The in vivo rabbit eye test (TG 405) has been the primary method utilized to assess the hazardous potential of chemicals to the eye. However, this has major ethical implications and therefore alternative methods of testing are desirable. The Short Time Exposure test (STE test) (OECD 491) is an in vitro method which can be utilized as a standalone test, or ideally as part of tiered testing strategy alongside other in vitro tests to categorize chemicals based on their potential to induce eye damage.

Traditionally, chemicals are categorized in one of three categories based on their potential to cause eye damage as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS):

• Chemicals which do not require classification for eye irritation or serious eye damage (UN GHS No Category)

• Chemicals which induce serious eye damage (UN GHS Category 1)

• Chemicals which induce eye irritation (UN GHS Category 2): Category 2A (eye irritation) or UN GHS Category 2B (mild eye irritation)

As a standalone test, the Short Time Exposure (STE) test can be utilized to identify chemicals which do not require classification for eye irritation or serious eye damage (UN GHS No Category) and Chemicals which induce serious eye damage (UN GHS Category 1). This test cannot identify UN GHS Category 2 chemicals on its own.

The STE test utilizes a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells seeded onto a 96 well plate and exposes the cells to a 5% and 0.05% W/W solution of chemical for 5 minutes. Following this, MTT (3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) (Thiazolyl Blue Tetrazolium Bromide) is applied to the cells and the cell viability assessed as a % compared to the appropriate solvent control. The results are then categorized as such:

_{1) Scott L, et al. A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches. Toxicol In Vitro. 2010 Feb;24(1):1-9. doi: 10.1016/j.tiv.2009.05.019. Epub 2009 May 31. PMID: 19490935
2) OECD (2020), Test No. 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/978926....}