Methomyl

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

metabolism

toxicokinetics

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

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

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

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

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.

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.

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).

Type:

metabolism

Results:

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

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.

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%).

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.

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.

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.

Reference 2

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

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Radio-labelled test substance Name: [1-13C/14C]methomyl
Lot#: [1-14C]methomyl: 2449-040; [1-13C]methomyl: 2565-151
Radiochemical Purity: [1-14C]methomyl: >97%; [1-13C]methomyl: 99.9%

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 0.1 M Sodium acetate

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Preliminary studies:

Pilot study was conducted with two rats (1 male and 1 female) received single oral doses of 14C-test substance. The rats were placed into separate metabolism chambers for collection of urine, faeces and expired air. Urine samples were collected separately in containers cooled in solid CO2 at 6, 6-24 hours and at 24-hour intervals for 120 hours. Faeces were collected separately at 24-hour intervals for 120 hours.
The results showed that over half of the dose was excreted in the urine within 24 hours (51% male; 57% female), with a total of 55% and 60% of the dose excreted by 120 hours in male and female rats respectively. Only 1-2% of the dose was excreted in the faeces and approximately of the dose remained in the carcass. Significant amounts of radioactivity were recovered in traps monitoring expired air. Radioactivity recovered in methanol traps for organic volatiles accounted for 13% (male) and 12% (female) of the dose and radioactivity recovered in sodium hydroxide traps for acidic volatiles accounted for approximately 16% (male) and 22% (female) of the dose (14C-acetonitrile and 14CO2 were determined to be the organic and acidic volatiles respectively. Overall material balance was excellent with approximately 95% (male) and 105% (female) of the administered dose recovered.

Type:

absorption

Results:

Absorption appeared to be complete as only about 2% of the dose was found in faeces.

Type:

distribution

Results:

Based on mean amount of radioactivity as percent of test substance in tissues, the concentration of radioactivity was highest in blood cells (3-4 µg equiv./kg), plasma (0.7-0.9 µg equiv./kg) equating to whole-blood concentrations of about 2 µg equiv./g.

Type:

metabolism

Results:

The one major metabolite identified in the urine was methomyl(L)-N-acetyl-S-[1-[[(methylamino)carbonyl] oxy] imino]-ethyl] cysteine] which accounts for 18% of the dose.

Type:

other: Elimination (Metabolism + Excretion)

Results:

The overall elimination half life was estimated to be approximately 5 hours.

Type:

excretion

Results:

The major route of elimination was via urinary excretion accounting for approximately half (~53%) of the administered dose.

Details on absorption:

At sacrifice 168 hours after dosing, 8-9% of the dose was retained in the carcass and tissues.

Details on distribution in tissues:

Mean amounts of radioactivity reported as a percent of dose in the tissues. Concentrations of radioactivity were highest in blood cells (3-4 µg equiv./g) and in plasma (0.7- 0.9 µg equiv./g). Assuming a blood packed cell volume of 0.48, whole-blood concentrations are calculated to be 1.9-2.3 µg equiv./g. Concentrations of radioactivity in all other tissues were lower than whole-blood, or blood cells or plasma alone. Tissues and organs highly perfused with blood (lung, spleen, heart, liver) generally had higher concentrations (0.4 - 0.7 µg equiv. /g) than organs less perfused (gonads, bone, fat (0.2- 0.3 µg equiv./g). There were no appreciable differences in the concentrations in tissues between male and female rats .

Details on excretion:

Male rats excreted a total of 52.9 ± 6.8% in the urine, 2.9 ± 1.2% in faeces, 21.8 ± 1.4% as 14CO2 and 12.1 ± 3.3% as [1-14C] acetonitrile. Female rats excreted a total of 52.7 ± 4.2% in the urine, 2.1 ± 0.3% in faeces, 23.1 ± 1.0% as 14CO2 and 12.9 ± 1.4% as [1-14C] acetonitrile. 8-9% of test substance was excreted in the carcass and tissues in both male and female rats. There were no statistical differences (P >0.05) in the excretion rate between the sexes. Although approximately 80% of the radioactivity was eliminated within the first 24 hours, it was necessary to keep animals in the metabolism cages for 168 hours to obtain 90+% excretion of the administered dose.

Key result

Test no.:

#1

Toxicokinetic parameters:

other: Overall elimination half life is estimated to be approximately 5 hours.

Metabolites identified:

yes

Details on metabolites:

Test substance metabolite profiles in the urine were nearly identical between male and female rats. Only one major metabolite was found in the urine, which was isolated and identified by mass spectral analyses as a mercapturic acid derivative of methomyl(L)-N-acetyl-S-[1-[[(methylamino)carbonyl] oxy] imino]-ethyl] cysteine]. More than ten additional minor urine metabolites were also observed and characterized by several chromatographic means (reverse phase HPLC, ion partition HPLC, normal phase TLC). Small amounts of acetonitrile (~2% of dose), acetate (~2% of dose) , a sulphate conjugate of test substance oxime (~3-4%) and acetamide were observed in the urine. No appreciable change in urine radioactivity was evident after treatment with ß-glucuronidase/sulphatase. No acetohydroxamic acid (a putative metabolite of acetamide) was observed in the urine, either before or after glucuronidase incubation. Acid treatment of the urine decomposed the major and most minor metabolites to 14C-acetate demonstrating that the minor metabolites shared a common two carbon fragment.
Three major metabolite fractions were separated by HPLC in blood cells, plasma and liver extracts. Qualitatively liver extract metabolite profiles appeared similar to those of blood extracts, suggesting that residual blood radioactivity in the liver is the major contributor to tissue radioactivity. 14C-Acetonitrile was the major component in blood and liver, comprising of 1/4 to 1/3 of the tissue radioactivity. Another major metabolite fraction appeared to be a glucuronide based on its apparent hydrolysis and change in polarity after incubation with ß-glucuronidase. The released radioactivity was very polar and did not appear to resemble test substance or test substance oxime in polarity. No appreciable amount of blood or liver radioactivity was characterized as acetamide or acetohydroxamic acid. No radioactivity in the blood or liver was released as acetohydroxamic acid after glucuronidase incubation. Most tissue residues could be decomposed to acetate upon heating with 6M HCI, indicating a common 2 carbon fragment in the major metabolites.

Conclusions:

- Absorption appeared to be complete as only about 2% of the dose was found in the faeces. At sacrifice 168 hours after dosing, 8-9% of the dose was retained in the carcass and tissues.
- Mean amounts of radioactivity reported as a percent of dose in the tissues. Concentrations of radioactivity were highest in blood cells (3-4 µg equiv./g) and in plasma (0.7- 0.9 µg equiv./g).
- The major metabolite was tentatively identified by mass spectral analyses as a mercapturic acid derivative of methomyl(L)-N-acetyl-S-[1-[[(methylamino)carbonyl]oxy]imino]-ethyl]cysteine] formed by displacement of the thiomethyl group. This metabolite accounted for approximately 18% of the dose. At least 10 minor metabolites were also found in the urine.
- The major route of elimination was via urinary excretion accounting for approximately half (~53%) of the administered dose. Significant amounts of radioactivity were expired in the air with ~22% of the dose collected as 14CO2 and ~13% collected as 14C-acetonitrile

Executive summary:

The study was conducted according to guideline EPA 85-1 to evaluate the absorption, distribution, metabolism and excretion of [1-14C] labelled test substance in male and female rats following a single oral dose of 5 mg/kg. This dose level produced clinical signs of cholinesterase poisoning such as tremors and humped posture with all animals recovering within 2 hours.

The major route of elimination was via urinary excretion accounting for approximately half (~53%) of the administered dose. Significant amounts of radioactivity were expired in the air with ~22% of the dose collected as 14CO2 and ~13% collected as 14C-acetonitrile. Absorption appeared to be complete as only about 2% of the dose was found in the faeces. At sacrifice 168 hours after dosing, 8-9% of the dose was retained in the carcass and tissues. Total material balance was excellent with an average of 99% of the administered dose recovered. There were no appreciable differences in the excretion rate or retention of radioactivity between male and female rats.

The metabolite profiles in the urine were comparable between male and female rats. The major metabolite was tentatively identified by mass spectral analyses as a mercapturic acid derivative of methomyl(L)-N-acetyl-S-[1-[[(methylamino)carbonyl]oxy]imino]-ethyl]cysteine] formed by displacement of the thiomethyl group. This metabolite accounted for approximately 18% of the dose. At least 10 minor metabolites were also found in the urine. Tentatively identified metabolites (as a % of dose) included a sulphate conjugate of test substance-oxime (~4%), acetate (1-2%) acetonitrile (~2%) and acetamide (~0.4%). No radioactive components in the urine corresponded to the test substance syn-methomyl, or its major hydrolysis product syn-methomyl oxime. Chromatographic evidence also excluded the presence of the anti-isomer form of test substance and anti-methomyl oxime as well as acetohydroxamic acid and urea as metabolites in the urine. Most minor metabolites appeared to share a common two carbon fragment, which upon acid hydrolysis released acetic acid.

Amounts of radioactive residues in the tissues at sacrifice were similar between male and female rats. Residues were highest in blood cells (3-4 µg equiv./g of test substance) and in plasma (0.7-0.9 µg equiv./g) equating to whole-blood concentrations of about 2 µg equiv./g. Concentrations of radioactivity were lower in all other tissues with tissue/ whole blood ratios less than one indicating no specific tissue retention. The major residue in blood and liver extracts was tentatively identified as acetonitrile based on its HPLC retention time and its volatility. The remaining blood and liver residues appeared to be more polar than acetonitrile and could be decomposed to acetic acid indicating a common two carbon fragment.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to determine the metabolism of 14C-labelled test substance in the lactating goat. A lactating goat was treated with 14C-labelled test substance for 10 days (2 doses per day) at a rate equivalent to 20 ppm in the diet. Daily samples of milk, blood, urine and feces were collected and analyzed for the presence of test substance.

GLP compliance:

no

Specific details on test material used for the study:

Radiolabelled test substance Name: 14C-Methomyl
Radiochemical purity: >99%

Radiolabelling:

yes

Species:

other: Goat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were maintained by Cannon laboratories Inc., Reading, Pennsylvania
- Diet: Afalfa hay and purina goat chow ad libitum
- Acclimation: Each goat is acclimatized in a metabolic cage before the treatment was started for 9 days.

Route of administration:

oral: feed

Vehicle:

other: Feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS AND DIET PREPARATION:
Goat I: 132 mg 14C-test substance and 132 mg analytical standard test substance were dissolved in 6.0 ml absolute ethyl alcohol. Aliquots (50 µl) of the resulting solution were each diluted to 10.0 ml in volumetric flasks and three 50 µl aliquots of each dilution were counted by liquid scintillation counting (LSC). From the resulting data, it was calculated that the methomyl in the original solution had a specific activity of 1.53 µCi/mg.
Aliquots (250 µI) of the concentrated solution were added to pulverized goat chow in 20 large gelatin capsules. Each capsule received 250 µl (11 mg, 16.8 µCi) which at two capsules per day was equivalent to 20 ppm test substance in the diet of a goat eating 1.1 kg feed per day. A radio-thin layer chromatographic (TLC) analysis of a portion of the diluted solution confirmed the identification of 14C-material as 14C-test substance with radiochemical purity >99%.
Goat Il: A solution was prepared with 150 mg sample of 14C-test substance and 270 mg analytical standard test substance in 6.0 ml absolute ethyl alcohol. Each treatment capsule received 250 µl (17.5 mg, 21.1 µCi) of this solution, which at 2 capsules per day was equivalent to 20 ppm in the diet of a goat eating 1.75 kg feed per day. Counting of aliquots of this solution showed that the diluted 14C-test substance in this treatment solution had a specific activity of 1.20 µCi/mg, and TLC analysis confirmed the identification of 14C-test substance with radiochemical purity >99%.
Goat Ill: A solution was prepared by dissolving 22.5 mg 14 C-test substance in 500 µl absolute ethyl alcohol. Counting of aliquots of this solution showed a specific activity of 3.40 µCi/mg for the 14C-test substance in this solution. A 400 µl aliquot (18 mg, 61.1 µCi) was added to pulverized goat chow in a single capsule.

Duration and frequency of treatment / exposure:

Goat I: 8 days, twice daily
Goat II: 10.5 days, twice daily
Goat III: single dose

Dose / conc.:

20 ppm

No. of animals per sex per dose / concentration:

Three goats

Control animals:

no

Details on study design:

- Rationale for animal assignment: Three goats were used in these experiments. During the acclimatization period, the milk production of the first animal hereafter designated Goat I, was stable. However, milk production, rapidly dropped when the animal was subjected to the stress of catheterization and twice daily oral dosing. Milk production decreased to the point that the last samples of milk were obtained on Day 7 of the treatment period. Therefore, oral dosing was discontinued after administration of the sixteenth dose, making the total treatment period 8 days. The animal was sacrificed two days after the last treatment. Examination at sacrifice revealed that the animal was pregnant. Examination of organs revealed no abnormalities except for the bladder, which was irritated due to the presence of the catheter. No analyses were conducted on samples from Goat I, except for monitoring of total radiocarbon levels in milk and urine during treatment and depuration phase of the experiment. Because the required data could not be obtained from Goat I, the entire experiment was set up again using a second animal, Goat II. This animal was dosed successfully for 10.5 days and sacrificed 24 hours after administration of the last dose. The only problem which arose in this experiment was a low total recovery of radioactivity. Because of this low radioactive materials balance, linked with evidence for the generation of volatile metabolites in numerous fractions, a special single dose test was set-up with a third animal, Goat III. The only objective of the experiment with Goat III was to try to establish where the losses from the Goat Il experiment had occurred.

Details on dosing and sampling:

TOXICOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Milk, urine, feces, blood, organs, tissues
- Time and frequency of sampling: Milk, urine, feces samples were collected daily. At sacrifice, blood, tissues and organs were collected

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Milk, urine, feces, blood, tissues
- Time and frequency of sampling: Milk, urine, feces samples were collected daily. At sacrifice, blood, tissues and organs were collected
- Method type(s) for identification: Liquid scintillation chromatography (LSC), High performance liquid chromatography (HPLC), Gas chromatography/Mass spectroscopy (GC/MS), Thin layer chromatography (TLC)
- Limits of detection: 0.002 ppm

Type:

other: Elimination (Metabolism + Excretion)

Results:

Test substance was eliminated rapidly. Approximately 16%, 8%, 7% of total dose was eliminated in urine, milk and feces respectively.

Type:

excretion

Results:

Total 48% of radioactivity was accounted for in urine, feces, expired air and milk.

Type:

metabolism

Results:

56%, 48% and 45% of the radioactivity in Day 3, Day 4, and Day 5 milk has been accounted for as lactose, triglycerides fats and casein.

Type:

distribution

Results:

The level of carbon-14 in tissues at sacrifice in tissues like liver, kidney, heart, pancreas, brain was 1.1, 0.03, 0.05, 0.01 and 0.01% respectively.

Type:

absorption

Results:

The levels of carbon-14 in blood gradually increased upto 1.1 ppm of test substance after 10 days of first dose.

Metabolites identified:

no

Details on metabolites:

No test substance or oximino compound was detected in any samples. Complete disintegration of the test substance structure occurred with reincorporation of carbon-14 into normal natural products, like lactose, fatty acids of lipids, and casein.

Examination of urine and feces:

When a sample of Day 7 urine was extracted with ethyl acetate, only 10% of the total radioactivity was extracted into the ethyl acetate. Of this organo-soluble material, 87% was lost when the combined extracts were concentrated by a factor of 93%. Although no further analyses were made of this volatile material, the behavior again is typical of acetonitrile.

When the Day 6 feces were extracted with methanol (five times), 56% of the radioactivity was recovered in the extracts while the remainder appeared bound in the insoluble material. The last extract recovered only 0.03% of the total. Furthermore, when the extracts were combined and concentrated, 86% of the carbon-14 was retained in the concentrate, When partitioned between ethyl acetate/ water, < 2% of the radioactivity from the methanol extract passed into the ethyl acetate. This means that neither test substance nor the oximino compound passes into the feces .

Radioactive components of milk:

Lactose, recovered from the methanol and aqueous extracts of freeze-dried whole milk, was consistently found to be radio-labelled by recrystallization to constant specific activity and melting point. The percentages of the total milk radioactivity accounted for by 14C-lactose was 11, 13 and 11% for Day 3, Day 4, and Day 5 milk, respectively. This is definite proof that the test substance has broken down completely with subsequent incorporation of carbon-14 into a natural product. The hexane extracts of the Day 3, Day 4 and Day 5 milk contained 37, 26 and 26%, respectively, of the total milk radioactivity for these days. When the residues in the hexane extracts were saponified, all the radioactive material behaved like the triglycerides. In all three samples, an average of 93% of the total radioactivity was recovered with the fatty acid fraction, and subsequently was esterified quantitatively by reaction with diazomethane. However, the specific activity, 30 dpm/mg, was too low for further identification of individual fatty acids. The small amount of radioactivity not included in the fatty acid fraction remained in the aqueous phase containing glycerol. This is further evidence for complete breakdown of the test substance with subsequent incorporation of carbon-14 into a normal milk constituent. Casein, the principal protein of milk, makes up 80% of the total protein. Casein, isolated by a standard method from all three samples of goat milk, was also found after repeated purification to be radiolabeled. The amount of total milk radioactivity accounted for as 14C-casein was 8, 9, and 8% for Day 3, Day 4 and Day 5, respectively.

 

Radioactive components of blood:

When a composite of daily blood samples was extracted three times with ethyl acetate, 43% of the total radioactivity was recovered in the ethyl acetate extract, 2% in the sublimate after feeze-drying of the aqueous phase, and 55% in the freeze-dried aqueous phase powder. When 250 ml of this ethyl acetate extract was concentrated to 12 ml, only 8% of the extracted radioactivity remained in the concentrate. When the entire concentrate was analyzed by TLC, no test substance or oximino derivative was found ( < 0.002 ppm). Similarly, when an aliquot of blood taken at sacrifice was extracted with ethyl acetate, 55% of the total radioactivity was found in the extracts, and 45% was recovered in the aqueous phase after extraction. However, when the combined extracts were concentrated, from 94 ml to 5.0 ml, only 7% of the extracted radioactivity remained in the concentrate.

Radioactive components of tissues:

Samples of liver, heart, and kidney tissues were freeze dried and then extracted in sequence with ethyl acetate and methanol. Because the ethyl acetate extracts would contain any test substance and/or oximino compound present in the tissue, the levels of carbon-14 found in these tissues indicates a test substance/oximino compound level of <0.02 ppm for heart and kidney. In the case of liver, the crude ethyl acetate extracts contain somewhat more radioactivity than this. However, when this material is redissolved in cyclohexane and partitioned between cyclohexane/water, more than 98% of the radioactivity remained in the organic phase. This is the reverse behavior that test substance or its oximino compounds would exhibit and therefore, the level of radioactivity attributable to these compounds is also <0.02 ppm for liver tissue as well. The insoluble material remaining after extraction of liver, kidney and heart was incubated with Pronase to dissolve and hydrolyze the protein. In each case, most of the radioactivity was solubilized, but less than of the total was extractable into ethyl acetate, indicating no liberation of test substance or oximino compound. The specific activity of the water-soluble material was too low for further characterization.

Conclusions:

- No test substance was detected in any samples collected.
- Complete disintegration of test substance occurred with reincorporation of carbon-14 into natural products lactose, fatty acids of lipids and casein.

Executive summary:

The study was conducted to determine the metabolism of 14C-labelled test substance in the lactating goat. A lactating goat was treated with 14C-labelled test substance for 10 days (2 doses per day) at a rate equivalent to 20 ppm in the diet. Daily samples of milk, blood, urine and feces were collected. One day after cessation of treatment, the animal was sacrificed and selected tissues taken for analysis. No test substance or oximino compound was detected in any samples. Complete disintegration of the test substance structure occurred with reincorporation of carbon-14 into normal natural products, like lactose, fatty acids of lipids, and casein.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to evaluate the metabolism of test substance in rats when administered orally to male rats at a nominal concentration of 1.2 mg of 14C-radiolabelled test substance in peanut oil.

GLP compliance:

no

Specific details on test material used for the study:

Test substance Name: S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate
Purity: Not specified

Radiolabelling:

yes

Species:

rat

Strain:

other: ChR-CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 169 g
- Diet: Purina Laboratory Chow

Route of administration:

oral: gavage

Vehicle:

other: Peanut Oil

Details on exposure:

Control Phase animal: Male rat, weighing 174 grams, was given a diet composed of ground Purina laboratory chow to which had been added 1% corn oil and 200 ppm test substance. 8 days later, when the weighed 230 grams, he was given by intragastric intubation 2 ml of peanut oil containing 1.2 mg of test substance and immediately placed in a glass metabolic unit.
Phase I: Male rat, weighing 169 grams, was given a diet composed of ground Purina laboratory chow to which had been added 1% corn oil and 200 ppm test substance. 8 days later, when the weighed 237 grams, he was given by intragastric intubation 2 ml of peanut oil containing 1.2 mg of C-14 labelled test substance and immediately placed in a glass metabolic unit.
Phase II: Male rat, weighing 172 grams, was given a diet composed of ground Purina laboratory chow to which had been added 1% corn oil and 200 ppm test substance. 7 days later, when the weighed 225 grams, he was given by intragastric intubation 2 ml of peanut oil containing 1.2 mg of C-14 labelled test substance and immediately placed in a glass metabolic unit.

Duration and frequency of treatment / exposure:

Single

Dose / conc.:

1.2 other: mg

No. of animals per sex per dose / concentration:

3 Male rats

Control animals:

no

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, tissues, blood, carcass and expired CO2
- Time and frequency of sampling: Control Phase : Samples of urine, feces. and expired CO2 were collected at 24-hour intervals for three days. After 72 hours, the animal was taken from the metabolism chamber and lightly anesthetized with CHCl3, blood was removed by syringe and needle from pumping heart. The animal was then killed with an overdose of CHCl3 and the organs and tissues surgically placed in plastic vials, and frozen are brain, lungs, heart, liver, spleen, kidneys, testes, gastrointestinal tract, and portion of muscle and fat. In addition, the carcass was placed in a plastic bag and frozen.
Phase I: Samples of urine, feces were collected at 24-hour intervals for three days. Expired CO2 was collected at 0-7 hours and 7-24 hours. After 72 hours, the animal was taken from the metabolism chamber and lightly anesthetized with CHCl3, blood was removed by syringe and needle from pumping heart. The animal was then killed with an overdose of CHCl3 and the organs and tissues surgically placed in plastic vials, and frozen are brain, lungs, heart, liver, spleen, kidneys, testes, gastrointestinal tract, and portion of muscle and fat. In addition, the carcass was placed in a plastic bag and frozen.
Phase II: Samples of urine, feces were collected at 24-hour intervals for three days. Expired CO2 was collected at 0-7 hours and 7-24 hours. The same procedure is followed here as in test phase I, except that the test was terminated after 24 hours.
- From how many animals: No
- Method type(s) for identification: Liquid Scintillation chromatography (LSC), Gas chromatography, Thin layer chromatography (TLC)

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): ß-Glucuronidase-aryl sulfatase treatment of the urinary metabolite fraction for detection of test substance or its metabolites. For identification of 14CO2 as a metabolite, the aliquots of the sodium hydroxide solutions from the first trap were treated with equal volimes of 20% barium chloride solution.

Type:

metabolism

Results:

All radioactivity was rapidly eliminated within 24 hours as 1 part of 14C-CO2, 2 parts of 1-14C-acetonitrile and 1 part of urinary metabolite

Type:

metabolism

Results:

No test substance or S-methyl 1-14C-N-hydroxythioacetimidate was detected.

Metabolites identified:

yes

Details on metabolites:

The chemical identity of the radiolabeled material excreted in the urine has not been established. However, no test substance or S-methyl N-hydroxythioacetimidate was detected. ß-Glucuronidase-aryl sulfatase treatment of the urinary metabolite fraction was without effect, indicating that there are none of the usual urinary type conjugates of these compounds present either. Finally, the urinary metabolite fraction was shown to be devoid of either the S-oxide or S,S-dioxide of test substance.

Identification of 14CO2 as a metabolite: When 10-ml aliquots of the most radioactive sodium hydroxide solutions from the first trap were treated with equal volumes of 20% barium chloride solution and centrifuged, it was found that 93% of the radioactivity was precipitated. It was concluded that the radioactivity in the first trap was essentially all the result of [14C] carbon dioxide.

Identification of Neutral volatile metabolite: Aliquots of the liquid nitrogen trap contents and of the dry ice trap contents were injected on a 4-ft X. 1/4-in. diameter Porapak Q gas chromatographic column. The separation was carried out using a flow of 50 cc/min of helium with column temperature held at 70°C for 2 min, then programmed at 5°/min to 220°. The radioactivity of both samples was recovered quantitatively in a single peak, with a retention time of 16 min. When the radioactive peak from a 50-µl aliquot of the liquid nitrogen trap contents was trapped on carbon, and a mass spectrum obtained, the mass spectrum was found to be identical with that of acetonitrile. The retention time of acetonitrile on the column described was found to be 16 min.

Table 1: Distribution of Radioactivity after treatment of Rats with S-methyl 1-14C-N-[(methylcarbamoyl)oxy]-thioacetimidate

	Rat (3 days)		Rat (1 day) (Phase I)		Rat (3 days) (Phase II)
	µCi	% Original treatment	µCi	% Original treatment	µCi	% Original treatment
External fractions
Carbon dioxide	1.5	23	0.94	15	1.03	17
Neutral volatile metabolite	ND		ND		2	33
Urinary metabolites	1.59	24	1.75	27	0.95	16
Feces	0.13	2	0.02	<1	ND
Body fractions
Blood	0.084		0.034		ND
Brain	0.003		0.003		ND
Fat	0.000		0.002		ND
Gastrointestinal tract	0.122		0.167		ND
Heart	0.005		0.003		ND
Hide	0.169		0.105		ND
Kidneys	0.007		0.010		ND
Liver	0.035		0.042		ND
Lungs	0.004		0.005		ND
Muscle	0.003		0.004		ND
Spleen	0.003		0.003		ND
Testes	0.005		0.006		ND
Carcass	0.234		0.227		ND
Body Total	0.674	10	0.611	9
Recovery		60		52

ND: Not determined

Conclusions:

All radioactivity was rapidly eliminated within 24 hours as 1 part of 14C-CO2, 2 parts of 1-14C-acetonitrile and 1 part of urinary metabolite. However, S-methyl 1-14C-N- [(methyl carbamoyl) oxy] thioacetimidate or S-methyl 1-14C-N-hydroxythioacetimidate was detected.

Executive summary:

The study was conducted to evaluate the metabolism of test substance in rats when administered orally to male rats at a nominal concentration of 1.2 mg of 14C-radiolabelled test substance in peanut oil. Essentially all the radioactivity was rapidly eliminated from the animal body within 24 hours in the ratio of 1 part 14C-CO2, 2 parts of 1-14C-acetonitrile, and 1 part urinary metabolite. The chemical Identity of the radiolabeled material excreted in the urine has not been established. However, no S-methyl 1-14C-N- [(methyl carbamoyl) oxy] thioacetimidate or S-methyl 1-14C-N-hydroxythioacetimidate was detected.

 

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to examine expired air for acetonitrile after administration of test substance to monkeys. The test substance was given to monkeys orally through drinking water. The expired air was collected at a rate of 8 to 10 liters/min for 2 hours. These samples were rinsed with acetone and analyzed for acetonitrile using gas chromatography.

GLP compliance:

no

Specific details on test material used for the study:

Test substance Name: Lannate methomyl
Purity: Not reported

Radiolabelling:

no

Species:

monkey

Strain:

other: Rhesus

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.6 to 3.1 kg

Route of administration:

oral: drinking water

Vehicle:

other: Distilled water

Dose / conc.:

20 other: mg/kg (Drinking water)

No. of animals per sex per dose / concentration:

3 monkeys

Control animals:

no

Details on study design:

Three rhesus monkeys were placed in restraining chairs and fitted with face masks to collect the expired air. The air was passed through a four-inch column of indicating silica gel and trapped in 1/8 inch glass U-tubes immersed in a -78°C dry ice-acetone bath. Control samples were collected for 30 minutes at the rate of 8 to 10 liters/minute. The animals were then given atropine in saline intravenously (2 mg/kg) to prevent salivation caused by test substance. After 5 minutes, the test substance was administered in distilled water via stomach tube at a level of 20 mg/kg, and the expired air collected at a rate of 8 to 10 liters/minute for 2 hours. Each sample was rinsed with acetone into a 25 ml. volumetric flask and brought to volume. 1 aliquot was analyzed for acetonitrile using gas chromatography

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Expired air
- Time and frequency of sampling: Expired air was collected at a rate of 8 to 10 liters/minute for 2 hours.
- Method type(s) for identification: Gas chromatography using flame ionization detector
- Limits of detection and quantification: LOD: 25 ng of acetonitrile

Type:

metabolism

Results:

The amount of acetonitrile identified through gas chromatography was less than 2% of total amount of test substance administered to each monkey.

Metabolites identified:

yes

Details on metabolites:

Analytical results showed that all samples of expired air contained a component chromatographically identifiable as acetonitrile. In all cases, the amount found was less than 0.6 mg per sample or less than 2% of the total amount of test substance administered to each monkey.

Conclusions:

The amount of acetonitrile identified through gas chromatography was less than 2% of total amount of test substance administered to each monkey.

Executive summary:

The study was conducted to examine expired air for acetonitrile after administration of test substance to monkeys. Three rhesus monkeys were placed in restraining chairs and fitted with face masks to collect the expired air. The air was passed through a four-inch column of indicating silica gel and trapped in 1/8 inch glass U-tubes immersed in a -78°C dry ice-acetone bath. Control samples were collected for 30 minutes at the rate of 8 to 10 liters/minute. The animals were then given atropine in saline intravenously (2 mg/kg) to prevent salivation caused by test substance. After 5 minutes, the test substance was administered in distilled water via stomach tube at a level of 20 mg/kg, and the expired air collected at a rate of 8 to 10 liters/minute for 2 hours. Each sample was rinsed with acetone into a 25 ml. volumetric flask and brought to volume. 1 aliquot was analyzed for acetonitrile using gas chromatography.

The results showed that all samples of expired air contained a component chromatographically identifiable as acetonitrile. In all cases, the amount of acetonitrile was less than 0.6 mg per sample, or less than 2% of the total amount of test substance administered to each monkey.

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to evaluate the levels of 14C-metabolites in rat blood after dosing with 14C-labelled test substance and the percentage of radioactivity of test substance in blood was extracted using ethyl acetate at 15 min, 1, 2, 4, 24, 48 and 72 hours.

GLP compliance:

no

Specific details on test material used for the study:

Radiolaebelled test substance name: S-Methyl N-[(methylcarbamoyl)oxy]thioacetimidate
Purity: Not reported

Non-radiolabelled test substance name: Methomyl
Purity: Not reported

Species:

rat

Strain:

other: CR-CD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 340 g

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For each rat, average weight 340 g, the required dose was 1.35 mg of 14C-labelled test substance in 4.3 ml of aqueous solution.

PRECONDITIONING:
Rats were preconditioned for 3 weeks on a normal diet containing 100 ppm non-radiolabeled test substance. After preconditioning, the rats in each group were weighed and the seven most uniform were selected for treatment.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

4 mg/kg bw/day

No. of animals per sex per dose / concentration:

10 male rats

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Blood, carcass, urine and feces
- Time and frequency of sampling: 15 min, 1, 2, 4, 24, 48 and 72 hours after treatment
- Other: Urine and feces were collected but not analyzed. Two aliquots (200 µL each) of each blood sample were analyzed for total carbon-14 content by combustion in a Packard Model 306 Sample Oxidizer. Then the entire remaining blood sample (approx. 10 ml) was acidified with hydrochloric acid and diluted to 40 mL with distilled water. Ultrasonic vibration was required to completely disperse the blood because some samples were partially coagulated despite the heparin treatment.
- Method type(s) for identification: Liquid scintillation counting (LSC), Thin layer chromatography (TLC)

Type:

absorption

Results:

15 minutes after treatment, 55% of radioactivity in the blood from test substance treated rat was extractable with ethyl acetate.

Type:

absorption

Results:

The levels of circulating 14C-labelled test substance in the blood rose rapidly during the first four hours after treatment and decreased thereafter.

Details on absorption:

The percentage of radioactivity in the blood from a test substance treated rat that was extractable with ethyl acetate is 55, 42, 37, 30, 20, 12 and 7% at exposure time of 15 min, 1, 2, 4, 24, 48 and 72 hours respectively.

- Concentration of the ethylacetate extracts of blood at 15 min, 1, 2 and 4 hours gave volatility loses of 47, 54 and 88 and 94% respectively.

Conclusions:

The levels of circulating 14C-labelled test substance in the blood rose rapidly during the first 4 hours after treatment and decreased thereafter.

Executive summary:

The study was conducted to evaluate the levels of 14C-metabolites in rat blood after dosing with 14C-labelled test substance. When 14C-labelled test substance was administeed orally to rats at a dose of 4 mg/kg, the percentage of radioactivity in the blood that was extractable with ethyl acetate is 55, 42, 37, 30, 20, 12 and 7% at exposure time of 15 min, 1, 2, 4, 24, 48 and 72 hours respectively. The levels of circulating 14C-labelled test substance in the blood rose rapidly during the first four hours after treatment and decreased thereafter.

Reference 7

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, Draft OECD Guideline, Dermal Delivery and Percutaneous Absorption: In Vitro Method (June 1996)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Name of radiolabelled test substance: S-methyl[1-14C]-N-[(methylcarbamoyl)oxy]thioacetimadate
Lot#: 312
Radioactive purity: >98%

Name of Non-radioactive test susbtance: Methomyl technical, Lannate 90
Lot#: Methomyl technical (101697-01), Lannate 90 (T101397-00)
Proportion of methomyl in lannate 90: 88.6%

Radiolabelling:

yes

Species:

other: Rat, Rabbit, Human skin epidermal membrane

Strain:

New Zealand White

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS USED FOR EPIDERMAL MEMBRANE PREPARATION
- Source: Charles river laboratories, United Kingdom
- Age at study initiation: Rat: 25 to 27 days old
- Weight at study initiation: Rabbit: 2 to 3 kg

Doses:

- Nominal doses: 440, 44, 4.4 and 0.4 µg.cm-2

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: The conventional formulation of Lannate 90 contains test substance and Hi-Sil 233. The test doses were formulated to mimick the conventional Lannate 90 formulations. The paste formulation of Lannate 90 was prepared by combining Hi-Sil 233 with radio-labelled and technical test substance followed by the addition of water. The final concentration was targeted at 450 g/kg of test substance of formulation. A visual assessment of the prepared test paste dose was compared to a prepared commercial Lannate 90 paste using a microscope and graticule. The grains were similar in size for both preparations.
For the preparation of the high aqueous dilution (44 g/L), radiolabelled test substance was blended with Lannate 90 and water. Production of the medium aqueous dilution 'a', 4.4 g/L, was achieved by mixing Hi-Sil 233 with radio-labelled methomyl; for the mid-aqueous formulation 'b', Hi-Sil 233 was combined with radio-labelled and technical test substance followed by the addition of water. The low aqueous dilution was prepared by combining Hi-Sil 233 with radio-labelled test substance and water to achieve the target solution concentration of 0.4 g/L. Nominally, the ratio of Hi-Sil 233 to test substance was 1 part Hi-Sil 233 to 9 parts test substance in all formulated doses. The formulations of test substance is given in Table-1 in "Any other information on materials and methods incl. tables section".

APPLICATION
14C-labelled test substance in appropriate formulation was applied over the stratum corneum surface using an M25 Gilson microman positive displacement pipette set at 3.2 µl. During each experimental period, 6 mock doses were collected into scintillation vials and counted by LSC (Liquid Scintillation Chromatography). The donor chambers were left open to the atmosphere.

SAMPLING
Receptor fluid was collected in hourly fractions from 0-6 h post dose, then 2 hourly fractions from 6-24 h post dose. At the end of the 24 h period, each cell was disconnected from the saline pump lines. The underside of the skin was washed (receptor rinse) with ca 2 washes of ca 1 ml saline, which was mixed with ca 10 ml scintillant and analyzed by liquid scintillation chromatography (LSC). The receptor rinse represented absorbed material which was in the receptor chamber, but had not been collected into the 24 h receptor fluid fraction.
The skin surface was washed (skin wash) with ca 4 washes of ca 2% soap solution using a Gilson pipetteman P5000 set at 2.5 ml. Each wash was aspirated with a 1 ml disposable pipette. This wash was collected into a preweighed pot labelled skin wash. The disposable pipette was transferred to a pot labeled cell wash. The total mass of wash was determined and triplicate ca 1 ml samples were weighed into scintillation vials for LSC analysis.
The receptor chamber was removed from the donor chamber and the skin taken and laid onto a piece of tissue paper. The donor and receptor chambers were transferred to the cell wash pot. Water, ca 40 ml, was added to this pot and weighed and the pot gently mixed. Triplicate ca 1 ml weighed aliquots were taken for LSC counting. The skin was dried with small pieces of tissue paper, which were collected into a pot labelled skin wipes. Water, ca 10 ml, was added, weighed, the sample mixed and triplicate ca 1 ml weighed aliquots were taken for LSC counting.
The remaining skin was transferred to a scintillation vial to which ca 1 ml Soluene-350 was added. The samples were left to solubilize at room temperature prior to addition of ca 10 ml scintillant.
The material found in the skin represented both unabsorbed (associated with the stratum corneum) and absorbed (remaining skin) material. Since material associated with the stratum corneum and remaining skin were not separately analyzed, then this fraction was included with the receptor fluid and receptor rinse as total absorbed material. The material in the skin wash, cell wash and skin wipes together represented unabsorbed material.

ANALYSIS
- Method type(s) for identification: Liquid scintillation chromatography (LSC)
- Liquid scintillation counting results d.p.m to cummulative d.p.m: The individual d.p.m in each receptor fluid fraction was added from each timepoint to give cummulative d.p.m.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human skin samples (11 breast and 1 abdomen) were obtained from the Plastic surgery Unit, St Johns Hospital NHS Trust, Livingston, United kingdom; Abdominal skin from juveline rats and rabbits from Charles river laboratories, united kingdom.
- Preparative technique:
Human epidermal membranes: Full thickness skin was obtained and cleaned of subcutaneous fat and connective tissue using scalpel blades, scissors and blue roll tissue paper. The skin was then cut into smaller pieces, wrapped in aluminum foil, put into self sealing plastic bags and stored at ca -20°C until required. When required, the skin samples were removed from storage and allowed to thaw. Epidermal membranes were prepared by immersing the full thickness skin sample in water heated to ca 60°C for ca 1 min. On removal, the epidermis was teased away using forceps, floated on water and picked up onto aluminum foil and these epidermal membranes were either used immediately, or stored at ca 4°C for up to 5 days prior to use.
Rat epidermal membranes: Abdominal skin was obtained from juvenile male rats which were 25 to 27 days old. The rats were held in the laboratory environment for 3 to 5 days prior to sacrifice by CO2 narcosis and cervical dislocation. The fur on the abdominal skin was clipped using small animal clippers (Oster), the clipped area was washed with acetone and the skin excised. When required, the skin sample was removed from -20°C freezer storage and allowed to thaw at room temperature. The skin was immersed in 2 M sodium bromide solution stratum corneum surface facing downwards, for ca. 18 h. The skin was blotted dry and the epidermis peeled away from the underlying dermis using forceps. Membranes damaged during this procedure were discarded. The prepared membranes were used immediately.
Rabbit full thickness membrane: The rabbits were held in the laboratory environment for 2 days prior to sacrifice by anesthetic overdose. The fur on the abdominal skin was clipped using small animal clippers (Oster) and excised.
- Thickness of skin: The surface area of exposed skin within cells was 0.32 cm2.
- Membrane integrity check: The integrity of each skin preparation was assessed prior to the application of dose by measuring its permeability to tritiated water. Samples were rejected from the study because their permeability to tritiated water was unacceptably high.
- Storage conditions: Human epidermal membranes: Skin which are cut in to small pieces are wrapped in aluminium foil and put into self sealing plastic bags and stored at -20°C untill required; Rat epidermal membranes & Rabbit epidermal membrane: The skin was stored at -20°C untill use.


PRINCIPLES OF ASSAY
- Diffusion cell: Automated flow-through diffusion cell apparatus.
- Receptor fluid: The receptor fluid used for the water and test substance permeability measurements was physiological saline (0.9% NaCl) solution (Baxter Sterile Intravenous Infusion Saline, Code B1323).
- Static system: Epidermal membrane
- Flow-through system: The flow-through cells were placed in an aluminum manifold heated via a circulating water bath to maintain skin surface temperature at ca 30-32°C. The cells were connected to multi-channel peristaltic pumps from their afferent ports, with the receptor fluid effluent dropping directly into vials on a fraction collector. The surface area of exposed skin within the cells was 0.32 cm2. The receptor chamber volume was 0.14 ml. The peristaltic pumps were adjusted to maintain a flow rate of ca 1.5 ml/h.
- Test temperature: 30 to 32°C

Signs and symptoms of toxicity:

no effects

Remarks:

All animals showed no extemal signs of ill health prior to skin excision and no gross lesions or abrasions were observed on rat or rabbit skin.

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
High aqueous dilution (44 g/L test substance): Following a single topical application of Lannate 90 diluted with water to a concentration of ca. 44 g test substance per liter of solution, the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 17.2, 65.2 and 16.4 respectively. Absorption of [14C]-test substance through rat skin was approximately 3.8-fold greater compared to human skin; the mean cumulative flux of [14C]-test substance through rabbit and human skin was comparable. The mean cumulative percent absorbed of [14C]-test substance (receptor fluid only) was 4.03, 15.3 and 3.81% of the applied dose for human, rat and rabbit respectively.
Overall, following a single topical dose of Lannate 90 diluted with water to a concentration of 44 g test substance per liter of solution, the cumulative flux (over 24 h) and receptor fluid (percent absorbed) for rat skin was greater when compared to rabbit and human skin. The 24 h cumulative flux and percent of absorbed dose in the receptor fluid for human and rabbit skin was comparable. The rank order of total absorbed (receptor fluid, receptor rinse and skin) was rat > rabbit > human.
Medium Aqueous Dilution (4.4 g/L of test substance): Following a single topical application of Lannate 90 diluted with water to a concentration of ca 4.4 g test substance per liter of solution, the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 7.95, 24.7 and 9.99 respectively. Absorption of [14C]-test substance through rat skin was approximately 3.1-fold greater compared to human skin; the mean cumulative flux of [14C]-test substance through rabbit and human skin was comparable. The mean cumulative percent absorbed of [14C]-test substance (receptor fluid only) was 17.5, 54.8 and 21.5% of the applied dose for human, rat and rabbit respectively.
Overall, following a single topical dose of Lannate 90 diluted with water to a concentration of 4.4 g test substance per liter of solution, the cumulative flux (over 24 h) and receptor fluid (percent absorbed) for rat skin was greater when compared to rabbit and human skin. The 24 h cumulative flux and percent of absorbed dose in the receptor fluid for human and rabbit skin was comparable. The rank order of total absorbed (receptor fluid, receptor rinse and skin) was rat > rabbit > human.
Low Aqueous Dilution (0.4 g/L of test substance): Following a single topical application of Lannate 90 diluted with water to a concentration of ca 0.4 g test substance per liter of solution, the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 1.55, 2.45 and 0.60 respectively. The rank order for mean cumulative flux of [14C]-test substance through rat, rabbit and human skin was rat > human > rabbit. The mean cumulative percent absorbed of [14C]-test substance (receptor fluid only) was 40.5, 63.0 and 16.0% of the applied dose for human, rat and rabbit respectively.
Overall, following a single topical dose of Lannate 90 diluted with water to a concentration of 0.4 g test substance per liter of solution, the rank order of cumulative flux, percent of absorbed dose in the receptor fluid and total absorbed (receptor fluid, receptor rinse and skin) was rat> human > rabbit.
Lannate 90 paste (450 g/kg of test substance): The mean cumulative percent absorbed of [14C]-test substance (receptor fluid only) was 1.54, 10.6 and 0.83% of the applied dose for human, rat and rabbit respectively. The percent dose applied contained in the skin was 0.24, 0.73 and 3.94% for human, rat and rabbit respectively, full thickness rabbit skin provided a larger reservoir for the applied dose and therefore may explain why it contained higher residue levels when compared to human and rat skin epidermal membranes. Independent of species, a significant portion of the applied dose was contained in the skin wash (83-92%).
Overall, following a single topical dose of Lannate 90 diluted with water to give a paste with a concentration of 450 g test substance per kilogram of paste, the rank order of cumulative flux (over 24h) and receptor fluid (percent absorbed) was rat > rabbit > human.

- Skin preparation (in vitro test system):
High aqueous dilution ( 44 g/L test substance): The percent dose applied contained in the skin was 0.53, 2.40 and 5.38% for human, rat and rabbit respectively. Full thickness rabbit skin provided a larger reservoir for the applied dose and therefore may explain why it contained higher residue levels when compared to human and rat skin epidermal membranes. Independent of species, a significant portion of the applied dose was contained in the skin wash (72-95%).
Medium Aqueous Dilution (4.4 g/L of test substance): The percent dose applied contained in the skin was 3.12, 3.70 and 14.2% for human, rat and rabbit respectively. Full thickness rabbit skin provided a larger reservoir for
the applied dose and therefore may explain why it contained higher residue levels when compared to human and rat skin epidermal membranes. A significant portion of the applied dose was contained in the skin wash for human(69.3%) and rabbit (56.6%) skin, with only 26.2% in the rat skin.
Low Aqueous Dilution (0.4 g/L of test substance): The percent dose applied contained in the skin was 3.50, 7.12 and 22.9% for human, rat and rabbit respectively. Full thickness rabbit skin provided a larger reservoir for the applied dose and therefore may explain why it contained higher residue levels when compared to human and rat skin epidermal membranes. A significant portion of the applied dose was contained in the skin wash for human (45.9%) and rabbit (47.6%) skin, with only 15.6% in the rat skin.
Lannate 90 paste (450 g/kg of test substance): Following a single topical application of Lannate 90 as a paste prepared with water (1:1 v/v) at a concentration of ca 450 g test substance per kilogram of paste, the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 68.2, 469 and 36.5 µg equiv.test substance.cm-2 respectively. Absorption of [14C]-test substance through rat skin was approximately 6.9-fold greater compared to human skin; the mean cumulative flux of [14C]-test substance for human skin was approximately 2-fold greater than rabbit skin.

Total recovery:

- Total recovery: High aqueous dilution (44 g/L test substance): At 24h, total recovery in human, rat and rabbit is 100.6, 96.7 and 99.4% of applied dose respectively
Medium Aqueous Dilution (4.4 g/L of test substance): At 24h, total recovery in human, rat and rabbit is 95.6, 95.3 and 95.5% of applied dose respectively
Low Aqueous Dilution (0.4 g/L of test substance): At 24h, total recovery in human, rat and rabbit is 44.6, 71.1 and 39.0% of applied dose respectively
Lannate 90 paste (450 g/kg of test substance): At 24h, total recovery in human, rat and rabbit is 94.8, 97.6 and 92.1% of applied dose respectively

Key result

Time point:

24 h

Dose:

Lannate 90 paste (450 g/L)

Parameter:

percentage

Remarks on result:

other: The mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 1.85, 11.62 and 4.79% respectively.

Key result

Time point:

24 h

Dose:

High Aqueous Dilution (44 g/L)

Parameter:

percentage

Remarks on result:

other: The mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 4.71, 18.14 and 9.32% respectively.

Key result

Time point:

24 h

Dose:

Medium Aqueous Dilution (4.4 g/L)

Parameter:

percentage

Remarks on result:

other: The mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 23.56, 62.88 and 36.19% respectively.

Key result

Time point:

24 h

Dose:

Low Aqueous Dilution (0.4 g/L)

Parameter:

percentage

Remarks on result:

other: The mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 44.60, 71.14 and 38.98% respectively.

Key result

Time point:

24 h

Dose:

Lannate 90 paste (450 g/L)

Parameter:

amount

Remarks on result:

other: The mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 68.17, 469.10 and 36.46 respectively.

Key result

Time point:

24 h

Dose:

High Aqueous Dilution (44 g/L)

Parameter:

amount

Remarks on result:

other: The mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 17.2, 65.2 and 16.4 respectively.

Key result

Time point:

24 h

Dose:

Medium Aqueous Dilution (4.4 g/L)

Parameter:

amount

Remarks on result:

other: The mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 7.95, 24.7 and 9.99 respectively.

Key result

Time point:

24 h

Dose:

Low Aqueous Dilution (0.4 g/L)

Parameter:

amount

Remarks on result:

other: The mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 1.55, 2.45 and 0.60 respectively.

Conclusions:

- Radiolabeled test substance was applied to human and rat epidermal and rabbit full thickness skin membranes in vitro. Broadly, for all species (human, rat, rabbit), for a reduction in the dose level, there was an increase in the percent of absorbed dose and a decrease in the cumulative flux.
- Rat skin is a poor model for the penetration of test substance through human skin.
- When examining cumulative flux (receptor fluid only), the rabbit is a good model for human skin at all dose levels except for the low aqueous dose. However, when comparing the total percent absorbed (receptor fluid, receptor rinse and skin) the rabbit is only a good predictor of human skin at the low aqueous dose.

Executive summary:

The study was conducted to evaluate the in vitro dermal absorption of the test substance. [1-14C]-labelled test substance was applied, at 4 dose levels over a 1000 fold range, to human and rat epidermal and rabbit full thickness skin membranes in vitro using a flow through diffusion cell system. The high aqueous dose (ca. 44 g/L) represented ca. 80% of the highest solubility of test substance in water. The medium and low aqueous doses were applied at 4.4 and 0.4 g/L respectively. The high (1:1 w/w) paste was applied at ca. 450 g/L. Receptor fluid (saline, 0.9% sodium chloride) was collected hourly for 0-6 h post dose and every other hour from 6-24 h post dose. The underside of the membrane was washed with saline to remove absorbed material which had not been collected into the receptor chamber. The unabsorbed material was collected using ca. 4 washes of ca. 2.5 ml 2% soap solution. The donor and receptor chambers were also rinsed with water. The skin surface was swabbed dry with tissue swabs, which were collected and rinsed in water to extract any residual material. The remaining skin was solubilized using soluene-350 tissue solubilizer. Total unabsorbed material was a sum of the skin wash, cell wash and tissue swabs. Total absorbed material was the sum of the 0- 24 h receptor fluid, receptor rinse and material associated with the skin.

Total percent absorbed [1-14C]-labelled test substance was highest for all dose levels in the rat. However, the total percent absorbed and flux for the rabbit full thickness skin were very similar to the human epidermis for each dose level.

In high paste dose (450 g/L), the mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 1.85, 11.62 and 4.79% respectively and the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 68.17, 469.10 and 36.46 respectively. In High aqueous dilution (44 g/L), the mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 4.71, 18.14 and 9.32% respectively and the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 17.2, 65.2 and 16.4 respectively. In Medium aqueous dilution (4.4 g/L), the mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 23.56, 62.88 and 36.19% respectively and the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 7.95, 24.7 and 9.99 respectively. In low aqueous dilution, the mean total percent of test substance absorbed in human, rat and rabbit epidermal membranes was 44.60, 71.14 and 38.98% respectively and the mean cumulative flux (µg.cm-2) of [14C]-test substance absorbed through human, rat and rabbit skin (over 24 h) was 1.55, 2.45, and 0.60 respectively.

These results conclude that broadly, for all species, for a reduction in the concentration of test substance in water, there was an increase in the proportion of the total absorbed dose (as a percent of the applied dose) and a decrease in the cumulative flux.

Rat skin is a poor model for the penetration of test substance through human skin.

When examining cumulative flux (receptor fluid only), the rabbit is a good model for human skin at all dose levels except for the low aqueous dose. However, when comparing the total percent absorbed (receptor fluid, receptor rinse and skin) the rabbit is only a good predictor of human skin at the low aqueous dose.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

80

Absorption rate - dermal (%):

45

Additional information

Methomyl was readily absorbed from the gastrointestinal tract and rapidly eliminated within 24 hours from dosing (80% in the rat and 63% in the monkey). Urinary excretion accounted for 53% of the

administered dose in rats and 32% in monkeys. Expired air accounted for approximately 33% of the administered dose in rats and 32% in monkeys. No specific bioaccumulation was noted with the

exception of some radioactivity in red blood cells. In rats the major urine metabolite is the mercapturic acid derivative. Acetonitrile is the major residue in blood and liver. In the monkey, over 18 metabolites were observed, none of which were greater than 4% and included those metabolites common with the rat. The monkey excretes more 14CO2 and less 14C-acetonitrile than the rat in expired air, and excretes considerably less of the mercapturic acid derivative of methomyl in urine (0.8% in monkey, 18% in rat); the monkey excreted a greater number of urinary metabolites.