Methomyl

Dermal absorption

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

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

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:

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

Test animals

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

Administration / exposure

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

Results and discussion

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

Percutaneous absorption

Percutaneous absorption 1

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.

Percutaneous absorption 2

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.

Percutaneous absorption 3

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.

Percutaneous absorption 4

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.

Percutaneous absorption 5

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.

Percutaneous absorption 6

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.

Percutaneous absorption 7

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.

Percutaneous absorption 8

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.

Applicant's summary and conclusion

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.