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Understanding OECD TG 439: The In Vitro Skin Irritation Test (SIT)

As regulatory toxicology continues to evolve, the OECD Test Guideline 439 stands as a cornerstone in the global shift toward non-animal toxicology testing— providing a humane, efficient, and scientifically validated alternative to traditional animal-based tests like the Draize rabbit test.

What is OECD TG 439?

OECD TG 439, adopted in 2010 and updated in subsequent years, describes a Reconstructed Human Epidermis (RhE) test method to assess the skin irritation potential of chemicals, formulations, and finished products. Skin irritation refers to the production of reversible damage to the skin occurring after exposure to a substance or mixture as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The basis of this method measures tissue viability, using MTT, after exposure to a test material.

OECD Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Skin Corrosion and Irritation includes the Skin Irritation Test (SIT; OECD TG 439) as a suitable method that can contribute to a weight-of-evidence (WoE) approach, in combination with other in vitro or in vivo methods, to support skin irritation or corrosion hazard classification.

How the SIT Test Works

  1. Tissue Preparation
    Commercially available RhE models are equilibrated in 6-well plates.
  2. Application of Test Substance
    A fixed dose is applied directly to the surface of the epidermal model.
  3. Exposure and Post-Incubation
    After a defined exposure period, the tissues are rinsed and placed back into incubation.
  4. Viability Assessment
    Tissue viability is measured using MTT, which quantifies the metabolic activity of viable cells. A reduction in relative viability less than or equal to 50% indicates that the substance is an irritant.
  5. Classification
    • Viability > 50%: Not irritating (GHS No Category)
    • Viability ≤ 50%: Irritating (GHS Category 1 or 2)

Advantages of The In Vitro Skin Irritation Test (SIT; OECD TG 439)

  • Human-Relevant: Uses human-derived reconstructed human epidermis, improving predictivity and relevance
  • Accepted Globally: Recognized by EU REACH, US EPA, and other regulatory authorities
  • Reproducible and Scalable: Study is designed in a way which allows for the testing of multiple materials simultaneously

Limitations

  • Cannot Distinguish between GHS Category 1 and 2: TG 439 is not designed to identify GHS Category 1 (corrosive) or GHS Category 2 (irritant) substances. 
  • Not Suitable for All Substance Types: Test materials which absorb light in the same range as MTT formazan and test materials which can directly reduce MTT may interfere with tissue viability measurements
  • Supplementary Data May Be Required: Subsequent runs may be necessary in the case of borderline results.

Regulatory Applications

The In Vitro Skin Irritation Test (SIT; OECD TG 439) is commonly used in safety evaluations for:

  • Cosmetics and personal care products
  • Commercial Products
  • Chemicals under REACH
  • Agrochemicals

Conclusion

The OECD TG 439 Skin Irritation Test represents a time-tested and major advancement in non-animal safety assessment. By offering a standardized, scientifically validated in vitro method, companies and regulators can ensure product safety while promoting animal welfare and scientific innovation.

Understanding OECD TG 439: The In Vitro Skin Irritation Test (SIT) 1