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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Recent well described and well conducted study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Principles of method if other than guideline:
Skin absorption and elimination kinetics were studied using human skin from the region of thorax of 40-50-years old Caucasian women, mounted on flow-through Teflon diffusion cells. 500 mg alpha-pinene was applied and after 1 to 4-h exposure, the content in the stratum corneum layers (separated by a tape-stripping method) and in the epidermis/dermis was determined using GC. Similarly, the elimination kinetics in the skin were analysed during 4 h following 1 h absorption.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): alpha-pinene, (-)-alpha-pinene
Analytical purity: > 99%
Source: Fluka, Buchs, Switzerland
Radiolabelling:
no

Test animals

Species:
human
Sex:
female
Details on test animals and environmental conditions:
Human cadaver skin was obtained from the region of thorax of 40-50-years old Caucasian women. The subjects did not have skin diseases. Before the experiment, the skin was stored frozen at -20 °C.

Administration / exposure

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Duration of exposure:
1, 2 or 4 h
Doses:
500 mg on 0.65 cm²
No. of animals per group:
4 for each time point
Control animals:
no
Details on study design:
No data
Details on in vitro test system (if applicable):
Diffusion cell: flow-through Teflon diffusion cell (Crown Glass, USA)
The diffusion area of the skin was 0.65 cm². The donor compartment was occluded with Parafilm (Sigma-Aldrich, Steinheim, Germany), and the system was maintained at temperature 37 ± 0.5 °C. An isotonic pH 7.3 phosphate buffer, 10 mL, preserved with 0.005% sodium azide (Fluka, Buchs, Switzerland) was recirculated beneath the skin with a constant rate 10 mL/h. The two-phase acceptor fluid protected against evaporation was used and sink conditions were ensured for all steps of the study. It was obtained by addition of 5 mL of methylene chloride to the vial served as reservoir of the buffer. The skin was only in contact with the aqueous phase. The experiment was terminated by removing terpenes from the skin surface and very short rinsing with methanol. The stratum corneum layers were separated by a tapestripping method, using 21 fragments of an adhesive
tape (3M Medica Pharma, St. Paul, USA). Collected samples were divided into three fractions (SC I-III). Each fraction, as well as the remaining viable epidermis with dermis (ED) was extracted with methanol (HPLC-grade, P.O.Ch., Gliwice, Poland).
In the elimination studies, the terpenes were applied only for 1 h and next, after removing terpenes from the donor chamber as described above, the skin was left in the chambers for 1, 2, 3 or 4 h. The acceptor medium was replaced by a fresh portion and its circulation was maintained. After the specified time, the skin was rinsed, removed and separated as described above. The terpenes in the extracts were analysed by GC with the detection limit 0.5 mg/mL.

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
not specified
Absorption in different matrices:
The dermal penetration of pure terpenes was studied during 4 h. The terpenes were present on the skin in infinite doses and the system was protected against evaporation. During that time no terpenes were detected in the acceptor fluid but extensive accumulation in the skin tissue occurred (see table 1).
Analysis of stratum corneum (SC) collected with an adhesive tape and merged into three groups demonstrates cumulation of terpenes in the outer (SC I), middle (SC II) and inner (SC III) layers. The results demonstrate rapid penetration of terpenes not only to the SC I layers but also to viable epidermis and dermis. The distance-dependent decreasing gradient of concentration for all terpenes is observed, although the concentrations were not normalized in respect of the collected SC mass. A steady-state concentration of terpenes in the SC can be assumed as soon as after 1 h. Maximum concentration in the SC was achieved as soon as after 1 h and did not further increase in the course of the study.
All studied terpenes are absorbed in high amounts in the viable epidermis with dermis (ED), however penetration into this layers is time-dependent process, constantly increasing during 4 h.
Total recovery:
No data
Conversion factor human vs. animal skin:
None

Any other information on results incl. tables

Table 1: Absorption of alpha-pinene (mg/cm2) into human skin layers (mean ± S.D., n = 4)

Skin layer

1-h exposure

2-h exposure

4-h exposure

SC I

4.3 ± 0.8

3.4 ± 0.4

7.7 ± 4.3

SC II

4.0 ± 0.5

3.2 ± 0.6

6.5 ± 4.1

SC III

3.0 ± 0.4

3.4 ± 0.8

6.0 ± 4.0

SC total

11.3 ± 4.7

10.0 ± 2.1

18.2 ± 12.1

ED

66.4 ± 16.8

147.2 ± 25.9

313.7 ± 38.3

Skin total

77.7 ± 14.5

157.2 ± 25.8

331.9 ± 34.7

Table 2: Elimination of alpha-pinene (mg/cm2) from human skin layers following 1 h absorption (t = 0) (mean ± S.D., n = 4)

 

Time after 1-h exposure

Skin layer

0

1

2

3

4

SC I

4.3 ± 0.8

3.2 ± 1.9

2.0 ± 1.8

0.7 ± 0.6

0.6 ± 0.7

SC II

4.0 ± 0.5

0.9 ± 1.0

0

0

0

SC III

3.0 ± 0.4

0.2 ± 0.2

0

0

0

SC total

11.3 ± 4.7

4.3 ± 3.0

2.0 ± 1.8

0.7 ± 0.6

0.6 ± 0.7

ED

66.4 ± 16.8

45.7 ± 13.0

35.9 ± 8.2

37.3 ± 3.6

33.9 ± 5.7

Skin total

77.7 ± 14.5

50.0 ± 10.1

37.9 ± 6.8

38.0 ± 3.3

34.5 ± 5.1

Applicant's summary and conclusion

Conclusions:
alpha-Pinene absorption into the different skin layers is rapid (steady-state concentrations in the skin obtained after 1-h exposure) but do not permeate through the skin to the acceptor medium due to large accumulation into the skin tissue.
Executive summary:

Skin absorption and elimination kinetics were studied using human skin from the region of thorax of 40-50-years old Caucasian women, mounted on flow-through Teflon diffusion cells. alpha-Pinene (500 mg) was applied onto the human skin (0.65 cm²), and after 1 to 4-h exposure, the content in the stratum corneum layers (separated by a tape-stripping method) and in the epidermis/dermis was determined using GC. Similarly, the elimination kinetics in the skin were analysed during 4 h following 1 h absorption. Quadruplicates were used for each time point.

The results demonstrate rapid penetration of terpenes not only to the first stratum corneum layers but also to viable epidermis and dermis (steady-state concentrations assumed to be obtained at 1-h exposure). However, alpha-pinene did not permeate across the skin to the acceptor medium due to large cumulation in the skin tissue. Two mechanisms of elimination process of terpenes from the SC are suggested: evaporation and slightly progressive penetration from inner layer into dermis.