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TEST METHOD
Using MTT ET-50 Protocol to categorize eye irritation of biocides, chemicals and cosmetics via in-vitro models
The Neat Protocol MTT ET50 Assay is an advanced in vitro method for assessing eye irritation potential without the need for animal testing. This assay utilizes the EpiOcular model, a Reconstructed Human Cornea-Like Epithelium (RhCE), to evaluate ocular irritation based on exposure time and cell viability reduction to 50% (ET50 values). The ET50 value (in minutes or hours) determines the eye irritation potential and can also be used for EPA hazard classification.
The diagram below provides a summary of the test method.
Understanding MTT ET-50 Testing
Application areas
The Neat Protocol MTT ET50 Assay is designed for the hazard identification of chemicals, biocides, and cosmetics. For medical devices, eye irritation testing may still be required, although there is no dedicated ISO 10993 standard for in vitro eye irritation testing. Therefore, this method may be used to screen the eye irritation potential of medical devices, depending on the applicable regulatory requirements.
Test Protocol
Step 1: 0.1 mL of the test sample is applied to the EpiOcular RhCE tissue for exposure time of 3, 30 and 60 minutes.
Step 2: The EpiOcular RhCE tissues are rinsed with DPBS.
Step 3: 0.3 mL of MTT solution is added to each well and incubated for 3 hours. The formazan formed is then extracted using isopropanol.
Step 4: 0.2 mL of the extracted formazan solution is transferred to a 96-well plate, and the absorbance is measured at 570 nm.
Testing Criteria
For products not expected to cause eye irritation, OECD 492 may be used to confirm that the product is non-irritating.
For products that may cause eye irritation, the Neat Protocol MTT ET50 Assay evaluates ocular irritation potential based on ET50 values. These values help determine the severity of eye irritation and support product classification according to the EPA hazard classification.
Exposure Time and Conditions
The standard method uses exposure times of 3, 30, and 60 minutes. For EPA hazard classification, exposure times of 2, 15, 45, and 90 minutes may be used.
Passing Criteria
Based on the standard method:
Based on EPA classification:
**Disclaimer: NO ANIMAL TESTING. TECOLAB is committed to ethical and cruelty-free testing practices. We do not conduct any form of testing on animals and all our biocompatibility tests are performed on in-vitro models.
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