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

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP / guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28 for the conduct of skin absorption studies, March 2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

604-351-6

Cas Number:

143390-89-0

Molecular formula:

C18 H19 N O4

IUPAC Name:

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

Details on test material:

1) Radiolabeled test substance
- Name of test material (as cited in study report): 14C-BAS 490 F (Kresoxim-methyl); test-substance No.: 08/0003-1; IUPAC-name: methyl 2-[o-(o-methylphenoxymethyl)phenyl]-2-(methoxyimino)acetate; label: benzyl-ring-U-C14
- Analytical purity: 99.2%
- Lot/batch No.: 613-2101
- Specific activity (if radiolabelling): 13.1 MBq/mg active ingredient
2) Non-labeled test substances
a) Technical active ingredient
- Name of test material (as cited in study report): BAS 490 F (Kresoxim-methyl); test-substance No.: 06/0625-2
- Analytical purity: 97.8%
- Lot/batch No.: COD-000225
b) Formulation
- Name of test material (as cited in study report): BAS 490 02 F (test substance number: 07/0746-1)
- Analytical purity: BAS 490 F (active ingredient Kresoxim-methyl) 48.2%
- Lot/batch No.: 3197
c) Vehicle (blank formulation)
- Name of test material (as cited in study report): BAS 490 AA F (Leerformulierung); test-substance No.: 08/0001-1
- Purity test date: formulation; information on composition of the test substance which appropriately defines the test batch, was given by the sponsor, and the sponsor holds this responsibility.
- Lot/batch No.: Ans.-Nr.: 992003

- Stability under test conditions: the stability of the test substance under storage conditions over the test period is guaranteed by the sponsor, and the sponsor holds this responsibility
- Other:
Homogeneity: the test substance was homogeneous by visual inspection.

Radiolabelling:

yes

Test animals

Species:

other: Human skin membranes

Strain:

other: Human skin membranes

Sex:

not specified

Details on test animals or test system and environmental conditions:

In vitro test

Administration / exposure

Type of coverage:

other: Diffusion cell

Vehicle:

other: tap water was used as vehicle for preparing the spray dilutions

Duration of exposure:

After a 6-hours exposure period the Teflon® discs were removed and the surface of the skin membranes was washed. The adhesive fleece cover of the donor compartment was reconstituted and the study period continued up to 24 hours. During the study period amounts of the receptor fluid were collected from each cell at several time points in order to determine kinetic parameters (lag phase, absorption rate and permeability constant). At the end of the study period the remaining test substance was recovered from all compartments of each diffusion cell. Times of sampling of receptor fluid were 0.5, 1, 2, 4, 6, 10 and 24 hours after application.

Doses:

- Nominal doses: (0.6 (low dose), 8 (mid dose) and 2500 (high dose) µg/cm2 (see Table 1&2)
- Dose volume: 10 µl/cm2
- Rationale for dose selection: test substance preparations corresponding to a 50% aqueous suspension of the formulation in powder form (maximally technically applicable concentration = high dose (concentrate, concentration of active ingredient about 250 mg/mL) and two representative aqueous spray dilutions for field use (a 1/625 aqueous dilution containing about 0.8 mg/mL BAS 490 F and a 1/8333 aqueous dilution containing about 0.06 mg/mL of BAS 490 F (supposed to have the density of water)) were applied.

No. of animals per group:

each dose was performed in independent run in 5 cells equipped with human skin

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Laboratory Glass Apparatus Inc, U.S.A. or BASF AG (Skin membranes of 4 donors (2 donors for each dose) were used within the study)
- Type of skin: Modified Franz cell consisting of an upper donor compartment and a wide opening at the top and a lower receptor compartment. After introducing the skin membrane, the two compartments were assembled and fixed with a stainless steel clamp and four screws. The receptor volume was about 4 mL. Sampling and replacement of the removed receptor fluid were carried out via an outlet (bottom) and inlet (top) pipe connection. The exposed area of the skin was about 1 cm².
- Preparative technique: Human skin membranes (Surgically removed skin from abdomen) were supplied dermatomed by BIOPREDIC (BIOPREDIC International, Rennes, France).
- Thickness of skin (in mm): 350 – 450 μm
- Membrane integrity check: the integrity of the skin membrane was determined by measuring its electrical resistance with a LCR bridge (LCR 400, Thurlby Thandar Instruments, England). Measured values above 1 kOhm were expected for intact skin membranes.
- Storage conditions: maximum storage time of 3 days in a refrigerator or 12 months at -20°C. Multiple freezing is omitted. Further details are given in the following sections (on receipt they were vacuum-wrapped, labeled appropriately and stored frozen at –20°C until use).

PRINCIPLES OF ASSAY
- Diffusion cell: the diffusion cells were assembled using the receptor compartment, the donor compartment, the stainless steel clamp as well as two three-port valves and the skin membrane. For this purpose, the receptor compartment equipped with the three-port valves, were filled with physiological saline for determination of electrical resistance for assessment of skin membrane integrity. Each skin membrane was hydrated in physiological saline for about 10 minutes before mounting to the diffusion cells. Subsequently, the skin membrane, with the external surface of the stratum corneum upper most, was placed on the fluid avoiding air bubbles underneath. The donor compartment was carefully placed on top so that the skin sample was stretched between the donor and receptor compartments. The setup was stabilized by means of the metal clamp and the diffusion cell was sealed in this way.
- Receptor fluid: Ethanol/tap water (1+1; V/V) for the high dose, Ethanol/tap water (3+7; V/V) for the mid dose and pure tap water for the low dose
- Solubility of test substance in receptor fluid: the solubility of BAS 490 F was determined in different ethanol/water mixtures (1+1; 3+7 and 1+9, V/V). After 65 hours it was at a maximum of 121 mg/L in ethanol/water (1+1; V/V), 22 mg/L in ethanol/water (3+7; V/V) and at a minimum of 3 mg/L in ethanol/water (1+9; V/V) (for details see analytical results of the sponsor). The maximum solubility of the test substance in water is reported to be 2 mg/L
- Static system: Soluene®-350 (Perkin Elmer) and ethanol (extraction media)
- Flow-through system: after the complete connection, the system was checked with several test runs. The desired flow rate (about 0.3 mL / 30 seconds) was adjusted by selecting appropriate tubes and rotation speed of the pump as well as by individually varying the contact pressure of the tube beds. The test runs also helped to detect any defects of the tube connections and insured the presence of pure receptor fluid in the receptor compartment.
- Test temperature: the receptor system was brought to 32 ± 1°C by means of the water bath thermostat and equilibrated for about 15 minutes.

Results and discussion

Absorption in different matrices:

ABSORPTION KINETICS (Table 4)
The mean absorption rate of all cells corresponding to the slope of the cumulative absorbed dose curve over time was about 0.9 μg/(cm²*h) in the high dose, 0.08 μg/(cm²*h) in the mid dose and 0.003 μg/(cm²*h) in the low dose groups. This is equivalent to a permeability constant of about 0.34*10-5 cm/h, 10*10-5 cm/h and 5.16*10-5 cm/h, respectively, taking into account the applied concentration. These rates are considered to indicate a very slow diffusion speed in the high and low dose group, and a moderate one in the mid dose group. Furthermore the evaluation of the absorption kinetic indicated a mean lag time of about 0.7, 0.8 and 1.2 hour, respectively for the high, mid and low dose groups.

The cumulative absorbed dose found in the receptor fluid after the 24-hour exposure period was about 0.2%, 5% and 3.9% of the applied doses, respectively for the high, mid and low dose groups.

The mean cumulative absorbed dose curves show sigmoid shapes with the range of the steepest slopes up to 2 hours after application for the mid dose and up to 4 hours for the high and low dose. Thereafter considerable decreases in absorption rates were observed with the curves of the high dose reaching a virtual plateau at 10 hours after application and those of the mid and low dose very shallow slopes.
The concentration ratio between high and mid or low dose (313- or 4167-fold difference) was not well reflected by the ratio of the mean maximum absorption rates (10.9- and 274-fold difference). The permeability constants (Kp) calculated for the mid and low dose from the absorption rates and the applied concentrations thus are different to that of the high dose. In contrast the ratio of the absorption rates from the mid and low dose (25.2-fold) compares rather well to the dilution factor between these doses (13.3- fold) resulting in more comparable permeability constants.
No huge differences in the cumulative absorbed relative dose were found for the mid and low dose, which however was considerably lower for the high dose.

Total recovery:

RECOVERY (Table 3)
The mean total recovery measured at the high dose fulfilled the OECD quality criteria in the high dose (105.1%) and the mid dose groups (108.8%), but not in the low dose group (108.0%). The individual values ranged 103-107% for the high dose, 102-118% for the mid dose (with the exception of one cell with total recovery of 118.2%) and 98-120% for the low dose.
Nearly the total amount of test substance was recovered from the skin membrane washings and tape strips (about 105% for the high dose, about 100% for the mid dose and about 101% for the low dose). Below 0.1% (for the high dose), about 2.6% (for the mid dose), and about 1.5% (for the low dose) of the test substance was associated with the skin membrane. About 0.2%, 6.5% and 5.3%, respectively was recovered from the receptor fluid, receptor chamber washing and from the receptor samples including wash out, which is considered to represent the absorbed dose. The amount recovered from skin and the absorbed dose sum up to about 0.3%, 9.1% and 6.8%, respectively.

Any other information on results incl. tables

Table 4: Mean recovery rates and absorption kinetic parameters

Species		Human
Dose Group		1	2	3
Target Applied Dose of Test Preparation	[mg or μL/cm²]	10	10	10
Target Applied Dose of Test Substance	[μg/cm²]	2500	8	0.6
Mean Nominal Applied Dose of Test Substance	[μg/cm²]	2652	8.07	0.628
Recovery
Mean Total Recovery Rate	[% of applied dose]	105.1	108.8	108.0
Mean Non-absorbed Dose	[% of applied dose]	104.9	99.7	101.3
Mean Amount associated with Skin	[% of applied dose]	0.047	2.59	1.45
Mean Absorbed Dose	[% of applied dose]	0.215	6.54	5.30
Absorption Kinetics
Kp	[*10-5 cm/h]	0.341	10.00	5.16
Absorption Rate	[μg / (cm²*h)]	0.903	0.0830	0.0033
Lag Time	[ h ]	0.7	0.8	1.2