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

Dermal absorption

Administrative data

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

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

603-923-2

Cas Number:

135590-91-9

Molecular formula:

C16H18Cl2N2O4

IUPAC Name:

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

Radiolabelling:

yes

Test animals

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

Administration / exposure

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.

Results and discussion

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

Percutaneous absorption

Percutaneous absorption 1

Dose:

0.53 mg/cm²

Parameter:

percentage

Absorption:

0.4 - 6 %

Remarks on result:

other: 0.5 - 24 h

Remarks:

direct method

Percutaneous absorption 2

Dose:

0.053 mg/cm²

Parameter:

percentage

Absorption:

1.2 - 27.9 %

Remarks on result:

other: 0.5 - 24 h

Remarks:

direct method

Percutaneous absorption 3

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

Any other information on results incl. tables

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