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Toxicological information

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:
2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Recent well described study
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
publication
Title:
Unnamed

Materials and methods

Principles of method if other than guideline:
Terpenes were evaluated as enhancers to improve the skin permeation of therapeutically active agents (tea catechins and theophylline). In vitro permeation was determined by Franz cells. The skin deposition and subcutaneous amounts of drugs sampled in vivo were evaluated by microdialysis. Macroscopic perturbation of the stratum corneum and the biological reaction in viable skin as evaluated by TEWL and colorimetry.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Female Wistar rats (180—200 g)

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: terpenes
Duration of exposure:
See details on exposure
Doses:
See details on exposure
No. of animals per group:
See details on exposure
Control animals:
no
Details on study design:
This study utilized Franz cells to explore the influence of terpenes on the in vitro skin permeation of drugs. The amount of drug retained within the skin reservoir was also determined in vitro and in vivo. The in vivo transcutaneous drug amount was determined using a microdialysis technique. The mechanisms of the enhancing ability of terpenes were investigated by TEWL and colorimetry.
Details on in vitro test system (if applicable):
In vitro skin permeation was carried out using a Franz diffusion assembly. The shaved back skin of female Wistar rats or a cellulose membrane (SelluSep® T2, MW cutoff of 6000—8000, Membrane Filtration Products, U.S.A.) was mounted on the receptor compartment with the stratum corneum (SC) side facing upwards into the donor compartment. The donor medium was 1 mL of 25% ethanol/pH 7.4 buffer containing 3.44mM drug with or without 3% terpenes. The receptor medium was 5mL of pH 7.4 citrate-phosphate buffer. The available diffusion area between compartments was 0.785 cm2. The stirring rate and temperature were kept at 600 rpm and 37 °C, respectively. At appropriate intervals, 300-mL aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug retained in the skin was determined at the end of the in vitro experiment (24 h) if necessary. The site of application on the skin was washed 10 times with a cotton cloth immersed in double-distilled water. A sample of skin was weighed, cut with scissors, positioned in a glass homogenizer containing 1 mL of 0.1 M HCl, and ground for 5 min with an electric stirrer. The resulting solution was centrifuged for 10 min at 10000 rpm and then filtered through a PVDF membrane (with a pore size of 0.45m m, Millipore, U.S.A.). All samples in the in vitro experiment were analyzed by HPLC.

Results and discussion

Signs and symptoms of toxicity:
not specified
Dermal irritation:
yes
Absorption in different matrices:
See other information on results
Total recovery:
See other information on results

Any other information on results incl. tables

Table 1: The Flux, Skin Deposition, and Enhancement Ratio of (+)-Catechin across Skin after in Vitro Permeation for 24 h Duration

 

Flux (nmol/cm2/h)

ERFlux a)

Skin deposition (nmol/mg)

ERDeposition b)

Control

0.58 ± 0.18

-

0.48 ± 0.22

-

Alpha -Terpineol

217.50 ± 13.55

375.00

5.08 ± 0.55

10.58

a) The enhancement ratio (ERFlux) was the (+)-catechin flux with terpene treatment/(+)-catechin flux of control group.b) The enhancement ratio (ERDeposition) was the (+)-catechin deposition in skin with terpene treatment/(+)-catechin deposition in skin of control group. All the values of terpenes (flux and skin deposition) were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4).

Table 2: The Flux (nmol/cm2/h) of (+)-Catechin, (-)-Epicatechin, EGCG, and Theophylline across Skin after in Vitro Permeation for 24 h Duration

 

(+)-catechin

(-)-Epicatechin

EGCG

Theophylline

Control

0.58 ± 0.18

0.62 ± 0.12

0

11.38 ± 5.68

Alpha -Terpineol

217.50 ± 13.55

214.19 ± 31.05

33.11 ± 6.53

233.673 ± 30.56

All the flux values of terpenes were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4).

Table 3: Skin/Vehicle Partition Coefficient of (+)-Catechin and Theophylline by Treating Skin in 3% Terpenes

 

(+)-catechin

Theophylline

Control

2.51 ± 0.36

0.23 ± 0.18

Alpha -Terpineol

9.43 ± 3.30

0.84 ± 0.09

All the partition coefficient values of terpenes were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=4)

Table 4: In Vivo TEWL and Erythema of Rat Skin after Treatment of 3% Terpenes for 24 h

 

ΔTEWL (g/m2/h)

Δa* (arbitrary unit)

Control

2.66 ± 0.65

-1.06 ± 0.73

Alpha -Terpineol

4.82 ± 1.21a)

1.95 ± 0.22a)

a) The value significantly higher than the control (p<0.05). Each value represents the mean ± S.D. (n=6)

Table 5: Skin Deposition (nmol/mg) of (+)-Catechin, (-)-Epicatechin, EGCG, and Theophylline after in Vivo Application for 6 h Duration

 

Control

Alpha-terpineol

(+)-catechin

0.11 ± 0.03

0.46 ± 0.16

(-)-Epicatechin

0.26 ± 0.09

0.59 ± 0.18

EGCG

0.003 ± 0.001

0.55 ± 0.14

Theophylline

0.11 ± 0.08

0.37 ± 0.13

All the partition coefficient values of alpha-terpineol were significantly higher than the control (p<0.05). Each value represents the mean±S.D. (n=6).

Applicant's summary and conclusion

Conclusions:
alpha-Terpineol was found to be the best enhancer for catechins and theophylline. The high enhancement by alpha terpineol was due to macroscopic perturbation of the stratum corneum (SC) and the biological reaction in viable skin as evaluated by transepidermal water loss (TEWL) and colorimetry.
Executive summary:

Using in vitro and in vivo techniques, terpenes were evaluated as enhancers to improve the skin permeation of therapeutically active agents derived from tea, including tea catechins and theophylline. The in vitro permeation was determined by Franz cells. The skin deposition and subcutaneous amounts of drugs sampled in vivo were evaluated by microdialysis. In vivo, terpenes promoted the skin uptake but not the subsequent subcutaneous concentration of (-)-epigallocatechin gallate (EGCG). Both increased skin/vehicle partitioning and lipid bilayer disruption of the stratum corneum (SC) contributed to the enhancing mechanisms of terpenes for topically applied tea catechins and theophylline based on the experimental results from the partition coefficient and transepidermal water loss (TEWL). alpha-Terpineol was found to be the best enhancer for catechins and theophylline. The high enhancement by alpha terpineol was due to macroscopic perturbation of the SC and the biological reaction in viable skin, as evaluated by TEWL and colorimetry.