Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1983
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study precedes guideline setting. It is well documented but some methodological aspects make it not reliable: the low temperature (21°C) used in this experiment, the fact that amount within the epidermis was not determined, and the potential loss of test substance through its determination by extraction process rather than radiolabelling may have contributed to the very low penetration rate reported. The integrity of the skin was not checked but in any case a loss of integrity would overestimate the penetration rate.It is therefore considered that the absolute penetration level may not be accurate.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1983

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In an in vitro skin absorption study, penetration of the test material through excised human epidermis was examined.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): acetyl eugenol
- Source: Inoue Koryo
- Analytical purity: 99.9%
Radiolabelling:
no

Test animals

Species:
other: Human cadaver skin

Administration / exposure

Doses:
0.2 mL
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Human cadaver
- Ethical approval if human skin: No data
- Preparative technique: Human lower abdominal skin was excised from a cadaver during autopsy and kept at -20 °C and thawed prior to examination. Subcutaneous tissue was removed using Cooper's scissors. Epidermis was separated from dermis by a modification of Baumberger's method (1942). Full thickness skin was placed dermis side down on a metal plate heated to 60 °C for a period of 10 minutes. The epidermis was separated from the dermis using forceps.
- Storage conditions: -20 °C

APPLICATION OF TEST MATERIAL.
- The glass chamber used in this study is described as follows: the upper surface of the epidermis was fixed to the lowest part of a glass tube (a), using adhesive. The glass tube (a) was then placed inside one arm of the U-shaped glass-chamber. Another glass tube (b) was withdrawn from the other side of the glass-chamber, and approximately 5 mL of saline was poured into the chamber until it came into complete contact with the bottom of the epidermis. The glass tube (b) was then replaced into the chamber.
- 0.2 mL of test material was applied to the top of the epidermis attached to the glass tube (Ea) by using a micropipette.
- The top of the glass tube (Ea) was covered with parafilm in order to avoid evaporation of the test sample.
- The chamber was kept in a thermostatically controlled cabinet (HAM-40 type, Seiwa Riko Company) at 21 °C and 55 % relative humidity for 72 h.

MEASUREMENT OF PENETRATION
- Seventy-two hours after application of test material, the glass tube (a) was removed. The saline from the chamber was poured into a test tube. The chamber and the bottom of the epidermis attached to the glass tube (a) were both washed 3 times with saline, poured into the same test tubethe measurement of penetration was performed. This saline (approximately 10 mL) was then poured into a 100mL flask.
- 10 mL of saturated salt water and 25 mL of ether were added and mixed vigorously.
- The compound was extracted in ether and. A An additional 25 mL of ether was added to the water fraction remaining after extraction, in order to insure complete extraction.
- The resultant 50 mL of ether, after the two extractions, was dehydrated by adding approximately 2 g of anhydrous Na2SO4. In order to remove the anhydrous Na2SO4, the ether fraction was filtered with filter paper and then condensed to 1 mL of ether by using a Kderna-Danish condenser.
- Then 2 µL of the condensed sample was injected into a Shimazu GC-6A gas chromatograph under following specifications:Detector: Flame ionization detector Column: 10 % FEAP on Gasport H60-80 mesh 2 mm 1.0 x 2.0 m (glass) Column temperature: 150-250 °C Injection temperature: 280 °C Detector temperature: 280 °CSensitivity: 10^3 x 32 – 10^4 x 16 mVΩ Carrier gas: N2 at 30 mL/minute
- The peak area on the gas chromatograms was compared with that of a standard sample, in which the concentration of the material tested was known.
- The amount of the test material which penetrated the human epidermis was calculated by the following formula:Amount of test material which penetrated human epidermis (µg) = (Peak area of test sample/ peak area of standard sample) x (concentration of test material in standard sample (µg/mL)) x (volume of test sample (mL))
- Percentage penetration was calculated using following formula:Percentage penetration = [Amount penetration (µg) / (200 µL x specific gravity x 10^3)] x 100 - The experiment was repeated 6 times.

Results and discussion

Percutaneous absorption
Dose:
0.2 mL
Parameter:
percentage
Absorption:
0.092 %
Remarks on result:
other: 72 h
Remarks:
penetration through excised human epidermis
Conversion factor human vs. animal skin:
Not applicable

Any other information on results incl. tables

Percentage penetration:

Penetration of 0.2 mL of test material through excised human epidermis after 72 h was found to be 0.092 ± 0.017 %.

 

Solubility in water and octanol:

Test material was freely soluble in octanol. Test material exhibited very slight solubility in water (2 to 5 ppm).

A high correlation between water solubility and percentage penetration was observed (r = 0.900, t = 12.040).

Applicant's summary and conclusion

Conclusions:
Under the test condition, penetration of test material through excised human epidermis after 72 h was found to be 0.092 ± 0.017 %. Also, high correlation between water solubility and percentage penetration through human epidermis of test material was observed. However due to significant methodological deficiencies, it is considered that the absolute penetration level may not be accurate.
Executive summary:

In an in vitro skin absorption study, penetration of test material through human epidermis was examined.  0.2 mL of test material was applied to the epidermis attached to the glass tube in a thermostatically controlled cabinet at 21 °C and 55 % relative humidity for 72 h. Seventy-two hours after application of test material, the measurement of penetration was performed by gas chromatography. the solubility of test material in water and octanol was also studied.

Penetration of test material through excised human epidermis after 72 h was found to be 0.092 ± 0.017%. Test material was freely soluble in octanol. Test material exhibited very slight solubility in water (2 - 5 ppm). A high correlation between water solubility and percentage penetration was observed.

This study precedes guideline setting. It is well documented but some methodological aspects make it not reliable: the low temperature (21°C) used in this experiment, the fact that amount within the epidermis was not determined, and the potential loss of test substance through its determination by extraction process rather than radiolabelling may have contributed to the very low penetration rate reported. The integrity of the skin was not checked but in any case a loss of integrity would overestimate the penetration rate.It is therefore considered that the absolute penetration level may not be accurate.