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Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Sep 2001 to 01 Oct 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
2000
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes
Remarks:
[Oxadin-4-14C]

Test animals

Species:
other: rat and human
Strain:
other: Hanlbm: WIST (SPF) (rats) and Caucasian donors (human)
Remarks:
male rats and male/female human
Sex:
male/female
Details on test animals and environmental conditions:
- RAT SKIN: Epidermal membranes were prepared from approximately 9 week old, male Hanlbm: WIST (SPF) rats.
- HUMAN SKIN: Abdominal cadaver skin from Caucasian donors (one male and one female) was obtained from the Institut für Pathologie, Kantonsspital Basel, Basel, Switzerland.

Administration / exposure

Type of coverage:
other: permeable tape (non-occluded)
Duration of exposure:
24 h
Doses:
- Nominal doses: 176.6 and 3450 µg of test material cm-2
- Actual doses: 20 g a.i./L and 350 g a.i./L
- Actual doses calculated as follows: not specified
- Dose volume: 6 µL
- Rationale for dose selection: not specified
Control animals:
no
Details on study design:
DOSE PREPARATION
The two doses were prepared to mimic the commercial 350 g/L formulation and the in use concentration of 20 g/L using the technical material, [14C]-labelled test material and formulation blank. The formulated test substance was checked for stability by TLC. The test material represented at least 98.4% and 97.9% of the radioactivity at the low and high dose levels, respectively.

APPLICATION OF DOSE:
- Those cells with skin membranes having acceptable Kp values in the integrity test were arranged on the manifolds and then a 6 µL aliquot of the formulated test substance was applied to the surface of each skin membrane. - - The donor chamber was covered with a permeable tape (non-occluded conditions). There were seven human skin samples and seven rat skin samples tested at each concentration.

REMOVAL OF TEST SUBSTANCE
Finally, the cells were washed with ethanol/water (1/:1, v/v) and the radioactivity in the cell wash was determined by LSC.

SAMPLE COLLECTION
- Duration of exposure and sampling: The skin was exposed to the test preparations for 24 hours during which time samples of receptor fluid were taken at suitable intervals (0-6 hours, every hour; 6-24 hours every 2 hours) to allow adequate characterisation of the absorption profile.
- Terminal procedures: Twenty four hours after application the skin membrane surface was rinsed with ethanol/water (1:1, v/v) and the radioactivity in the skin rinse was determined by LSC. The skin membrane was removed from the in-line cells and dissolved in tissue solubilizer prior to LSC.

SAMPLE PREPARATION
The two doses were prepared to mimic the commercial 350g/L formulation and the in use concentration of 20 g/L using the technical material, [14C]-labelled test material and formulation blank. The formulated test substance was checked for stability by TLC. The test material represented at least 98.4% and 97.9% of the radioactivity at the low and high dose levels, respectively.

ANALYSIS
All components of the test system (e.g. receptor fluid, skin wash, donor chamber) were analysed by LSC and the recovery determined.

OTHER:
- Definition of absorbed test material: The absorbed (systemically available) dose is considered to be the test material detected in the receptor fluid. Material removed from the surface of the epidermis by the washing procedure is regarded as unabsorbed.. In vivo, the majority of the dose in the epidermis, especially that in stratum corneum, would eventually be lost by desquamation.






Details on in vitro test system (if applicable):
SKIN PREPARATION
- Epidermal membranes were prepared from approximately 9 week old, male Hanlbm: WIST (SPF) rats. Abdominal cadaver skin from Caucasian donors (one male and one female) was obtained from the Institut für Pathologie, Kantonsspital Basel, Basel, Switzerland. The skin samples were wrapped in tinfoil and stored at -18°CC.
- Prior to use, the skin samples were removed from the freezer and allowed to thaw at room temperature. The subcutaneous fat was carefully removed from the full thickness skin and pieces of about 4 x 5 cm2 were stretched evenly over a cork block, with stratum corneum uppermost. Skin sections of about 200 µm thickness were cut off from the top using a dermatome.
- Pieces of skin membranes (approx. 1.8 x 1.8 cm2) were mounted in diffusion cells between the donor and receptor chamber so the stratum corneum was exposed to air and the basal part in contact with the receptor fluid. 0.9% NaCl was pumped through the receptor chamber for an equilibrium period of 0.5-1.0h.
- The integrity of the membranes was checked by applying tritiated water to the skin membrane surface under occluded conditions.
- The cumulative penetration was determined over 6 hours by collecting hourly fractions. The permeability coefficient (Kp) of each skin membrane was calculated for the 3-6 hours interval. Rat skin membranes with Kp > 3.5-10^-3 cm h-1 and human skin membranes with Kp > 2.5.10^-3 cm h-1 were excluded from the subsequent experiment.

PRINCIPLES OF ASSAY
- Diffusion cell: Diffusion of the test material into and across the skin to a receptor fluid was measured using glass diffusion cells in which the epidermis formed a horizontal membrane and provided an application area of 0.64cm2. Seven flow-through diffusion cells were used per aluminium manifold which was connected to a water bath to maintain the temperature of the skin membranes at 31 - 33°C. The receptor chambers were connected to a multi-channel peristaltic pump, with a pump speed of approximately 3 mL/h.
- Receptor fluid: The receptor fluid (50% ethanol in water) was chosen to ensure that the test substance would freely partition into this from the skin membrane and never reach a concentration that would limit its diffusion.
- Temperature: Throughout the experiment the receptor fluid was stirred and the epidermal membranes were maintained at a normal skin temperature of 32 ± 1°C in a water bath.


Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
RAT SKIN MEMBRANE
- After application of the test material at the low dose level, 0.82% of the applied dose penetrated through the rat skin membrane within 24 hours. At the high dose level the portion penetrating through the rat skin membrane within 24 hours accounted for 1.18% of the dose. The total amount which penetrated within this period accounted for 1.45 µg cm-2 at the low dose level and 40.8 µg cm-2 at the high dose level.
- The flux, which reflects the penetration rate under steady state conditions, was calculated to be 0.062 µg cm-2.h-1 at the low dose level and 1.18 µg cm-2.h-1at the high dose level. The time interval of steady state conditions varied at both dose levels between the individual cells from 1-24 hours and 6-24 hours. The 19-fold higher concentration of the undiluted formulation led to a corresponding 19-fold higher penetration rate as compared to the diluted formulation.

HUMAN SKIN MEMBRANE
- Within 24 hours, 0.19% of the applied low dose and 0.44% of the applied high dose penetrated through the human skin membrane, corresponding to 0.34 µg cm-2 and 15.0 µg cm-2, respectively. The calculated flux, under steady state conditions, accounted for 0.013 µg cm-2.h-1 and 0.20 µg cm-2.h-1 at the low and high dose levels, respectively. The time interval for steady state conditions was between 1-24 and 3-24 hours at the low dose level and between 1-24 and 12-24 hours at the high dose level. Again, the 19-fold higher concentration at the high dose level led to a corresponding 16-fold higher penetration rate (flux) as compared to the low dose level.

See Table 1 at "Any other information on results incl. tables" for further details.
Total recovery:
RAT SKIN MEMBRANE
The mean recovery was 97.2% of the applied radioactivity at the low dose level and 89.2% of the applied radioactivity at the high dose level.

HUMAN SKIN MEMBRANE
The mean recovery was 100.4% and 89.6% at the low and high dose level, respectively.

ANALYSIS OF SKIN RINSE
At both dose levels and in both species, at least 96.5% of the radioactivity was found as unchanged test material. Hence, it was concluded that at both dose levels and in both species the test substance remained stable for 24 hours on the skin membrane.

See Table 2 at "Any other information on results incl. tables" for further details.
Percutaneous absorptionopen allclose all
Key result
Time point:
24 h
Dose:
176.6 µg cm^-2
Parameter:
other: Flux
Absorption:
0.062 other: µg cm^-2 h^-1
Remarks on result:
other: Rat skin membrane
Key result
Time point:
24 h
Dose:
3451 µg cm^-2
Parameter:
other: Flux
Absorption:
1.179 other: µg cm^-2 h^-1
Remarks on result:
other: Rat skin membrane
Key result
Time point:
24 h
Dose:
176.6 µg cm^-2
Parameter:
other: Flux
Absorption:
0.013 other: µg cm^-2 h^-1
Remarks on result:
other: Human skin membrane
Key result
Time point:
24 h
Dose:
3451 µg cm^-2
Parameter:
other: Flux
Absorption:
0.204 other: µg cm^-2 h^-1
Remarks on result:
other: Human skin membrane

Any other information on results incl. tables

Table 1: Summary of test material absorption through rat and human skin membranes

Test System

Rat skin membrane

Human skin membrane

Dose level

low

high

low

high

Applied dose (µg cm-2)

176.6

3451

176.6

3451

Applied volume (µL)

6

6

6

6

Application area (cm2)

0.64

0.64

0.64

0.64

Concentration (mg cm-3)

18.8

368.1

18.8

368.1

Penetration within:

µg cm-2

% of dose

µg cm-2

% of dose

µg cm-2

% of dose

µg cm-2

% of dose

6 hour

0.39

0.22

19.4

0.56

0.10

0.06

8.2

0.24

12 hour

0.78

0.44

28.8

0.83

0.17

0.10

12.8

0.37

24 hour

1.45

0.82

40.8

1.18

0.34

0.19

15.0

0.44

Flux (µg cm-2h-1)

0.062

1.179

0.013

0.204

Table 2: Summary of test material recovery (% of dose)

Test System

Rat skin membrane

Human skin membrane

Dose level

low

high

low

high

Applied dose (µg cm-2)

176.6

3451

176.6

3451

Perfusates

0-24 hour

0.82

1.18

0.19

0.44

Remaining dose

cell wash

0.19

0.43

0.08

2.42

 

skin rinse

95.71

75.19

99.92

69.00

 

skin membrane

0.50

12.40

0.23

17.73

 

subtotal

96.40

88.02

100.24

89.15

Recovery

97.22

89.20

100.24

89.59

Applicant's summary and conclusion

Conclusions:
The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation.
Executive summary:

The percutaneous penetration of the test material was determined in vitro using split-thickness skin membranes from rat and human skin. The skin membranes were set up in flow-through diffusion cells, the formulated [14C] radiolabelled test substance was applied onto the skin membranes and then the perfusates were collected at defined time intervals (0-6 hours at 1 hour intervals and 6-24 hours at 2 hour intervals). Two dose levels were used, i.e. a low dose level of nominal 0.2 mg test material / cm2 and a high dose level of nominal 3.5 mg test material/cm2. The low dose of 20 g a.i./L reflects concentrations recommended for use in the field, the high dose represents the undiluted formulation (350 g a.i./L). 24 hours after application, the skin membrane surface was rinsed with ethanol/water (1:1) and the radioactivity in the skin rinse was determined by LSC. The skin membrane was removed from the in-line cells and dissolved in tissue solubiliser prior to LSC. Finally, the cells were washed with ethanol/water (1:1) and the radioactivity in the cell wash determined.

During the 24 hours after application of formulated [Oxadiazin-4-14C] labelled test material, only 0.8% and 1.2% of the dose penetrated through rat split-thickness skin membranes at the low and high dose levels, respectively. The human split-thickness skin membranes showed a lower permeability of the test material. Within 24 hours after application, only 0.2% and 0.4% of the low and high doses, respectively, penetrated through human skin membranes.

A species difference in respect to the penetration of formulated test amterial was also reflected by the flux. The flux at steady state conditions was determined to be about 0.06 µg-cm-2 h-1 and 1.18 µg-cm-2 h-1 through rat skin membranes and about 0.01 µg.cm-2 h-1 and 0.20 µg.cm-2 h-1 through human skin membranes at the low and high dose level, respectively. This results in a human:rat ratio of the flux of about 1:4.8 at the low dose level and 1:5.8 at the high dose level.

The 19-fold higher concentration of the undiluted formulation led to a corresponding 19-fold and 16-fold higher penetration rate (flux) in rat and human skin membrane, respectively.

The radioactivity in the skin rinse at the end of exposure at both dose levels and for both species was analysed as unchanged test material. The experimental recoveries were between 89.2% and 100.4% of the applied dose. The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation.