The Porcine Corneal Opacity and Reversibility Assay (PorCORA) and Assessment of the Drivers of Classification with Regards to Ocular Damage

PorCORA - Corrosive or Severe Eye Irritant?
PorCORA – Corrosive or Severe Eye Irritant?

Eye Irritation Testing

The Draize Rabbit Eye Test (DRET) is the regulatory ‘Gold Standard’ for  assessing and categorizing ocular irritation/corrosivity.  The DRET focuses attention on damage to a number of different ocular structures, which are scored and weighted based on toxicological importance. The structures are as follows: Cornea (CO) – 80 points, Conjunctiva (Conj) – 20 points, and Iris (IR) – 10 points.

The heaviest weighting is on corneal damage, which is 80 points out of the total 110-point scale devised by Draize.  Corneal irritation scoring is based on assessment of opacities on the cornea, Conj irritation is assessed by increased vascularization, and iridial damage is assessed by function of the iris (ability to constrict or dilate pupil)and deepening of the rugae.

PorCORA – Corneal Irritation Focus

Since the CO scores have the heaviest weight, and most often are the drivers of eye irritation, we developed the Porcine Cornea Opacity Reversibility Assay (PorCORA), an ex vivo corneal model. The PorCORA can distinguish between a material’s potential to cause severe (reversible) versus corrosive (irreversible) damage. Excised cultured porcine corneas are topically dosed with a test material and kept in culture for 21 Days.  Corneal damage is visually assessed and scored.  If CO reverse and are not visible by Day 21, the test material is categorized as a non-corrosive material and deemed to not cause permanent ocular damage.

In internal validation studies, we tested 56 chemicals and dilutions of chemicals ranging from corrosive (GHS category 1) to non-irritating (GHS not categorized). Using Cooper Statistics, we arrived at an accuracy of 89% with a positive and negative predictivity of 85% (cat. 1) and 93% (not cat.1), respectively.

Drivers of Classification Concept

To determine if these Cooper Statistics could be improved, we used the drivers of classification concept based on Barroso et al., 2016*. Upon re-examination of our data based on this published database and methodology, we found that four chemicals (1,2,4-Triazole, N-Butanol, 2,5-Dimethyl-2,5-Hexanediol, and Potassium Cyanate) had invalid tests (animals euthanized prior to day 21) or produced irritation not driven by corneal opacities. These chemicals were removed from our dataset. Without these four chemicals the accuracy improved to 92%. Moreover, the major change was in our positive predictivity, which increased to 91%. The negative predictivity for this subset of chemicals remained the same.
Lastly, PorCORA’s predictivity was assessed based on CO persistence; i.e., materials that produced low CO scores (from Draize Rabbit Eye Tests). Of the 56 chemicals tested, 40 had low severity (i.e., CO mean scores < 3). The accuracy for persistence of low CO scores was 88% with positive and negative predictivity of 79%, and 92%, respectively.

* Barroso, J., Pfannenbecker, U., Adriaens, E., Alépée, N., Cluzel, M., De Smedt, A., Hibatallah, J., Klaric, M., Mewes, K., Millet, M., Templier, M., McNamee, P.  2016.  Cosmetics Europe compilation of historical serious eye damage/irritation in vivo data analysed by drivers of classification to support selection of chemicals for development and evaluation of alternative methods/strategies: the Draize eye test Reference Database (DRD).  Arch. Toxicol.  91:521-547.

Conclusions

Based on these data, the PorCORA was proven to be a valid test to assess a material’s potential to distinguish reversible versus irreversible eye damage.

Appropriate choices of validation chemicals, based on the drivers of classification, have proven valuable and should be applied to replacement ocular assays that previously failed to meet acceptance criteria.

PorCORA testing results are encouraging and future studies are planned that will pursue mixtures and chemical substances of unknown or variable composition, complex reaction products and biological materials.  PorCORA

If you would like to find out more about PorCORA and other in vitro/alternative toxicology testing methods, please click here.

 

In Vitro Eye Irritation Testing

In Vitro Eye Irritation Testing-MB Research Labs
In Vitro Eye Irritation Testing- MB Research Labs

In Vitro Eye Irritation Testing

Currently, it is generally accepted that no single in vitro eye irritation test will be able to replace the in vivo Draize eye test
to predict across the entire range of irritation for different chemical classes. Several labs (including MB Research) have been working on strategic combinations of several in vitro and alternative test methods within a (tiered) testing strategy that could replace the Draize Rabbit Eye Test.

Available In Vitro & Alternative Eye Irritation Test Methods

  • OptiSafe™In Vitro Eye Irritation Test
  • 3D Human Ocular Tissue Equivalent Systems – MatTek, EpiSkin
  • HET-CAM – Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM)
  • Bovine Corneal Opacity and Permeability Assay (BCOP)
  • Porcine Cornea Reversibility Assay (PorCORA)
  • Chorioallantoic Membrane Vascular Assay (CAMVA)
  • Replacement Ocular Battery (ROBatt)
  • Keratinocyte/Fibroblast Viability Assay (Neutral Red or MTT)
  • Keratinocyte Proliferation (turnover) Assay

For more info on in vitro and alternative toxicology methods, please see: www.mbresearch.com.

OptiSafe – Non-animal Ocular Irritation Test

OptiSafe™ – Non-animal Ocular Irritation Test 

OptiSafe Eye Irritation TestingOptiSafe™ Ocular Irritation Test

MB Research Labs is now offering the new OptiSafe™ – an in vitro test method in which a test substance is applied to a semi-permeable membrane. Damage to macromolecules in the membrane is measured to assess the test substance’s potential to cause eye irritation.

OptiSafe™ Background Information

OptiSafe™ is an acute in vitro irritation test method used to determine the ocular irritation potential based of a test substances utilizing a set of biochemical tests (mostly performed in parallel; “multiplexed”) which evaluate the potential of the test substance ability to disrupt, denature and degrade biological molecules. These measured values are relevant to GHS and EPA standard ocular safety classification systems.

Evaluated Mechanisms of Ocular Injury and Prediction Models

Different ocular irritants including, acids/bases, alcohols/aldehydes/ketones, strong oxidants and reactive chemicals/mixtures, damage the cornea and other tissues through additive and/or synergistic chemical reactions and biophysical effects.

Evaluated mechanisms of injury include:

  • Denaturation of specific water insoluble polymers that model the phospholipid bilayer of cells (which can occur at the corneal epithelium and conjunctiva).
  • Direct denaturation of macromolecules that model ordered collagen (which can occur at the corneal stroma).
  • Indirect denaturation of molecules across a membrane via osmotic effects (osmotic effects across the corneal epithelium and stroma can damage the cornea).
  • Potential to damage tissue via excessive oxidation and reactivity (which can occur at the epithelium, stroma, conjunctiva and iris).
  • Potential to damage tissues via extreme buffering (which can occur at the epithelium, stroma, conjunctiva and iris).

NICEATM reviewed a validation study conducted by the OptiSafe™ test method developer, Lebrun Labs, and concluded that the study data indicated that the OptiSafe™ method compared favorably to other in vitroocular toxicity testing methods. NICEATM is currently coordinating a validation study of the OptiSafe™ test method to demonstrate the reproducibility of the method among Lebrun Labs and two naïve laboratories. The study received support from an NIEHS Small Business Innovation Research grant. The study is ongoing and is expected to be completed in mid-2018.

MB Research Labs is a primary testing source using OptiSafe™ and has been deeply involved in the validation of OptiSafe™.

Utility of OptiSafe™

OptiSafe™ can be used to determine the irritation potential of cosmetics, creams, and a wide variety of consumer products. Results are presented as GHS, EPA classifications, an ocular irritation score and class.

OptiSafe™ can be used for:

  • Screening Finished Products for Ocular Irritation.
  • Ocular safety testing during product development for knowledge based formulation. (Products that irritate do not last long in the marketplace).

The Advantages of OptiSafe™ include:

  • Accurate Ocular Irritancy Test
  • High Sensitivity for Mild and Moderate Irritants
  • Fast Results
  • 1 year shelf life
  • Neither uses nor harms animals

MB Research Labs performs many other in vitro eye and dermal irritation tests for screening of cosmetics, consumer products, pharmaceuticals and chemicals.

www.mbresearch.com

OptiSafe™ is a trademark of Lebrun Labs, LLC and used here with permission.