Assessment of skin irritation potential

Human skin model testing for medical device extracts

At Eurofins Human Safety Testing the human skin model test can be used for detection of skin irritation potential of medical devices. The prediction and classification can be done by the assessment of the effect on EpiDermTM model or SkinEthicTM model.

The modified human skin model test for medical devices is a reliable in vitro test method and is performed in accordance with the draft ISO guideline 10993-10 Part 10 at our laboratory1.

Assessment of skin irritation potential of medical devices

  • Acute irritation characterises a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances2.
  • The EpiDermTM and the SkinEthicTM model 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 the test item is extracted and applied directly on the skin tissue surface. The cell viability is measured by MTT assay.
  • The Skin Irritation Test can be used as a reliable in vitro test method to identify chemicals in extracts from medical devices as "irritant" or "non-irritant"3.

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skin-irriation_md

Acute irritation characterises a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances2.

Procedure: Principles of the human skin model test with medical devices

Protocol

    
Model

EpiDermTM (Reconstructed human skin model) supplied from MatTek

SkinEthicTM (Reconstructed human skin model) supplied from EpiSkin
Analysis
  • skin irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control tissues)
  • optional: Interleukin-1α (IL-1α) release into the tissue culture medium
Test chemical concentrations
  • 100 µL undiluted extract in 0.9% NaCl (polar)
  • 100 µL undiluted extract in sesame oil (non-polar)
Extraction and absorption capacity
  • extraction at 37 ± 1 °C for 72 ± 2 h in 0.9% NaCl or sesame oil with continuous agitation/shaking
  • determination of the absorption capacity of the test item in NaCl (polar) or in sesame oil (non-polar)
Exposure time 18 ± 0.5 h in the incubator 24 ± 2 h in the incubator
Quality controls
  • positive control: 1% SDS in NaCl and sesame oil
  • negative control: DPBS
  • vehicle controls:
  • → 0.9% NaCl as solvent for polar extracts
  • → sesame oil as solvent for non-polar extracts
Pre-tests

to determine if additional controls are needed:

NSMTT*: mixing of test item extract with MTT medium to determine if test item alone can reduce MTT

→ blue colouring: in main experiment two killed tissues treated with test item and two untreated killed tissues were added as controls

NSCliving*: mixing of test item extract with H2O or isopropanol to determine if strong own colour of test item can discolour at contact with this liquids

→ optical discolouring (measuring of spectrum): in main experiment two living tissues without incubation with MTT medium were added as controls

NSCkilled*: if the two other controls were determined

→ in main experiment two killed tissues without incubation with MTT medium were added as controls
Application
  • direct topical application of extracts on skin tissues
  • three tissue replicates per dose group
Data delivery
  • optical density (OD) value with microplate spectrophotometer at 570 nm
  • tissue viability of each dose group
Prediction model  mean tissue viability (% negative control):
  • ≤ 50%: Irritant; (IL-1α > 60 pg/mL)
  • > 50%: Non-Irritant; (IL-1α ≤ 60 pg/mL)

*NSMTT: non-specific MTT reduction
#NSCliving: non-specific colouring of living tissues
§NSCkilled: non-specific colouring of killed tissues

References

  1. ISO 10993-10, 2010(E), “Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization“. Annex D: In vitro tests for skin irritation
  2. UN (2015). United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), sixth revised edition, UN New York and Geneva
  3. DeJong W., Hoffmann S., Lee M., Kandárova H., Pellevoisin, C., Haishima Y., Rollins B., Zdawczyk A., Willoughby J., Bachelor M., Schatz T., Skoog S., Parker S., Sawyer A., Pescio P., Fant K., Kim, KM., Kwon JS., Gehrke H., Hofman-Hüther H., Meloni, M., Julius C., Briotet D., Letasiova S., Kato R., Miyajima A., De La Fonteyne L., Videau C. Tornier C., Turley A.P., Christiano N., Rollins T.S. and Coleman K.P., (2018) Round robin study to evaluate the reconstructed human epidermis (RhE) model as an in vitro skin irritation test for detection of irritant activity in medical device extracts. Toxicology in Vitro.