kresoxim-methyl (ISO);methyl (E)-2-methoxyimino-[2-(o-tolyloxymethyl)phenyl]acetate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: (GLP, QA)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1993

Materials and methods

Objective of study:

distribution

Principles of method if other than guideline:

Determination of tissue distribution of radio-labeled test substance by quantitative whole-body autoradiography (QWBA) in male and female Wistar rats after a single oral administration of test substance.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

EC Number:

604-351-6

Cas Number:

143390-89-0

Molecular formula:

C18 H19 N O4

IUPAC Name:

methyl (2E)-2-methoxyimino-2-[2-[(2-methylphenoxy)methyl]phenyl]acetate

Details on test material:

- Name of test material (as cited in study report): Non-radiolabelled Reg No 242 009 and Reg No 242 010 and labelled Reg No 242 009
- Molecular weight (if other than submission substance): 313.34
- Physical state: beige powder
- Analytical purity: 98.7% and 97.7% (see below) for non labelled test substance (HPLC)
- Lot/batch No.: N21 (ZST No 90/577) and L 48/92 for non labelled and 353-01 for labeled test substance
- Radiochemical purity (if radiolabelling): 99.2% for the labelled test substance (thin layer chromatography (TlC) and high performance liquid chromatography (HPLC)). Following chromatography, the areas of radioactivity on the TLC plate were located and quantified using a RITA linear analyser (Lablogic Systems Ltd, Sheffield, England). The non-radiolabelled standard was visualised by irradiation with UV light at 254 nm. Non-radiolabelled Reg No 242 009 and Reg No 242 010 were used as authentic standards.
- Specific activity (if radiolabelling): 96.38 pCi/mg (3.566 MBq/mg) and 30.2 mCi/mMol (1117.4 MBq/mMol)
- Locations of the label (if radiolabelling): 2-[2-(2-methylphenoxy)-methyl]-phenyl[phenyl-U-14C]-2-(methoxyimido) acetic acid methyl ester
- Stability under test conditions: the stability of the formulated test article was investigated over 4 h using the HPlC and TlC systems
- Storage condition of test material: radiolabelled and non-radiolabelled test articles were stored at approximately -20 and +4°C, respectively

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:(WI)BR strain rats were obtained from Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: approximately 8 weeks of age on arrival
- Weight at study initiation: the weight of the animals at the start of treatment was 207 to 216 g and 161 to 182 g for males and females respectively
- Fasting period before study: the diet was removed overnight prior to dosing and returned approximately 4 h after administration of the radiolabelled test article
- Housing: five per cage according to sex, in wire floor polypropylene cages suspended over polypropylene dirt trays containing soft white wood sawdust (Special Diet Services, Stepfield, Witham, Essex)
- Individual metabolism cages: no
- Diet (e.g. ad libitum): animals were allowed free access to commercial pellet diet, SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diet Services, Stepfield, Witham, Essex)
- Water (e.g. ad libitum): animals were allowed free access to mains water from glass bottles attached to the cages. The contents of the bottles were changed daily.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 air changes/h
- Photoperiod (hrs dark / hrs light): 12 h light (0600 to 1800 h) and 12 h darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was prepared as a suspension in 1% (w/v) aqueous carboxy methylcellulose to provide a nominal dose volume of 5 mL/kg body weight

(14C)-Reg No 242 009 was administered orally, by gavage, at a dose level of 50 mg/kg body weight corresponding to a nominal radioactive dose of 20 pCi (740 kBq) per animal

Duration and frequency of treatment / exposure:

the animals were treated once

Doses / concentrations

Remarks:

Doses / Concentrations:
50 mg/kg body weight

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

none

Details on study design:

Oral administration was selected because this is the administration route employed in the pertinent toxicology studies.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (distribution)
- Tissues and body fluids sampled (delete / add / specify): whole body
- Time and frequency of sampling: at the following intervals post-dose, one animal of each sex was killed by asphyxiation in a rising concentration of carbon dioxide: Time points: 0.5, 2, 8, 24 and 96 h
- Other:
After sacrifice, each rat was pinned out on a board and rapidly frozen by total immersion in a mixture of solid CO2/hexane (ca -80 DC) for approximately 30 min.
After trimming off the legs and tail, the frozen carcass was set in a block of aqueous 2% w/v carboxymethylcellulose and mounted onto the stage of a PMV 450MP cryo-microtome maintained at about -20 DC (Thermometric Ltd, Northwich, Cheshire). Sagittal sections (about 25pm) were then obtained at 6 levels through the carcass: Level A, Exorbital lachrymal gland (males), ovary (females); Level B, Intra-orbital lachrymal gland; Level C, Harderian gland; Level D, Adrenal gland; Level E, Thyroid; Level F, Brain and spinal cord.
The sections were mounted on Cellux tape, were freeze-dried in and placed in contact with Hyperfilm B-max. After 21 and 49 days of exposure at ca -20°C, the autoradiograms were developed in and fixed Y-ray liquid fixative. These autoradiograms were used in the evaluation of the results. One freeze-dried section at each level was mounted and used for reference purposes when evaluating the autoradiograms. Enlargements of areas of interest were prepared where appropriate.

- Image analysis of whole-body autoradiograms: in addition to the usual visual evaluation, the autoradiograms were analysed using the validated Seescan Solitaire Plus Quantitative Whole-Body Autoradiography System (QWBAS) to get quantitative or semi-quantitative information. The autoradiograms (21 and 49 days exposure) were placed on a light box and the image captured using a charged coupled device camera. The Amersham (14C)-Microscales on each film were then used to calibrate the camera over a range of optical densities.

For each tissue measured, the maximum possible area was defined and quantified. Exceptions were muscle, where an area of ca 1 cm2 was measured in the lower back; skin, which was measured from the neck; blood, which was measured only in the heart and bone and bone marrow which were measured in the pelvic girdle.

- Determination of radioactivity (performed in duplicate): radioactivity was measured for 10 min or for 2 sigma %using LKB, Beckman or Packard Tri-Carb liquid scintillation counters with the facilities for computing quench-corrected disintegrations per minute (dpm).
Efficiency correlation curves were prepared for organic sample types and were routinely checked by the use of (14C}-n-hexadecane standards. The spectrometer was reca1ibrated when a deviation of greater than 3% was observed when counting quality control standards.

- Limit of detection: the limit of detection for liquid scintillation counting was taken as twice the background disintegration rate obtained from the measurement of blank samples of the same type.

Statistics:

-

Results and discussion

Preliminary studies:

none

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Not applicable

Details on distribution in tissues:

The highest levels of radioactivity were associated with the contents of the gastro-intestinal tract. Lower concentrations were found in liver (5.269 and 4.939 µg equiv/g at 0.5 and 2 h respectively) and kidney (6.483 and 6.514 µg equiv/g respectively).

Of the remaining tissues where radioactivity was present, i.e., blood, bone, bone marrow, brain, eye (including the uveal tract), Harderian gland, lung, muscle (including the myocardium), salivary glands, gonads (of male rats) thymus and thyroid, concentrations were below 1.6 µg equivalents of radio-labeled test substance/g of tissue.

At 24 h post-dose, radioactivity was detected only in the contents of the gastro-intestinal tract (at moderate levels) and in the liver and skin at low levels. After 96 h, radioactivity persisted at low levels in the contents of the intestinal tract, in both sexes as well as in or on the skin of the females.

Details on excretion:

not applicable

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

not applicable

Any other information on results incl. tables

RADIOCHEMICAL PURITY

The radiochemical purity of radio-labeled test substance was 96.6% using the HPLC system. When the peak corresponding to the Z isomer (Reg No 242 010) was included, the radiochemical purity was 99.2%. The mean radiochemical purity of radio-labeled test substance was 99.5% using two TLC systems (these systems could not resolve the Z isomer from the main peak). The overall mean value was 99.4%.

 

SPECIFIC RADIOACTIVITY

The mean specific radioactivity was 96.45 µCi/mg (3.569 MBq/mg) equivalent to 30.22 mCi/mMol (1118 MBq/mMol).

 

FORMULATION STABILITY

The stability of the formulation was assessed at 0 and 4 h after preparation. Comparison of the respective chromatograms showed no significant degradation of the radio-labeled test substance over this period.

 

CLINICAL OBSERVATIONS

During the course of the study, no overt pharmacological or toxicological signs were observed in the test animals which could have been attributed to the administration of the test substance.

 

WHOLE BODY AUTORADIOGRAPHY

Following a single oral administration of radio-labeled test substance, peak concentrations of radioactivity were attained in most tissues at 0.5 or 2 h post-dose in both male and female animals.

With the autoradiograms used in this study, the highest standard corresponded to a radioactivity concentration of 1104 nCi/g of tissue (675.2 µg equivalents of test substance/g of tissue). No tissues were found to have a concentration higher than this. Standards at the lower end of the acceptable range of optical densities corresponded to a radioactivity concentration of 5.7 or 2.6 nCi/g of tissue (3.486 or 1.590 µg equivalents of test substance/g of tissue) depending on the optical characteristics of the film. Between the highest and lowest standards detailed above, mean values were within ±5% of the actual value and all values were within a 95% confidence interval of the mean. Between the lower standards detailed above and optical densities corresponding to radioactivity concentrations of 2.6 or 1.1 nCi/g of tissue (1.590 or 0.673 µg equivalents of test substance/g of tissue), the optical density readings were within ±20% of the mean. Below these limits, optical density readings were outside ±50% of the mean and these results are reported as not quantifiable.