Human Skin Model Test: In Vitro Skin Irritation

**Human Skin Model Test: In Vitro Skin Irritation**

(EpiDerm™ and EPISKIN-SM™)

The prediction and classification of skin irritation potential of substances can be performed by the assessment of the effect on EpiDerm™ and EPISKIN-SM™ models.

The human skin model test is validated by the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) and is performed in accordance with the OECD guidance OECD 439 at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of Skin Irritation Potential

Acute irritation characterizes a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances².
The EpiDerm™ and EPISKIN-SM™ represent a reconstituted three-dimensional human epidermis (RhE) model which consists of human epidermal keratinocytes. This in vitro model mimics biochemical and physiological properties of the upper human skin.
To determine skin irritation potential substances are applied directly on the skin tissue surface and the cell viability is measured by the MTT assay.
The EpiDerm™ and EPISKIN-SM™ human skin model test can be used as a reliable in vitro method to identify substances as non-classified ("No Category") or they can be classified into UN GHS "Category 2". A positive result will further require a skin corrosion test (OECD 431) for a final classification into "Category 1"¹.
The skin irritation potential of medical device extracts can also be determined by using a modified EpiDerm™ human skin model test.

Procedure

Principles of the Human Skin Model Test

Protocol
Model	EpiDerm	EpiSkin
Supplier	MatTek	Skin Ethic
Analysis	Skin irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control)	Skin irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control) optional: Interleukin-1α (IL-1α) release into the tissue culture medium
Test chemical concentrations	Liquids: 30 µL (undiluted) Solids: 25 mg + 25 µL DPBS	Liquids: 10 µL (undiluted) Solids: 10 mg + 25 µL DPBS
Exposure time and incubation periods	60 min incubation with dose groups 24 h post-incubation and further 18 h incubation in media	15 min incubation with dose groups 42 h post incubation
Quality controls	Positive control: 5% SDS Negative control: DPBS
Pre-tests	To determine if additional controls are needed: NSMTT: mixing of test item with MTT medium to determine if test item alone can reduce MTT à blue colouring: in main experiment two killed tissues treated with test item and two untreated killed tissues are added as controls NSC_living^#: mixing of test item with H₂O or isopropanol to determine if strong colour of test item can discolour at contact with this liquids à optical discolouring (measuring of spectrum): in main experiment two living tissues without incubation with MTT medium are added as controls NSC_killed^§*: if the two other controls were determined à in main experiment two killed tissues without incubation with MTT medium are added as controls
Application	Direct topical application of chemicals on skin tissues Three tissue replicates per dose group
Data delivery	Optical density (OD) value with microplate spectrophotometer at 570 nm
Prediction model	Mean tissue viability (% negative control): ≤ 50%: Irritant (I): UN GHS "Category 2" (IL-1α > 60 pg/mL) > 50%: Non-Irritant (NI): UN GHS "No Category" (IL-1α ≤ 60 pg/mL)

*NSMTT: non-specific MTT reduction

^#NSC_living: non-specific colouring of living tissues

^$NSC_killed: non-specific colouring of killed tissues

Data

Eurofins Data for demonstration technical proficiency of the Human Skin Model Test (EpiDerm™ and EPISKIN-SM™)

Chemical	UN GHS category	EF category for EpiDerm™	EF category for EPISKIN-SM™
Non-Classified Chemicals (UN GHS "No Category")
Naphthalene acetic acid	No Category	No Category	No Category
Isopropanol	No Category	No Category	No Category
Methyl stearate	No Category	No Category	No Category
Heptyl butyrate	No Category	No Category	No Category
Hexyl salicylate	No Category	No Category	No Category
Classified Chemicals (UN GHS "Category 2")
Cyclamen aldehyde	Category 2	Category 2	Category 2
1-Bromohexane	Category 2	Category 2	Category 2
1-Methyl-3-phenyl-1-piperazine	Category 2	Category 2	Category 2
Heptanal	Category 2	Category 2	Category 2

EF = Eurofins Munich GmbH

Table 1: Eurofins data of in vitro skin irritation with EpiDerm™ and EPISKIN-SM™ of nine tested proficiency chemicals compared to the data of the OECD guideline 439¹.

In Table 1 the obtained data from the in vitro skin irritation with EpiDerm™ and EPISKIN-SM™ of five non-classified and four classified ("Category 2") chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 439.

References

OECD (2015). OECD Guideline for the Testing of Chemicals No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. 28 July 2015
UN (2015). United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), sixth revised edition, UN New York and Geneva