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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

other: guidelines of the USEPA

Principles of method if other than guideline:

According to Schmidt, R.D. (1989). Guidance on when and how to conduct livestock and metabolism studies. United States Environmental Protection
Agency Memorandum, July 1989.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C

Species:

other: goat

Strain:

other: British Saanen

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: M.G. and H.A. Supton, Goat Farmer, Lappages Farm, Rettendon Common, Chelmsford, Essex, UK
- Weight at study initiation: 59 kg on receipt
- Housing: loose in a pen for a period of acclimatisation and then transferred to a metabolism crate
- Individual metabolism cages: yes, metabolism crate
- Diet (e.g. ad libitum): coarse goat mix and lamb and ewe mix (Clark and Butcher Ltd., Lion Mills, Soham, Ely, Cambridgeshire) provided twice daily. The daily feed intake was measured.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5-21
- Humidity (%): 60-69
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSES:
Radiolabelled HOE 107892 was accurately weighed directly into gelatin capsules which were sealed and stored at + 4 °C in uniquely numbered vials.

DIET PREPARATION
- Rate of preparation of diet (frequency): once (by HMR, Preclinical Development of Hoechst AG, Pharmacokinetics, Frankfurt)
- Mixing appropriate amounts with (Type of food): weighed directly into gelatin capsules; the goat was given a daily single oral dose by administration of one or two capsules per day, depending upon the feed intake of the goat, so a dose level of nominally 10 ppm in the diet was achieved.
- Storage temperature of food: + 4 °C

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The purity and stability of the dose material were determined using HPLC. The radiochemical purity was shown to be 98.76% which is the same as the
radiochemical purity of the dose material (98.7%) determined prior to the start of the study. Therefore the dose material was stable under the conditions of storage at + 4°C for the duration of the study.

Duration and frequency of treatment / exposure:

7 days, once daily

Dose / conc.:

10 ppm (nominal)

Remarks:

mean of 11.23 ± 2.27 (equivalent to a mean dose level of 18.79 ± 2.38 mg/day); total dose of 131.51 mg/kg bw/day, equivalent to 7.956 mCi

No. of animals per sex per dose / concentration:

1 female

Control animals:

no

Details on study design:

- Dose selection rationale: The study was performed following the guidelines of the USEPA which outline the requirement for a minimum dose level of 10 ppm in the feed to enable quantification and where appropriate identification of the residues in milk and edible tissues. A goat, such as the one used in this study, will consume 1.5 kg diet/day, therefore to achieve a minimum dose level of 10 ppm, a nominal dose of 15 mg per day was administered to the goat.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces (including crate washes), blood, plasma, milk, heart, kidney, liver, lung, muscle (hindquarter, flank and loin), fat (subcutaneous, perirenal and omental), bile, rumen and abomasal fluid
- Time and frequency of sampling: milk twice daily (0830 and 1600 h), urine and faeces separately at 24 h intervals, blood samples were obtained by venepuncture of the jugular vein prior to dosing, then at 0.5, 1, 2, 3, 4, 6, 8, 24 and 30 hours after administration of the initial dose and twice daily (1000 and 1600 h) thereafter until the termination of the study when blood was obtained immediately prior to necropsy. At approximately twenty-three hours after administration of the final dose, the goat was killed by stunning with a captive bolt, pithed followed by exsanguination, and samples of the aforementioned tissues were collected.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine, heart, kidney, liver, subcutaneous fat, renal fat, omental fat, milk
- Time and frequency of sampling: The same samples were used as for the study of pharmacokinetic parameters.
- From how many animals: 1
- Method type(s) for identification: HPLC-MS-MS, Liquid scintillation counting, TLC
- Limits of detection and quantification: see table

Statistics:

The concentration of tissue residues in all samples was calculated. Means and standard deviations for each analysis and for individual values in each group were also calculated. All tissue residue calculations were carried out automatically by the system. All calculations relating to analysis of tissue extracts were made manually.

Details on absorption:

The residue levels of [14C]-HOE 107892 and/or its metabolites in the whole blood and plasma sampled at intervals throughout the duration of the study were relatively low (0.001-0.038 in blood and 0.001-0.048 mg equivalents/kg in plasma) and reached a plateau in both by day 5 of dosing.

Details on distribution in tissues:

Of the edible tissues, the highest residues of the radiolabelled test article and its metabolites were seen in the kidney (0.170 mg equivalents/kg) and liver (0.061 mg equivalents/kg). The residue levels in the heart and 3 types of fat (subcutaneous, omental and perirenal) were low (0.010-0.016 mg equivalents/kg) and in the three types of muscle (loin, flank and hindquarter) examined were almost an order of magnitude lower (0.003-0.004 mg equivalents/kg). The lungs, which are not classed as an edible tissue, were shown to have a low residue level (0.017 mg equivalents/kg). The abomasal and rumen fluid and bile residue levels were considerably greater (0.322-0.592 mg equivalents/kg) which showed that some remnants of the dose were still passing through the gut. The residue levels of HOE107892 and/or its metabolites in the milk were extremely low (<0.001 mg equivalents/kg) and were shown to reach a plateau, of 0.001 mg equivalents/kg, by day 3 of dosing, even though the residue levels in blood and plasma continued to rise until day 5 and were consistently an order of magnitude greater than in the milk.

Details on excretion:

The major route of excretion of the dose was via the urine (mean percentage of daily dose 62.71 ± 14.77 %) with faecal elimination accounting for considerably less (mean percentage of daily dose 12.51 ± 6.91 %), resulting in a mean total percentage of dose of 75.22 ± 17.65 % (Table 1). The residue levels of the metabolites in the urine reached a plateau on day 5 of dosing with a maximum concentration of 16.130 mg equivalents HOE 107892/kg urine, no parent compound was excreted via the urine. Overall, the major metabolite excreted via the urine was HOE113225 (89-93 % of the total radioactivity in the urine) with small amounts of HOE109453 (4-7 % of total radioactivity) and HOE094270 (3-4 % of total radioactivity) (Table 4). Faecal elimination of the dose was very slow on days 1 and 2 of the study but increased on day 3 and remained at that level for the remainder of the study. A very small percentage of the daily dose (<0.01 %) was eliminated daily in the milk (Table 2). Overall, 81.1 % of the total radiolabel given to the goat was accounted for in the urine, faeces, milk, whole organs and fluids which were analysed at necropsy (Table 3).

Metabolites identified:

yes

Details on metabolites:

Three different metabolites of the test article in goat were identified, namely HOE 113225, the carboxylic product, HOE 94270, the pyrazole, and HOE 109453, the dicarboxylic acid. Depending on the type of tissue investigated, their amount was variable (Table 4). The metabolism of HOE 107892 in the goat resulted in the successive de-ethylation of the parent molecule to form HOE 113225 followed by HOE 109453 and finally decarboxylation of one carboxylic acid group with aromatisation of the heterocyclic ring to form HOE 094270. The major metabolite seen in milk, urine and all tissues, except the subcutaneous fat, was HOE 113225. Trace amounts of parent compound was shown to be present in kidney, liver and heart.

Table 1: Elimination of radioactivity following single daily oral doses at a dose rate equivalent to nominally 10 ppm in the diet for 7 days:

	Percentage of daily radioactive dose
Day	Urine	Faeces (including crate wash)	Total excreted
1	43.82	0.72	44.52
2	73.02	5.48	78.50
3	44.47	18.82	63.29
4	60.09	14.41	74.50
5	78.69	19.03	97.72
6	78.38	13.16	91.54
7	60.53	15.94	76.47
% recovery of total dose	63.20	13.17
Mean	62.71 ± 14.77	12.51 ± 6.91	75.22 ± 17.65

Table 2: Elimination of radioactive dose in the milk following single daily oral doses at a dose rate equivalent to nominally 10 ppm in the diet for 7 days:

Time of milking after first dose (hours)	Percentage of daily radioactive dose
6	0.0004
24	0.0018
30	0.0017
48	0.0036
54	0.0012
72	0.0037
78	0.0022
96	0.0048
102	0.0036
120	0.0051
126	0.0030
144	0.0048
150	0.0032
168	0.0046

Table 3: Recovery of total dose administered over a 7 day period:

Tissue/excreta	Percentage recovery of total dose administered
Urine	63.20
Faeces	13.17
Milk	0.006
Abomasal fluid	0.299
Rumen fluid	4.333
Bile	0.018
Heart	0.002
Kidneys	0.023
Liver	0.049
Lungs	0.006
Total	81.106

Table 4: Extraction and characterisation/identification of the radiolabelled residues in the tissues, milk and urine:

Tissue	Residue level mg equiv/kg	% Organo-extractable	% Characterised/identified
			HOE 107892	HOE 113225	HOE 94270	HOE 109453	Unknowns	Non-polar unknowns
Kidney	0.170	94.34	1.46	82.68	4.86	-	-	-
Liver	0.061	64.38	2.07	41.20	10.80	-	2.20*	-
Heart	0.011	84.46	5.44	65.81	-	-	-	-
Subcut fat	0.016	100	-	-	-	-	-	100**
Perirenal fat	0.011	70.47	-	52.02	-	-	-	-
Omental fat	0.010	46.01	-	14.4	-	-	-	-
Milk	0.001	78.90	17.1	38.1	-	-	-	-
Urine	NA	100	-	89-93	3-4	4-7	-	-
Lungs	0.017	NA	NA	NA	NA	NA	NA	NA
Muscle hindquarter	0.004	NA	NA	NA	NA	NA	NA	NA
Muscle flank	0.004	NA	NA	NA	NA	NA	NA	NA
Muscle loin	0.003	NA	NA	NA	NA	NA	NA	NA
Abomasal fluid	0.322	NA	NA	NA	NA	NA	NA	NA
Rumen fluid	0.592	NA	NA	NA	NA	NA	NA	NA
Bile	0.573	NA	NA	NA	NA	NA	NA	NA

NA – Not available

*Unknowns each <0.001 ppm

** Four non-polar unknowns each <0.01 ppm

HOE 107892: Diethyl-1-(2,4-dichlorophenyl)-5-methyl-2-pyrazoline-3,5-dicarboxylate (test article)

HOE 113225: 1-(2,4-dichlorophenyl)-5-ethoxycarbonyl-5-methyl-2-pyrazoline-3-carboxylic acid

HOE 94270: 1-(2,4-dichlorophenyl)-5-methyl-pyrazole-3-carboxylic acid

HOE 109453: 1-(2,4-dichlorophenyl)-5-methyl-2-pyrazoline-3,5-dicarboxylic acid

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

toxicokinetics

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: about 6 - 7 weeks (male), about 8-10 weeks (female)
- Weight at study initiation: 214 ± 5 g (male) mean, 199 ± 8 g (female) mean
- Housing: individually, expired air: two animals per cage
- Individual metabolism cages: yes, metabolism box with device for separate collection of urine and faeces
- Diet (e.g. ad libitum): commercial ready-mixed diet (Altromin 1321, produced by Altromin GmbH, Lage/Lippe, FR Germany), ad libitum
- Water (e.g. ad libitum): tap water (municipal supply), ad libitum
- Acclimation period: about 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 25-48
- Photoperiod (hrs dark / hrs light): 12/12

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 mixed in each case immediately prior to application.

VEHICLE
- Justification for use and choice of vehicle (if other than water): sesame oil
- Concentration in vehicle: about 0.2 and 20 mg/g formulation
- Amount of vehicle (if gavage): 0.88-0.96 g solution/animal


HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The test substance solution was stable after an investigating time of 24 h. Before application, 2 samples were taken from the preparation to determine the concentration. The preparations were shown to be homogeneous since the results of both measurements were identical.

Duration and frequency of treatment / exposure:

single gavage

Dose / conc.:

1 mg/kg bw/day (nominal)

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:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood, plasma (100 mg/kg group only), other tissues (Pancreas, Kidneys, Gonads, Liver, Heart, Lungs, Skeletal muscle, Subcutaneous fat, Retroperiton. fat, Brain, Bones), cage washes, expired air
- Time and frequency of sampling: Sampling intervals for urine: 0 - 2, 2 - 4, 4 - 8, 8 - 24, 24 - 48, 48 - 72, 72 - 96, 96 - 120, 120 - 144, 144 - 168 hours; for cage washings: 0 - 24, 24 - 72, 72 - 120, 120 - 168 hours; for faeces: in 24-hour intervals up to 168 hours after application; blood and tissue samples were taken 168 h after application; continuous suctioning of 0.2 m³ cage air per hour.

Statistics:

The pharmacokinetic parameters were analyzed by graphical methods and split up into different exponential processes (feathering). The biological half-lives (t1/2) were calculated by least square straight line fitting. Areas under the curve (AUC) were estimated applying the trapezoidal rule using a desk-top computer (model 9845 B, Hewlett-Packard, Bad Homburg, FR Germany). The square of the correlation coefficient was taken as a main criterion in ascertaining the limits of validity. The detection limit was determined using the individual blank value for the corresponding biological
material by the following formula: Detection limit (µg/g) = background (Bq)/[sample weight (g) x specific radioactivity (Bq/µg].
The concentrations calculated in (µg equivalents/g) are considered significant only if the double blank value has been measured.

Preliminary studies:

not performed

Details on absorption:

Not examined.

Details on distribution in tissues:

The concentrations in the organs and tissues and blood/plasma at the end of the study (7 days after dosing) were on average 2 to 3 times higher in the females than in the males, regardless of dose. The highest radioactivity concentrations were found in the plasma and the blood, those in the plasma being higher by a factor of 1.6-1.8 than those in the blood in animals of either sex and at either dose. Detectable radioactivity in bones occurred only among the females. No radioactivity at all was detected in the central nervous system in either sex or at either dose. Following administration of 1 mg/kg, the concentrations ranged from 0.0035 (skeletal muscle) to 0.063 µg equivalents/g (plasma) in the females, and from 0.0017 (skeletal muscle) to 0.021 µg equivalents/g (plasma) in the males.

In the following presentation, the values for the females are always given first, followed by those for the males. The values for plasma (0.063/0.021 µg equivalents/g) and blood (0.036/0.012 µg equivalents/g) was followed by those for lungs with 0.028/0.012 µg equivalents/g. Of the other organs and tissues, the ovaries showed concentrations of 0.019 µg equivalents/g, which were (dose corrected) 4.8 times higher than those in the testes.
The next highest concentrations were in fat, the values of 0.015/0.0047 µg equivalents/g in subcutaneous fat being higher than the 0.0071/0.0034 µg equivalents/g in retroperitoneal, perirenal fat.

The concentrations in the other organs/tissues (skeletal muscle, pancreas, liver, heart muscle, bones and kidneys) ranged in the females from 0.0035 to 0.011 µg equivalents/g, and in the males (testes included) from 0.0017 to 0.0039 µg equivalents/g.

Following administration of approx. 100 mg/kg, the mean concentrations (corrected for dose) were higher than after 1 mg/kg by a factor of 38 (± 10) in the females and 32 (± 10) in the males. The measured concentrations ranged from 0.12 µg equivalents/g (bone) to 1.96 µg equivalents/g (plasma) in the females and from 0.058 µg equivalents/g (skeletal muscle) to 0.59 µg equivalents/g (plasma) in the males.
The order was the same as at the lower dose: plasma (1.96/0.59 µg equivalents/g), blood (1.19/0.38 µg equivalents/g), lungs (0.75/0.31 µg equivalents/g), ovaries (0.63 µg equivalents/g) and fat, where again the concentrations in the subcutaneous fat (0.61/0.21 µg equivalents/g) were somewhat higher than those in the retroperitoneal fat (0.45/0.21 µg equivalents/g).

The concentrations in the other examined organs/tissues (skeletal muscle, pancreas, liver, heart muscle, bone, kidneys) ranged between 0.12 and 0.34 µg equivalents/g in the females and 0.058 and 0.16 µg equivalents/g in the males.

Details on excretion:

Investigation of the cage air in which expired air is included, showed that no volatile radioactivity was present in the study period of 24 hours after dosing.

The main route of radioactivity excretion was the urine, where, after administration of the low dose of 1 mg/kg, 76.80 ± 8.77 % of the dose was recovered for the males and 88.22 ± 2.80 % for the females. The comparative values for the high dose of 100 mg/kg were 67.66 ± 6.18 % for the males and 79.48 ± 9.34% for the females.
Following administration of 1 mg/kg, the percentage of the dose recovered in faeces was 29.61 ± 7.43 % for the males and 13.59 ± 2.78 % for the females. Following administration of 100 mg/kg, the faecal portion was 32.09 + 5.30 % for the males and 23.85 +9.07 % for the females.
One day after treatment, between 83 and just on 91 % of the applied radioactivity had been excreted, depending on the animal group. Two days after treatment, this amounted to between 91 and over 99% of the dose.

Excretion was biphasic. The biological half-lives for excretion via urine for males and females at the low and high dose were between 4 and 7 h for the rapid phase I, about 5 h on average, and at a mean value of 27 h (23-31 h), i.e. just over one day, for the terminal phase II. The values for radioactivity elimination via faeces were similar, i.e. 6 - 7 h, with an average of 6 h for phase I and on average 1.5 days (31-41 h) for phase II.

The autoradiographic investigations were also carried out 7 days after treatment in one male and one female rat. The objective was to investigate the distribution of the low dose of 1 mg/kg. In the female, blood showed the highest densities, followed by lungs. High concentrations were also present in the subcutaneous and mesenteric fat, but not in brown fat. More radioactivity was found in the liver and in connective tissue structures (e.g. fasciae, intestinal walls, walls of the eyes), and also, in lower concentrations, in the kidneys. No density distinguishable from zero effect was present in the central nervous system. In the male, the autoradiograms of the radioactivity in the organism showed such slight densities that it was impossible to arrive at any reliable evaluation. A faint representation of radioactivity could be discerned only in the region of the epididymides and in some blood vessels.

Reference 3

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

Objective of study:

excretion

metabolism

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

Radiolabelling:

yes

Remarks:

14C

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

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

Dose / conc.:

1 mg/kg bw/day (nominal)

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.

Preliminary studies:

not performed

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.

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.

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.

Reference 4

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 85-3 (Dermal Penetration)

Deviations:

no

Principles of method if other than guideline:

The objective of the study was to investigate the penetration and absorption pattern of Hoe 107892 in rats following dermal application and to determine the kinetics of the absorption and elimination process in skin, blood and carcass. The study also included quantitative measurements of the products excreted by faeces and urine.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: about 6-9 weeks
- Weight at study initiation: 225 - 250 g
- Fasting period before study:
- Housing: individually in rat-specific plastic metabolism boxes with device for separate collection of urine and faeces
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): milled commercial ready-mixed diet (Altromin® 1324/1321 Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water (e.g. ad libitum): tap water (municipal supply), ad libitum
- Acclimation period: approx. 1 day before start of study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 35 - 55
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

water

Duration of exposure:

0.5, 1, 2, 4, 10, 24 h

Doses:

- Nominal doses: 0.22, 2.22, 22 mg/kg (corresponding to approx. 0.0052 mg/cm², 0.052 mg/cm², 0.52 mg/cm²)
- Actual doses: determined for each rat
- Actual doses calculated as follows: The exact dose was measured by weighing the syringe before and after application. The administered dose was calculated on the basis of the active ingredient concentration and the amount of preparation administered
- Rationale for dose selection: According to information supplied by the sponsor the doses are within, or considerably above, the range likely for maximum operator exposure (5.3 µg/cm²).

No. of animals per group:

4

Control animals:

yes

Remarks:

1 animal

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The radioactive test substance was dissolved in a blank formulation, which then received the code Hoe 046360 24 EW14 A7. This contained 75 g Hoe 107892/1 and 69 g Hoe 046360/1. Registration of this formulation has been applied for. The RCL obtained the blank formulation without added Hoe 107892 from the formulation department of AgrEvo. The radioactively labelled Hoe 107892-14C was then added in the RCL.The preparation was produced before the start of the study and was used over a period during which its stability was guaranteed.
low dose: ca. 0.75 mg/g preparation
middle dose: ca. 7.5 mg/g preparation
high dose: ca. 75 mg/g preparation

The preparation for the high dose was produced first. This was then diluted with water (aqua fontana) to produce the middle and low doses ("spray liquids").
- Method of storage: no data


APPLICATION OF DOSE:
approx. 0.3 g preparation/kg body weight ( = ca. 0.07 g/animal) was administered evenly with a 1 ml disposable syringe


TEST SITE
- Preparation of test site: The hair on the animals' backs had been removed with a clipper 24 h before application, care being taken to ensure that the skin was not damaged. The skin was then wiped clean with acetone and a plastic application ring had been attached to the rump with a non-irritating cyanacrylate adhesive.
- Area of exposure: 10.2 cm²
- Type of cover / wrap if used: After the substance had dried, a perforated cover permitting the air to circulate freely was placed over the treated skin area in such a way as to avoid any contact by rubbing, scratching, licking, etc.
- Time intervals for shavings or clipplings: 24 h before application


SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: plastic application ring and perforated cover


REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: before each animal was killed
- Washing procedures and type of cleansing agent: the skin on the application site was washed several times with balls of cottonwool wrapped in gauze and soaked in a solution of aqua fontana and LUZIL "prima" washing-up liquid at body temperature (about 37 °C). This was followed by repeated rinsing with water (4 times). The radioactivity in this sample contains the portion of the dose which has not penetrated.
- Time after start of exposure: 0.5, 1, 2, 4, 10, 24 hours


SAMPLE COLLECTION
- Collection of blood: Blood samples were not taken separately. The blood removed at killing was used in each case.
- Collection of urine and faeces: Urine and feces were collected up to the time of sacrifice. The urine in the bladder at this time was counted as part of the urine fraction, faeces in the intestine at this time were counted as part of the faecal fraction.
- Collection of expired air: not done
- Terminal procedure: no data
- Analysis of organs: blood, faeces (including intestinal content), urine (including bladder content), liver, kidney, skin (untreated area), skin (treated area), carcass, rinsing water used for application ring; radioactivity measured here counted as portion of dose which had not penetrated. Before the carcass was homogenised the remaining skin had to be removed. This was weighed and submitted for examination.


SAMPLE PREPARATION
- Preparation details: The urine obtained at any one collection interval was filled up with water and weighed. One ml or 2 ml aliquots were directly measured after addition of commercial scintillation cocktail. The feces accumulated during each of the collection intervals were dried at room temperature and pulverised with normal commercial kitchen mixers. Aliquots of this were then weighed out and combusted. The resulting CO2 was absorbed with Carbo-Sorb (Canberra-Packard) and the samples were measured after addition of the scintillator. To investigate tissue distribution, larger organs and tissues were homogenised with Ultra-Turrax-appliances after addition of a mixture of ethanol and deionised water (1:1), the amount of which depended on the consistency of the tissue. Smaller tissues were finely cut. The samples were dissolved in volumetric flasks at 60 - 70 °C in Digestin (Merck, Darmstadt, Germany). Addition of approx. 0.2 ml Perhydrol (Merck) was sufficient to remove discolourations. Measurements were then performed after addition of the scintillator. Samples of the blood obtained at sacrifice were absorbed in cut-to-size filter paper, weighed, dried at room temperature and combusted, after which the resulting 14CO2 was absorbed with Carbo-Sorb (Canberra Packard). The radioactivity was determined after addition of the scintillator. The radioactivity of the cotton swabs and of the application ring was extracted with Acetic acid ethyl ester. Measurements were then performed after addition of the scintillator.


ANALYSIS
- Method type(s) for identification: Liquid scintillation counting
- Liquid scintillation counting results (cpm) converted to dpm as follows:
- Validation of analytical procedure: blank values were also measured in biological material and subtracted from the measured values. These blank values had been obtained from biological material collected from the animals prior to administration (urine, faeces). For distribution in organs and tissues, identical material from an untreated animals was measured at the same time and subtracted from values measured in the samples.
The results are given in µg equivalents Hoe 107892/g and excreted amounts as percentages of administered radioactivity, i.e. they represent the sum of the original compound and/or radioactively-labelled metabolites. To obtain a unit independent of metabolisation, the test preparation concentrations in µg equivalents/g can be converted to nmol by the following formula: 1µg is equivalent to 2.68 nmol Hoe 107892.
- Limits of detection and quantification: The detection limit was determined using the individual blank values for the corresponding biological material by the following formula: Detection limit = background [Bq]/(sample weight [g] * specif. radioactivity [Bq/µg]).
The concentrations (in µg equivalents/g) were considered significant only if the double blank value had been measured. The measured impulses had to be above the limit of exclusion given in each particular case.

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

Value ranges over the different exposure times are given for the high, mid and low dose groups (in this order):
- Non-occlusive cover + enclosure rinse (application device): 7.67-28.84%, 2.89-15.39 , 2.53-7.06%
- Skin wash: 55.44-74.86%, 47.52-75.58%, 44.44-66.89%
- Skin test site: 0.78-5.28%, 4.03-7.71%, 7.05-12.98%
- Skin, untreated site: 0.04-0.42%, 0.12-1.62%, 0.44-2.50%
- Blood: 0.034-0.55 %, 0.23-2.02%, 0.75-3.26%
- Carcass (and tissues): 0.20-1.68 %, 0.87-6.44%, 3.07-10.32%
- Urine + cage wash + urinary bladder content: 0.065-2.87%, 0.037-12.32%, 0.049-18.57%
- Faeces (and intest. content): 0.016-0.90%, 0.054-7.12%, 0.31-4.48%

Total recovery:

- Total recovery: ranging from 76.72 - 94.45 % for all dose groups and all timepoints
- Recovery of applied dose acceptable: yes

Dose:

0.53 mg/cm²

Parameter:

percentage

Absorption:

0.4 - 6 %

Remarks on result:

other: 0.5 - 24 h

Remarks:

direct method

Dose:

0.053 mg/cm²

Parameter:

percentage

Absorption:

1.2 - 27.9 %

Remarks on result:

other: 0.5 - 24 h

Remarks:

direct method

Dose:

0.0053 mg/cm²

Parameter:

percentage

Absorption:

4.2 - 30.8 %

Remarks on result:

other: 0.5 - 24 h

Remarks:

direct method

Conversion factor human vs. animal skin:

no data

Of the radioactivity on the skin at the application site, an average of between 44.4% and 75.6% was removed in the washings. Taken together with the 2.5% to 28.8% in the washings from the application ring, the non-penetrated portion of the dose amounted to between 49% and 84.3%. This portion was comparable at all doses, but also decreased at all doses in proportion to the length of exposure.

In the blood, the lowest concentrations after the short exposures were 0.043, 0.014 and 0.183 µg equivalents/g. They increased with longer exposure times and reached their maximum at the high and middle doses only after 24 h (2.51 and 0.996 µg equivalents/g, corresponding to 0.6 and 2.02 %, respectively, of administered dose), whereas at the low dose the maximum of 0.179 µg equivalents/g (3.3 % of administered radioactivity) was already reached after 10 h exposure. The maximum concentrations did not show a linear dose relationship.

After the longest exposure period (24 h), the portion of administered radioactivity excreted was 3.8 % (2.9 % urine, 0.8 % feces) at the highest dose, 19.4 % (12.3 % urine, 7.1% feces) at the middle dose, and 23.1 % (18.6% urine, 4.5% feces) at the low dose. The renal portion was invariably higher than the faecal. In general there was at first a rapid, then a considerably reduced increase in the concentrations in the excreta of the animals from the high and middle dose groups up to the longest exposure period, whereas the increase was at first rapid and then largely uniform in the case of the animals from the low dose group.

The highest concentrations in the examined organs and tissues were found at all examination times in the skin at the application site. The mean values for all animals were 65.2 µg equivalents/g (4.17%) in the high dose group, 11.3 µg equivalents/g (6.03%) in the middle dose group, and 2.0 µg equivalents/g (9.26%) in the lowest dose group. No relation was observed between the concentrations and the length of exposure.

The radioactivity which had penetrated the skin was measured not only in the excreta but also in the internal organs and tissues. Concentrations higher than those in the blood were found to some extent in the kidney and liver after shorter exposure periods, but after 24 h exposure the concentrations in the blood at all doses were higher than in the examined organs and tissues.

Using the direct method, the absorption rate was determined by adding the portions of radioactivity in the examined organs and tissues, the carcass, the faeces (+ intestinal contents) and the urine (+ bladder content and cage washings).

Absorption quotas determined by the direct method (% of administered dose):

	Duration of exposure (h)
Nominal dose (mg/cm²)	0.5	1	2	4	10	24
0.52	0.35	0.43	0.49	0.78	1.31	6.00
0.052	1.19	2.96	3.07	7.05	11.06	27.90
0.0052	4.18	13.17	18.46	17.70	36.04	30.85

Description of key information

Oral absorption, rat: > 65-72%, assumed almost complete after longer intervals

Excretion rat: 78-99% (65-88% urine, 13-32% feces)

Metabolites: 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]

Tissue distribution (in the order of magnitude): plasma, blood, lungs, ovaries, subcutaneaous fat, retroperitoneal fat

Rate for dermal absorption: rat, 0.5 to 24 h exposure: 0.35 to 30.85% (depending on exposure time and dose)

Additional information

The information provided demonstrate absorption via the gastrointestinal tract to be the major route with at least 65 - 72 % after only 48 hours, and is assumed to be almost complete after longer time intervals. The rapid and nearly complete excretion mainly via the urine and to a much lesser extent the feces and the absence of the unchanged parent compound in the urine demonstrate that the test compound and its metabolites are not retained for long in the animal organism and, consequently, that enterohepatic circulation does not play a significant role.

Based on the findings in urine and feces the metabolism of the test compound 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 aromatisation of the pyrazoline ring. The radioactivity in the blood plasma could be attributed exclusively to the relatively polar dicarboxylic acid, the main residue component in the urine.

Another study demonstrated dermal absorption ranging from 0.4 to 30.8 % measured between 0.5 and 24 hours after exposure, depending on the dose applied. Excretion via urine and feces ranged from 3.8 % for the high dose to 23.1 % for the low dose of the administered radioactivity, the highest concentrations in the examined organs and tissues were found at all examination times in the skin at the administration site, demonstrating that even at higher doses and longer times not all administered radioactivity is absorbed through the skin.

The radioactivity which had penetrated the skin was not only measured in the excreta but also in the internal organs and tissues. Concentrations higher than in blood were found to some extent in the kidney and liver after shorter exposure periods, but after 24 h exposure the concentrations in blood at all doses were higher than in the examined organs and tissues.

Discussion on bioaccumulation potential result:

There are 2 studies available performed in rats, one focusing on absorption, excretion and metabolite formation, the other one addressing tissue distribution and excretion. Therefore, both studies taken together give a good overview of the complete situation and are considered as key and supporting study.

Both studies demonstrated comparable numbers for excretion ranging between 78 - 99 % within 48 hours, of which 65 - 88 % are excreted via urine, and 13 - 32 % via feces. Absorption via the gastro-intestinal tract was estimated to be at least > 65 - 72 %, based on urine excretion levels within 48 hours in that study. Longer collection intervals would even result in higher levels. Therefore, absorption can thus be assumed to be almost complete.

High excretion levels 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 enterohepatic circulation does not play a significant role. This conclusion is confirmed by the relatively low portions excreted with the feces.

The following metabolites were identified:

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 could be evidently observed. No parent compound was detected in the urine, whereas it was the main portion in the feces of the males. 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.

The highest radioactivity concentrations were found in the plasma and the blood, followed by those in the lungs, the ovaries, the subcutaneous and the retroperitoneal fat.

A third study is available that was included 7 daily applications to a goat. The results in principle reflected what was found in the rat, main excretion route was via the urine with 63 % the feces with 13 % of the daily dose, the same metabolites were identified. Slight differences were only observed in the tissue distribution of radioactivity with clearly less radioactivity found in the plasma and the blood of the goats, compared to rats, a considerably high aount could be recovered from the rumen fluid. Only less than 0.01 % of the daily dose wa eliminated daily in the milk.

Discussion on absorption rate:

Of the radioactivity on the skin at the application site, an average of between 44.4 % and 75.6 % was removed in the washings. The non-penetrated portion of the radioactivity on the skin at the application site amounted to between 49 % and 84.3 %. This portion was comparable at all doses, but also decreased at all doses in proportion to the length of exposure.

Blood concentrations increased with longer exposure times and reached their maximum at the high and middle doses only after 24 h (0.6 and 2.02 %, respectively, of administered dose), whereas at the low dose the maximum 3.3 % of administered radioactivity was already reached after 10 h exposure. The maximum concentrations did not show a linear dose relationship.

After the longest exposure period (24 h), the portion of administered radioactivity excreted ranged from 3.8 % (2.9 % urine, 0.8 % feces) at the highest dose to 23.1 % (18.6 % urine, 4.5 % feces) at the low dose. The renal portion was invariably higher than the faecal. In general there was at first a rapid, then a considerably reduced or uniform increase in the concentrations in the excreta.

The highest concentrations in the examined organs and tissues were found at all examination times in the skin at the application site. The mean values for all animals were 4.17 % in the high dose group, 6.03 % in the middle dose group, and 9.26 % in the lowest dose group. No relation was observed between the concentrations and the length of exposure.

The radioactivity which had penetrated the skin was measured not only in the excreta but also in the internal organs and tissues. Concentrations higher than those in the blood were found to some extent in the kidney and liver after shorter exposure periods, but after 24 h exposure the concentrations in the blood at all doses were higher than in the examined organs and tissues.

Using the direct method, the absorption rate was determined by adding the portions of radioactivity in the examined organs and tissues, the carcass, the faeces (+ intestinal contents) and the urine (+ bladder content and cage washings), ranging from 0.35% to 30.85%.