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An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model
Published on: July 13, 2009
Normal human keratinocyte assay to predict the human irritancy response
Richard Clothier1, Nancy Khammo
1Fund for the Replacement of Animals in Medical Experiments and School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, UK. Richard.Clothier@nottingham.ac.uk
Nature Protocols
|April 5, 2007
Summary
This study presents a new in vitro assay for assessing skin irritation potential, crucial for chemical registration and EU REACH regulations. The assay uses human keratinocytes and macrophages, offering a faster, animal-free alternative for safety testing.
Area of Science:
- Toxicology
- Dermatology
- In vitro assays
Background:
- Chemical safety assessments require evaluating skin irritation potential.
- EU REACH regulations mandate specific data for chemical registration.
- Existing animal testing methods face ethical and regulatory scrutiny.
Purpose of the Study:
- To develop and validate an in vitro assay for predicting human skin irritation.
- To provide an alternative to animal testing for regulatory compliance.
- To assess the utility of human keratinocytes and macrophages in irritation testing.
Main Methods:
- Utilized human passage 1-3 primary keratinocytes and macrophages.
- Employed a two-tiered approach: direct keratinocyte assay and mediator analysis.
- Measured keratinocyte activity using the resazurin assay.
- Assessed mediator release (nitric oxide, IL-1alpha) from stimulated macrophages.
Main Results:
- The assay successfully measures keratinocyte activity and mediator release.
- The assay can be completed within 3-7 days post-exposure.
- Identified nitric oxide and IL-1alpha as key mediators, with others potentially involved.
Conclusions:
- The developed in vitro assay offers a viable, animal-free method for assessing skin irritation.
- This assay supports regulatory requirements for new chemical registration under EU REACH.
- Further research may elucidate the full spectrum of mediators involved in the skin irritation response.
