Dermal Irritation/Dermal Toxicity Study
Materials Required
Principle
This protocol assesses dermal irritation using reconstructed human epidermis (RhE) models such as EpiDerm, EPISKIN, and SkinEthic RHE, in which a test substance is applied topically and tissue viability is measured after exposure; reduced viability reflects cytotoxic injury associated with skin irritation potential[1][2]. The primary readout is MTT reduction, where viable cells convert tetrazolium salt into colored formazan measured by spectrophotometry; this signal is used as a quantitative viability endpoint for classifying irritant versus non-irritant responses[2][3]. The historical in vivo comparator is the Draize rabbit skin irritation method, which scores erythema and edema after topical exposure, but validated RhE assays were developed to replace or reduce reliance on this animal-based endpoint[1][4].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use the test substance neat when compatible with the RhE protocol, Dulbecco’s phosphate-buffered saline or equivalent buffer as negative control, and 5% sodium dodecyl sulfate as positive control when using the EpiDerm SIT protocol[2].
• Use MTT reagent for viability measurement and an appropriate extraction solvent for solubilizing formazan before spectrophotometric reading[2][3].
• MTT is the required viability dye because the validated irritation readout is based on conversion of MTT by viable tissue[2][3].
• Use validated RhE tissues maintained in culture inserts, sterile culture plates, a humidified cell-culture incubator, sterile pipettes, a timer, and a microplate spectrophotometer to culture tissues, apply test substances, control exposure timing, and quantify extracted formazan absorbance[2][3].
• Use MTT reagent for viability measurement and an appropriate extraction solvent for solubilizing formazan before spectrophotometric reading[2][3].
• MTT is the required viability dye because the validated irritation readout is based on conversion of MTT by viable tissue[2][3].
• Use validated RhE tissues maintained in culture inserts, sterile culture plates, a humidified cell-culture incubator, sterile pipettes, a timer, and a microplate spectrophotometer to culture tissues, apply test substances, control exposure timing, and quantify extracted formazan absorbance[2][3].
Experimental Procedure
• Equilibrate RhE tissues in assay medium before dosing, maintain tissues under aseptic culture conditions, and assign replicate tissues to test substance, negative control, and positive control groups; the EpiDerm SIT protocol reports three tissues per test chemical and per control[2].
• Prepare liquid, semisolid, solid, or waxy test substances for direct topical application according to the tissue-model protocol; the EpiDerm SIT protocol was described for chemicals including cosmetic and pharmaceutical ingredients[2].
• For the EpiDerm SIT example, pre-incubate tissues, expose the tissue surface to the test substance for 60 minutes, remove the test substance, post-incubate tissues for 42 hours, then perform the MTT viability assay[2].
• For SkinEthic RHE studies, published protocols used direct topical application or in vitro patch formats and assessed MTT viability, histology, and IL-1α release as endpoints; reported protocol formats varied across studies, so model-specific timing should be taken only from the selected validated model protocol[5].
• After post-incubation, incubate tissues with MTT, extract the reduced formazan, and read absorbance using a microplate spectrophotometer; the absorbance reflects viable-cell-dependent MTT reduction[2][3].
• Calculate relative tissue viability by normalizing each treated tissue to the negative-control tissue response; the EpiDerm SIT protocol uses MTT viability to discriminate GHS Category 2 irritants from non-irritants[2].
• Interpret the positive control as an assay-performance control and the negative control as the baseline viability control; published EpiDerm SIT protocol reporting used three replicate tissues for each test chemical and each control[2].
• If a material is colored, a nanoparticle, or otherwise likely to interfere with optical or MTT-based readouts, include only literature-supported interference controls or omit interpretation when interference cannot be resolved; nanoparticle studies using reconstructed skin models specifically assessed corrosion/irritation with MTT-based viability and related controls[6].
• Prepare liquid, semisolid, solid, or waxy test substances for direct topical application according to the tissue-model protocol; the EpiDerm SIT protocol was described for chemicals including cosmetic and pharmaceutical ingredients[2].
• For the EpiDerm SIT example, pre-incubate tissues, expose the tissue surface to the test substance for 60 minutes, remove the test substance, post-incubate tissues for 42 hours, then perform the MTT viability assay[2].
• For SkinEthic RHE studies, published protocols used direct topical application or in vitro patch formats and assessed MTT viability, histology, and IL-1α release as endpoints; reported protocol formats varied across studies, so model-specific timing should be taken only from the selected validated model protocol[5].
• After post-incubation, incubate tissues with MTT, extract the reduced formazan, and read absorbance using a microplate spectrophotometer; the absorbance reflects viable-cell-dependent MTT reduction[2][3].
• Calculate relative tissue viability by normalizing each treated tissue to the negative-control tissue response; the EpiDerm SIT protocol uses MTT viability to discriminate GHS Category 2 irritants from non-irritants[2].
• Interpret the positive control as an assay-performance control and the negative control as the baseline viability control; published EpiDerm SIT protocol reporting used three replicate tissues for each test chemical and each control[2].
• If a material is colored, a nanoparticle, or otherwise likely to interfere with optical or MTT-based readouts, include only literature-supported interference controls or omit interpretation when interference cannot be resolved; nanoparticle studies using reconstructed skin models specifically assessed corrosion/irritation with MTT-based viability and related controls[6].
Troubleshooting
Problem: Positive-control response is weak or inconsistent.
• Possible Cause: Tissue handling, culture condition, or exposure timing deviated from the validated protocol.• Literature-supported Solution: Repeat the assay using aseptic handling, the validated exposure/post-incubation schedule, and replicate control tissues as described for EpiDerm SIT[2].
Problem: Test substance produces abnormal absorbance unrelated to tissue viability.
• Possible Cause: The material may be colored, may reduce MTT directly, or may interfere optically.• Literature-supported Solution: Use literature-supported interference controls for such materials, and do not classify irritation if corrected viability cannot be reliably obtained[6].
Problem: Results differ between RhE models.
• Possible Cause: RhE models can require model-specific adaptations despite using related irritation principles.• Literature-supported Solution: Use the protocol and prediction model validated for the specific RhE model rather than transferring parameters without validation[1][5].
References:
- [1]. Spielmann H, Hoffmann S, Liebsch M, Botham P, Fentem JH, Eskes C, et al. The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the skin integrity function test. Altern Lab Anim. 2007;35(6):559-601. [Content Brief]
- [2]. Kandarova H, et al. An in vitro skin irritation test (SIT) using the EpiDerm reconstructed human epidermal (RHE) model. J Vis Exp. 2009;(29):e1366. [Content Brief]
- [3]. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. [Content Brief]
- [4]. Draize JH, et al. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharmacol Exp Ther. 1944;82(3):377-390.
- [5]. Kandarova H, et al. Assessment of the skin irritation potential of chemicals by using the SkinEthic reconstructed human epidermal model and the common skin irritation protocol evaluated in the ECVAM skin irritation validation study. Altern Lab Anim. 2006;34(4):393-406. [Content Brief]
- [6]. Kim H, et al. Skin corrosion and irritation test of nanoparticles using reconstructed three-dimensional human skin model, EpiDerm. Toxicol Res. 2016;32(4):311-316. [Content Brief]
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