TOXIKON FINAL GLP REPORT: G4 MOUSE LYMPHOMA MUTAGENESIS ASSAY OECD. Test Substance OIS Author Devaki Sadhu, Ph.D.

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1 TOXIKON FINAL GLP REPORT: G4 MOUSE LYMPHOMA MUTAGENESIS ASSAY OECD Test Substance OIS 1220 Author Devaki Sadhu, Ph.D. Final Report Date June 6, 2008 COMPLIANCE OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring 21 CFR, Part 58 Good Laboratory Practice for Non Clinical Laboratory Studies MANAGEMENT OF THE STUDY Performing Laboratory Toxikon Corporation 15 Wiggins Avenue Bedford, MA Sponsor Oculus Innovative Sciences 1135 North McDowell Boulevard Petaluma, CA 94954

2 TABLE OF CONTENTS Title Page Table of Contents Study Summary Quality Assurance Statement Study Director Signature and Verification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification of Test and Control Substances 5.0 Identification of Test System 6.0 Justification of Test System 7.0 Experimental Design and Dosage 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality Agreement Table 1: Table 2: Table 3: Table 4: TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Non Activated Assay) TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Activated Assay) TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Non Activated Assay) Confirmatory Assay TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Activated Assay) Confirmatory Assay Page 2 of 15

3 STUDY SUMMARY The test substance, OIS 1220, was evaluated for its potential to induce a statistically significant increase in the number of homozygous thymidine kinase mutants (TK / ) over the background frequency in a mutant mouse lymphoma L5178Y cell line, heterozygous at the thymidine kinase locus (TK +/ ). An initial range finding assay was performed to determine cytotoxicity and select test concentrations of the test substance for the definitive assay. The range finding assay was conducted by exposing L5178 cells to the neat, 1:2, 1:4, 1:8, 1:16, and 1:32 dilutions of the test substance (made with Sterile Water for Injection (SWFI)) for four hours in the absence of metabolic activation. Results showed that there was complete toxicity (100%) at the neat concentration whereas the 1:2 and 1:4 dilutions induced partial toxicity showing 27.5 and 45.8 % survival, respectively. No significant reduction in the viable cells was observed in any of lower dilutions of the test substance treatment groups compared to the negative control. Based on the results of the range finding assay, the definitive assay was conducted exposing cells to the 1:2, 1:4, 1:8, and 1:16 dilutions of the test substance in the presence and absence of metabolic activation. The results of the definitive assay showed that there was no significant increase in the incidence of homozygous mutants in any of the test substance treatment groups compared to the respective negative control groups in both non-activated and activated conditions. There was a statistically significant increase in the incidence of homozygous mutants in cells exposed to the positive control substances in both non activated and activated conditions, validating the functioning of the assay. A confirmatory assay was performed to verify the results of the definitive assay and the results were consistent with the negative results of the definitive assay. Based on the criteria of the study protocol, the test substance is considered non mutagenic in the Mouse Lymphoma Mutagenesis Assay. Page 3 of 15

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6 1.0 PURPOSE This assay evaluated the ability of the test substance to induce an increase in the formation of homozygous thymidine kinase mutants (TK / ) over the background rate in a mouse lymphoma cell line, in the presence and absence of a metabolic activation system. The mutant cell line, L5178Y, carries the mutation in the thymidine kinase locus (TK +/ ). 2.0 REFERENCES The study was based upon the following references: 2.1 OECD 476, Organization for Economic Co Operation and Development (OECD) Guidelines for the Testing of Chemicals, In vitro Mammalian Cell Gene Mutation Test, adopted 21 July ICH Harmonized Tripartite Guideline. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, S2A, 1995, FDA: Published in the Federal Register, Vol. 61, April 24, 1996, page ICH Harmonized Tripartite Guideline; Genotoxicity: A Standard Battery for Genotoxicity Testing for Pharmaceuticals, S2B, 1997, FDA: Published in the Federal Register, 21 November Turner, N.T., A.G. Batson and D. Clive. Procedures for the L5178 TK +/ Mouse Lymphoma Cell Mutagenicity Assay, Handbook of Mutagenicity Testing Procedures. Eds. Kilbey, B.S., M. Legator, W. Nichols and C. Ramel, BV:Elsevier Science Publishers, ISO/IEC 17025, 2005, General Requirements for the Competence of Testing and Calibration Laboratories. 3.0 COMPLIANCE The study conformed to the current FDA 21 CFR, Part 58 Good Laboratory Practice for Non Clinical Laboratory Studies and OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring guidelines (OECD ENV/MC/CHEM(98)17). 4.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES The Sponsor supplied the following information on a test requisition form or other correspondence, wherever applicable (excluding confidential or trade secret information). The Sponsor was responsible for all test substance characterization data as specified in the GLP regulations. 4.1 Test Substance: Test Substance Name: OIS 1220 CAS/Code #: Not Supplied by Sponsor (N/S) Lot/Batch #: 08B0008-RD Page 6 of 15

7 Physical State: N/S Color: N/S Expiration Date: N/S Density: N/S Stability: N/S Solubility: N/S ph: N/S Storage Conditions: Room Temperature Safety Precautions: Standard Toxikon Laboratory Safety Precautions 4.2 Control Substance(s) (Toxikon Supplied, unless specified by the Sponsor): Negative Control Substance Name: Fischer s Cell Culture Medium Toxikon QC #: LPR GT Physical State: Liquid Color: Pink Stability: Stable Storage Conditions: 4 ± 2 C Safety Precautions: Standard Laboratory Safety Precaution Positive Control Substance Name: Methylmethanesulfonate (MMS) Toxikon QC #: CSC GT Physical State: Liquid Color: Colorless Stability: Stable Storage Conditions: 4 ± 2 C Safety Precautions: Known mutagen. Appropriate Laboratory Safety Practices Apply Positive Control Substance Name: Dimethylbenzanthracene (DMBA) Toxikon QC #: CSC GT Physical State: Solid Color: Yellow Stability: Stable Storage Conditions: 4 ± 2 C Safety Precautions: Known mutagen. Appropriate Laboratory Safety Practices Apply. 5.0 IDENTIFICATION OF TEST SYSTEM The L5178Y (TK +/ ) mouse lymphoma cells are obtained from the American Type Culture Collection (Manassas, Virginia), Cell Line # CRL The test substance was administered in vitro, through a solvent compatible with the test system. This is the optimal route of administration available in this test system, per Sponsor specification. Page 7 of 15

8 6.0 JUSTIFICATION OF TEST SYSTEM Historically, the L5178Y (TK +/ ) Mouse Lymphoma Mutagenesis Assay has been demonstrated to be effective in detecting the mutagenic activity of chemicals. This assay evaluated the ability of the test article to induce an increase in the formation of homozygous thymidine kinase mutants (TK / ) over the background rate in a mouse lymphoma cell line, in the presence and absence of a metabolic activation system. The L5178Y is a mutant cell line, heterozygous at the thymidine kinase locus (TK +/ ). 7.0 EXPERIMENTAL DESIGN AND DOSAGE 7.1 Preparation of Test Cultures: The L5178Y (TK +/- ) cells were routinely grown in Fischer s cell culture medium, supplemented with 10% heat inactivated horse serum and 0.1% pluoronic F680 (complete medium). The incomplete medium (serum free) was designated as F 0 P and complete medium as F 10 P. Subcultures of L5178Y (TK +/- ) mouse lymphoma cells were periodically cleansed of spontaneously occurring TK -/- cells by growing the cells in F 10 P containing a mixture of thymidine, hypoxanthine, methotrexate, and glycine (THMG). The THMG was removed, and the cells were grown for an additional 24 hours in culture medium containing thymidine, hypoxanthine, and glycine (THG). Cultures of recently cleansed cells were used in the Range Finding Assay and the Mouse Lymphoma Assay. The test substance at neat and various lower concentrations was combined with 2 X F 0 P at 1:1 ratio and used for exposure. 7.2 Mouse Lymphoma Assay Procedure: Range Finding Assay: An initial range finding assay was performed to evaluate toxicity of the test substance using neat, 1:2, 1:4, 1:8, 1:16, and 1:32 dilutions. The toxicity was evaluated based on relative total growth (RTD) after a 4 hour exposure in the absence of metabolic activation Definitive Assay: Based on the results of the range finding assay showing complete toxicity at the neat concentration and partial toxicity at 1:2 and 1:4 dilutions with 27.5 and 45.8% survival respectively, the definitive assay was conducted using 1:2, 1:4, 1:8, and 1:16 dilutions of the test substance. Non activated dosing solutions contained the test substance or controls in serum free medium only. The activated dosing solutions contained the test substance or controls in serum free medium and the metabolic activation mixture. Cultures were exposed at 37 ± 1 C, 4 6% CO 2, in a humidified atmosphere for 4 hours. After the 4 hour exposure period, the cells were pelleted by centrifugation. The test substance was removed by aspirating the supernatant. The cells were rinsed with serum free medium, resuspended in 20 ml of culture medium, and incubated at 37 ± 1 C. Page 8 of 15

9 7.2.3 Metabolic Activation (S 9): An S 9 microsome fraction from the livers of Aroclor 1254 induced Sprague Dawley rats was obtained from Moltox Inc, (Boone, NC). The S9 was stored at 80 C until use. The S 9 fraction is combined with cofactors and culture medium to form the metabolic activation system (activation mixture) Expression: At 20 and 44 hours post exposure intervals, an aliquot of each culture was removed and the cell population density was determined. A trypan blue exclusion method enumerated the viable cell number. The test cultures were measured at 20 and 44 hours post exposure for cell concentrations and adjusted to cells/ml. After the two day expression period, cultures having a population density of cells/ml or greater were selected for cloning Selective Growth: Following the expression period, cells were grown in cloning medium in the presence of a selective agent to quantitate the mutation frequency. The cloning medium contains F 20 P (Complete medium containing 20% heat inactivated horse serum or other appropriate serum), 0.38% soft agar and 1 µg/ml of the selective agent, trifluorothymidine (TFT). A total of six dishes were used for each dose level at a cell density of cells/dish. The cultures were incubated for days at 37 ± 1 C to allow colonies to develop Parallel Cloning Efficiency: Concurrently, cells were grown in cloning medium without the selective agent to determine the cloning efficiency. A total of 6 dishes were plated for each dose level at a density of 200 cells/dish. The cultures were then incubated as described above for days to allow colony formation. The relative cloning efficiency (RCE) was determined for each culture. 7.3 Controls: Negative Control: The L5178Y (TK +/- ) cells were dosed with the negative control (extracting media: cell culture medium) in the presence and absence of a metabolic activation system Positive Control: MMS served as the positive control for the non activated assay. It was added directly to the dosing solution at a concentration of 0.5 µl/ml. DMBA served as the positive control for the activated assay. It was dissolved in an appropriate solvent and added directly to the dosing solution at a concentration of 6 µg/ml. 8.0 DOSAGE: Based upon dose range finding studies, test concentrations (namely 1:2, 1:4, 1:8, and 1:16 dilutions) of the test substance were used to expose the cells in the mutagenicity experiment. The test substance was combined with 2 X F 0 P at 1:1 ratio and used for exposure. Page 9 of 15

10 9.0 EVALUATION CRITERIA 9.1 Evaluation of Test Results: The results of the Mouse Lymphoma Mutagenesis Assay will be evaluated on the basis of the number of TFT resistant mutants per surviving cells. The significance of the test results will be determined by: Statistical Analysis: The test results will be analyzed using a statistical methods such as the t-test using Graph Pad Prism Software by Analytical Software, Inc. (Analyzing Data with Graph Pad Prism, Harvey Motulsky). This statistical method determines if there is a significant (p < 0.05) increase in the mutation frequency of the test article compared to the negative control group. A dose related response will be determined, if appropriate, by a linear regression analysis Positive Response: Single Test Dose: The test substance would be considered to have caused a positive response in the assay if a reproducible and statistically significant increase in the number of mutants over its concurrent negative control is observed Multiple Test Doses: The test substance would be considered to have caused a positive dose response in the assay if a dose-related increase in the number of mutants with r 0.95 (obtained from the Linear Regression data analysis) is observed, with at least one test substance dose showing a reproducible and statistically significant increase (p 0.05) over the negative control. 9.2 Confirmatory Assay: The confirmatory assay will be conducted as described for the mutagenesis assay (Section 7.2) in the case of negative results. 9.3 The study and its design will employ methodology to minimize uncertainty of measurement and control of bias for data collection and analysis RESULTS 10.1 Range Finding Assay: The results of the range finding assay showed that there was complete toxicity (100%) at the neat concentration whereas 1:2 and 1:4 dilutions induced partial toxicity showing 27.5 and 45.8% viability, respectively. No significant reduction in the viable cells was observed in any of lower dilutions of the test substance treatment groups compared to the negative control Mouse Lymphoma Assay: There was no significant increase (p > 0.05) in the frequency of homozygous mutants in any of the test substance treatment groups compared to the respective negative control groups in both non activated and activated conditions (Tables 1 2). Page 10 of 15

11 10.3 Confimatory Assay: The results of the confirmatory assay were consistent with the negative results of the definitive mouse lymphoma assay (Tables 3 4) CONCLUSION The test substance, OIS 1220, was evaluated for its potential to induce a statistically significant increase in the number of homozygous thymidine kinase mutants (TK / ) over the background frequency in a mutant mouse lymphoma L5178Y cell line, heterozygous at the thymidine kinase locus (TK +/ ). An initial range finding assay was performed to determine cytotoxicity and select test concentrations of the test substance for the definitive assay. The range finding assay was conducted by exposing L5178 cells to the neat, 1:2, 1:4, 1:8, 1:16, and 1:32 dilutions of the test substance (made with Sterile Water for Injection (SWFI)) for four hours in the absence of metabolic activation. Results showed that there was complete toxicity (100%) at the neat concentration whereas the 1:2 and 1:4 dilutions induced partial toxicity showing 27.5 and 45.8 % survival, respectively. No significant reduction in the viable cells was observed in any of lower dilutions of the test substance treatment groups compared to the negative control. Based on the results of the range finding assay, the definitive assay was conducted exposing cells to the 1:2, 1:4, 1:8, and 1:16 dilutions of the test substance in the presence and absence of metabolic activation. The results of the definitive assay showed that there was no significant increase in the incidence of homozygous mutants in any of the test substance treatment groups compared to the respective negative control groups in both non-activated and activated conditions. There was a statistically significant increase in the incidence of homozygous mutants in cells exposed to the positive control substances in both non activated and activated conditions, validating the functioning of the assay. A confirmatory assay was performed to verify the results of the definitive assay and the results were consistent with the negative results of the definitive assay. Based on the criteria of the study protocol, the test substance is considered non mutagenic in the Mouse Lymphoma Mutagenesis Assay RECORDS 12.1 Original raw data are archived at Toxikon Corporation A copy of the final report and any report amendments is archived at Toxikon Corporation The original final report, and a copy of any protocol amendments or deviations, is forwarded to the Sponsor All unused test article shall be returned to the Sponsor by Toxikon, per Sponsor s request CONFIDENTIALITY AGREEMENT Statements of confidentiality were agreed upon prior to study initiation. Page 11 of 15

12 Test Substance : OIS 1220 Lot/Batch #: TABLE 1 TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Non-Activated Assay) 08B0008-RD Selective Growth Groups Average Negative Normalized* Positive Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Groups Average Surviving Fraction** Negative Positive Test Substance: 1: Test Substance: 1: Test Substance: 1: Test Substance: 1: * Normalized to Surviving Fraction: (Number of Cells/Surviving Fraction). Mutants per 1 x 10 6 cells. ** Number of cells surviving/number of cells seeded. Parallel Cloning Efficiency Page 12 of 15

13 Test Substance : OIS 1220 Lot/Batch #: TABLE 2 TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Activated Assay) 08B0008-RD Selective Growth Groups Average Negative Normalized* Positive Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Parallel Cloning Efficiency Groups Average Surviving Fraction** Negative Positive Test Substance: 1: Test Substance: 1: Test Substance: 1: Test Substance: 1: * Normalized to Surviving Fraction: (Number of Cells/Surviving Fraction). Mutants per 1 x 10 6 cells. ** Number of cells surviving/number of cells seeded. Page 13 of 15

14 Test Substance : OIS 1220 Lot/Batch #: TABLE 3 TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Non-Activated Assay) - Confirmatory Assay 08B0008-RD Selective Growth Groups Average Negative Normalized* Positive Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Groups Average Surviving Fraction** Negative Positive Test Substance: 1: Test Substance: 1: Test Substance: 1: Test Substance: 1: * Normalized to Surviving Fraction: (Number of Cells/Surviving Fraction). Mutants per 1 x 10 6 cells. ** Number of cells surviving/number of cells seeded. Parallel Cloning Efficiency Page 14 of 15

15 Test Substance : OIS 1220 Lot/Batch #: TABLE 4 TFT Resistant Mutant Frequency and Parallel Cloning Efficiency of the Negative Control, Positive Control, and Test Substance (Activated Assay) - Confirmatory Assay 08B0008-RD Selective Growth Groups Average Negative Normalized* Positive Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Test Substance: 1: Normalized* Groups Average Surviving Fraction** Negative Positive Test Substance: 1: Test Substance: 1: Test Substance: 1: Test Substance: 1: * Normalized to Surviving Fraction: (Number of Cells/Surviving Fraction). Mutants per 1 x 10 6 cells. ** Number of cells surviving/number of cells seeded. Parallel Cloning Efficiency Page 15 of 15