2, absorption appeared to reach a maximum after 4 hours and plateau at 9.81 % of the applied dose. At a dose  level of 0.015 mg/cm2, absorption was minimal through 10 hours when the average absorption was 2.16 % of the dose; absorption increased to approximately 12 % after an exposure period of 24 hours. At a dose level of 0.15 mg/cm2, absorption reached a maximum of approximately  0.90 % at 30 minutes, which suggested that this dose level represented an infinite dose of the test substance. Greater than 92 % of the applied doses were recovered at each exposure period, washes. At least 80 % of the recovered radiolabel was found in skin. Templates and nonocclusive patches contained only small amounts of the doses. Radiolabel in skin was relatively constant at any dose level and, in general, the percent of the dose in skin appeared to decrease as dose level increased. Carcasses contained a minimum of 77 %, but usually greater than 90 % of the absorbed dose at each exposure period and at all dose levels. Less than 2 % of the dose was excreted by 24 hours at the two lower dose levels and less than 0.1 % was excreted at the highest dose level. The data from this dermal absorption study suggest that the test substance in Sentinel 40WG is not extensively absorbed at any dose level from  0.0025 to 0.15 mg/cm .  Maximum absorption appeared to be approximately 1 % to 12 % of the dose at dose levels of 0.0025 and 0.015 mg/cm2. Absorption at the high dose level of 0.167 mg/cm2 was amounting to 2.94 % including the dose retained in skin after 24 hours. Absorption at the mid dose level of 0.0182 mg/cm2 was low, amounting to 14.31 % including the dose absorbed and the doser etained in skin after 24 hours. At the low dose level of 0.0025 mg/cm2, absorption reached a plateau level of approximately 16 % including the dose absorbed and the dose retained in skin after 10 hours.

Description of key information

- (14C)-test substance was rapidly and almost completely excreted mainly via the faeces and to a lesser extent through the urine; the compound was extensively metabolised and analysis showed complex metabolic patterns; OECD TG 417, Hassler 2003

- Oral absorption rate: ≥ 86 %; OECD TG 417; Schweitzer 1987

- Dermal absorption rate: 16 % (10 hr, 0.0025 mg/cm2), in vivo dermal absorption study in rats; EPA OPPTS 870.7600; Andre 1993

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

86

Absorption rate - dermal (%):

16

Absorption rate - inhalation (%):

100

Additional information

Experimental toxicokinetic data are available for assessing adsorption, distribution, metabolism and excretion of the substance (Schweitzer 1987; Hassler 2003; Andre 1993). The test substance is expected to be readily absorbed via the oral route with absorption rate of 86 % and to a small extent by the dermal route (16 %). The substance is readily excreted mainly via faeces and secondly, urine. After seven days the compound is almost completely eliminated from the body, residues in tissues and organs were very low, however at the high dose and in experiments where the animals were administered the test substance by i.v there was an apparent increase in residue retention in the tissues of females in comparison to the males. Residues were predominately associated with organ involved in elimination, including the kidney, renal fat and liver as well as the adrenal glands.

 

Absorption

Oral route: The absorption was almost complete (at least 86%) independently from the dose level and regimen. It was delayed after high doses but remained linear in the investigated dose range. The dose normalized maximal blood levels did not differ significantly from one experiment to the other except in the high dose study (males) due to delayed absorption. They amounted to, about 2.2 µg/mL for a 10 mg/kg dose. The average AUC were however slightly higher by the female than male rats.

In the supporting studies, the test item (radiolabelled) was found to be rapidily and extensively adsorbed at the low dose (10 mg/kg) with maximal blood levels reached between 1.5-7 hours post dosing. Rats administerdadministered the high dose (130 mg/kg) displayed a slower but equally extensive abdsorption, with maximal blood concentrations reached between 24-48 hours. Based on bile duct cannulated experiment, the test item had a total bioavailability of > 86 %. Repeated administration resulted in an increased absorption with levels in the blood reaching a maximum after 8 days despite further administration. Based on the absorption studies provided there was no evidence of accumulation in any tissues of the rat.

Dermal route: In the in vivo dermal absorption study in rats, the absorption at the high dose level of 0.167 mg/cm2 was minimal, amounting to 2.94 % including the dose retained in skin after 24 hours. Absorption at the mid dose level of 0.0182 mg/cm2 was low, amounting to 14.31 % including the dose retained in skin after 24 hours. At the low dose level of 0.0025 mg/cm2, absorption increased from 4.15 % including the dose retained in skin after 0.5 hours to a plateau level of approximately 16 % including the dose retained in skin after 10 hours. At all three dose levels investigated, the majority of the applied test substance was readily washed from the application site skin after the exposure period. The absorbed dose was excreted mainly in urine and to a lesser extent in faeces.

 

Distribution

The excretion of the compound derived materials was almost completed at the end of the investigation, although radioactive residues were found in most tissues at this time. The highest levels were observed in the adrenal cortex and liver after i.v. dosing (1.2 µg/g) followed by renal fat, kidney, spleen. The tissue concentrations were the slightly higher in the females than in males in all experiments. However there was no unusual retention of drug derived materials in the rats and multiple dosing did not influence the distribution patterns. In conclusion, the test substance was well absorbed in the rat and almost completely excreted within 168 hrs post-dose. Although residues were still detected in the animals at the end of the investigation period, there was no unusual retention of the compound and/or its metabolites in this species under the testing conditions.

In the supporting studies, the test item was found to have a rapid and extensive volume of distribution as evidenced by maximal blood levels being reached between 1.5-7 hours and with most tissues having higher residue levels than found in the blood by 3 hours. The test item was found to be predominately associated with the organs of elimination (kidney, liver and pancreas) as well as the spleen and adrenal glands. Following repeated administration a similar pattern of distribution was observed, with the test item accumulating in all tissues up to 7 days after which point no further accumulation was observed despited further dosing. Upon cessation of treatment, the test item was rapidly eliminated from all tissues indicating so signs of residue retention.

 

Metabolism

In the 14-day repeated oral dose study in rats, 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.

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 the supporting studies, the test item was rapidly adsorbed and widely distributed within the liver and adrenals showing the highest levels of radioactivity in the rat after a single oral dose and there was no sign of unusual retention of the test item-derived material. The test item was extensively metabolised by the rat regardless of the route of administration or the dosage regimen and of the sex of the test animal. Metabolism profiles for the different scenarios for both males and females were found to be almost identical with minor quantitative differences. One test item’s diastereomer was found to be more extensively metabolized than the other diastereomer. The metabolic pathway of the test item in the rat resulted to be similar to the one of the goat. In vitro data showed that the pattern of major metabolites formed from rat liver fractions and mouse liver fractions are essentially the same, with some quantitative and qualitative differences.

 

Elimination and excretion

In the study by Hassler, 2003 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 %). However, it is known from another study (Schweitzer 1987) that the administered test substance was almost completely absorbed and about 60 % of the dose re-entered the intestinal tract by biliary excretion. Ultimately this part was excreted with the faeces.

In sum 56 % and 40 % of the totally administered dose was excreted with the faeces and the urine, respectively.

In the supporting studies, the major route of elimination of the test item in the rat was predominately found to occur via the bile, accounting for approximately 75 % in males and 59 % in females. Elimination via the urine occurred to a greater extent in females (26.8 %) than males (9.5 %), which accounted for the difference in elimination in the bile. Faecal elimination accounted for less than 5 % of the administered dose. The majority of the test substance was eliminated via the urine and bile within the first 48 hours post administration irrespective of the route of administration. However some of the test substance may be reabsorbed from the bile and excreted via the urine. Over 85 % of the test item was eliminated within 144 hours. Repeated administration had no significant effect upon the routes and rates of elimination compared to a single oral dose. The elimination from most tissues occurred rapidly, following monophasic kinetics. Biphasic elimination was observed for adrenals with a first phase half-life similar to the other tissues and a slower second phase. There was no sign of an unusual retention of test item-derived material in the rat after a single oral dose.