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 Hermann 1 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 LESIONS 1 (1) CORNEA: (A)Opacity: Degree of density (area most dense taken for reading): No ulceration or opacity0 Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible1 2 Easily discernible translucent area, details of iris slightly obscured2 2 Opalescent areas, no details of iris visible, size of pupil barely discernible3 2 Opaque cornea, iris not discernible through the opacity4 2 (B)Area of cornea Involved: One quarter (or less) but not zero1 Greater than one-quarter, but less than one-half2 Greater than one-half, but less than three-quarters3 Greater than three quarters up to whole area4 SCORE EQUALS A x B x 5Maximum Total80 (2) IRIS: (A)Values: Normal0 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)1 2 No reaction to light, hemorrhage, gross destruction (any or all of these)2 2 SCORE EQUALS A x 5Maximum Total10 (3) CONJUNCTIVAE: (A)REDNESS (refers to palpebral and bulbar conjunctivae excluding cornea & iris): Blood vessels normal0 Some blood vessels definitely hyperemic (injected)1 More diffuse, deeper crimson red, individual vessels not easily discernible2 2 Diffuse beefy red3 2 (B)CHEMOSIS No swelling0 Any swelling above normal (includes nictitating membranes)1 Obvious swelling with partial eversion of lids2 2 Swelling with lids about half closed3 2 Swelling with lids more than half closed4 2 (C)DISCHARGE No Discharge0 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 lids2 Discharge with moistening of the lids and hairs and considerable area around the eye3 SCORE EQUALS (A+B+C)x2Maximum Total20 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

7. Comparison of Response of Several Species to Eye Irritants Test AgentDecreasing Order of Sensitivity of Species Tested Reference Liquid DetergentsRabbit, Dog a, MonkeyBeckley (1965) NS b Rabbit, Dog, MonkeyCarter and Griffith (1965) NSRabbit, Dog, MonkeyGovacchini (1972) Commercial ShampoosRabbit, Hamster, Mouse, Rat, Guinea Pig, Dog, Cat, Monkey, Chicken Gershein and McDonald (1977) Cationic DetergentsMouse, Hamster, Rabbit, Guinea Pig, Rat, Dog, Cat, Monkey, Chicken Gershein and McDonald (1977) GriseinRabbit, Guinea Pig, RatMelnikova and Rodianov (1979) a – Corneal effects were greatest in the dog. b – Not Specified.

8. 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

9. “…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.”

10. 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.”

11. “Those Were The Draize”

12. 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 Y X Z RC50 > XRC50 < X IVS > Y IVS < YNo. dead cell/vol > ZNo. dead cell/vol < Z

13. 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

14. 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

15. Rabbit Conjunctiva Chorioallantoic Membrane 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

16. CAM-VA: Procedures

17. CAM-VA: Normal CAM-VA: Ghost Vessels CAM-VA: Capillary InjectionCAM-VA: Moderate Hemorrhage CAM-VA: Scoring Responses

18. Sub-Aim 1A – Develop conjunctival analog scoring criteria using a set of 50 validation chemicals with CAMVA (X) 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. 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.

19. Uses excised Bovine Corneas normally discarded as waste Measures two endpoints: 1)Changes in light transmission through the cornea 2)Changes in corneal cell structure which may not affect light transmission BCOP Bovine Cornea Opacity/Permeability Test NormalOpacity

20. 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.

21. Regulatory Classification HMIS 2GHS 2AEPA 3 “corneal Irritation clearing in 8-21 days” “fully reverses within 21 days” “corneal involvement or other eye irritation clearing in 7 days or less” Moderate Irritant

22. 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

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

24. Regulatory Classification Reg. ClassConditions HMIS IIIIrreversible destruction of eye tissue; corneal irritation persists for more than 21 days. GHS 1Corneal effects do not fully reverse within 21 days. EEC R41Risk of serious damage to eyes (moderate but persistent lesions, eye corrosion) EPA 1Irreversible corneal involvement persisting for more than 21 days. Reg. ClassConditions HMIS IICorneal irritation clearing in 8 to 21 days GHS 2A“fully reverses within 21 days” EEC R36Irritating to eyes (moderate eye irritation reversible < 21 days) EPA 2Corneal involvement clearing in 8 to 21 days Ocular Corrosive Severe Irritant

25. 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 Y will be evaluated with PorCORA to distinguish if the chemical is a Severe Irritant or an Ocular Corrosive.

26. 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

27. 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.

28. Regulatory Classification Reg. ClassConditions HMIS ISlight to mildly irritating but reversible within 7 days GHS 2BCorneal effects fully reverses within 21 days. Reg. ClassConditions HMIS 0Essentially non-irritating with effects clearing in less than 24 hrs EPA 4Minimal effects clearing in less than 24 hrs Slight Irritant Non-Irritant

29. 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)

30. 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

31. 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

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