Methomyl

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Objective of study:

metabolism

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methomyl

EC Number:

240-815-0

EC Name:

Methomyl

Cas Number:

16752-77-5

Molecular formula:

C5H10N2O2S

IUPAC Name:

(E)-[1-(methylsulfanyl)ethylidene]amino N-methylcarbamate

Test material form:

solid: crystalline

Specific details on test material used for the study:

Radio-labelled test substance Name: [1-13C/14C]methomyl
Lot#: [1-14C]methomyl: 2729-122; [1-13C]methomyl: 2565-151
Radiochemical Purity: [1-14C]methomyl: >97%; [1-13C]methomyl: 99.9%

Non-radiolabelled test substance name: DPX-X1179
Lot#: X1179-379
Purity: 98.9%

Radiolabelling:

yes

Test animals

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shamrock (GB) Ltd., Sussex, United Kingdom
- Age at study initiation: 1-2.5 years
- Weight at study initiation: 2.040 to 2.246 kg
- Housing: Monkeys were housed individually in labelled stainless steel metabolism cages fitted with perspex splash fronts immediately after dosing to facilitate the separate collection of urine and faeces
- Diet: Standard primate diet (Special Diets Services Ltd., Witham, Essex, United Kingdom)
- Water: ad libitum
- Acclimation period: Atleast 2 months

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 26°C
- Humidity: 45 to 75%
- Air changes (per hr): Approximately 15
- Photoperiod: 12 hrs dark / hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.1 M Sodium acetate buffer

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Nominal level of radiolabelled test substance at dose level of 5 mg/kg bw were prepared with nominal dose volumes of 4 mL/kg bw in a solution of 0.1M sodium acetate buffer previously adjusted to pH 5 with acetic acid. The dose solution were prepared within 2 hoursvof dosing.

Duration and frequency of treatment / exposure:

Single oral dose

Doses / concentrations

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

5 males

Control animals:

no

Details on study design:

- Dose selection rationale: The 5 mg/kg dose level was selected because it has been shown to produce signs of acetylcholinesterase inhibition (mild miosis) in previous primate studies without producing severely toxic effects that would compromise a meaningful evaluation of the data.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, expired air, cage washes, heart, lungs, spleen, brain, testes, gastro-intestinal tract, kidneys, liver and samples of fat, muscle and skin. Femurs were removed for bone and bone marrow samples.
- Time and frequency of sampling: Urine samples were collected separately in containers cooled in solid CO2 at 0-6, 6-24 hours and at 24-hour intervals for 168 hours. Faeces and cage debris were collected separately from each animal during 24-hour intervals up to 168 hours. After each 24-hour collection period the cages were with water and the washings were retained. Expired air was collected for the first 48 hours and traps changed at 0-6, 6-24 and 24-48 hours.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine
- Time and frequency of sampling: Equal proportions of the volumes of 0-6 and 6-24 hour urine were pooled from individual monkeys to provide 0-24 hour samples (representing >90% urine radioactivity) and were analyzed directly without further clean-up.
- From how many animals: Urine samples are pooled from all animals.
- Method type(s) for identification: Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), Liquid scintillation countimg (LSC)

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
Samples of pooled urine were adjusted to pH 5 with 0.2M sodium acetate buffer solution. Samples were incubated for ca. 18 hours at 37°C in the presence of the ß-glucuronidase/sulphatase for enzymic hydrolysis.
In all cases the samples were shown to have ß-glucuronidase activity by further incubation of subsamples in the presence of phenolphthalein glucuronide for 30 minutes at 37°C. The formation of a red color on addition of 1M sodium hydroxide confirmed ß-glucuronidase activity. Incubates were analyzed directly by TLC or HPLC.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Absorption appeared to be complete; only about 3% of the dose was found in the faeces. At sacrifice, 168 hours after dosing, 4 to 5% of the dose was found to remain in the tissue.

Main ADME results 2

Type:

distribution

Results:

Concentrations of radioactivity were highest in liver (0.7-0.9 µg equivalents/g), fat (0.4-0.7 µg equivalents/g) and kidney (0.4-0.5 µg equivalents/g).

Main ADME results 3

Type:

metabolism

Results:

18 radioactive metabolites were found in the urine with no individual metabolite accounting for greater than 4% of the dose.

Main ADME results 4

Type:

excretion

Results:

Test substance metabolites were eliminated mainly in expired air and urine, with greater than 50% of the administration dose excreted during the first 24 hours.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:

Intermediate concentrations of radioactivity (0.1-0.5 µg equivalents/g) were found in other tissues and were generally higher than concentrations in the blood (0.1-0.2 µg equivalents/g).

Details on excretion:

Male monkeys excreted a total of 32.2 ± 4.3% dose in urine (including cage wash), 3.2 ± 0.4% in faeces, 33.6 ± 3.2% as 14CO2 and 5.4 ± 1.4% [1-14C]acetonitrile. Greater than half of the dosed radioactivity (~63%) was excreted during the first 24 hours. At sacrifice, approximately 5% of the radioactivity was retained in the tissues, mostly in fat (1.6%) and muscle (1.4%).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Test substance is rapidly and extensively metabolized in monkeys. A combination of HPLC and TLC was used to characterize and quantitate the 18 radioactive metabolites found in the urine. Qualitatively the urine metabolite profiles from each monkey were similar with no individual metabolite accounting for greater than 4% of the dose. Small amounts of acetonitrile (1-3% dose), acetate (0.2-0.6% dose), acetamide (0.3-0.5% dose) and methomyl oxime sulphate dose) were found in the urine. The mercapturic acid derivative of test substance (a major rat metabolite) accounted for about 1% of the dose. No appreciable changes in the proportions of radioactive components in the urine after treatment with ß-glucuronidase/sulphatase relative to the control samples were observed. In addition, no acetohydroxamic acid (N-hydroxy acetamide, a putative metabolite of acetamide) was observed in the urine before or after glucuronidase incubation. Chromatographic analysis confirmed the absence of test substance and the following potential test substance metabolites in the urine like anti-methomyl, syn- or anti-methomyl oxime, methomyl sulphoxide, methomyl sulphone, methomyl oxime sulphoxide and hydroxy methyl methomyl.

Any other information on results incl. tables

Recovery:

Overall, the material balance was 80-82% of the administered dose for 3 of the 4 animals, with a slightly lower recovery of 74% for fourth animal. This lower recovery may be due to interruption of the collection system. Volatiles were not collected after 48 hours in order not to unduly stress monkeys in the later part of the study. This may have been a factor in not obtaining 100% material balance in this study.

Proposed metabolic pathways of test substance in monkeys:

Two major metabolic pathways are evident in monkeys one resulting in the formation of 14CO2 and one resulting in the formation of 14C-acetonitrile. In the 14CO2 pathway, the carbamate ester is cleaved, releasing the oxime portion of the molecule. The oxime was not observed in the urine, which suggests that the oxime is readily metabolized. Since 14CO2 has been shown to be a major metabolite of syn-methomyl oxime when orally administered to rats, syn-methomyl oxime is likely to be the major precursor in the formation of 14CO2 which comprised at least 34% of the dose administered in this study.

 In the 14C-acetonitrile pathway, the test substance isomerizes in vivo to form the anti-methomyl isomer (not detected). This anti-isomer, possibly along with the corresponding hydrolysis product, anti-methomyl oxime, undergo a Beckman-type rearrangement and elimination reaction to form acetonitrile. The resultant acetonitrile is mostly found in the expired air of the monkeys (approximately 5% of the administered dose). In addition, other metabolic pathways resulting in the formation of numerous polar urinary metabolites were present. Minor urinary metabolites tentatively identified included the mercapturic acid derivative of test substance (a major rat metabolite), acetamide and acetic acid (minor rat metabolites). Many of unidentified urinary metabolites (none of which represented >4 % of the applied dose) had similar chromatographic behaviour to minor metabolites found in an earlier rat metabolism study and are probably the result of extensive metabolism of primary metabolites.

Applicant's summary and conclusion

Conclusions:

- Absorption appeared to be complete; only about 3% of the dose was found in the faeces. At sacrifice, 168 hours after dosing, 4 to 5% of the dose was found to remain in the tissue.
- Tissue concentrations presented as µg equivalents/g fresh tissue based on radioactivity. Concentrations of radioactivity were highest in liver (0.7-0.9 µg equivalents/g), fat (0.4-0.7 µg equivalents/g) and kidney (0.4-0.5 µg equivalents/g).
- Extensive metabolic degradation of 14C-labelled test substance was observed, at least 18 metabolites were found in the urine, none of which individually accounted for more than 4% of the dose. Tentatively identified metabolites in urine (as a percentage of the dose) included the mercapturic acid derivative of test substance (0.4-1.2%), acetonitrile (1-3%), acetate (0.2-0.6%), and acetamide (0.3-0.5%).
- Male monkeys excreted a total of 32.2 ± 4.3% dose in urine (including cage wash), 3.2 ± 0.4% in faeces, 33.6 ± 3.2% as 14CO2 and 5.4 ± 1.4% [1-14C]acetonitrile. Greater than half of the dosed radioactivity (~63%) was excreted during the first 24 hours.

Executive summary:

The study was conducted according to guideline EPA 81-5 to evaluate the absorption, distribution, metabolism and excretion of [1-14C]-labelled test substance and its metabolites in four male monkeys following a single oral dose. Specific analyses for acetamide in the urine were also included to determine whether or not the metabolism of test substance to acetamide was a significant pathway in the monkey.

Clinical signs of cholinesterase poisoning in the monkeys were not observed at the 5 mg/kg dose level used in this study. Cholinesterase inhibition would be expected based on results seen in other studies, however, it was not monitored.

The excretion rate and retention of radioactivity did not vary appreciably between individual animals. Test substance metabolites were eliminated mainly in expired air and urine, with greater than 50% of the administration dose excreted during the first 24 hours. During the first 48 hours after dosing, an average of 34% dose was eliminated as 14CO2 and 5% dose was eliminated as 14C-acetonitriIe. An average of 32% dose was eliminated via the urine (including cage wash) during 0-168 hour after dosing. Absorption appeared to be complete; only about 3% of the dose was found in the faeces. At sacrifice, 168 hours after dosing, 4 to 5% of the dose was found to remain in the tissue.

Extensive metabolic degradation of 14C-labelled test substance was observed, at least 18 metabolites were found in the urine, none of which individually accounted for more than 4% of the dose. Tentatively identified metabolites in urine (as a percentage of the dose) included the mercapturic acid derivative of test substance (0.4-1.2%), acetonitrile (1-3%), acetate (0.2-0.6%), and acetamide (0.3-0.5%). The low levels of acetamide were similar to those levels found in rat urine in another rat metabolism study, demonstrating that the metabolic conversion of test substance to acetamide is not a major metabolic pathway in primates nor is it species-specific. No radioactive components in the urine corresponded to the test substance, or its major hydrolysis product, methomyl oxime. Chromatographic evidence also confirmed the absence of following potential test substance metabolites in the urine: anti-methomyl, anti-methomyl oxime, acetohydroxamic acid (N-hydroxy acetamide), methomyl sulphoxide, methomyl sulphone, methomyl oxime sulphoxide, and hydroxymethyl methomyl. No significant changes in the urine metabolite profiles were observed after ß-glucuronide/sulphatase incubation indicating an absence of glucuronide or sulphate conjugates in the urine.