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Ex Vivo Eye Irritation Test (EVEIT)

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Biospotter

The BioSpotter presented here is capable of toxicologically automated use of the EVEIT system.

The three-axis robot is equipped with a dispensing mechanism that allows – similar to an inkjet printer – to place up to five different substances in the nanolitre range on the corneal surface in a location-precise, volume-calibrated and rapid manner.

A stroboscopic calibration system is used to precisely set the volume to be applied. An integrated high-resolution camera allows documentation of macroscopic and fluorescein-stainable changes of the corneal surface.

The BioSpotter can repeatedly target positions on the corneal surface with high accuracy.

The total volume to be applied can be applied sequentially to the corneal surface in the form of repeated partial volumes within fractions of a second.

3R-Principle: Replace, Reduce, Refine

ACTO Offers Alternatives to Animal Testing

If you are interested in testing your substances, formulations, or chemicals in the EVEIT system, please feel free to contact us at: info@acto.de

Test Principle

The EVEIT system, in particular, embraces the generally recognized ethical necessity of using alternatives to animal testing.

In 1959, the zoologist William Russel and the microbiologist Rex Burch first formulated the aim of a “humane science” in their book “The Principles of Humane Experimental Technique”. Today it is known as the 3R-Principle, which stands for Replace (animal tests by alternatives), Reduce (the number of test animals), and Refine (alleviate the suffering of test animals by less harmful tests which yield more conclusive results).

Based on the combination of cornea cultivation and imaging techniques, ACTO aims to establish a method which forms an innovative and universal platform for alternative research without animal testing and which enables prescribed studies within the scope of the new EU-Chemical Regulation (REACH) and the EU-Directive on the Evaluation of Cosmetics and Pharmaceuticals. The EVEIT system replaces animal experiments, delivers better results, and as a whole, can reduce animal experiments.

Since the EVEIT system provides more detailed and more reproducible results than animal experiments, altogether it can reduce and replace animal experiments in ophthalmology and toxicology (DRAIZE test). Eyes of slaughtered rabbits are treated so that they can be cultivated for several weeks. As a result, a testing method is available that makes live animal testing obsolete and yields results that are equally or even more robust than those of animal tests. The essential criterion of this method is the in vitro healing of wounds and chemical injuries.

These corneal tests help researchers to recognize the effect of and tolerance to phosphate-containing eye drops and any possible side effects like calcifications before these eye drops are applied on humans. During these studies, the cornea can be evaluated microscopically and biochemically at any time and longitudinal analyses over time can be taken. This system allows tracing the smallest changes that, in people, may lead to changes of the eye only after years.

By combining the biological EVEIT system with optical coherence tomography (OCT)— introduced by the RWTH Aachen University Institute for Semiconductor Technology (IHT) in a joint research project (cf. 2. BMBF Funding) — another non-invasive analysis method now exists in which even the slightest corneal changes in vitro can be imaged. ACTO e. V. and the IHT have been honored with various prizes for this innovative alternative method to animal testing.

Ex Vivo Eye Irritation Test (EVEIT) means:

  • More conclusive results than the unspecific DRAIZE Test
  • Standardized exposures
  • Reproducible results
  • Highest analytical precision
  • Predictive results
  • Healing and release of inflammatory messengers

The effects on the eye can be subdivided in:

  • Pharmaceutical effects on the eye
  • Acute changes through toxins, caustic substances, mechanical and physical influences
  • Long-term changes after a single- and repeated contact
  • Microbiological effects.

Currently animal tests are conducted to examine tolerance to drugs, medical products and chemicals. Even though there are alternative testing methods, they can only detect acute toxicity.

There is no procedure that

  • Chronic toxicity
  • Repeated application
  • Long-term adverse effects, and
  • may represent a possible cure.

In the “EVEIT-acute” system, corneal clouding, changes of the surface and of the deeper corneal tissue are detectable through special investigative methods (microscopy, OCT, biochemistry and histology). Long-term changes after such a single contact with a caustic substance and especially the question whether the tissue can be regenerated can only be evaluated when the cornea is kept vital.

In the “EVEIT long-term” system, the cornea is kept alive for 20 days. Here the cornea is nourished with a nutrient medium and is kept moist in a special chamber that mimics the eye. Under these conditions, it can be seen after a single contact but also after multiple contacts with substances, whether the cornea completely heals or whether only the defect heals or no corneal healing takes place.

The question arises of how a cornea reacts after repeated exposure to a substance, e.g., eye drops or cosmetics, which are typical cases in point. Yet after a single exposure to a substance, not all conclusions can be drawn which would be relevant to the exposed person or patient.

In the EVEIT system, however, up to 100 applications of a substance per day can be placed on the cornea for several days in succession. This would be impossible in tests on live animals or even on volunteers. Thus, the EVEIT system enables presenting the temporally compressed reaction to chronic irritations.

The EVEIT multi-local system allows the miniaturized study of chronic and acute risk of chemical substances on one single cornea at various sites of the eye surface. In this way, several tests can be grouped together on one eye. This offers the unique opportunity to test a reference substance as well as a variety of substances or different concentrations of one substance in just one batch on the eye. With this approach, large test series can be condensed and internally referenced. The existing individuality of such a corneal explant simultaneously becomes a reference, and comparisons of the severity of an ocular injury can be systematically delineated in a single corneal section.

This both simplifies and accelerates the testing procedure.

Possible tests in the EVEIT system are: multi-local exposures, eye-drop applications, applications of low-dosed or high-dosed recurring chemical substances, liquids, gases but also particles.

READ MORE

Examples of the effects cited here: EVEIT-fundamental publication

Applied Methods

Freshly explanted eyes of dead rabbits from a rabbit slaughterhouse are used for all the planned and conducted experiments in the EVEIT system.

For the EVEIT-acute test, the freshly explanted rabbit eyes are fixed in a holder and are then applied with the corresponding chemical substance to be tested. Then the eye and its reaction to the chemical substance are carefully observed by means of microscopy and optical coherence tomography.

In the EVEIT-long-term test, the corneas are prepared out of the freshly explanted eyes and are transferred to an artificial eye chamber where the eyes are nourished and microscopically monitored. After 24 hours, the state of the cornea is examined based on biochemical measurement values, the testing of clarity, stability of the epithelium (outer layer) as well as on the functionality of the endothelium (inner layer). Only upon fulfilling these criteria — which allow drawing clear conclusions about the cornea’s vitality — can the corneas be subjected to special tests in an incubator for 3 to 20 days.

During this study, the cornea can be repeatedly evaluated microscopically, and longitudinal analyses over this period can be performed via OCT. The EVEIT system allows tracing the slightest changes that may lead to changes in the human eye only after years. The possibility to conduct many applications within a short time leads to a kind of fast-motion production of effects on the cornea.

We can prove these effects on hand of many experiments and publications. Currently, the project is being prepared for the validation of the replacement of animal tests at the European Centre for the Validation of Alternative Methods (ECVAM).

Corneal Inflammation

At present, ACTO e.V. is further developing the EVEIT system to show that an induced corneal inflammation can be healed.

Simulation of the Dry Eye

ACTO is also further developing the EVEIT system to enable a simulation of the “Dry Eye Syndrome” so that the same aforementioned experiments can be conducted under these conditions.

Review:
To date, the important projects in the development of the EVEIT System

The following two projects have relevantly contributed to the development of the EVEIT system as an alternative method to animal testing:

The Institute for Semiconductor Technology at RWTH Aachen University has contributed a special optical technique, optical coherence tomography (OCT), to this project. For the first time, this measurement technique enables an objective documentation and quantification of the effect of chemicals to the cornea. Thus, subjective evaluation of the animal eyes is replaced by objective laboratory parameters.

The funding of about Euro 500,000 yielded new results on the reaction within the test system. This funding was used for a series of repeated experiments on 32 reference substances, earmarked by an advisory board from industry, ECVAM, the German Federal Institute for Risk Assessment (BfR) and various research institutes. Altogether the test system shows high accuracy in assessing the effect of chemicals on the eye. Twenty-seven (27) of 32 substances were correctly assigned into categories I, II and III according to the Globally Harmonized System of Classification (GHS). In 5 cases the test’s predictability fell short.

Within the project, the development of simplified preparation and analytic procedures in the EVEIT system could be boosted.

Ex vivo Tests for Cosmetics

Animal testing-free experiments ought to replace cosmetic tests on live animals. With respect to the 2009-enacted prohibition of animal testing in the EU, COLIPA (Cosmetic, Toiletry and Perfumery Association), the umbrella association of the European cosmetic manufacturers, has commissioned top scientists to investigate alternative methods.

The COLIPA project, followed by ACTO in cooperation with the RWTH Aachen University Hospital, is a key project for the entire research in chemical burns. Within the scope of this project, ACTO could develop an innovative system with isolated rabbit eyes that can detect healing, pathological healing and the response of the eye to repeated use of substances. In the near future, this will do away with the DRAIZE-test, which is painful for the living test animal. The employed corneas originate from slaughtered animals not killed for this purpose.

This system results in a strongly further developed version of the Ex Vivo Eye Irritation Test (EVEIT):

  • In the tests, it has been proven that the healing process of the corneal epithelium, a critical factor in eye irritations, can be measured.
  • The monitoring of glucose, lactate and the pH-value all lend reliable certainty about the survival of the corneas in a culture system.
  • The amount of inflammatory messengers was found to be proportional to the concentrations of irritants.
  • The cornea also survived repeated ex vivo exposures to chemical substances in the culture chamber.
  • The healing of the corneal epithelium is a decisive parameter in evaluating the toxicity of new substances.

ACTO is convinced that the experiments on rabbit corneas in a culture system, possibly combined with the array technique on human corneas that are not suited for transplantation but that are available for research, might lead to one system that can replace the DRAIZE-test. This new system will allow predictions about the course of eye.

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