This cell-based method addresses the effects of a test compound on intracellular metabolism and extracellular acidification rate, and can be used as a measure of eye irritancy potential.
The method was validated in an EURL ECVAM retrospective validation study and is ready for consideration for regulatory use within a top-down and/or bottom-up approach in tiered eye irritation testing strategies.
As part of a wider testing strategy this means it can be used to identify potential ocular corrosives and severe irritants and/or non-irritants for certain chemical classes.
Read more about the Cytosensor Microphysiometer (CM) toxicity test method on ICCVAM-NICEATM website here.
[collapsed]Eye irritation refers to (negative) changes in the eye following the application of a test substrate to the front (anterior) of the eye.
According to animal-based methods for assessing eye irritation, four representative in vivo end-points (corneal opacity, iritis (inflammation of the iris of the eye), conjunctiva chemosis, and conjunctiva redness) are scored and graded 24, 48 and 72 hours after installation of the test material in the eye sack of three different animals.
The test material is then classified as 'Category 1' if effects have not reversed or are not expected to reverse within 21 days of observation in at least one animal. If all effects have reversed within 21 days, the substance is classified as 'Category 2'.
Further sub-classification options are available within this second category. Materials that fall in neither categories are not classed as eye irritants.
This approach has been defined in various guidelines, directives and legislation (OECD test guideline 405, the United Nations Globally Harmonised System (UN GHS), and EU Directive 1272/2008 (European Union regulation on classification, labelling and packaging, EU CLP).
In terms of in vitro methods, a variety of end-points are used as proxy measures of eye irritation.[/collapse]
The Cytosensor Microphysiometer (CM) toxicity test method
[collapsed]The Cytosensor Microphysiometer (CM) toxicity test method is a cell-based in vitro assay that was developed as a partial replacement of the Draize rabbit eye irritation test.
The method consists of a monolayer of adherent mouse L929 fibroblast cells that are cultured on a polycarbonate cell culture insert. Within the system there is also a light-sensitive pH sensor.
The ocular toxicity of test substances is evaluated according to reductions in metabolic rate of the cells following exposure (as assessed by changes in the rate of reduction of pH in the cell culture medium). The cells have a natural rate of metabolism that results in the production of extra-cellular acids. This rate is expected to vary following exposure to potentially toxic substances.[/collapse]
Animal testing replacement
[collapsed]The test was developed as a partial replacement of the Draize rabbit eye irritation test. According to the outcome of the retrospective validation test conducted by EURL ECVAM, the approach can be used to assess irritation potential of certain classes of chemicals. In particular it can be used as part of wider testing strategies to identify ocular corrosives and severe irritants or substances that are non-irritants.
However, the method does not correctly identify moderate or mild ocular irritants. Also, the method can only be used to evaluate water-soluble substances since characteristics of the system, in particular small tubing diameters, means that only solutions can be evaluated.[/collapsed]
[collapsed]The retrospective validation study of four cytotoxicity and cell function-based assays, i.e. the neutral red release test, the red blood cell test, the fluorescein leakage assay and the cytosensor microphysiometer test, took place between May 2006 and October 2008.
The study was based on the retrospective collection of existing data compiled according to the EURL ECVAM modular approach to validation and weight-of-evidence principles.[/collapse]
Validation study outcomes
[collapsed]After peer review, two assays, the cytosensor microphysiometer test and the fluorescein leakage assay, were scientifically validated for being used as an initial step within a top-down approach to identify ocular corrosives and severe irritants from all other classes for the chemical applicability domain of water soluble chemicals.
In addition, the cytosensor microphysiometer test was scientifically validated for being used as an initial step within a bottom-up approach useful to identify non-irritants from all other classes for water-soluble surfactants, and water-soluble surfactant-containing mixtures.[/collapsed]
EURL ECVAM recommendations
[collapsed]You can read the full statement of ESAC relating to this method in the section below. In short EURL ECVAM recommended that the cytosensor microphysiometer test can be used to identify either substances that are severe ocular irritants/corrosives or non-irritants depending on the testing strategy applied i.e. as part of a top-down or bottom-up testing approach.
Based on the assessment we made, the method is not likely to fully replace the animal-based Draize test and is likely only of use if combined with further in vitro tests.[/collapse]