# In Vitro Skin Irritation Protocol: RhE Tissue Model Guide
> Learn the 5-step SOP for In Vitro Skin Irritation testing using Reconstructed Human Epidermis (RhE). Covers MTT assay, GHS categories, and regulatory standards.

Tags: in-vitro-testing, skin-irritation, rhe-tissue-model, mtt-assay, laboratory-protocol, un-ghs, toxicology
## In Vitro Skin Irritation Protocol Overview
* **Goal:** Quantify skin irritation potential without animal testing using Reconstructed Human Epidermis (RhE).
* **Mechanism:** Mimics biochemical and physiological properties of human skin.

## Step 1: Tissue Receipt & Conditioning
* Initial incubation: 1 hour upon receipt.
* Overnight incubation with media change.
* Conditions: 37 ± 1°C, 5 ± 1% CO2 in a humidified atmosphere.

## Step 2: Tissue Treatment
* Topical treatment with test substance in triplicate.
* Controls: Included concurrent Negative Controls (NC) and Positive Controls (PC).

## Step 3: Rinsing & Post-Expression
* Complete removal of test substance.
* Initial Incubation: 24 ± 1 hour.
* Second Incubation: Media change, then 42 ± 2 hours to capture delayed effects.

## Step 4: MTT Reduction Assay
* Tissues incubated with MTT solution.
* Viable cells reduce MTT into blue/purple formazan precipitate via active metabolism.

## Step 5: Extraction & Quantification
* Isopropanol extraction of purple formazan.
* Quantification via spectrophotometry at 570 ± 30 nm Optical Density (OD).

## Prediction Model & Regulatory Standards
* **Non-Irritant (NI):** Mean tissue viability > 50%.
* **Irritant (I) / GHS Cat 2:** Mean tissue viability ≤ 50%.
* **Compliance:** Aligns with UN GHS standards for Safety Datasheets (SDS) and occupational safety labeling.
---
This presentation was created with [Bobr AI](https://bobr.ai) — an AI presentation generator.