Coffee, bean, roasted, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Are coffee silverskin extracts safe for topical use? An in vitro andin vivo approach.

Author:

Rodrigues F et al.,

Year:

2015

Bibliographic source:

Industrial Crops and Products 63 (2015) 167–174

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

not specified

Remarks:

Study was performed in a research laboratory, probably not GLP compliant.

Test material

Test material information

Constituent 1

Reference substance name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

Molecular formula:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

IUPAC Name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: coffee silver skin samples were provided by a national coffee roaster (BICAFÉ, Portugal).
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: coffee silver skin was milled to particle size of approximately 0.1 mm using a A11 basic analysis mill (IKA Wearke, Staufen, Germany) and stored in silicone tubes at room temperature (25–28ºC) until extract preparation. Samples (1 g) were submitted to solvent extraction by maceration with 20 mL of water, ethanol: water (1:1 v/v) or ethanol for 30 min at 40ºC. The three different extracts obtained were filtered through Whatman No. 1 paper filter and the filtrates collected.
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The in vitro reconstructed human skin tissue (EpiSkin) method, proposed to replace animal testing for skin corrosivity and skin irritation is based on determining cell viability, and cytokine release (IL-1a) as an additional endpoint. The reconstructed 0.38 cm2 skin inserts were used and the assay was performed according to the manufacturer instructions and protocol.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After exposing to the extracts and controls for 15 min, the epidermis samples were washed with sterile PBS and then incubated in the maintenance medium. After 42 h, the medium was collected and frozen at -20ºC for further determination of IL-1a.

Number of replicates:

3 replicates per coffee sliverskin extract

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

water extract

Value:

117.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

water/ethanolic extract

Value:

105.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

ethanolic extract

Value:

117.8

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The results of skin irritability assay. Cell viability was assessed by MTT while the post-exposure basal media was analyzed for IL-1a release (pg/mL)

Coffee silverskin extract	Viability (%)	IL-1a (pg/mL)
Water	117.7 ± 11.7	8.5 ± 0.1
Water/ethanol (1:1 v/v)	105.9 ± 15.9	76.4 ± 2.0
Ethanol	117.8 ± 9.8	17.6 ± 3.2
Positive control (SLS)	11.6 ± 2.4	552.9 ± 32.1
Negative control (PBS)	100 ± 9.7	28.4 ± 2.6

EpiSkin method distinguishes between irritants and non-irritants. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels of 50%. The three coffee silverskin extracts were not considered skin irritants, as the viability in all cases was higher than 50%, with values ranging between 105.9 ± 15.9 and 117.8 ± 9.8.

The low irritating potential was also confirmed by the low release of IL-1a, a highly active and pro-inflammatory cytokine produced by keratinocytes.

Additionally, histological analysis did not reveal any morphological differences between extracts and negative control, in contrast to the positive control, where the adverse effects were observed in all the epidermal layers.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results from the in vitro reconstructed human epidermis test, the coffee silverskin extracts did not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008.

Executive summary:

The reconstructed skin inserts (EpiSkin method) were exposed to three types of coffee silverskin extracts (water, water/ethanol, ethanol) and controls for 15 min. The cell viability was determined by MTT assay, while the post-exposure basal media were analyzed for IL-1a release. Three extracts were not considered skin irritants in this test as the cell viability in all the cases was above 50% with the values ranging between 105 ± 15.9 and 117 ± 9.8 %. This potential was also confirmed by the low release of IL-1a, a highly active and pro-inflammatory cytokine produced by keratinocytes. Additionally, no morphological differences were observed between extracts and negative control (PBS), in contrast to the positive control (SLS) where the adverse effects were observed in all the epidermal layers.

Under the conditions of this assay, the coffee silverskin extracts did not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008.