In Vitro Skin Irritation Protocol: RhE Tissue Model Guide

In Vitro Skin Irritation Protocol

Standard Operating Procedure for RhE Tissue Models

Protocol Overview

The In Vitro Irritation test generally utilizes Reconstructed Human Epidermis (RhE) designed to mimic the biochemical and physiological properties of human skin. This protocol quantifies skin irritation potential without animal testing.

Step 1: Tissue Receipt & Conditioning

Initial incubation for 1 hour immediately upon receipt to equilibrate tissues.

Overnight incubation follows (with media change).

Environment: Standard culture conditions (37 ± 1°C, 5 ± 1% CO2 in humidified atmosphere).

Step 2: Tissue Treatment

Tissues are treated topically with the test substance to mimic real-world exposure.

Performed in triplicate tissues for statistical reliability.

Includes concurrent Negative Controls (NC) and Positive Controls (PC).

Step 3: Rinsing & Post-Expression

Tissue Rinsing

Remove test substance completely.

Initial Incubation

24 ± 1 hour post-treatment incubation.

Second Incubation

Fresh media change, then 42 ± 2 hours incubation to capture delayed effects.

Step 4: MTT Reduction Assay

Tissues are incubated with MTT solution after the post-treatment period.

Mechanism: Viable cells with active metabolism reduce MTT into a blue/purple formazan precipitate.

Non-viable cells do not convert the dye, remaining distinct.

Step 5: Extraction & Quantification

Isopropanol Extraction

Extract the purple formazan from tissues using isopropanol.

Spectrophotometry

Measure the Optical Density (OD) at 570 ± 30 nm.

Protocol Diagram Summary

From receipt to quantification, the entire process takes approximately 3-4 days including all incubation periods.

Prediction Model & Results

Non-Irritant (NI)

Mean tissue viability > 50%

Irritant (I) / GHS Cat 2

Mean tissue viability ≤ 50%

Regulatory Conclusion

This method aligns with UN GHS (Globally Harmonized System) standards.

Classifies substances into 'Category 2' (Irritant) or 'No Category' (Non-irritant).

Critical for safety datasheets (SDS) and occupational safety labeling.