Hesperidin

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hesperidin

EC Number:

208-288-1

EC Name:

Hesperidin

Cas Number:

520-26-3

Molecular formula:

C28H34O15

IUPAC Name:

5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

orange/brown powder
Batch# 120323
Purity: 91.15% based on dry mass

Test animals

Species:

other: seeded human epidermal keratinocytes

Strain:

other: supplied by SkinEthic Laboratories, Nice, France

Details on test animals or test system and environmental conditions:

The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:

other: EPISKIN model

Preparation of test site:

not specified

Controls:

other: sodium dodecyl sulphate (SDS) 5% as positive control

Amount / concentration applied:

Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Observation period:

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 ºC, 5% CO2 in air for 42 hours.
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was
transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination. 2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO 2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Number of animals:

not relevant, measurements were made in triplicates for negative control, positive control and test item.

Details on study design:

Absorbance/Optical Density measurement at day 6:
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Relative mean viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: viability of negative control. Time point: 6 days following incubation. Max. score: 0.0. Reversibility: no data. Remarks: Max score is theoretical for no viability. (migrated information)

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.7%. The positive control acceptance criterion was therefore satisfied. The mean OD₅₄₀ for the negative control treated tissues was 0.841 and the standard deviation value of the percentage viability was 7.0%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 6.0%. The test item acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item hesperidin was considered to be Non-Irritant (NI) in this human epidermis skin model test.

Executive summary:

The relative mean viability of the test item treated tissues was 100.0% after a 15-Minute exposure period. Thus, hesperidin had no effect on viability and the substance is considered non-irritant.