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Replacement Ocular Battery (ROBatt)

Replacement Ocular Battery (ROBatt). 01-U01-NS073481-01 Dan Cerven, MS MB Research Labs. METHODS FOR STUDY OF IRRITATION AND TOXICITY OF SUBSTANCES APPLIED TOPICALLY TO THE SKIN AND MUCOUS MEMBRANES. JOHN H. DRAIZE, GEOFFREY WOODARD AND HERBERT O. CALVERY

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Replacement Ocular Battery (ROBatt)

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  1. Replacement Ocular Battery (ROBatt) 01-U01-NS073481-01 Dan Cerven, MS MB Research Labs

  2. METHODS FOR STUDY OF IRRITATION AND TOXICITY OF SUBSTANCES APPLIED TOPICALLY TO THE SKIN AND MUCOUS MEMBRANES. JOHN H. DRAIZE, GEOFFREY WOODARD AND HERBERT O. CALVERY From the Division of Pharmacology, Food and Drug Administration, Federal Security Agency, Washington D.C. Received for publication, November 2. 1944

  3. ACCORDING TO DRAIZE: “In the measure of injury to the eye a modification of the scoring system of Friedenwald, Hughes and Hermann1 is used. Injuries to the cornea, conjunctival and palpebral mucosae and the iris are scored separately. In testing the materials (liquids, solutions and ointments) 0.1 ml is instilled in the conjunctival sac. Readings are usually made at 1, 24 and 48 hours after instillation of the agent into the eye……. Readings are also made after 96 hours if residual injury is present.”

  4. SCALE FOR SCORING OCULAR LESIONS1 (1) CORNEA: (A) Opacity: Degree of density (area most dense taken for reading): No ulceration or opacity 0 Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 12 Easily discernible translucent area, details of iris slightly obscured 22 Opalescent areas, no details of iris visible, size of pupil barely discernible 32 Opaque cornea, iris not discernible through the opacity 42 (B) Area of cornea Involved: One quarter (or less) but not zero 1 Greater than one-quarter, but less than one-half 2 Greater than one-half, but less than three-quarters 3 Greater than three quarters up to whole area 4 SCORE EQUALS A x B x 5 Maximum Total 80 (2) IRIS: (A) Values: Normal 0 Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof), iris still reacting to light (sluggish reaction is positive) 12 No reaction to light, hemorrhage, gross destruction (any or all of these) 22 SCORE EQUALS A x 5 Maximum Total 10 (3) CONJUNCTIVAE: (A) REDNESS (refers to palpebral and bulbar conjunctivae excluding cornea & iris): Blood vessels normal 0 Some blood vessels definitely hyperemic (injected) 1 More diffuse, deeper crimson red, individual vessels not easily discernible 22 Diffuse beefy red 32 (B) CHEMOSIS No swelling 0 Any swelling above normal (includes nictitating membranes) 1 Obvious swelling with partial eversion of lids 22 Swelling with lids about half closed 32 Swelling with lids more than half closed 42 (C) DISCHARGE No Discharge 0 Any amount different from normal (does not include small amounts observed in inner canthus of normal animals) 1 Discharge with moistening of the lids and hairs just adjacent to lids 2 Discharge with moistening of the lids and hairs and considerable area around the eye 3 SCORE EQUALS (A+B+C)x2 Maximum Total 20 The maximum total score is the sum of all scores obtained for the cornea, iris and conjunctivae. 1Draize, J. H. et al. J. Pharm. Exp. Ther. 82:377-390, 1944. 2Indicates a positive response ULTRAVIOLET FLUORESCEIN SCAN SCORING CODE: 0 = Negative 1 = Positive with an area 1/4 or less 2 = Positive with an area >1/4 but <1/2 3 = Positive with an area >1/2, but <3/4 4 = Positive with an area >3/4, up to entire area

  5. Comparison of Response of Several Species to Eye Irritants

  6. Study of Intra- and Interlaboratory Variability in the Results of Rabbit Eye and Skin Irritation Tests. CARROL S. WEIL AND ROBERT SCALA Mellon Institute, Carnigie-Mellon University, Pittsburg, PA And Esso Research and Engineering Company, Medical Research Division, Linden, NJ October 12, 1970

  7. “…it is suggested that the rabbit eye and skin procedures currently recommended by the Federal Agencies for use in delineation of irritation of materials should not be recommended as standard procedures in any new regulation.”

  8. Rabbit Eye Irritation Test: Collaborative Study Frank N. Marzulli and Dennis I. Ruggles Division of Toxicology, Food and Drug Administration, Washington D.C. October 1973 “when performed according to the directions cited, the Draize rabbit eye test for irritation is capable of providing reliable and reproducible information about the eye irritant capacity of test materials.”

  9. “Those Were The Draize”

  10. Project Overview • Two year research project to validate this tiered testing strategy for regulatory ocular irritation screening using alternative toxicology methods that do not utilize live animals • Validation consists of four main studies in which alternative test methods with varying degrees of sensitivity will be used to categorize known corrosives and severe, moderate, slight and non-irritating materials RC50 < X RC50 > X X IVS < Y No. dead cell/vol > Z No. dead cell/vol < Z IVS > Y Z Y

  11. Project Scope • Two Main Milestones • Milestone 1: • X – CAMVA RC50 cutoff • Y – BCOP In Vitro Score cutoff • Z – PorFocal cutoff • Milestone 2: • PorCORA - time to clear for evaluated chemicals • Publish and Present to ICCVAM/ECVAM • Commercialization

  12. Specific Aims • Development of scoring criteria for the battery assays that are to be used in the Replacement Ocular Battery Decision Tree • SubAim 1A – Develop conjunctival analog scoring criteria using a set of 50 validation chemicals with the CAMVA (X) • SubAim 1B – Develop initial corneal irritation scoring creiteria using a set of 30 validation chemicals with the BCOP (Y) • SubAim 1C – Determine Severe Irritancy or Ocular Corrosivity potential using PorCORA test scoring criteria and a set of 10-20 validation chemicals • Sub-Aim 1D – Develop irritation scoring criteria (Z) used to discriminate between non-irritating and slightly irritating test articles using PorFocal and a set of approximately 20 chemicals

  13. Chorioallantoic Membrane Vascular Assay (CAM-VA) • X - Initial Screen for Ocular Irritation • Discriminates Severe-Moderate and Slight-Non-Irritating Materials Methods • Dose CAMs; “Topically” Endpoint: Hemorrhage/Blood flow • Dose Response Curve of % positive • Calculate RC50 • Correlation: 84–92% rabbit Draize • Rapid, Inexpensive Rabbit Conjunctiva Chorioallantoic Membrane

  14. CAM-VA: Procedures

  15. CAM-VA: Scoring Responses CAM-VA: Ghost Vessels CAM-VA: Normal CAM-VA: Capillary Injection CAM-VA: Moderate Hemorrhage

  16. Sub-Aim 1A – Develop conjunctival analog scoring criteria using a set of 50 validation chemicals with CAMVA (X) The Decision Tree variable of X will be in the form of an RC50 – the calculated concentration that produces a positive response in the CAM of 50% of treated eggs. We will utilize an existing CAMVA protocol to evaluate 50 validation chemicals chosen from the ECETOC databank with an MMAS between 0 and 110. Once all validation chemicals have been assayed, the RC50s will be evaluated and a threshold RC50 (X) will be created which will indicate if the chemical should be evaluated with the BCOP or PorFocal.

  17. BCOPBovine Cornea Opacity/Permeability Test Normal Opacity • Uses excised Bovine Corneas normally discarded as waste • Measures two endpoints: • Changes in light transmission through the cornea • Changes in corneal cell structure which may not affect light transmission

  18. Sub-Aim 1B – Develop initial corneal scoring criteria using a set of ~30 validation chemicals with the BCOP (Y) The ROBatt Decision Tree variable Y will be expressed as the In Vitro Score, which is calculated using both the opacity and permeability response of excised bovine corneas for the BCOP assay. The In Vitro Score is based upon the assessment of a change in light transmission and sodium fluorescein permeability. The validation chemicals will be selected from the RC50s determined by CAMVA. Those with an RC50 < X will be used in the BCOP assay.

  19. Regulatory Classification Moderate Irritant

  20. PorCORA – Porcine Cornea Opacity and Reversibility Assay • Severe Ocular Irritation vs. Corrosivity • Multiple Topical Dosing Possible • Damage assessed by retention of fluorescent stain • Up to 21 days

  21. Score Corneal Grading Criteria1 0 No Stain Retention. 1 One Quarter (or less) but not zero. 2 Greater than one quarter, but less that one half 3 Greater than one half, but less than three quarters. 4 Greater than three quarters up to the whole area. Corneal Scoring and Staining • Draize,J.H., et al., J. Pharm. Exp. Ther., 82:377-390, 1944.

  22. Regulatory Classification Ocular Corrosive Severe Irritant

  23. SubAim 1C – Determine Severe Irritancy or Ocular Corrosivity potential using PorCORA test scoring criteria and a set of 10-20 validation chemicals The validation chemicals with an RC50 < than X and an In Vitro Score > Y will be evaluated with PorCORA to distinguish if the chemical is a Severe Irritant or an Ocular Corrosive.

  24. PorFocal – Porcine Confocal Assay • Adaptation of PorCORA – Air Interface Culture Method • Uses confocal microscopy and Ethidium homodimer dead cell staining kits to assess tissue viability • Optically “sections” tissue through epithelial layers • Dosing 1x per day for 7 days • Highly sensitive – • quantification of individual cell death per volume of tissue • additive effects from multiple doses over a week

  25. Sub-Aim 1D – Develop Irritation-scoring criteria used to discriminate between non-irritating and slightly irritating test articles This test will consider (slight to non-irritating) substances with the RC50s >X as determined by CAMVA. ~20 compounds from the range of 0 – 15 MMAS (Draize, ECETOC) will be assayed using the PorFocal method. According to historical Draize information (ECETOC database), predicted outcomes of these chemicals would be slightly irritating. Once all validation chemicals have been evaluated, the results of the Dead tissue staining will be tabulated and a cutoff score (Z) will be determined that will be used to discriminate which chemicals can be classified as non-irritating and slightly irritating.

  26. Regulatory Classification Slight Irritant Non-Irritant

  27. Perceived Technical Challenges • Cost effective procurement of confocal microscope system capable of imaging corneas to a depth of 50µm. • Quantification of X, Y, Z for different classes of chemicals • Commercialization • Mixtures • Client Interest (Participation)

  28. Perceived Administrative Challenges • Budgetary Adjustments • Additional Funds Available to address unforeseen technical issues? • Timeline/Milestone Adjustments • Scope Impact? • Requests for Extension • Replacement of Key Personnel • Post Grant Validation • Inter-Lab Validations • Acceptance by ICCVAM/ECVAM

  29. Project Progress • How many CAMVAs (X) completed - 30 • How many BCOPs (Y) completed - 6 • Confocal Microscope Demo’d • Scheduled - Olympus, Nikon • Other - TBD • Ordered BCOP Opacitometer

  30. Thank You Dan Cerven, MS Director of Laboratories MB Research Labs, Email: mbinfo@mbresearch.com Tel: 215-536-4110

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