Cocoa, powd., alkalized

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Multi-centre comparative study based on OECD Guidelines; not all variables controlled. Reasonably well-reported publication. Study carried out with caffeine, a key component of cocoa.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

GLP compliance:

yes

Remarks:

Two/10 participating laboratories were GLP-compliant, the others (n=8) “adhered to this quality system as much as possible”.

Test material

Test material information

Constituent 1

Reference substance name:

3,7-Dihydro-1,3,7-trimethyl-1H-purine-2,6-dione

IUPAC Name:

3,7-Dihydro-1,3,7-trimethyl-1H-purine-2,6-dione

Constituent 2

Reference substance name:

Caffeine

EC Number:

200-362-1

EC Name:

Caffeine

Cas Number:

58-08-2

Molecular formula:

C8H10N4O2

IUPAC Name:

1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione

Constituent 3

Reference substance name:

1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl-

EC Number:

619-173-4

IUPAC Name:

1H-Purine-2,6-dione, 3,7-dihydro-1,3,7-trimethyl-

Details on test material:

- Name of test material (as cited in study report): Caffeine
- Molecular formula (if other than submission substance): C8H10N4O2
- Molecular weight (if other than submission substance): 194.2
- Smiles notation (if other than submission substance): c12c(n(=O)n(c1=O)C)C)ncn2C
- InChl (if other than submission substance): 1S/C8H10N4O2/c1-10-4-9-6-5(10)7(13)12(3)8(14)11(6)2/h4H,1-3H3
- Structural formula attached as image file (if other than submission substance): see Fig. 1
- Substance type:
- Physical state: solid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Radiochemical purity (if radiolabelling): no data
- Specific activity (if radiolabelling): 51.2 mCi/mmol
- Locations of the label (if radiolabelling): 1-methyl-14C
- Expiration date of radiochemical substance (if radiolabelling): no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Radiolabelling:

yes

Remarks:

In 7 cases

Test animals

Species:

human

Strain:

other: Not applicable

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Administration / exposure

Type of coverage:

other: Not applicable

Vehicle:

other: ethanol:water, 1:1 v/v

Duration of exposure:

24 hours

Doses:

4 mg/ml concentration: application of 25 µL/cm2 skin surface, therefore 100 µg/cm2 [0.32-3.14 cm2 exposed]

Control animals:

no

Remarks:

Not applicable

Details on study design:

No data

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: surgical waste/post-mortem
- Ethical approval if human skin: in accordance with national guidelines
- Type of skin: full thickness/dermatomed from breast, leg or abdomen
- Preparative technique: cleaned of subcutaneous fat
- Thickness of skin (in mm): 0.3 – 1.8
- Membrane integrity check: visual/tritiated water/capacitance
- Storage conditions: -20oC (-70oC for 2 participants)
- Justification of species, anatomical site and preparative technique: to study robustness of methodology

PRINCIPLES OF ASSAY
- Diffusion cell: static (4 labs) and flow-through (6-labs)
- Receptor fluid: saline (0.9% NaCl)
- Solubility of test substance in receptor fluid: no data
- Static system: receptor compartment volume 5.0 – 17.7 ml with stirrer bar
- Flow-through system: receptor compartment volume 0.25 – 3.5 ml with/without stirrer bar; flow rate approx 1.5 ml/h
- Test temperature: no data
- Humidity: no data
- Occlusion: yes
- Reference substance(s): not applicable
- Other: Exposed skin area 0.64 – 3.14 cm2 (static cells); 0.32 – 0.95 cm2 (flow-through).
12-22 replicates/laboratory (9 labs).
Repeated sampling of receptor fluid, at a minimum of 1,2,4,8 and 24-hours. Test material remaining at 24-hr was removed with cotton swabs (5) soaked in EtOh/H20 (1:1, v/v) and a dry cotton swab.

Results and discussion

Signs and symptoms of toxicity:

not specified

Remarks:

Not applicable

Dermal irritation:

not specified

Remarks:

Not applicable

Total recovery:

Human skin at 24h – 24.5 ± 11.6 % dose (9 participants)
Rat skin at 24h – 53.7 ± 2.9 % dose (1 participant)

Human skin at 24h – 66.4 – 100.6 % dose (6 participants)
Rat skin at 24 h – 99.5 ± 0.2 % dose (1 participant)

Any other information on results incl. tables

 

Percutaneous absorption rate

Time point:

Max. absorption rate : 1.2 – 5.2 h (Human)

 

Rate:

Mean max. rate : 2.24±1.43µg/cm²/h

 

Percutaneous absorption rate[multiple blocks possible]

Time point:

Max absorption rate : 1.2± 0.2 h (Rat)

 

Rate:

Mean max. rate : 6.82±0.82µg/cm²/h

 

Remarks:

 

Percutaneous absorption rate[multiple blocks possible]

Time point:

24 h          

 

Dose:

4 mg/ml

 

Absorption (%):

>

13

<

45

 

Remarks:

For human skin, total penetration was determined to be 13.1 to 44.5% (in 6 labs)

 

Percutaneous absorption rate[multiple blocks possible]

Time point:

24 h

 

Dose:

4 mg/ml

 

Absorption (%):

ca

61

 

Remarks:

For rats, total penetration was determined to be about 61.3% (in 1 lab)

Applicant's summary and conclusion

Conclusions:

In a multi-centre comparative in vitro skin absorption study, the total penetration in 24-hr of caffeine across human skin was calculated to be about 13-45%, whilst rat skin was about 61%.

Executive summary:

In a study to investigate the robustness of in vitro percutaneous absorption methodology, the rate of absorption of caffeine through human and rat skin preparations was determined by 10 participating laboratories and the extent of absorption by 7 laboratories. Experiments with human skin preparations were carried out to a common protocol based on OECD Guideline 428, using preparations derived from stored post-mortem or surgical waste samples from both male and female subjects. Skin samples varied in thickness from 0.3 to 1.8 mm and were used in either static or flow-through cells. Full-thickness rat skin preparation from young male Sprague-Dawley rats were also used. Caffeine was applied at a dose of 100µg/cm²and levels determined in receptor fluid at intervals up to 24 hr. After washing, the residual levels in skin were also determined.

 

The maximum rate of percutaneous absorption of caffeine through human skin was about 2.2µg/cm²/hr and through rat skin was about 6.8µg/cm²/hr and the time to maximum absorption was between 1.2-5.2 hr in humans and about 1.2 hr in rats. Recovery in receptor fluid at 24 hr amounted to about 25% of the dose for human skin and 54% for rat skin and mean recovery in the washed skin at 24 hr varied from 1 to 12% of the dose for human skin and about 8% for rat skin. Overall recovery of the applied dose varied from 66 to 100% for human skin and 99.5% for rat skin.

 

In conclusion, the total penetration in 24-hr of caffeine across human skin was calculated to be about 13-45%, whilst rat skin was about 61%.