TOXICITY TESTING FOR OCULAR DRUG PRODUCTS *

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1 24 TOXICITY TESTING FOR OCULAR DRUG PRODUCTS * Javier Avalos, Abigail Jacobs, and Jonathan K. Wilkin Division of Derrnato\ogic and Dental Drug Products Center for Drug Evaluation and Research Food and Drug Administration Rockville, Maryland 1. INTRODUCTION Regulatory agencies world wide have implemented guidelines and recommendations in order to promote and protect the health of its citizens. Agencies like the European Economic Community (EEC), Organization for Economic Cooperation and Development (OECD), and the Japanese Ministry of Health and Welfare are involved in the regulation of toxic chemicals. In the United States, four agencies bear most of the direct responsibility for the regulation of these chemicals. These are the Consumer Product Safety Commission (CPSC), Environmental Protection Agency (EPA), Occupational Safety and Health Administration (OSHA), and the Food and Drug Administration (FDA). Each agency makes a risk/benefit analysis for a given compound that is to be marketed and transported or could potentially contaminate the environment by establishing a safety profile. At the Food and Drug Administration, the Agency evaluates drug products and devices that are to be marketed in the United States. For a drug product or device, a safety profile is obtained after conducting appropriate toxicological evaluations. Drug products are submitted to one of several centers within the FDA for evaluation. At the Center for Drug Evaluation and Research (CDER), the mission is to assure that safe and effective drug products are available to the American people. Every effort is made by the Center to use the most appropriate means in establishing the safety profile of these products. In the safety profile, the toxicological evaluations submitted in each application are primarily in vivo studies. However, examples do exist where in vitro methods have been used to address a specific issue in the safety profile of a drug product. * This statement is an informal communication under 21 CFR (b)(9) that represents the best judgment of the Division of Dermatologic and Dental Drug Products at this time. This document does not necessarily represent the formal position of the Center for Drug Evaluation and Research or the Food and Drug Administration, and does not bind or otherwise obligate the Center or Agency to the views expressed. Advances ill Ocular Toxic%gy, edited by Green e/ 0/. Plenum Press, New York,

2 262 J. Avalos et al. The focus of this chapter will be ocular drug products. Regulations that address the safety profile, commonly submitted assays that are part of the safety profile, and potential alternatives for evaluating ocular irritation for an ocular drug product to be marketed will be discussed. Additionally, the current role and advantages of these alternatives in the drug development process will be emphasized. 2. REGULATORY CONSIDERATIONS Several regulations have been established specifically to address drug products to be marketed in the United States. Title 21 of the Code of Federal Regulations pertains to the Food and Drug Administration. Within this series of volumes, general guidance is contained addressing regulations for food additives, good laboratory practices, good manufacturing practices, drugs for human use, and medical devices. In parts 312 and 314 of Title 21, procedures and requirements governing the use of investigational new drugs (IND) and the submission of new drug applications (NDA) are given I. The procedures identified in parts 312 and 314 are to be followed if there is an intention to conduct a clinical investigation with a new drug product or to seek approval to go to market. It is part (a)(8) that specifically addresses the safety profile of the investigational drug product. In this section, the content of the safety profile is provided in general terms. Section (a)(8)(ii)requires submission of "an integrated summary of the toxicological effects of the drug in animals and in vitro.,,1 This description allows inclusion in the application of the detailed results of acute, subchronic, chronic toxicity tests, reproductive toxicity tests, and "any in vitro studies intended to evaluate drug toxicity". As affirmed in (a)(8)(ii), the Agency will accept in vivo studies as well as in vitro studies in order to establish a safety profile. Currently, the Agency has not identified a single in vitro assay which would be capable of replacing an in vivo assay. The significance, however, of the regulations is to emphasize that any information which will address the potential toxicity of a given drug product will be accepted by the Agency for review and consideration Commonly Submitted Non-Clinical Toxicology Studies The Agency evaluates the risk/benefit for each product prior to its introduction to humans and its subsequent marketing. As part of the risk/benefit analysis, a safety profile is determined. Several factors must be considered in determining the number of non-clinical safety studies necessary to provide an adequate safety profile. The most critical is the intended human use of the drug product. The intended dose, indication, number of applications per day, route of exposure, whether male and female volunteers will be exposed, intended patient population, exclusion and inclusion criteria, and anticipated length of clinical exposure will determine the recommended number, type, and duration of nonclinical studies. In addition, the number of non-clinical studies will be dependent on whether the drug product is a new molecular entity or a reformulation. To capture the potential toxicity of an ocular drug product, studies evaluating the potential effects of the drug product on the ocular system as well as on the rest of the animal are usually needed. However, some of the studies discussed in this section may be unnecessary for certain products. In general, the type of studies usually recommended would be single or repeat toxicity, reproductive toxicity, genotoxicity, pharmacokinetic, and special toxicity studies. Other toxicity studies could include ocular and dermal irritation, and dermal sensitization studies. Pharmacokinetic studies are important in evaluating the sys-

3 Toxicity Testing for Ocular Drug Products 263 temic exposure of ophthalmic drug products. In order to initiate the clinical studies, toxicity studies via the intended route that are of equal or longer duration than the proposed clinical study are recommended. Reproductive toxicity studies are also recommended for drug products intended for intermediate-term or chronic use. Chronic non-clinical studies would include the one year in duration or lifetime carcinogenicity studies. However, systemic absorption of the drug products would be critical in determining the need for the carcinogenicity studies. The non-clinical studies should include both genders and two species (one rodent and non-rodent). In addition, the studies should be conducted in accordance with current Good Laboratory Practices (21 CFR 58)2. The Sponsor is encouraged to contact the FDA Division as early in the development of the drug product as possible to obtain guidance and comment on protocols. 3. MODIFIED DRAIZE ASSAY As part of the safety profile, ocular and dermal drug products, as well as ocular devices, are evaluated for their potential to cause ocular lesions. A current method for determining ocular irritation is the modified Draize assay (16 CFR )3. This protocol is very similar to the protocol proposed by Draize et at. (1944t The main differences in the protocol proposed by the CPSC in 1979 and the original protocol were a decrease in rabbits (9 to 6), limited observation periods, inclusion of a rinsing procedure, and modification of the scoring system. More importantly, a provision for testing solid materials was included, as well as a suggestion for the use of a slit lamp, binocular loupe, and fluorescein dye, to facilitate the detection of corneal injury. The original use of this protocol was to determine if accidental exposure to agents could induce injuries to the eye General Conduct of Assay As described in the Code of Federal Regulations (16 CFR ), the test agent (0.1 ml) is instilled into the conjunctival cul-de-sac of six albino rabbits 3. Following instillation, treated eyes are either rinsed or not rinsed. The treated eye is then observed at 1, 24, 48 hours and up to 7 days following exposure. Treated eyes are evaluated for severity and reversibility of injury to the iris, cornea, and conjunctiva. The degree or extent of injury is noted using the scoring system described in the modifications made by CPSC (16 CFR )3. A positive ocular reaction to a drug product occurs if the exposure elicits any corneal ulceration or opacity greater than a fine stippling, corneal opacification greater than a loss of normal luster, any iridial inflammation greater than a slight deepening of the iridial rugae, conjunctival swelling sufficient to cause partial eversion of the eyelids, or a diffuse redness of the conjunctiva which obscures the details of individual blood vessels. The product would be considered an irritant if four or more of the six animals had a positive response. Positive responses in two or three animals of the six would warrant a second administration to six more rabbits. 4. ALTERNATIVES TO THE MODIFIED DRAIZE ASSAY An interest in reducing the number animals used in non-clinical testing, refining procedures that would allow for a decrease in discomfort to the animal, or replacing the standard in vivo test has led to the development of a variety of alternative assays. These

4 264 J. Avalos et al. assays and the advances made in biotechnology may provide useful information in the ocular safety assessment process. Living cells, tissues, or recently enucleated eyes are being employed in many of these assays. Although several assays have been proposed as substitutes for the Draize ocular assay, none of the alternatives may be capable of rep lacing the in vivo assay due to the complexity, structurally and functionally, of the eye. Perhaps the assays would be of greater value as complements to the in vivo assays or assist in defining a product's potential biological response. In addition, the assays could provide methodologies for addressing the potential biotransformation end-products or characterizing a mechanism of action for the product. The use of these alternatives as prescreens or part of tier testing systems has been proposed by several investigators5-9. In the approach by Hurley et al. (1993), in vitro alternatives were described as part of a tier testing system for assessing eye irritations. The tier testing system is illustrated in Figure 1. The authors, members of the Interagency Regulatory Alternatives Group (IRAG), proposed as their final report a stepwise progression of screens so that little or no animal testing would be required for agents meeting the criteria identified in the report. The first parameter proposed as a screen was measurement of the ph of the test agent. Murphy et al. (1982) reported the effects of ph on ocular irritation 10. Acids with a ph below 2.5 and alkalies with a ph above 11.5 produce severe damage to the corneal stroma of rabbit eyes that had been denuded of the corneal epithelium. However, preliminary information indicates that the total alkalinity of the test agents may be a better predictor of irritation than ph'o.". Structure-activity relationship may also be used as a screen. Comparison of the structural moieties of the test agent to known irritant structural moieties would provide insights into the agent's potential to cause ocular irritation. Similarly, the requirements for ocular testing may be waived for reformulations which contain known irritants. The third step in this tier testing system was the use of in vitro tests. In the following section, some of the advantages and disadvantages are discussed. As part of the proposed tier testing system, a battery of these assays may assist in identifying those compounds that are likely to be severe irritants to the eye. Severe irritants or corrosive agents to the Drug Product PhYSicochemical Properties (e.g., ph) SAR In Vitro Observations Acute Dermal TOxicity Findings Draize Modified Assay Figure 1. Tier testing system recommended for the assessment of ocular irritation of drug products. Historical ocular irritation data of drug products and ingredients are first obtained. The physicochemical properties of active ingredient and other ingredients of the drug product are determined. If necessary, the material is then evaluated in a battery of in vitro assays. After the in vitro assays, either the acute dermal toxicity or primary dermal irritation assays could be conducted. Then, the modified Draize assay could be conducted, if necessary. (Modified from Hurley et a/,r)

5 Toxicity Testing for Ocular Drug Products 265 skin are also most often irritating to the eye. The use of rabbits in the primary dermal irritation assay or a dermal toxicity study would provide valuable information in assessing tbe potential ocular irritation of a drug product. Additionally, these in vivo assays would provide systemic toxicity data for review in the drug development process. As part of the tier testing approach, dermal irritation (PDII > 5) or acute toxicity (LD50 <200 mg/kg) would preclude any ocular irritation testing. If after using this approach an ocular irritation assay is deemed necessary, a couple of options for the ocular irritation assay are available. The current modified Draize as described in (3.1.) could be employed. A second alternative would be the use of a more recently modified version of the Draize assay. This later method would use a similar method of exposure, observation periods, and scoring system as described earlier. However, the number of animals treated with test material would be reduced to three animals. Because of the lower number of animals, the interpretation of the assay will also differ. In the report of the IRAG symposia, a reduction in animals used in the ocular irritancy assay was proposed l2. Two options were given. Both options would decrease the number of animals originally proposed by CPSC from six to three. In the first option, two animals would be tested. If positive findings were reported, then no further testing would be necessary. If a negative response was obtained, a third animal would be treated with test material. In the second option, three animals would be used. If two of three animals had a positive response, the material would be considered an irritant. With the first option, an accuracy of 97% was given with low false positive «3%) and false negative (0%) responses l2 For the second option, an accuracy of 94% was noted with a very low false negative rate of 1 % and an acceptable false positive rate of <5% 12. Another option would be to treat one animal first. If severe reactions are noted, no further testing would be recommended. However, if mild or no response is reported, then two more animals are treated and the findings noted. Using this method, two of three animals would need to have positive findings for designation as an irritant. Additionally, the dose employed would be 0.01 ml of a liquid or O.OI-ml equivalent weight dosage of a solid if the material is suspected to be a severe ocular irritant. If not, then the dose would also be similar as described in (3.1). This low-dose volume was also proposed at the IRAG symposia \ In Vitro Assays The utilization of cell cultures, tissues organs, or enucleated eyes to assess the irritant potential of substances offers many advantages over conventional animal tests. These include reducing the number of animals and cost, using quantifiable objective end-point measurements and tissue from the target species, being reproducible, consuming less time, and being easier to perform. With such advantages, a greater number of chemical agents could conceivably be evaluated for their potential toxic effects. Unfortunately, none of the in vitro alternative assays directly measures the extent of irritation that can be produced. Instead, the in vitro models measure a variety of cellular end-points, like morphology, membrane integrity, viability, function, inflammatory mediator release, and recovery and repair Other assays evaluate Tetrahymena motility, protein denaturation, and ocular wound healing. Although the mechanism of irritation still remains unclear, cytokines likely playa pivotal role in the inflammation response. Physical or chemical injury to the eye is the initial event in the inflammatory response. Incorporation of the assays which measure the release of the inflammatory mediators would be the more optimal method for assessing the irritant potential of substances. These systems are measuring part of the pathogenic proc-

6 266 J. Avalos et al. ess and, thus, would be expected to provide a better estimate of the irritant potential of chemical agents. Even though the identical end-point can be determined in either the cell culture or organ test systems, the evaluation of chemical agents would be less expensive and more versatile with the cell culture system. Use of end-points like cytotoxicity (membrane integrity or cell adhesion), cell function (cell metabolism), or cell growth with the cell culture systems would provide a less expensive, but reproducible, robust, and adaptable model when compared to the cytokine release assay. Even though these assays do not directly assess the irritant reaction, valuable information can be attained in regards to the possible mechanism of action of the test substance. In addition, a prioritization scheme for further analysis can be generated from these readily adaptable assays. Ranking of chemical-induced in vitro cytotoxicity, cytokine release, and cellular function have correlated well with historical in vivo ocular irritancy data for certain chemical classes l 4-l7. The correlation of the end-points was dependent on the concentration of test material, duration of exposure, and class of compound. However, the studies have not been extensive, and more data are required before this correlation can be accepted, especially for different classes of chemicals. Tissue organ end-points like alteration in smooth muscle contractility of isolated rabbit ileum or evaluation of electrical conductivity in rat skin slices provide a more direct evaluation of tissue rather than isolated cells. However, such tissues have little relationship to ocular toxicity. The use of enucleated eyes for evaluating changes in corneal opacity or permeability would be a more direct measure of ocular toxicity Drawbacks of Assays As already briefly described, the criteria for the validation of such assays have not been generally accepted. Validation would require that an assay initially have an in vivo predictive value, and be intralaboratory and interlaboratory validated to statistically predict the sensitivity of the in vitro assay relative to the expected in vivo response. Formal validation of an in vitro assay would depend on the intended use of the assay. In turn, an assay for the complete replacement of a widely accepted in vivo procedure like the Draize test would require a much more formalized validation prior to its routine use. Currently, no linkage between the action of the assays and the response of the human eye has been established. Additionally, many of these assays have not employed large numbers and a variety of materials in the development of the assay. Other practical limitations exist in collecting tissue for the alternative. Severe limitations also exist in assessing the effects of the drug product on the anterior portion of the eye, evaluating the reversibility of the lesion in the eye, or assessing any potential systemic toxicity following an ocular exposure. The eye is a very complex organ which a single in vitro alternative assay would likely be too simple to mimic. By using the in vitro assays in combination, the specific biological effects of a drug product or class of chemicals might be assessed. Nevertheless, the best assays currently available to determine the potential irritation potential of a chemical agent are still the animal models. The major advantage of developing an in vitro alternative for ocular irritation is the possibility of ultimately replacing the in vivo standard. Utilization of the assays as screens could minimize the number of chemicals to be evaluated in vivo and the reduce the number of animals used in safety testing. These assays, with time, will also generate databases that assess the predictive characteristics ofthe in vitro tests. Also with time, the development of more sophisticated organ systems that incorporate corneal epithelium, stromal fibroblasts, and endothelial cells will provide a more robust for in vitro ocular safety assessment.

7 Toxicity Testing for Ocular Drug Products SUMMARY As part of the safety assessment, the Food and Drug Administration evaluates dermal and ocular drug products for their potential ocular toxicity. Several factors (ph, structure-activity relationships, in vitro alternatives, primary dermal irritation index, and acute dermal toxicity) should be considered for the drug product prior to conducting an ocular irritation assay. The drug product should not be tested further if the drug product has a ph of 2.5 or below or 11.5 or above. The assay should also not be conducted if the active ingredient(s) have analogous chemically active moieties that are known irritants or if the reformulation contains known irritants. Additionally, the assay should not be conducted if the drug product has an acute dermal toxicity lethality at or below 200 mg/kg or a primary dermal irritation index score of 5 or above. The findings from in vitro systems like Corrositex with ocular and dermal drug products should also be considered prior to performing an irritation assay. Strongly positive findings in these assays could preclude the need for a modified Draize assay. Other alternatives could be used to complement the in vivo studies, assist in defining a mechanism of action, or identify possible biotransformation metabolites of the drug products. When animal testing is to be conducted, a low volume dosing (0.0 I ml or g) in one animal may be performed for suspected severe irritants. If the product is not classified as an irritant or moderate irritant, then 0.1 ml or 0.1 g of material should be instilled in two or three more rabbits. The responses are scored according to the modified Draize scoring system at 24, 48, and 72 hours, and, if necessary, at 7 days to assess the potential recovery from the insult. In summary, the Agency encourages the use of alternative tests prior to conducting a modified Draize assay for ocular irritation. Additionally, the Agency encourages the Sponsor to contact the Agency as early as possible for guidance in the development of the drug product. Such interaction could conserve valuable resources and potentially reduce the time to market for the drug product. 6. ACKNOWLEDGMENT JA would like to thank Dr. Wiley Chambers for his support and critical review of the manuscript. 7. REFERENCES I. Code of Federal Regulations Vol 21 parts 312 and Code of Federal Regulations Vol 21 part Codc of Federal Regulations Vol 16 part Draize. JH. Woodard. G. Calvery, HO. Method for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharm Exp Ther 1944;82: Frazier. JM. Gad. Sc, Goldberg, AM, McCulley, JP. A critical evaluation of alternatives to acute ocular irritation testing. In: Goldberg AM. ed, Alternative Methods in Toxicology Vol 4, Mary Ann Liebert, New York. 6. Wilcox, OK, Bruner, LH. AT LA 1990; 18: Bruner, LH, Shadduck, J, Essex-Sorlie, D. Alternative methods for assessing the effects of chemicals. In: Hobson OW, ed. Dermal and Ocular Toxicology: Fundamentals and Methods, , CRC Press, Inc., Boca Raton, FL. 8. Hurley, PM, Chambers, WA, Green, S, Gupta, KC, Hill, RN, et al. Screening procedures for eye irritation. Food Chern Toxicol 1993;31 :87-94.

8 268 J. Avalos et al. 9. Chan, P., Hayes, AW. Acute toxicity and eye irritancy. In: Hayes AW, ed. Principles and Methods of Toxicology 3rd edition, , Raven Press, New York. 10. Murphy, JC, Osterberg, RE, Seabaugh, VM, Bierbower, BW. Ocular irritancy responses to various phs of acids and bases with and without irrigation. Toxicology 1982;23 : II. Booman, KA, DeProsp, J, Demetrulias, J, et al. The SDA alternatives program: comparison of in vitro data with Draize test data. J Tox Cut Ocular Toxicol 1988;8: Springer, JA, Chambers, WA, Green, S, Gupta, KC, Hill, RN, et al. Number of animals for sequential testing. Food Chern ToxicoI1993;31: Lambert, LA, Chambers, WA, Green, S, Gupta, KC, Hill, RN, et al. The use of low-volume dosing in the eye irritation test. Food Chern ToxicoI1993;31: Borenfreund, E, Borrero, O. In vitro cytotoxicity assays: potential alternatives to the Draize ocular irritancy test. Cell Bioi ToxicoI1984;1: Borenfreund, E, Shopsis, C. Toxicity monitored with a correlated set of cell culture assays. Xenobiotica 1985;15: Demetrulias, J, North-Root, H. Prediction of the eye irritation potential for surfactant-based household cleaning products using the SIRC cell toxicity test. In: Goldberg AM, ed, Alternative Methods in Toxicology Vol. 6, Mary Ann Liebert, New York. 17. Shadduck. J, Everitt, J, Bay, PHS. Use of in vitro cytotoxicity to rank ocular irritation of six surfactants. In: Goldberg AM, ed, Alternative Methods in Toxicology Vol 3, , Mary Ann Liebert, New York.