MODULE TOXICOLOGY WRITTEN SUMMARY

Transcription

1 MODULE TOXICOLOGY WRITTEN SUMMARY

2 TABLE OF CONTENTS PAGE 1. BRIEF SUMMARY SINGLE DOSE TOXICITY Introduction Rat Intramuscular and subcutaneous administration Dog Oral administration Monkey Oral administration Intramuscular and subcutaneous administration REPEAT DOSE TOXICITY Introduction Mouse Oral administration day study week study Rat Oral administration day study week toxicity study week toxicity study Monkey Oral administration day study week toxicity study week toxicity study GENOTOXICITY Introduction In Vitro Studies (Dolutegravir) Non-mammalian cell systems Ames assay Mammalian cell systems Mutation test at the thymidine kinase locus in mouse lymphoma L5178Y cells In Vivo Studies (Dolutegravir) Oral micronucleus test in the rat Potential Impurities gsk001* Standard Ames test Mouse lymphoma assay gsk002* Standard Ames test gsk003* Standard Ames test * 新薬承認情報提供時に置き換え

3 4.5. Route Assessment for Genotoxic Impurities CARCINOGENICITY Introduction Mouse Oral administration Carcinogenicity study Rat Oral administration Carcinogenicity study REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Introduction Fertility and Early Embryonic Development Rat Embryofetal Development Studies Rat Oral administration Rabbit Oral administration Pre-Natal and Post-Natal Development, Including Maternal Function Rat Juvenile Toxicity Studies Rat Tolerability Dose range study Definitive Study LOCAL TOLERANCE Introduction Dermal Irritancy In vitro Rabbit Ocular Irritancy In vitro Rabbit Skin Sensitisation Potential Mouse OTHER TOXICITY STUDIES Introduction Immunotoxicity T-cell dependent antibody response DISCUSSION AND CONCLUSIONS REFERENCES...74 APPENDIX 1 CARCINOGENICITY TABLES...75 APPENDIX 2 ADDITIONAL INFORMATION

4 LIST OF TABLES PAGE Table 2.1 Table 2.2 List of Single Dose Toxicity Studies Performed with Dolutegravir...11 Batch Numbers of Dolutegravir and Formulations Used in Single Dose Toxicity Studies...16 Table 3.1 List of Repeat Dose Toxicity Studies Performed with Dolutegravir...18 Table 3.2 Table 4.1 Table 4.2 Table 5.1 Table 5.2 Table 6.1 Table 6.2 Table 7.1 Table 7.2 Batch Numbers of Dolutegravir and Formulations Used in Repeat Dose Toxicity Studies...27 List of Genotoxicity Studies Performed with Dolutegravir and Potential Impurities...29 Batch Numbers of Dolutegravir and Formulations Used in Genotoxicity Studies...35 List of Carcinogenicity Studies Performed with Dolutegravir...37 Batch Numbers of Dolutegravir and Formulations Used in Carcinogenicity Studies...40 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir...42 Batch Numbers of Dolutegravir and Formulations Used in Reproductive and Developmental Toxicity Studies...51 List of Local Tolerance Studies Performed with Dolutegravir...53 Batch Numbers of Dolutegravir and Formulations Used in Local Tolerance Studies...57 Table 8.1 List of Other Toxicity Studies Performed with Dolutegravir...59 Table 8.2 Table 9.1 Table 9.2 Table 9.3 Batch Numbers of Dolutegravir and Formulations Used in Other Toxicity Studies...61 Principal Toxicological Findings in Rats and Monkeys Following Oral Administration of Dolutegravir...68 Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir...69 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M 2 ) Following Oral Administration of Dolutegravir in the 4 and 26 week Rat Toxicology Studies

5 Table 9.4 Table.1 Table.2 Table.3 Table.4 Table.5 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M 2 ) Following Oral Administration of Dolutegravir in the 14 Day and 4 and 38 Week Monkey Toxicology Studies...73 List of Tissues Studied in Carcinogenicity Studies...76 Carcinogenicity Study in Mice - Tumor Incidence...77 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence...86 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence...87 Carcinogenicity Study in Mice - Summary of Survival and Fate of Animals...89 Table.6 Carcinogenicity Study in Rats - Tumor Incidence Table.7 Table.8 Table.9 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence Carcinogenicity Study in Rats - Summary of Survival and Fate of Animals

6 1. BRIEF SUMMARY Dolutegravir has been tested in a comprehensive toxicology development program. Single dose intramuscular and subcutaneous studies have been performed in the rat and monkey, and repeat oral dose studies for durations of up to 26 weeks in the rat and up to 38 weeks in the monkey have been performed. The potential for genetic toxicity of dolutegravir has been investigated in the Ames test, mouse lymphoma assay, and micronucleus test, and the tumorigenic potential has been studied in oral carcinogenicity studies in the mouse and the rat. A range of reproductive toxicity studies have been performed in the rat and rabbit following oral administration. Additionally, studies investigating local tolerance and immunotoxicity have been performed. A listing of all of the pivotal toxicology studies performed with dolutegravir is given in Table 1.1. Table 1.1 Listing of Pivotal Toxicology Studies Performed With Dolutegravir Study Type and Duration Repeat Dose Toxicity Up to 26 weeks Up to 38 weeks Genotoxicity Ames test Mouse lymphoma assay Micronucleus Route of Administration Oral Oral In vitro In vitro Oral Species Rat Monkey Bacterial cells L5178Y cells Rat Carcinogenicity Oral Mouse and Rat Reproductive and Developmental Fertility/early embryonic development Embryofetal development Pre- and post-natal development Juvenile toxicity Oral Oral Oral Oral Rat Rat, Rabbit Rat Rat Immunotoxicity Oral Rat Local Tolerance Dermal irritancy Ocular irritancy Local lymph node assay In vitro In vivo In vitro In vivo Topical Human cell model Rabbit Human cornea Rabbit Mouse Studies were carried out by the oral route of administration as this is the proposed therapeutic route in humans. The species and strain used in these investigations were selected on the basis of similarities in the pharmacokinetic and metabolic handling of 6

7 dolutegravir between the selected species and humans. Furthermore, for all of the species and strains used in the toxicological evaluation of dolutegravir, considerable data on the background pathology and the response to a wide variety of pharmaceuticals are available to assist in the evaluation of the findings and extrapolation of their relevance to humans. All studies described in this section were performed using the sodium salt of dolutegravir (unless otherwise indicated) which is the form proposed for use in humans; however, all dosages and concentrations quoted in this summary are expressed in terms of the parent compound (referred to simply as dolutegravir). All definitive studies were performed in full compliance with Good Laboratory Practice (GLP) regulations. Studies undertaken to establish suitable dose levels were performed in accordance with the general principles of GLP regulations. A list of studies that were undertaken with dolutegravir but are not included in this submission is presented in Appendix 2. The impurity profiles of the drug substance batches used in the nonclinical toxicology studies were comparable to the impurity profile of the material used in clinical investigations and that proposed for use in the marketed product. There are 7 specified impurities in the proposed dolutegravir Drug Substance Specification [m3.2.s.4.5]. Drug substance batch numbers and details of the formulations used in the toxicology studies are presented. A genotoxic assessment of the dolutegravir drug substance impurities is provided in Section 4.5. A brief summary of the important findings from the toxicology studies is provided below. Details of the study designs and important findings are presented in Sections 2 to 8, and in m2.6.7, Table 5.1 to Table A critical assessment of all of the toxicology data is presented in Section 9, Discussion and Conclusions. Additional tables for carcinogenicity studies are presented in Appendix 1. Single Dose Toxicity Single dose oral acute toxicity studies have not been conducted with dolutegravir; however, the potential for acute toxicity was assessed in repeat dose studies at the highest possible systemic exposure based on saturation of absorption (rat) or highest tolerable dose (monkey). Repeat Dose Toxicity The primary finding from repeat dose toxicity studies up to 26 weeks in rats and 38 weeks in monkeys with dolutegravir was gastrointestinal (GI) toxicity, which is believed to be the result of local drug administration at the mucosal surface of the gut following oral dosing, rather than systemic toxicity. GI toxicity in animals did not translate to an increased risk for clinical adverse events at dolutegravir doses of 50 mg QD or 50 mg BID. Liver effects were seen in the 14-day monkey study in male monkeys that received a dose that exceeded the maximum tolerated dose (1000 mg/kg/day) and consisted of hepatocellular single cell necrosis and diffuse hepatocellular hypertrophy and/or vacuolation. Additional changes included transient ALT increases at 7

8 300 mg/kg/day, increased AST, bilirubin, GTP, and triglycerides at 1000 mg/kg/day and decreased total cholesterol at 1000 mg/kg/day. Human subjects were carefully monitored for liver effects and cumulative data to date suggests a hepatic safety profile for dolutegravir that is comparable to raltegravir and efavirenz, the comparators used in the Phase III studies. Genotoxicity Dolutegravir was negative in in vitro and in vivo genetic toxicology assessments and there are no impurities of mutagenic concern, indicating that dolutegravir does not pose a genotoxic risk in humans. Carcinogenicity Dolutegravir was not carcinogenic to mice at doses up to 500 mg/kg/day or rats at doses up to 50 mg/kg/day following oral administration for 104 consecutive weeks. In both species, dolutegravir administration had no effect on survival, there were no treatment related clinical signs, and there were no neoplastic or non-neoplastic findings attributed to dolutegravir. Reproductive and Developmental Toxicity Dolutegravir had no effects on male or female fertility in rats and no effect on embryofetal development in pregnant rats or rabbits. Based on animal data, dolutegravir is not anticipated to increase the risk of adverse developmental (or reproductive) outcomes in humans when used in accordance with dosing information in the product label. Dolutegravir administration resulted in suppressed body weight gain and decreased food consumption during the lactation period in a pre- and postnatal development study in rat dams (F0) receiving 1000 mg/kg/day. Associated with the maternal toxicity, decreased body weights were noted in the offspring (F1) in the 1000 mg/kg group from pre-weaning until adolescence. The NOAEL for postnatal development of the offspring (F1) was 50 mg/kg/day. Based on the fact that effects on offspring body weights were noted at doses where maternal toxicity was observed, and the presence of considerable safety margins expected at the proposed clinical doses, there is minimal risk for adverse effects on post-natal development in offspring of mothers receiving dolutegravir. In a juvenile toxicity study in rats, dolutegravir administration resulted in two preweanling deaths at 75 mg/kg/day. There were no new target organs identified in juveniles compared to adults and the NOAEL in juvenile rats was 2 mg/kg/day. 8

9 Local Tolerance In vitro, dolutegravir is slightly/mildly irritating to skin and ocular model systems. There was no indication of contact sensitization in a mouse local lymph node assay when dolutegravir was administered topically. Immunotoxicity Dolutegravir was not immunotoxic, as assessed by T cell dependent antibody response (TDAR), in adult rats at doses 1000 mg/kg/day. In juvenile rats, there were no test article-related effects on TDAR, and no effects on lymphocyte subsets (T cells, both CD4 and CD8 subsets, and B cells) and CD4 or CD8 T cell receptor V usage in peripheral blood. 9

10 2. SINGLE DOSE TOXICITY In accordance with ICH M3 (R2), acute toxicity information can be obtained from appropriately conducted dose-escalation studies or short-duration dose-ranging studies that define an MTD in the general toxicity test species. Therefore, acute toxicity studies have not been conducted; however, a series of single dose toxicity studies were designed and conducted during the development of dolutegravir and these studies are described in this section. In addition, repeat dose oral toxicity studies investigated the effects of repeated high doses (up to 500 mg/kg/day in the rat and up to 1000 mg/kg/day in the monkey) and are discussed in Section Introduction Single dose toxicity studies were performed in order to assess the effects of administration of oral, subcutaneous and intramuscular doses of dolutegravir, and to compare the toxicokinetics for the different routes of administration. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 2.2 of this summary. A full listing of the single dose studies performed, together with the location of the reports within this submission and their GLP status, is provided in Table

11 Table 2.1 List of Single Dose Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Single Dose Rat (Sprague Dawley) 6M 6M Subcutaneous Intramuscular 2.5 Single No GSK RD2009/00921 (R42470) m Single Dose Rat (Sprague Dawley) 3M 3M Subcutaneous Intramuscular 2.5, 5 Single No GSK RD2009/00959 (R42475) m Single Dose Rat (Sprague Dawley) 3M Intramuscular 4, 7.3, 8.7 Single No GSK 2011N (R42826) m Single Dose Rat (Sprague Dawley) 3M Intramuscular 10 Single No GSK 2012N m Single Dose * Dog (beagle) 1F Oral 30, 100, 150, 250, 500 Single No RD2009/00963 m Single Dose Monkey (cynomolgus) 4F 4F 4F Oral Subcutaneous Intramuscular Single No GSK CD2009/00647 (D09113) m Single Dose Monkey (cynomolgus) 1F Oral (gavage) 50, 125, 250, 500 Single No RD2007/01184 (E TF-008-R) m Single Dose Monkey (cynomolgus) 3F Oral (gavage) 1, 3, 10, 50 Single No RD2008/01762 (S TB-44-R) m Note: All single dose oral studies were performed with dolutegravir, sodium salt form, while the subcutaneous and intramuscular studies were performed with the parent form of dolutegravir. * Dolutegravir is referred to in this report as MTS B. Testing Facility: GSK = GlaxoSmithKline = 11

12 2.2. Rat Intramuscular and subcutaneous administration The toxicokinetics and potential toxicity of dolutegravir (2.5 mg/kg, parent form) were investigated in male rats (n=6/group) following a single intramuscular or subcutaneous injection [Report RD2009/00921, m ]. Dolutegravir was formulated as a longacting parenteral wet bead-milled suspension in 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH 2 PO 4 H 2 O, 0.006M Na 2 HPO 4 and 0.83% NaCl to investigate the feasibility for administration as a parenteral formulation. Clinical observations, toxicokinetics, and body weights were evaluated. A tabulated summary of this study is presented in m2.6.7, Table 5.1. Plasma concentrations of dolutegravir were quantifiable up to Day 15 following the subcutaneous route and up to Day 8 following the intramuscular dose. Mean C max, AUC 0-24 and AUC 0-t values for dolutegravir were similar between both routes of administration. No test article-related clinical signs were observed and there was no evidence of injection site irritation. An additional study was conducted with single doses of dolutegravir (2.5 and 5 mg/kg, parent form) administered by both intramuscular and subcutaneous routes to male rats (n=3/group) [Report RD2009/00959, m ]. Dolutegravir was formulated as a homogenized suspension, at a larger particle size than had been previously administered, in 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH 2 PO 4 H 2 O, 0.006M Na 2 HPO 4 and 0.83% NaCl. Clinical observations, toxicokinetics, and body weights were evaluated. A tabulated summary of this study is presented in m2.6.7, Table 5.1. Mean dolutegravir C max, AUC 0-24, and AUC 0-t values for the 2.5 or 5 mg/kg doses were similar between subcutaneous and intramuscular administration. The mean AUC 0-t increased 2.3- to 3.2-fold with the increase in dose from 2.5 to 5 mg/kg. Both dose levels of dolutegravir were well tolerated. Groups of male rats (n=3/group) were administered single intramuscular doses of dolutegravir at 4, 7.3, or 8.7 mg/kg to assess the tolerability and toxicokinetics of three formulations [Report 2011N123574, m ]. The formulations were comprised as follows: 20 mg/ml polysorbate 20, 20 mg/ml polyethylene glycol 3350, 45 mg/ml mannitol in sterile water (Formulation 1); 20 mg/ml polysorbate 20, 20 mg/ml polyethylene glycol 3350, 45 mg/ml mannitol, 10 mg/ml sodium carboxymethylcellulose in sterile water (Formulation 2); or sesame oil (Formulation 3). A summary of the study design and the results from this study are presented in m2.6.7, Table

13 No injection site irritation was noted with dolutegravir in any of the formulations. None of the three formulations provided sustained concentrations of dolutegravir >60 ng/ml for at least 43 days. In another study, groups of male rats (n=3/group) were administered single intramuscular doses of dolutegravir at 10 mg/kg to assess the tolerability and toxicokinetics of multiple vehicles [Report 2012N136936, m ]. The following endpoints were evaluated: clinical observations, irritation scoring (Draize method; up to Day 14), body weights, and toxicokinetic evaluations were performed on plasma samples collected on Days 1 (0.5, 2, 4, 8, 12 hours post-dose), 2, 3, 4, 6, 8, 15, 22, 29, 36, and 43 post-dose. No injection site irritation (erythema or edema) was noted with any formulation. Body weights were slightly decreased on Day 43 (relative to controls) for rats given 10 mg/kg in microparticles with low drug loading of 40%, 10 mg/kg in microparticles with high drug loading of 70%, 10 mg/kg in microparticles with low drug loading of 40% and micronized free acid, and 10 mg/kg in micronized free suspension. Dosing dolutegravir in the in situ gel with slow release provided the longest period of plasma concentrations above μg/ml (PA-IC 90 [PA is protein adjusted]). The higher systemic exposure (mean C max and AUC 0-t ) values were achieved with the in situ gel with fast release, the in situ gel with slow release, or micronized free acid suspension Dog Oral administration In a toxicity and toxicokinetics study, single oral doses of dolutegravir at 30, 100, 150, 250 and 500 mg/kg were administered to female beagle dogs (n=1/group) [Report RD2009/00963, m ]. Body weights, clinical observations, food consumption, urinalysis and blood chemistry were determined. Toxicokinetic evaluation was performed on samples collected following each single dose. A tabulated summary of this study is presented in m2.6.7, Table 5.1. Vomiting or vomitus was observed in the animals given 150 mg/kg between thirty minutes and two hours after dosing. It was speculated that vomiting or vomitus had influenced exposure level of the animals given 150 mg/kg, and thus it was difficult to achieve an appropriate dose relationship. These results indicate the difficulty of evaluating the toxicity of dolutegravir in dogs Monkey Oral administration In a toxicity and toxicokinetics study, single oral doses of dolutegravir at 50, 125, 250 and 500 mg/kg were administered to fasted female cynomolgus monkeys (n=1/group) [Report RD2007/01184, m ]. Dolutegravir was formulated in 0.5% HPMC with 0.1% Tween 80. The following were evaluated: clinical observations, body weights, food consumption, hematology, clinical chemistry and toxicokinetic analysis. 13

14 Toxicokinetic evaluation was performed on samples collected following each single dose. A tabulated summary of this study is presented in m2.6.7, Table 5.1. In the toxicokinetic analysis, no dose correlation was noted in the C max or AUC 0-24 values. There were no apparent differences in the C max values between 125 and 500 mg/kg and the AUC 0-24 value at 500 mg/kg was approximately 1.4-fold the respective AUC at 125 mg/kg. No adverse effects on body weight, clinical observations or clinical pathology occurred in monkeys at single oral doses of up to 500 mg/kg. In a single dose toxicokinetic study conducted to aid in dose selection for the 38 week toxicity study, dolutegravir was administered orally to 3 female monkeys at doses of 1, 3, 10 and 50 mg/kg, with 7 days between doses [Report RD2008/01762, m ]. Dolutegravir was formulated in 0.5% HPMC with 0.1% Tween 80. Clinical observations, body weights, food consumption and toxicokinetics were evaluated. A tabulated summary of this study is presented in m2.6.7, Table 5.1. The exposure to dolutegravir in this study was generally dose-related. However, large inter-animal differences were noted at doses 10 mg/kg and higher. No treatment related effects on body weight, food consumption and clinical observations were noted in any animal or dose. Dolutegravir was also administered as a single oral dose to female monkeys (n=4) at a dose level of 3 mg/kg [Report CD2009/00647, m ]. Dolutegravir was formulated as a suspension in 0.5% HPMC and 0.1% Tween 80. Data were collected for clinical observations (including qualitative assessment of food consumption), toxicokinetics and body weights. A tabulated summary of this study is presented in m2.6.7, Table 5.1. The exposure values (toxicokinetics) collected after single oral doses were used for comparison with the exposure values obtained on the same study after single subcutaneous or intramuscular administration (see Section below for discussion of other routes of administration). Dolutegravir was well tolerated after a single oral dose of 3 mg/kg Intramuscular and subcutaneous administration Dolutegravir (parent form of the drug) was administered as a single intramuscular or subcutaneous injection to female monkeys (n=4/group) at a dose of 1 mg/kg [Report CD2009/00647, m ]. Dolutegravir was formulated as a suspension in 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH 2 PO 4 H 2 O, 0.006M Na 2 HPO 4 and 0.83% NaCl to investigate the feasibility of administration as a long acting parenteral formulation. Data were collected for clinical observations (including qualitative assessment of food consumption), toxicokinetics and body weights. A tabulated summary of this study is presented in m2.6.7, Table

15 Mean dose-normalized C max following single oral administration (3 mg/kg, from same study, discussed in Section 2.4.1) was higher than that following subcutaneous administration; however, the mean dose-normalized AUC 0-24 following oral administration was similar to the dose-normalized AUC 0-t following subcutaneous administration. There were no marked differences ( 2-fold) in the mean C max, AUC 0-24 or AUC 0-t values following subcutaneous or intramuscular administration. Dolutegravir was well tolerated and no clinical observations were noted at the injection sites. 15

16 Table 2.2 Batch Numbers of Dolutegravir and Formulations Used in Single Dose Toxicity Studies Batch or Lot Number(s) Route of Administration Species (strain) Duration of Dosing Report No. (Study No.) Concentration (mg/ml) Formulation N a IM, SC Rat (Sprague Dawley) Single RD2009/00921 (R42470) 20 2 N b IM, SC Rat (Sprague Dawley) Single RD2009/00959 (R42475) 20, 40 2 ee a, ee b, and ee c IM Rat (Sprague Dawley) IM Rat (Sprague Dawley) 05 Oral Monkey (cynomolgus) Single 2011N (R42826) 10, 20, 45 Multiple (refer to report) Single 2012N to 33 Multiple (refer to report) Single RD2007/01184 (E TF-008-R) 10, 25, 50, A7Z001 Oral Monkey Single RD2008/ , 0.03, 0.1, 1 (cynomolgus) (S TB-044-R) 0.5% w/v A7Z002 N Oral IM, SC Monkey (cynomolgus) Single CD2009/00647 (D09113) Note: The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. All single dose oral studies were performed with dolutegravir, sodium salt form, while the subcutaneous and intramuscular studies were performed with the parent form of dolutegravir. Key: Formulation Code: IM = Intramuscular. 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 SC = Subcutaneous. 2 = 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, a = Long-acting parenteral wet bead-milled suspension M NaH 2PO 4H 2O, 0.006M Na 2HPO 4 and 0.83%NaCl. b = Homogenized suspension, at a larger particle size

17 3. REPEAT DOSE TOXICITY 3.1. Introduction Repeat dose toxicity studies investigating the effects of repeated administration of dolutegravir have been performed in Sprague Dawley rats and cynomolgus monkeys. These were preceded, where appropriate, by preliminary studies to establish the maximum repeatable daily doses for use in the definitive studies. Repeat dose toxicity studies up to 13 weeks in duration have also been conducted in CD-1 mice to establish suitable dose levels for use in oral carcinogenicity studies. To support the long-term therapeutic use of Dolutegravir Sodium Tablets, studies were performed by the oral route of administration for periods up to 26 weeks in the rat and 38 weeks in the monkey. Sufficient animals were used in each study to allow meaningful evaluation of the data. Studies undertaken to establish suitable dose levels for use in repeat dose toxicity studies were performed in accordance with the general principles of Good Laboratory Practice (GLP) regulations. All definitive studies were performed in full compliance with GLP regulations. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 3.2 of this summary. A full listing of the repeat dose toxicity studies performed, together with the location of the reports within this submission and their GLP status, is provided in Table 3.1. An inter-species comparison of dolutegravir plasma concentrations following repeated administration in the definitive toxicity studies is presented in Section 9. A tabular summary of the repeat dose toxicokinetic data derived from the toxicity studies is presented in m2.6.7, Table

18 Table 3.1 List of Repeat Dose Toxicity Studies Performed with Dolutegravir Type of Study Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Species (Strain)/ Test System Mouse (CD-1) Mouse (CD-1) Rat (Sprague Dawley) Rat (Sprague Dawley) Rat (Sprague Dawley) Monkey (cynomolgus) Monkey (cynomolgus) Monkey No./Sex/ Group 10M/10F 10M/10F 10M/10F a 10M/10F a,b 12M/12F d 3M/3F 3M/3F c 7M/7F Method of Administration Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral Dose (mg/kg/day) or Concentration Duration of Dosing (cynomolgus) (gavage) Note: All studies were performed with GSK A, the sodium salt form, unless otherwise noted. No observed adverse effect levels (NOAEL) are bolded. Key: a = Additional 4M/4F per group included for toxicokinetic parameters only. b = An additional 5M/5F were included at 1000 mg/kg/day for a 4 week recovery investigation. c = An additional 2M/2F were included at 100 mg/kg/day for a 4 week recovery investigation. d = An additional 6M/6F per group included for toxicokinetic parameters only. e = An additional 10M/10F per group were killed after 17 weeks of treatment for interim examinations. f = An additional 6M/6F were added at 500 mg/kg/day group for a 4 week recovery period. g = An additional 2 to 3M/2 to 3F per group were killed after 17 weeks of treatment for interim examinations. h = An additional 2M/2F were added at 15 and 50/30 mg/kg/day group for a 4 week recovery period. GLP Testing Facility Report No. (Study No.) 10, 100, 500, days No RD2009/01546 (S TF-066-R) 10, 50, 500, weeks Yes RD2009/00028 (S TF-068-L) 50, 150, days Yes RD2007/01140 (E TB-012-L) 2, 10, 100, weeks Yes RD2008/01628 (E TB-043-L) 5, 50, weeks e,f Yes RD2009/00410 (SBL ) 100, 300, days Yes RD2007/01142 (E TF-029-L) 25, 50, weeks Yes RD2008/00107 ((E TF-036-L) 3, 10, 15, 50/30 38 weeks g,h (SBL ) Yes RD2009/00036 Testing Facility: = = = = Location in CTD m m m m m m m m

19 3.2. Mouse Oral administration day study In a 14 day oral repeat dose study, dolutegravir was administered to mice at doses of 0 (control), 10, 100, 500, or 1500 mg/kg/day [Report RD2009/01546, m ]. Dolutegravir was formulated with 0.5% HPMC and 0.1% Tween 80. Parameters that were evaluated during the study were viability, clinical observations, toxicokinetics, body weights, food consumption, hematology and clinical chemistry, organ weights, macroscopic observations and microscopic pathology. A tabulated summary of this study is presented in m2.6.7, Table 6.1. The toxicokinetics of dolutegravir was non-linear at the given dose levels. Systemic exposure in male and female mice increased with increasing dose and there was no evidence of accumulation of dolutegravir after dosing for 14 days. In general, there were no sex differences in exposure to dolutegravir. There were no treatment-related effects on body weights, food consumption, clinical signs, organ weights and histopathology. Clinical pathology changes suggestive of a subtle hematopoetic response were noted. In females, this consisted of increases in mean platelet volume at 100 mg/kg/day and red cell distribution width at 1500 mg/kg/day. More subtle effects occurred in males at 1500 mg/kg/day and these were marginally increased reticulocytes, platelets and total bilirubin. There were no adverse findings and the NOAEL was 1500 mg/kg/day week study In a 13 week oral repeat dose study, dolutegravir was administered to mice at doses of 0 (control), 10, 50, 500, or 1500 mg/kg/day [Report RD2009/00028, m ]. Dolutegravir was formulated with 0.5% HPMC and 0.1% Tween 80. Parameters that were evaluated during the study were viability, clinical observations, toxicokinetics, body weights, food consumption, hematology and clinical chemistry, organ weights, macroscopic observations and microscopic pathology. A tabulated summary of this study is presented in m2.6.7, Table 7.1. The systemic exposure to dolutegravir increased less than dose proportional with increasing doses on Days 1, 28 and 85. Systemic exposure increased 1.9-fold for a 30-fold increase in dose between 50 mg/kg/day and 1500 mg/kg/day and 1.2-fold for a 10-fold increase in dose between 500 mg/kg/day and 1500 mg/kg/day. No notable difference in systemic exposure to dolutegravir was observed among the sampling days or between the sexes at any dose level. There were no treatment-related effects on body weights, food consumption, clinical signs and organ weights. The main findings were limited to mice receiving the 19

20 1500 mg/kg/day dose level. This included minimal increases in alkaline phosphatase and total bilirubin in males and minimal to mild increases in aspartate aminotransferase and potassium in females. There was a minimal or slight increase of mucous neck cells in the glandular mucosa of the stomach, with occasional mucosal and/or submucosal eosinophilic and lymphocytic infiltrates in most animals given 1500 mg/kg/day. There were no adverse findings and the NOAEL was 1500 mg/kg/day Rat Repeated dose toxicity studies with dolutegravir have been carried out in the Sprague Dawley rat and dolutegravir was formulated in 0.5% HPMC and 0.1% Tween 80 for each of these studies Oral administration day study Groups of rats (10/sex/group) were given 0 (control), 50, 150 or 500 mg/kg/day of dolutegravir once daily for 14 days by oral gavage [Report RD2007/01140, m ]. An additional 4 rats/sex/group were included for toxicokinetic evaluation. The following evaluations were performed: clinical observations, body weight, food consumption, water consumption, auditory examination, ophthalmoscopy, urinalysis, hematology, blood chemistry, organ weight, gross and histopathology (including stage-dependent evaluation of spermatogenesis), and toxicokinetics. A tabulated summary of this study and noteworthy findings are presented in m2.6.7, Table 7.2. Systemic exposure (C max and AUC 0-24 ) to dolutegravir was generally less than doseproportional and no notable (>2-fold) sex related differences in at any dose or sampling occasion were observed. There were no deaths throughout the study. No treatment-related changes were observed in clinical observations, body weight, food consumption, water consumption, auditory examination, ophthalmoscopy, hematology, blood chemistry, organ weight, and spermatogenic staging. There were statistically significant increases in urine specific gravity in males given 500 mg/kg/day and in females given 50 and 500 mg/kg/day. Because no treatment-related microscopic changes were observed in the kidneys, the change was not considered toxicologically significant. Mild increased mucous neck cells, eosinophil infiltration to submucosa and focal edema in the glandular stomach occurred in males and females given 500 mg/kg/day. The gastric mucosal lesions in this study were judged to be mild, treatment-related irritation without clinical signs and macroscopic findings and, because they did not adversely affect the general condition or health of the animal and were not associated with other evidence of systemic toxicity, were considered non-adverse. The NOAEL was considered to be 500 mg/kg/day, which was associated with gender mean C max and AUC 0-24 of 116 g/ml and 1830 g.h/ml, respectively on Day

21 week toxicity study Groups of rats (10/sex/group) were given 0 (control), 2, 10, 100 or 1000 mg/kg/day of dolutegravir once daily for 4 weeks [Report RD2008/01628, m ]. An additional 5/sex were included at 0 (control) and 1000 mg/kg/day groups to assess the reversibility of potential drug-related effects following a 4 week recovery period. Data that were collected included toxicokinetics, clinical observations, body weights, food consumption, ophthalmoscopy, hematology, clinical chemistry, urinalysis, organ weights, macroscopic observations and microscopic examination. A tabulated summary of this study and noteworthy findings are presented in m2.6.7, Table 7.3. Systemic exposures (C max and AUC 0-24 ) increased with increasing dose although less than dose proportionally at 10 mg/kg/day. There were no obvious sex related differences in exposure and no difference in exposure between sampling days. Gastric mucosal changes were noted in both sexes at 100 mg/kg/day. The lesions were characterized by increased mucous neck cells with globule leukocytes infiltration, edema and eosinophilic infiltration in submucosa, as well as acanthosis with edema and mixed cellular infiltration of the limiting ridge. Hemorrhage was observed in the lamina propria of the mucosa at 1000 mg/kg/day. All gastrointestinal changes were considered to be due to irritation of the drug substance and, with the exception of hemorrhage, were considered to be nonadverse. All findings observed at 1000 mg/kg/day were reversible following a 4 week recovery period. Based on the stomach lesions observed at 1000 mg/kg/day, the NOAEL was 100 mg/kg/day which is associated with gender mean C max and AUC 0-24 of 55 g/ml and 751 g.h/ml, respectively, on Day week toxicity study Dolutegravir was administered orally once daily to rats at 0 (control), 5, 50 and 500 mg/kg/day for 17 weeks (n=10/sex/group) or 26 weeks (n=12/sex/group) with an additional 6/sex added at the 500 mg/kg/day group to evaluate the reversibility of toxicity following a 4 week recovery period after 26 weeks of dosing [Report RD2009/00410, m ]. The following data were collected: clinical signs, body weights, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, toxicokinetics, and macroscopic and microscopic observations. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 7.4. The mean C max and AUC 0-24 values for dolutegravir on Days 1, 30, 120 and 180 generally increased less than proportionally in both males and females, and the values of females tended to be greater than those of males and slightly increased with repeat dosing at the dose level of 5 mg/kg/day. There were no obvious sex differences in exposure to dolutegravir, nor significant drug accumulation after repeat dosing. Hemorrhage in the stomach was observed microscopically at 500 mg/kg/day in 1 male at the end of the 17 week dosing period and 1 male at the end of the 26 week dosing period but was not observed in animals after a 4 week recovery period. Macroscopic lesions (white or red focus) in the stomach were observed in 1 male at the end of the 4 month dosing period and 2 males and 2 females at 500 mg/kg/day group at the end of the 6 21

22 month dosing period. Animals given 5 mg/kg/day had non-adverse slight to very slight thickening of the forestomach limiting ridge mucosa, eosinophil infiltration in the glandular stomach submucosa and/or increased globule leukocytes and mucous neck cells in the glandular stomach mucosa. In recovery animals, thickening of the forestomach limiting ridge mucosa and increased globule leukocytes of the glandular stomach mucosa was observed at a reduced incidence. Based on the finding of hemorrhage in the glandular stomach mucosa in 2 males at 500 mg/kg/day, the NOAEL for the 26 week dosing period was 50 mg/kg/day (end of study gender averaged AUC and C max values of g.h/ml and 47.3 g/ml, respectively) Monkey Repeated dose toxicity studies with dolutegravir have been carried out in the cynomolgus monkey and dolutegravir was formulated in 0.5% HPMC and 0.1% Tween 80 for each of these studies Oral administration day study Groups of monkeys (3/sex/group) were given 0 (control), 100, 300 or 1000 mg/kg/day of dolutegravir once daily for two weeks by oral gavage to evaluate the potential toxicity and toxicokinetics of dolutegravir [Report RD2007/01142, m ]. The following evaluations were performed: clinical observations, body weight, food consumption, ophthalmoscopy, electrocardiography, urinalysis, hematology, blood chemistry, organ weight, bone marrow examination, gross and histopathology, hepatic drug-metabolizing enzyme activity, and toxicokinetics. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 7.5. Systemic exposure (C max and AUC 0-24 ) to dolutegravir was generally less than doseproportional and was impacted by repeated emesis resulting in a large degree of variability and some overlap in exposures between dose groups, which was most pronounced at 300 and 1000 mg/kg/day. No treatment-related effects were evident on ophthalmoscopy or electrocardiography. One female given 1000 mg/kg/day died on Day 13. Repeated daily emesis began on Day 2 and diarrhea on Day 5 with progression to decreased activity, lateral or crouching position, pale oral mucosa, and subnormal body surface temperature prior to death on Day 13. Hematology and clinical chemistry evaluations conducted on the day of death revealed an increase (2-fold change from control mean) in fibrinogen, marked increases (3-fold change from control mean) in ALT and urea nitrogen and decreases (0.8 to 0.9- fold from control mean) in sodium and chloride. Although the liver and digestive tract could not be examined thoroughly due to autolysis, histopathological findings that might cause death were not evident in the kidneys, heart or lungs. Although a direct cause of death was not determined, the deteriorated condition due to treatment-related effects on 22

23 the digestive tract (emesis, diarrhea, ulcer in colon) with significant change in blood electrolytes was believed to contribute to the death. Males and females given 300 and 1000 mg/kg/day exhibited emesis and diarrhea (loose, muddy or watery stools) from initiation of dosing and 2 males given 1000 mg/kg/day had decreased activity and lateral or crouching position from Day 13 of dosing. Persistent decreases (2 to 17% from pretest) in body weight or body weight gain occurred in 2 males and 2 females given 300 mg/kg/day and in 2 males and all females given 1000 mg/kg/day. Of these animals, 1 male and 2 females given 300 mg/kg/day and 2 males and 1 female (animal found dead on Day 13) given 1000 mg/kg/day had decreased food consumption. Hematological evaluations showed decreases in the reticulocyte count (0.2 to 0.5-fold control mean) in males and females in the 300 and 1000 mg/kg/day groups; decreases in the platelet count (0.5 to 0.7-fold control mean) and increases in fibrinogen (1.4 to 1.6- fold control mean) in males and females in the 1000 mg/kg/day group; a decrease in the reticulocyte ratio (0.3 to 0.5-fold control mean) and prolongation of APTT (1.1-fold control mean) in males in the 1000 mg/kg/day group; and a decrease (0.9-fold control mean) in the red blood cell count in females in the 1000 mg/kg/day group. Clinical chemistry evaluations showed increases in ALT (2.3-fold control mean) in males in the 300 mg/kg/day group and in males and females in the 1000 mg/kg/day group (5 to 10-fold control mean); increases in AST (2-fold control mean), total bilirubin (2 to 3-fold control mean), urea nitrogen (1.6 to 1.8-fold control mean) and creatinine (1.2 to 1.5-fold control mean) and decreases in sodium, chloride (0.87 to 0.95-fold control mean) and the A/G ratio (0.7-fold control mean) in males and females in the 1000 mg/kg/day group; increases in γ-gtp (1.3-fold control mean) and triglycerides (1.9-fold control mean) in males in the 1000 mg/kg/day group; and a decrease (0.7-fold control mean) in total cholesterol in females in the 1000 mg/kg/day group. Urinalysis revealed decreases in the urinary volume (0.3 to 0.6-fold control mean) and sodium and chloride excretion levels (0.2 to 0.3-fold control mean) in males and females and a decrease in the potassium excretion level (0.3-fold control mean) in females in the 1000 mg/kg/day group. Gross pathology revealed reddish spots in the stomach or colon, small thymus, brownish discoloration of the mesenteric lymph nodes and enlargement of the lymph nodes in the abdominal cavity in animals in the 300 and 1000 mg/kg/day groups, including the animal found dead on Day 13, and whitish protrusion in the esophagus and reddish spots in the ileum in the 1000 mg/kg/day group. Decreases in the weight of the thymus were noted in the 300 and 1000 mg/kg/day groups and increases in the weights of the liver and adrenals were noted in the 1000 mg/kg/day group. Histopathology revealed atrophy of the mucosal epithelium and cell debris from the crypts of the cecum, colon and rectum, hemorrhage in the mucosa of the colon, atrophy of acinar cells in the pancreas and parotid glands, decrease in lipid droplets in zona fasciculata cells in the adrenals, atrophy of the cortex of the thymus and decrease in lymphocytes in the paracortex of the mesenteric lymph nodes in monkeys given

24 and in the submandibular lymph nodes in monkeys given 1000 mg/kg/day groups. In the 1000 mg/kg/day group, dilatation of the tubules in the kidneys, gelatinous marrow, atrophy of the white pulp in the spleen were noted in males and females and hypertrophy, single cell necrosis and vacuolation of hepatocytes in the liver, hypertrophy of the zona fasciculata of the adrenals and atrophy of the mucosal epithelium and hemorrhage in the mucosa of the stomach were observed in males. In the bone marrow examinations, a decrease in the nucleated cell count was noted in one male in the 1000 mg/kg/day group. Based on these results, the NOAEL of dolutegravir was considered to be 100 mg/kg/day. Due to the large individual variation in systemic exposure, the lowest measured AUC 0-24 (41 μg h/ml) and C max (5.75 μg/ml) in the 100 mg/kg/day group was used to define the NOAEL exposure week toxicity study Groups of monkeys (3/sex/group, except the control and high dose groups, which were 5/sex/group) were given 0 (control), 25, 50 or 100 mg/kg/day dolutegravir once daily for 30 days by oral gavage [Report RD2008/00107, m ]. The two additional animals/sex from the control and 100 mg/kg/day groups were maintained for a 30 day recovery period to assess the reversibility of any effects. Evaluations were performed to assess clinical observations, body weights, toxicokinetics, hematology, clinical chemistry, organ weights, urinalysis, ophthalmology, electrocardiography, bone marrow examination, and macroscopic and microscopic pathology. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 7.6. In both sexes, the exposure values were nearly the same levels in all treated groups on all sampling days. Thus, dose-linearity was not observed at the tested dose levels. There were no obvious differences in exposure between the sexes. Clinical signs of vomiting and diarrhea were observed from initiation of dosing, and a crouching position was observed in one male and one female from Day 17 of dosing in animals given 100 mg/kg/day. Persistent decreases (as low as 0.78X) in body weight or body weight gain were noted in 2 males and 2 females given 100 mg/kg/day. Dolutegravir administration was associated with decreases in red blood cells (0.90X) in females at 100 mg/kg/day. Histopathological findings observed at 100 mg/kg/day consisted of slight inflammatory cell infiltration in the lamina propria of the cecum, colon and rectum; slight cell debris from the crypts of the cecum and colon; atrophy of the mucosal epithelium of the cecum and colon, atrophy of the thymus with acinar cells in the pancreas (considered to be associated with malnutrition and not directly attributed to treatment). All findings were reversible following a 30 day recovery period. Based on these results, the NOAEL was 50 mg/kg/day (gender mean AUC 132 g.h/ml, C max 17 g/ml). 24

25 week toxicity study Dolutegravir was administered orally once daily to monkeys at 0 (control), 3, 10, 15 and 50/30 mg/kg/day for 17 weeks (n=2 or 3/sex/group) or 38 weeks (n=3 or 4/sex/group) with an additional 2/sex/group added at 15 and 50/30 mg/kg/day to evaluate the reversibility of toxicity following a 4 week recovery period after 38 weeks of dosing [Report RD2009/00036, m ]. In animals given 50 mg/kg/day, the dose level was dropped to 30 mg/kg/day on Day 70 due to the moribund condition of some animals. Examinations and/or measurements were performed to determine clinical signs, body weights, food consumption, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, toxicokinetics, and macroscopic and microscopic pathology. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 7.7. Overall, the exposure values showed a tendency to increase with dose in both sexes throughout the study. There was no evidence for increased exposure values in males after repeat dosing and there was a slight tendency for increased exposure in females after repeat dosing. There were no other notable differences in exposure between sexes. Two males given 50 mg/kg/day died or were euthanized (on Day 55 and Day 59, respectively) with abnormal feces (diarrhea and/or soft stool), decreased food consumption and body weight loss. Microscopically, mononuclear cell infiltration and hemorrhage in the lamina propria in the cecum and colon was observed in one animal. Additional histopathology findings considered secondary to the moribund condition consisted of inflammatory cell infiltration in the epithelium in the esophagus and tongue, and kidney dilatation of renal tubules and cellular and hyaline casts. After Day 69, the 50 mg/kg/day was reduced to 30 mg/kg/day for the duration of the study. In surviving animals given 50 mg/kg/day prior to dose reduction, hematological changes consisted of increased monocyte count (2.02 to 2.36X) in 3 males on Days 30 and 69, increased WBC (1.64 to 2.26X), neutrophil count (2.30 to 11.9X) and/or ratio (1.41 to 6.21X) in 2 males and 1 female and decreased RBC (0.83X), hematocrit (0.82X), and hemoglobin (0.82X) and increase in fibrinogen (2.00X) in 1 female on Days 30 and 69, and prolonged APTT (1.13 to 1.35X) in 1 male and 1 female on Day 69. Statistically significant increases in monocyte (1.92X) and neutrophil count (2.06X) on Day 30 and increase in neutrophil count (2.06X) on Day 69 were noted in males, and prolonged APTT (1.19X) was also noted in females on Day 69. In blood chemistry, decreased glucose in 1 male and 1 female (0.47 to 0.48X), increased inorganic phosphorus in 1 male (1.36X) and increase in triglycerides in 1 female (5.3X) and decreased Cl (0.90 to 0.94X) in 1 male and 1 female were observed on Day 69. Following administration of 30 mg/kg/day on Day 70, diarrhea and/or soft stool were observed in 3 males and 2 females; however, these were transient changes, and no decreased food consumption or body weight was noted. Salivation was also observed in the 50/30 mg/kg/day group immediately after dosing. At necropsy at the end of the 38 week dosing period, red focus and recessed focus in the mucosa of the stomach body, multifocal mononuclear cell infiltration and slight hemorrhage in the lamina propria, very slight multifocal erosions and multifocal 25

26 epithelial regeneration in the stomach were observed in 1 female at 50/30 mg/kg/day. At the end of the 4 week recovery period, multifocal mononuclear cell infiltration and very slight hemorrhage in the lamina propria and multifocal epithelial regeneration in the stomach were observed in 1 female at 50/30 mg/kg/day. However, the changes in this animal were of lesser severity and there were no active erosions suggesting likely regression of changes upon cessation of treatment. In the 15 mg/kg/day group, abnormal feces (diarrhea and/or soft stool) were observed between Days 70 and 104 of dosing in 1 male, and transiently decreased food consumption was also noted. However, these changes recovered during the dosing period from Day 105 of dosing, no body weight change was noted, and the overall health of the animal was not adversely affected. No test article-related changes were observed in the 3 and 10 mg/kg/day groups. Based on the gastrointestinal intolerance leading to mortality and morbidity in 2 animals, decreased body weight accompanied by frequently observed abnormal feces and decreased food consumption at 50 mg/kg/day and adverse findings of slight hemorrhage and very slight multifocal erosions in the stomach of 1 female at 50/30 mg/kg/day, the NOAEL after 38 weeks of treatment was 15 mg/kg/day (end of study gender averaged AUC and C max values of 38.8 g.h/ml and 5.05 g/ml, respectively). 26

27 Table 3.2 Batch Numbers of Dolutegravir and Formulations Used in Repeat Dose Toxicity Studies Batch or Lot Study Type Species Duration of Report No. Concentration Formulation Number(s) Dosing (mg/ml) B86001 Repeat Dose Mouse 14 days RD2009/ , 10, 50, B86001 Mouse 13 weeks RD2009/ , 5, 50, MTS B-07 Repeat Dose Rat 14 days RD2007/ , 15 and 50 1 A7Z001 Rat 4 weeks RD2008/ , 1, 10, A7Z001 & B86001 Rat 26 weeks RD2009/ , 5, 50 1 R06001 Repeat Dose Monkey 14 days RD2007/ , 30, Monkey 4 weeks RD2008/ , 5, 10 1 A7Z001 & B86001 Monkey 38 weeks RD2009/ , 1.0, 1.5, 3, 5 1 Note: The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. Studies were performed with dolutegravir sodium salt form, unless otherwise noted. Formulation Code: 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 27

28 4. GENOTOXICITY 4.1. Introduction Dolutegravir has been evaluated for genotoxicity potential both in vitro and in vivo. Potential mutagenic activity has been assessed in bacteria cells in vitro by the standard Ames test. The genotoxic potential of dolutegravir has been assessed in mammalian cells in vitro in the L5178Y mouse lymphoma assay. Experiments were carried out both in the presence and absence of a rat liver post-mitochondrial metabolising system (S9-mix), together with appropriate vehicle and positive controls. The potential for clastogenic effects has also been assessed in vivo using the oral micronucleus test in the rat. In addition, an assessment of the route of synthesis for dolutegravir has been conducted to determine whether any impurities might be present which are known or suspected DNAreactive mutagens, and to assess the likelihood of any such impurities being present in final drug product. All studies described in this section were performed using the sodium salt of dolutegravir, which is the form proposed for use in humans; however, all concentrations and dosages quoted in this summary are expressed in terms of the parent compound (which is referred to as dolutegravir). The impurity profile of the batches of test material used in these investigations was consistent with that used in the clinical evaluation of dolutegravir and that proposed for use in the marketed product. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 4.2 of this summary. All definitive studies were conducted in full compliance with Good Laboratory Practice (GLP) regulations. All assays used internationally recognised and validated techniques in full accordance with the ICH Harmonised Tripartite Guidelines (S2A Guideline on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals and S2B A Standard Battery for Genotoxicity Testing of Pharmaceuticals) and with the general principle of the United Kingdom Environmental Mutagen Society (UKEMS) Subcommittee on the Guidelines for Mutagenicity Testing (Part I, 1983; Part I revised, 1990 and, where appropriate, Part II, 1993). A full listing of studies performed and the GLP status, together with the location of the reports within this submission, is provided in Table

29 Table 4.1 List of Genotoxicity Studies Performed with Dolutegravir and Potential Impurities Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Dolutegravir Ames test NA NA In vitro 5 to 849 g/plate a NA Yes WD2007/00514 (V27467) Screening mouse lymphoma assay Mouse lymphoma assay Rat micronucleus Impurities NA NA In vitro Up to 80 g/ml b NA No GSK WD2007/01581 (MLA-580) NA NA In vitro 5 to 85 g/ml a NA Yes WD2007/00515 (V27468) Rat (Sprague Dawley) 6M Oral (gavage) gsk019* NA NA In vitro Up to 5000 g/plate 50, 150, days Yes WD2007/00513 (R27469) m m m m NA No GSK 2012N m gsk002* NA NA In vitro Up to 500 g/plate NA No GSK 2010N m gsk001* NA NA In vitro Up to 5000 g/plate NA No GSK 2012N m gsk001* NA NA In vitro Up to 1000 g/ml NA No GSK WD2010/00483 m Note: All studies using dolutegravir were performed with GSK A, the sodium salt form. Key: a = Highest concentration limited by solubility. b = Highest concentration limited by either solubility or toxicity. NA = Not applicable. Testing Facility: = = GSK = GlaxoSmithKline. * 新薬承認情報提供時に置き換え 29

30 4.2. In Vitro Studies (Dolutegravir) Non-mammalian cell systems Ames assay Dolutegravir was tested in Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) or Escherichia coli strain WP2 uvra (pkm101) at concentrations ranging from 5 to 849 g per plate in the presence or absence of an in vitro metabolic activation system (S9-mix) [Report WD2007/00514, m ]. The maximum concentration tested was limited by solubility in dimethyl sulphoxide. In addition, there was clear evidence of bactericidal activity in the form of a diminution of the background bacterial lawn and/or a marked reduction in the number of revertant colonies in the presence and absence of S9-mix at 500 and/or 849 g per plate in strains TA100, TA1535 and TA1537. The negative and positive controls all produced acceptable responses. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 8.1. Dolutegravir was not mutagenic in the bacterial mutation assay in either the presence or absence of S9-mix Mammalian cell systems The mutagenic potential of dolutegravir has been assessed in mammalian cells in vitro in two mouse lymphoma assays. Appropriate vehicle and positive controls were included in all assays which were conducted in both the presence and absence of a rat liver postmitochondrial metabolising system (S9-mix). In both studies, the positive controls produced biologically significant increases in mutation frequency Mutation test at the thymidine kinase locus in mouse lymphoma L5178Y cells In a preliminary mouse lymphoma L5178Y TK +/- screen, dolutegravir was tested for 3 hours in the presence of S9-mix at the maximum solubility limit of 80 µg/ml and also was tested for 24 hours in the absence of S9-mix [Report WD2007/01581, m ]. The maximum test concentration in the 24 hour exposure was limited by cytotoxicity to 20 µg/ml. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 8.2. The results for the vehicle controls were within acceptable ranges, determined from laboratory historical data, and the positive controls induced clear unequivocal increases in the numbers of mutant colonies. In this screening assay, dolutegravir was negative when tested for 3 hours in the presence of S9-mix (the relative total growth was reduced to 54%). Dolutegravir was weakly positive, at highly cytotoxic (85% cytotoxicity) concentrations only, when tested for 24 hours in the absence of S9-mix. 30

31 In a definitive GLP mouse lymphoma L5178Y TK +/- assay, dolutegravir was tested up to a maximum solubility limit of 85 g/ml following both the 3 hour treatment in the presence and absence of S9 metabolic activation and 24 hour treatment without S9 metabolic activation [Report WD2007/00515, m ]. For this study, a tabulated summary with noteworthy findings is presented in m2.6.7, Table 8.3. The vehicle control data were within acceptable ranges and positive controls all produced acceptable responses, therefore, the performance of the vehicle and positive controls was consistent with a valid assay. Dolutegravir was not genotoxic at the maximum solubility limit of 85 g/ml following both the 3 hour treatment in the presence and absence of S9 metabolic activation (relative total growth reduced to 81% and 53%, respectively) and 24 hour treatment without S9 metabolic activation (relative total growth reduced to 26%). The results from this more robust definitive assay, using a fully characterized drug substance batch, indicate that dolutegravir does not present a genotoxic hazard to humans In Vivo Studies (Dolutegravir) Any clastogenic or spindle formation effect of dolutegravir on eukaryotic cells in vivo has been assessed using the micronucleus test performed by the oral route in the mouse and by the subcutaneous route in the rat Oral micronucleus test in the rat Dolutegravir was tested in vivo in the rat bone marrow micronucleus assay following 2 oral doses of 50, 150 or 500 mg/kg/day to male rats (n=6/group) given 24 hours apart [Report WD2007/00513, m ]. The highest dose tested was the dose producing maximum systemic exposure (C max and AUC) of 87.1 μg/ml and 1360 μg.h/ml, respectively (based on the Day 1 data from the rat 2 week oral toxicity study). All rats were euthanized 24 hours after the final dose and femoral bone marrow smears were prepared. Cyclophosphamide was used as the positive control. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table 9.1. The results for the vehicle control were within the ranges determined from laboratory historical data and the positive control induced clear unequivocal increases in micronuclei, therefore, the performance of the controls was consistent with a valid assay. Dolutegravir was not genotoxic in vivo in the oral rat micronucleus assay following two oral doses of 50, 150, or 500 mg/kg/day, given 24 hours apart. The highest dose tested was the dose producing the maximum systemic exposure. 31

32 4.4. Potential Impurities Standard Ames test A bacterial mutagenesis assay (Ames test) was conducted to assess the potential of gsk001*, an intermediate in the synthesis of dolutegravir, to induce gene mutations in vitro in Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2uvrA (pkm101) [Report 2012N153013, m ]. A single preincubation test was conducted for each tester strain both in the presence and absence of rat liver S9-mix, together with appropriate vehicle and positive controls. gsk001* produced a two-fold or more increase in the revertant colonies over the negative control group in TA98, TA100, TA1537 and WP2uvrA with and without S9- mix in the dose-finding study and the main study. The maximum concentration tested was 5000 g/plate in accordance with current guidelines Mouse lymphoma assay A study was conducted to assess the potential genotoxicity of gsk001*, a synthesis intermediate and potential impurity in the manufacturing process, in the mouse lymphoma assay [Report WD2010/00483, m ]. Three independent mutation assays were conducted: two in which cells were treated for 3 hours in the presence or absence of S9-mix, and one in which cells were treated for 24 hours in the absence of S9- mix. The maximum test concentration examined in each assay was 1000 μg/ml. Increases in mutant frequency were observed at concentrations of 900 μg/ml and above for the 3 hour treatment in the absence of S9-mix and at 1000 μg/ml for the 24 hour treatment in the absence of S9-mix. For the 24 hour treatment assay, the increase in the mutant frequency was predominantly due to small colonies. gsk001* was considered genotoxic in the mouse lymphoma assay Standard Ames test A bacterial mutagenesis assay (Ames test) was conducted to assess the potential of, an intermediate in the synthesis of dolutegravir, to induce gene mutations in vitro in Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2uvrA (pkm101) [Report 2010N105217, m ]. Single plate incorporation tests were conducted with using water as a vehicle and using dimethylcarbonate (DMC) as a vehicle for each tester strain both in the presence and absence of rat liver S9-mix. The maximum concentration analyzed was limited by toxicity to 500 and 150 g/plate in the presence and absence of S9-mix, respectively, for both vehicles. Using water as a vehicle, was mutagenic with 32 * 新薬承認情報提供時に置き換え

33 strain TA100 in the presence and absence of S9-mix at 150 g/plate (maximum fold increases of 2.7 and 3.6, respectively) and with strain TA1537 in the absence of S9-mix at 150 g/plate (maximum fold increase of 7.5). Using DMC as a vehicle, gsk002* was mutagenic with strain TA100 at 250 g/plate (maximum fold increase of 2.1) Standard Ames test A bacterial mutagenesis assay (Ames test) was conducted to assess the potential of gsk003*, an intermediate in the synthesis of dolutegravir, to induce gene mutations in vitro in Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2uvrA (pkm101) [Report 2012N150346, m ]. A single plate incorporation test was conducted for each tester strain both in the presence and absence of rat liver S9-mix, together with appropriate vehicle and positive controls. gsk003* was mutagenic in strains TA100, TA98 and WP2uvrA (pkm101) when tested in the presence and absence of S9-mix and strain TA1537 in the absence of S9-mix only. The maximum concentration tested was 5000 g/plate in accordance with current guidelines Route Assessment for Genotoxic Impurities The proposed drug substance specifications [see m3.2.s.4.5] for dolutegravir indicate that the specified impurities, gsk006*, gsk007*, gsk008*, gsk009*, gsk010*, gsk011* (enantiomer) and gsk012* (diastereomer) do not exceed the 0.15% w/w ICH qualification threshold [ICH Q3A(R2)]. All 7 impurities demonstrated no structural alerts for potential mutagenicity and carcinogenicity following in silico analysis, DEREK v13. An assessment of the commercial route of synthesis for dolutegravir has been conducted to determine whether any starting materials or intermediates might be present which are known or suspected to be DNA-reactive mutagens, and to assess the likelihood of any such impurities being present in final drug substance. This assessment included an evaluation for genotoxicity structural alerts (DEREK v13). The following synthetic intermediates and impurities were highlighted as potential genotoxins and were shown to be mutagenic in a bacterial assay: gsk001* (see Section ), gsk002* (see Section ), gsk003* (see Section ), gsk004* (NTP Reference), and gsk005* (Skopec, 1978). Additionally, gsk001* was considered genotoxic in a mouse lymphoma assay (see Section ). The structures of these impurities are provided in m3.2.s.4.5. A maximum recommended exposure concentration of 15 ppm for these impurities (excluding gsk020*) is based upon the TTC of 1.5 g/day with respect to a dolutegravir sodium daily dose of 100 mg. For gsk020*, a 2 ppm limit is applied based on ICH Q3C. These impurities are controlled by specifications of the intermediates or by control of the manufacturing process. Therefore, no specification is proposed in the final drug substance [m3.2.s.2.6, Section 6.1.5]. 33 * 新薬承認情報提供時に置き換え

34 In summary, there are no impurities of mutagenic concern at a level that would exceed the threshold of toxicological concern [TTC] as defined by the CHMP guidelines on the limits of genotoxic impurities (i.e., >1.5 g/day). 34

35 Table 4.2 Batch Numbers of Dolutegravir and Formulations Used in Genotoxicity Studies Batch or Lot Study Type Species (strain) or Duration of Report No. Concentration Formulation Number(s) Test System Dosing (Study No.) (mg/ml) R06001 Ames Test In vitro NA WD2007/00514 (V27467) U23359/4/1 Mouse Lymphoma In vitro NA WD2007/01581 (MLA-580) R06001 Mouse Lymphoma In vitro NA WD2007/00515 (V27468) R06001 Micronucleus Rat 2 days WD2007/00513 (R27469) 0.05 to to to , 15 and 50 1 Note: The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. Studies were performed with dolutegravir sodium salt form, unless otherwise noted. Formulation Code: 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 2 = Dimethyl sulfoxide (DMSO) 35

36 5. CARCINOGENICITY 5.1. Introduction Carcinogenicity studies of lifetime duration (104 weeks) were performed to determine the tumorigenic potential of dolutegravir and were carried out by the oral route in the CD-1 mouse and Sprague Dawley rat. These studies were preceded by preliminary studies to determine suitable high dosages for use in the main studies. Both of the main studies were conducted in accordance with GLP regulations. The mouse and the rat are the species of choice for this type of investigation and the strains used were selected because of the considerable amount of knowledge available to the investigators regarding both their background pathology and their reactions to a wide variety of drugs. Furthermore, information on the pharmacokinetic and metabolic handling of dolutegravir in these species and strains confirms their suitability for selection (see m2.6.4, Pharmacokinetics Written Summary). Sufficient animals were used in each study to allow meaningful evaluation of the data. All studies described in this section were performed using the sodium salt of dolutegravir, which is the form proposed for use in man; however, all dosages and concentrations quoted in this summary are expressed in terms of the parent compound (referred to simply as dolutegravir). The impurity profile of the batches of test material used in these investigations was consistent with that used in the clinical evaluation of dolutegravir and that proposed for use in the marketed product. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 5.2 of this summary. A full listing of studies performed and the GLP status of the studies, together with the location of the reports within this submission, is provided in Table 5.1. Additional tabulations of List of Tissues Studied, Tumour Incidence, Chronological Listing of Tumour Occurrence, and Survival and Fate of Animals are presented in Appendix 1. For the definitive 104 week carcinogenicity studies in mice and rats, the electronic SAS data files are located with their corresponding reports in Module 4 (m , rat SAS data and mouse SAS data). 36

37 Table 5.1 List of Carcinogenicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Carcinogenicity Mouse (CD-1) 65 Oral 45 TK (gavage) 7.5, 25, years Yes 2012N m Carcinogenicity Rat (Sprague Dawley) 65 Oral 12 TK (gavage) 2, 10, 50 2 years Yes 2012N m Key: TK = Satellite groups for toxicokinetics analysis. Testing Facility: = 37

38 5.2. Mouse Preliminary studies were performed to determine suitable dosages for use in a main oral carcinogenicity study investigating the carcinogenic potential of dolutegravir in the CD-1 mouse. The preliminary studies determined that steady state systemic exposure based on AUC 0-24 increased with increasing dose and was less than dose proportional in both the 2 week and 13 week studies. In the 2 week toxicity study, systemic exposure increased 1.5-fold for a 15-fold increase in dose between 100 mg/kg/day and 1500 mg/kg/day and 1.3-fold for a 3-fold increase in dose between 500 mg/kg/day and 1500 mg/kg/day. In the 13 week toxicity study, systemic exposure increased 1.9-fold for a 30-fold increase in dose between 50 mg/kg/day and 1500 mg/kg/day and 1.2-fold for a 3-fold increase in dose between 500 mg/kg/day and 1500 mg/kg/day Oral administration Carcinogenicity study Groups of CD-1 mice (65/sex/group) were administered dolutegravir via oral gavage once daily at dose levels of 0 (water), 0 (vehicle: aqueous 0.5% w/w HPMC with 0.1% w/w Tween 80), 7.5, 25, or 500 mg/kg/day for up to 104 weeks [Report 2012N152419, m ]. Satellite groups of toxicokinetic animals (20/sex/control groups, 45/sex/dolutegravir-treated groups) were also dosed with dolutegravir and blood was collected during Weeks 4 (Day 26) and 26 (Day 182) for toxicokinetic analysis. Other parameters evaluated during the study included viability, clinical observations, body weights, food consumption, hematology (termination), macroscopic observations and microscopic pathology. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table All surviving males were euthanized at the end of the dosing period (Week 105). Dosing was discontinued for the 500 mg/kg/day females at Week 101 due to declining survivorship ( 20 animals/group) and these animals were held until the termination of all surviving females during Weeks 102 and 103 when the water control group survival reached 20 animals. In general, male and female mice had quantifiable plasma concentrations of dolutegravir on Days 26 and 182. The systemic exposure (C max and AUC 0-24 ) to dolutegravir increased with increasing dose on Days 26 and 182. The systemic exposure to dolutegravir was equal to or less than proportional from 7.5 mg/kg/day to 25 mg/kg/day or to 500 mg/kg/day. No accumulation was observed from Day 26 to Day 182 in male or female mice. No marked gender differences (less than 2-fold) in exposure were observed on Days 26 and 182. Dolutegravir administered orally to mice at doses of 7.5, 25 or 500 mg/kg/day for up to 104 weeks had no effect on survival. There were no dolutegravir-related clinical signs or effects on body weight, food consumption and hematology. There were no neoplastic or non-neoplastic findings attributed to dolutegravir. The no observed adverse effect level 38

39 (NOAEL) for non-neoplastic findings after chronic oral administration was the high dose of 500 mg/kg/day Rat Oral administration Carcinogenicity study Sprague Dawley rats (65/sex/group) were orally gavaged once daily with dolutegravir at dose levels of 0 (water), 0 (vehicle; 0.5% w/w HPMC with 0.1% w/w Tween 80), 2, 10, or 50 mg/kg/day for up to 104 weeks [Report 2012N152418, m ]. Groups of satellite animals (4/sex/control groups; 12/sex/dolutegravir-treated groups) were similarly dosed and blood was collected at the end of Week 4 and Week 28 for toxicokinetic analysis. Other parameters evaluated during the study included viability, clinical observations, ophthalmology, body weights, food consumption, hematology and clinical chemistry (termination of dosing), urinalysis [Week 103 (males) and Week 94 (females)], macroscopic observations and microscopic pathology. A tabulated summary of this study, with noteworthy findings, is presented in m2.6.7, Table Surviving males were euthanized during Weeks 104 and 105 at the end of the 104 week dosing period. Dosing was discontinued for Groups 3 (2 mg/kg/day) and 5 (50 mg/kg/day) females in Week 90 and Week 88, respectively, due to declining survivorship ( 20 animals/group) and these animals were held until the termination of all females during Week 95 when the control (water) survival reached 20 animals. All male and female rats had quantifiable plasma concentrations of dolutegravir on Day 28 and Day 182. The systemic exposure to dolutegravir increased with increasing dose on Day 28 and 182. The increases in the systemic exposure were generally less than dose proportional from 2 mg/kg/day to 10 mg/kg/day or to 50 mg/kg/day on Day 28 and 182. No accumulation of dolutegravir was observed after repeated oral administration from Day 28 to Day 182. In general, the AUC 0-24 and C max values in males were comparable (on Day 28) to or less (on Day 182) than those values in females. Dolutegravir administered orally by gavage to rats at doses of 2, 10 and 50 mg/kg/day for up to 104 weeks had no effect on survival and there were no neoplastic or nonneoplastic findings attributed to the test article. There were no dolutegravir-related clinical signs and no effects upon body weight, food consumption, ophthalmology, hematology, clinical chemistry and urinalysis were noted. The NOAEL for non-neoplastic findings after chronic oral administration was the high dose of 50 mg/kg/day. 39

40 Table 5.2 Batch Numbers of Dolutegravir and Formulations Used in Carcinogenicity Studies Batch or Lot Study Type Species (strain) Duration of Dosing Report No. Concentration Formulation Number(s) or Test System (Study No.) (mg/ml) B86001 and B86001 and Carcinogenicity Mouse 104 weeks 2012N , 2.5, 50 1 Carcinogenicity Rat 104 weeks 2012N , 1, 5 1 Note: The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. Studies were performed with dolutegravir sodium salt form, unless otherwise noted. Formulation Code: 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 40

41 6. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY 6.1. Introduction A range of reproductive toxicity studies have been performed to investigate the consequences of repeated administration of large doses of dolutegravir on the mammalian reproductive processes. The effects of dolutegravir on fertility and general reproductive performance and peri- and post-natal development have been investigated in Sprague Dawley rats, while the effects on embryofetal development have been investigated in Sprague Dawley rats and Japanese white rabbits. Studies were conducted in Sprague Dawley rats to assess potential juvenile toxicity. To support the therapeutic use of dolutegravir sodium tablets in women of child-bearing age, studies were performed by the oral route of administration. Studies undertaken to establish suitable dose levels for use in repeat dose toxicity studies were performed in accordance with the general principles of the Good Laboratory Practice (GLP) regulations. All definitive studies were performed in full compliance with GLP regulations. All studies described in this section were performed using the sodium salt of dolutegravir, which is the form proposed for use in humans; however, all dosages and concentrations quoted in this summary are expressed in terms of the parent compound (referred to simply as dolutegravir). The impurity profile of the batches of test material used in these investigations was consistent with that used in the clinical evaluation of dolutegravir and that proposed for use in the marketed product. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 6.2 of this summary. A full listing of studies performed and the GLP status, together with the location of the reports within this submission, is provided in Table

42 Table 6.1 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Fertility and early embryonic development Rat (Sprague Dawley) 20M/20F Oral (gavage) 100, 300, 1000 Up to 66 days (M) Up to 42 days (F) Yes XD2009/00368 (S TF-063-L) m Dose range embryofetal development study Rat (pregnant) (Sprague Dawley) 7F Oral (gavage) 100, 300, days (gestation Days 6 to 17) Yes RD2008/01761 (S TB-051-L) m Embryofetal development study Rat (pregnant) (Sprague Dawley) 20F Oral (gavage) 100, 300, days (gestation Days 6 to 17) Yes XD2009/00367 (S TB-062-L) m Dose range study Rabbit (non-pregnant) (Japanese white) 3F Oral (gavage) 30, 100, 300, days No RD2008/01760 (S TF-052-L) m Dose range embryofetal development study Rabbit (pregnant) (Japanese white) 4F Oral (gavage) 100, 300, days (gestation Days 6 to 18) Yes RD2009/00186 (S TF-060-L) m Embryofetal development study Rabbit (pregnant) (Japanese white) 18 to 20F Oral (gavage) 40, 200, days (gestation Days 6 to 18) Yes XD2009/0366 (S TF-065-L) m

43 Table 6.1 (Continued) List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Pre- and postnatal development Rat (Sprague Dawley) 22F Oral (gavage) 5, 50, 1000 Gestation Day 6 to Lactation Day 20 Yes 2011N (SG10306) m Juvenile tolerability Rat (Sprague Dawley) (juvenile) 8M/8F Oral (gavage) 5, 50, 100, 500, days (Day 4 to Day 21 pp) No GSK CD2009/00409 (D09072) m Juvenile dose range Rat (Sprague Dawley) (juvenile) 20M/20F Oral (gavage) 2, 25, 75, days (Day 4 to Day 31 pp) No GSK CD2009/00770 (D09126) m Juvenile toxicity Rat (Sprague Dawley) (juvenile) 10M/10F Oral (gavage) 0.5, 2, 75 a 63 days (Days 4 to 66 pp) Yes GSK CD2010/00023 (G09229) m Note: All studies were performed with GSK A, the sodium salt form. No observed adverse effect levels (NOAEL) for definitive studies are bolded. a = Additional 30/sex/group were added for toxicokinetic analysis and 10/sex/group were added for T-cell-dependent antibody response assessments. pp = Post partum. Testing Facility: GSK = GlaxoSmithKline. = = 43

44 6.2. Fertility and Early Embryonic Development Rat In a definitive fertility and early embryonic development study, rats (20/sex/group) were administered oral doses of dolutegravir at 100, 300 and 1000 mg/kg/day for 63 to 66 days in males (28 days prior to mating, throughout mating until 1 day prior to necropsy) and for up to 42 days in females (14 days prior to mating and throughout mating until Day 7 of gestation) [Report XD2009/00368, m ]. For the males, clinical observations and measurements of body weights and food consumption were conducted periodically and the genital organs including the testes, epididymides, seminal vesicles and prostate were weighed post-mortem. Sperm evaluation was conducted using semen collected from the cauda epididymis. For the females, clinical observations, measurements of body weights and food consumption and observations of the estrous cycle were conducted. The males and females in the same group were co-housed from Day 15 of dosing for a maximum of 14 days. The mated females underwent Cesarean section and post-mortem examination of Day 13 of gestation. The numbers of corpora lutea, implantations and dead and live embryos were determined. A summary of the study design and the results from this study are presented in m2.6.7, Table There were no treatment-related effects and the NOAEL was considered to be 1000 mg/kg/day Embryofetal Development Studies Rat Oral administration In a dose range finding study in pregnant rats (n=7 females/group), dolutegravir was administered orally at dose levels of 100, 300, and 1000 mg/kg/day on gestation Days 6 to 17 [Report RD2008/01761, m ]. Animals were observed for effects on clinical signs, body weight, and food consumption. Rats were killed on gestation Day 21 for examination of embryos / fetuses. Dose levels were selected based on expected exposure and the results from the 4 and 26 week repeat dose toxicity studies. A summary of the study design and the results from this study are presented in m2.6.7, Table There were no deaths in any dams. No adverse effects were observed in clinical observations, body weight, food consumption, maintenance of pregnancy or gross findings in any dolutegravir-treated dams. No treatment-related changes were noted of fetal viability, sex ratio, fetal body weight, placental weight, external malformations and external morphology of placenta of live fetuses. In a definitive embryofetal development study in pregnant rats (20 females/group), dolutegravir was administered orally at 100, 300 and 1000 mg/kg/day on gestation Days 6 to 17 [Report XD2009/00367, m ]. Animals were observed for effects on clinical signs, body weights, and food consumption. Rats were killed on gestation Day 44

45 21 and the parameters that were determined included the numbers of corpora lutea and implantations, early and late resorptions, live and dead fetuses, and sex, body weight, placental weight, gross findings of placentae, and external, visceral and skeletal morphology of live fetuses. Visceral and skeletal examinations were conducted on the control and 1000 mg/kg/day groups. The dolutegravir concentration in plasma was determined on gestation Days 6 and 17. A summary of the study design and the results from this study are presented in m2.6.7, Table The mean exposure values in the 300 mg/kg/day group were almost the same levels as those of the 100 mg/kg/day group, while exposure values increased less than dose proportionally between the 300 and 1000 mg/kg/day groups on gestation Days 6 and 17. Repeat dosing did not cause any obvious effects in any toxicokinetics parameters. No adverse effects were observed and the NOAEL for maternal and fetal toxicity was determined to be 1000 mg/kg/day Rabbit Oral administration Dolutegravir was administered orally to non-pregnant rabbits (n=3/group) for 14 days at doses of 30, 100, 300 and 1000 mg/kg/day [Report RD2008/01760, m ]. The purpose of this study was to select dose levels for a dose range finding study to determine the effects of dolutegravir on embryofetal development in pregnant rabbits. Parameters that were determined were clinical observations, food consumption, gross lesions, and body weights and toxicokinetics. A summary of the study design and the results from this study are presented in m2.6.7, Table The mean C max values increased less than dose proportionally between 30 and 300 mg/kg/day, while values at 1000 mg/kg/day were approximately the same as the 300 mg/kg/day dose group. The mean AUC 0-24 values increased dose proportionally between 30 and 300 mg/kg/day, while values at 1000 mg/kg/day were approximately the same as at 300 mg/kg/day. Scant feces and urine, decreased food consumption (up to 39%) and suppressed body weight gain (0.27X on Day 15) were evident in 2 of 3 animals in the 1000 mg/kg/day dose group. There were no gross lesions noted in any animal at any dose level. The dose level of 1000 mg/kg/day was determined to be suitable as the high dose level for the next dose range finding study in pregnant rabbits. In a preliminary dose range finding study in pregnant rabbits (n= 4 or 5/group), dolutegravir was administered orally at dose levels of 100, 300 and 1000 mg/kg/day on Day 6 to 18 of gestation [Report RD2009/00186, m ]. Clinical observations, food consumption and body weights were determined for the dams. On Day 29 of gestation, further parameters were determined which included the numbers of corpora lutea, implantations and live and dead fetuses, and external morphology, sex, body weights and placental weights of live fetuses. A summary of the study design and the results from this study are presented in m2.6.7, Table

46 Food consumption was markedly decreased in 2 of 4 dams at 100mg/kg/day, 2 of 5 dams at 300 mg/kg/day, and in all 5 dams at 1000 mg/kg/day. One dam with decreased food consumption in the 100 mg/kg/day group delivered prematurely on Day 28 of gestation, while no abortions or premature deliveries occurred at higher doses. Scant/no feces or scant/no urine associated with decreased food consumption was observed for all groups, including the control group. Suppressed body weight gain was also noted in dams in the 1000 mg/kg/day group. No gross lesions were evident in any dam and no treatmentrelated effects were evident on the viability, growth or external morphology of embryos / fetuses at any dose level. The dose level 1000 mg/kg/day, at which some maternal toxicity is expected, was considered appropriate as the high dose level for the subsequent embryofetal development study in rabbits. In a definitive embryofetal development study, dolutegravir was orally administered at dose levels of 40, 200, 1000 mg/kg/day to pregnant rabbits (n=18 to 20/group) on Days 6 to 18 of gestation [Report XD2009/0366, m ]. Clinical observations, body weights, toxicokinetics and food consumption were conducted for the dams. On Day 29 of gestation, the dams underwent Cesarean section and necropsy to determine the following parameters: the numbers of corpora lutea, implantations and live and dead fetuses and body weights, placental weights, sex and external, visceral and skeletal morphology of live fetuses. A summary of the study design and the results from this study are presented in m2.6.7, Table The mean C max values increased less than dose proportionally between 40 and 1000 mg/kg/day. The mean AUC 0-24 values increased dose proportionally between 40 and 200 mg/kg/day, while those values increased less than dose proportionally between 200 and 1000 mg/kg/day. There was no evidence of increased exposure after repeat dosing. Suppressed body weight gain (13.6% on gestation Day19), decreased food consumption (up to 53%) and scant or no feces/urine associated with the decreased food consumption were noted in the 1000 mg/kg/day dams. One dam in the 40 mg/kg/day group aborted. Since no abortion was noted at the 200 or 1000 mg/kg/day dose levels, this was considered not to be treatment-related. No gross lesions were evident in any dam and no treatment-related effects were noted for the viability, growth or external, visceral or skeletal morphology of fetuses at any dose level. The NOAEL was 200 mg/kg/day for maternal general toxicity and 1000 mg/kg/day for maternal reproductive function and embryofetal development Pre-Natal and Post-Natal Development, Including Maternal Function Rat In a pre- and postnatal development study, dolutegravir was administered orally to female rats (22/group) at dose levels of 0, 5, 50 or 1000 mg/kg/day from Day 6 of gestation to Day 20 of lactation [Report 2011N121663, m ]. Clinical observations, body weights, food consumption and gross pathology at termination of the lactation period were conducted on the dams (F 0 ) to evaluate maternal toxicity. The following parameters 46

47 were evaluated for effects on the subsequent generation (F 1 ): status at birth (number of newborns or live newborns, delivery index, birth index or sex ratio of live newborns), clinical signs, viability index on Day 4 of lactation, weaning index, body weights, physical development (pinna unfolding, growth of hair, eruption of the upper incisors and eyelid opening), early behavior (back righting and negative geotaxis), sensory functions (visual placing response, pupillary reflex, Preyer s reflex and pain response), open field test, conditioned avoidance response (shuttle box test), genital development (preputial separation and vaginal opening), vaginal smears, mating ability, fertility, gross pathology, and implantation and viability of embryos (F 2 ) on Day 13 of gestation. A summary of the study design and the results from this study are presented in m2.6.7, Table Suppressed body weight gain and decreased food consumption were noted in dams (F 0 ) in the 1000 mg/kg/day group during the early stages of the lactation period. Decreased body weights were noted in the subsequent generation (F 1 ) in the 1000 mg/kg group from pre-weaning until adolescence. No treatment-related toxic effects were noted on any other parameters in this group and there were no treatment-related findings at 50 mg/kg/day. The NOAELs were 50 mg/kg/day for maternal general toxicity (F 0 ) and for development of the subsequent generation (F 1 ) and 1000 mg/kg/day for maternal reproductive functions (F 0 ) such as maintenance of pregnancy, delivery and nursing Juvenile Toxicity Studies Rat Tolerability In an initial tolerability study, dolutegravir was administered orally at 5, 50, 100, 500, and 1000 mg/kg/day to juvenile rats (n=8/sex/group) from Day 4 to Day 21 postpartum (pp) [Report CD2009/00409, m ]. The following examinations and endpoints were evaluated: survival, clinical observations, body weights, body length, toxicokinetics, and in-life photographs were obtained. A summary of the study design and the results from this study are presented in m2.6.7, Table Due to unscheduled deaths, neither C max nor AUC was reported for the 1000 mg/kg/day group. There were no marked sex differences ( 2-fold) observed in systemic exposure to dolutegravir. In plasma samples collected on Day 21 pp, systemic exposure increased much less than in proportion to the increase in dose. Systemic exposure at 500 mg/kg/day was similar to that following 100 mg/kg/day in either sex. Doses of 500 mg/kg/day were not tolerated due to deaths starting on Day 14 pp. Macroscopic observations included bright yellow discoloration of intestines, liver, and skin, and small adrenals. Clinical observations at 500 mg/kg/day included alopecia and loss of skin elasticity. There was growth retardation at levels 500 mg/kg/day, evidenced as marked decreases in body weight and body length. 47

48 Dose range study Dolutegravir was administered orally at 2, 25, 75 and 300 mg/kg/day to juvenile rats (n=20/sex/group) from Day 4 to Day 31 pp [Report CD2009/00770, m ]. The following examinations and endpoints were conducted on this study: survival, clinical observations, body weights, in-life photographs, post-mortem macroscopic evaluation of 8 rats/sex/group, histological examination of stomach, liver, spleen, lymph, thymus, clinical chemistry, hematology, toxicokinetics, and enumeration of B and T cells via flow cytometry. A summary of the study design and the results from this study are presented in m2.6.7, Table Toxicokinetic parameters were determined in plasma samples collected on Day 13 pp. There were no marked sex differences observed in systemic exposure to dolutegravir. The systemic exposure at 300 mg/kg/day was similar to or less than that at 75 mg/kg/day. The dose level 300 mg/kg/day was not tolerated, and due to mortality, all remaining animals were euthanized one week early. Other findings at 300 mg/kg/day included clinical observations of slightly decreased activity, various degrees of alopecia (hair loss) and moderate lack of skin elasticity in a few animals with marked decrease in mean body weights throughout the study. The only macroscopic observation was one male with a small thymus, although it was not possible to necropsy all of the animals that were found dead because of cannibalization or autolysis. Test article-related microscopic effects were observed in the lymphoid tissues (spleen, thymus, mesenteric and mandibular lymph nodes), glandular stomach and liver. Decreased lymphocytes of the splenic white pulp occurred in males and females with minimal increased extramedullary haematopoiesis in the splenic red pulp of these animals. Decreased lymphocytes of the thymus and mesenteric and mandibular lymph nodes occurred in males and females. There was minimal eosinophilic infiltrate in the mucosa of the glandular stomach, and minimal increased cytoplasmic rarefaction in the hepatocytes. The increased rarefaction (consistent with increased glycogen) in hepatocytes is likely related to the lack of fasting of this group of animals prior to euthanasia. Dose of 75 mg/kg/day were tolerated, although reductions in body weight gain were observed at 25 mg/kg/day. There were no macroscopic findings at 75 mg/kg/day, and microscopic finding were minimal decreased lymphocytes in a few animals isolated to the spleen (along with minimal increased extramedullary haematopoiesis), and lymph nodes as well as a few animals with minimal eosinophilic infiltrate in the glandular stomach mucosa. There were no remarkable hematology or clinical chemistry findings. In addition to standard hematology, peripheral blood lymphocytes were enumerated by flow cytometry. There was a non dose-related trend of a slight decrease in the number of peripheral blood T cells in both male and female rats given 25 mg/kg/day, with no effect on total B cells. The relationship of peripheral blood T cell changes to doluegravir is uncertain because of lack of dose-response relationship and no corresponding histopathologic changes in lymphoid tissues in rats given 25 mg/kg/day. 48

49 Definitive Study In a definitive juvenile toxicity study, juvenile rats (10/sex/group) were given dolutegravir orally at doses of 0.5, 2 or 75 mg/kg/day from Day 4 to 66 postpartum (pp) with an additional 30/sex/group added for toxicokinetic analysis and an additional 10/sex/group added for a T cell dependent antibody response (TDAR) study [Report CD2010/00023, m ]. The following examinations and endpoints were evaluated: survival, external morphology, clinical observations, body weights, food consumption, toxicokinetics, physical development (including vaginal opening in females and balanopreputial skin fold separation in males), hematology, coagulation, clinical chemistry, urinalysis, macroscopic and microscopic pathology, stage dependent evaluation of spermatogenesis, organ weights, femur length, T cell dependent antibody response (anti- KLH IgM on Day 50 pp and anti-klh IgG on Day 59 pp), immunophenotyping of lymphocyte subsets (T and B cell enumeration) and T cell receptor V beta usage in peripheral blood. A summary of the study design and the results from this study are presented in m2.6.7, Table The systemic exposure to dolutegravir was generally lower on Day 32 pp compared to that on Day 13 pp. The Day 32 pp C max or AUC 0-24 values ranged from 9 to 89% lower than those values on Day 13 pp. There was no apparent exposure difference between sexes. There were two preweanling deaths (both male pups) that were considered test article related at 75 mg/kg/day. One male was found dead on Day 12 pp and another male on Day 17 pp. Both rats demonstrated a lack of sufficient body weight gain (for juvenile animal of these ages) in the days prior to death. There were no clinical observations in the days before the death and a cause of death was not determined in either case as there were no findings and no evidence of gavage trauma at necropsy. Because i) systemic exposure to dolutegravir was higher on Day 13 pp in the younger pre-weanling pups compared to the more mature juvenile pups on Day 32 pp, and ii) the two deaths occurred only in the high dose group and at a similar age and presentation to the prior dose range study at higher doses, the possible relationship to treatment could not be excluded. Therefore these preweanling deaths are considered test article related. Also, there was one death in the control group that occurred during the toxicokinetic sampling procedure on Day 32 pp. There were test article-related effects on mean body weight in males and females at 75 mg/kg/day during the preweaning periods and predominantly in females during the postweaning period. Over the preweaning treatment period (Day 4 and 21 pp), mean body weight gain was decreased (0.86X control mean gain) for males and females. Unlike the males, the body weight effect persisted throughout the entire study for females during the postweaning period. Although the females at 75 mg/kg/day gained weighed at a comparable pace with the control group, the decrease in gain that occurred in the preweaning period was not regained; males at 75 mg/kg/day gained weight at a faster pace than the control group and their absolute body weight became comparable to the control group during the postweaning period. This resulted in a Day 66 pp mean body weight value of 257 g for females at 75 mg/kg/day (0.91X of mean control value) compared to a control mean value of 282 g at the end of the study. Although this body 49

50 weight effect in females lasted the duration of the study, it did not result in an overall effect on growth as measured by length of a long bone (right femur). Food consumption was not affected during the postweaning period. Thus, the body weight effect at this dose level is interpreted to be an effect of general toxicity of the compound and not an effect on developmental growth. There were microscopic findings including degeneration/regeneration of the olfactory and/or respiratory epithelium of the nasal cavity of male ( 0.5 mg/kg/day) and female (75 mg/kg/day) rats that were considered secondary to expelling of the gavaged material rather than a direct test article effect. From the nasal cavity there was expulsion of gavage material noted sporadically during the oral dosing procedure at all dose levels. This conclusion was supported by the overall low incidence, low severity, and lack of a linear dose response and clear pattern of microscopic distribution and is consistent with a local irritation effect. There were no test article-related differences among the groups for the age at which offspring attained physical signs of sexual maturation (vaginal opening or balanopreputial skinfold separation). There were no test article-related effects on hematology, urinalysis, clinical chemistry parameters, macroscopic findings or organ weights. There were no changes considered related to dolutegravir administration in stage-dependent evaluation of spermatogenesis. There were no test article-related effects on T cell dependent antibody response (TDAR) measured on Day 67, and no effects on lymphocyte subsets (T cells, both CD4 and CD8 subsets, and B cells) and CD4 or CD8 T cell receptor Vβ usage in peripheral blood. The no observed adverse effect level (NOAEL) in juvenile rats was considered to be 2 mg/kg/day; systemic exposure values in males on Day 13 pp were AUC 0-24 = 303 μg.h/ml and C max = 15.2 μg/ml and on Day 32 pp AUC 0-24 = 85.7 μg.h/ml and C max = 7.71 μg/ml, with no appreciable difference in gender. 50

51 Table 6.2 Batch Numbers of Dolutegravir and Formulations Used in Reproductive and Developmental Toxicity Studies Batch or Lot Study Type Species (Strain) Duration of Report No. Concentration Formulation Number(s) Dosing (Study No.) (mg/ml) B86001 Fertility and early Rat (Sprague Up to 9 weeks XD2009/ , 3, 10% w/v 1 embryonic development Dawley) (S TF-063-L) A7Z001 Dose range embryofetal Rat (Sprague 12 days RD2008/ , 3, 10% w/v 1 development study Dawley) (S TB-051-L) B86001 Embryofetal Rat (Sprague 12 days XD2009/ , 3, 10% w/v 1 development study Dawley) (S TB-062-L) A7Z001 Dose range study Rabbit (Japanese 14 days RD2008/ , 2, 6, 20% w/v 1 White) (S TF-052-L) A7Z002 Dose range embryofetal Rabbit (Japanese 13 days RD2009/ , 6, 20% w/v 1 development study White) (S TF-060-L) B86001 Embryofetal Rabbit (Japanese 13 days XD2009/ , 4, 20% w/v 1 development study White) (S TF-065-L) Pre- and postnatal Rat (Sprague Day 6 gestation 2011N , 0.5, 10% w/v 1 development Dawley) Day 20 lactation (SG10306) A7Z002 Juvenile tolerability Rat (Sprague 18 days CD2009/ , 10, 20, 100, Dawley) (D09072) A7Z002 Juvenile dose range Rat (Sprague Up to 4 weeks CD2009/ , 5, 15, 60 1 Dawley) (D09126) B87002 Juvenile toxicity Rat (Sprague Dawley) Up to 9 weeks CD2010/00023 (G09229) 0.1, 0.4, 15 1 Note: The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. Studies were performed with dolutegravir sodium salt form, unless otherwise noted. Formulation Code: 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 51

52 7. LOCAL TOLERANCE 7.1. Introduction Local tolerance studies with dolutegravir were carried out to assess worker health and safety hazards associated with manufacture of the drug product, or to assess irritancy potential. These studies included ocular and skin irritancy studies, as well as studies of the potential to cause allergic contact dermatitis (skin sensitization), performed in various models. All studies were performed in full compliance with Good Laboratory Practice (GLP) regulations. Except as noted, all studies described in this section were performed using the sodium salt of dolutegravir, which is the form proposed for use in humans; however, all dosages and concentrations quoted in this summary are expressed in terms of the parent compound (referred to simply as dolutegravir). The impurity profile of the batches of test material used in these investigations was consistent with that used in the clinical evaluation of dolutegravir and that proposed for use in the marketed product. Where noted, the parent or free acid of dolutegravir (GSK B) was used. A table listing the batches of dolutegravir together with information on method of formulation used in these investigations is presented in Table 7.2 of this summary. A full listing of studies performed and the GLP status, together with the location of the reports within this submission, is provided in Table

53 Table 7.1 List of Local Tolerance Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Skin irritancy study Reconstituted human skin (SkinEthic RHE) NA In vitro 25 mg/site NA Yes ED2010/00004 (1127/1884) m Skin irritancy study * Rabbit (Japanese white) 3M Topical 500 mg/site Single No RD2010/00201 (S TF-080-N) m Ocular irritancy study Reconstituted human corneal model (SkinEthic RHE) NA In vitro 30 mg/site NA Yes ED2010/00005 (1127/1885) m Ocular irritancy study * Rabbit (Japanese white) 6M Topical 100 mg/eye Single No RD2010/00202 (S TF-079-N) m Local lymph node assay * Mouse (CBA/Ca) 5F Topical 25 L/site (25% w/w) (sodium salt) 3 days Yes ED2009/00019 (1127/1834) m Local lymph node assay Mouse (CBA/Ca) 5F Topical 25 L/site (25% w/w) (parent) Note: * These studies were performed with GSK A, the sodium salt form, and the other studies used the free acid form, GSK B. 3 days Yes 2010N (1127/1886) Testing Facility: = = GSK = GlaxoSmithKline. m

54 7.2. Dermal Irritancy In vitro An in vitro study was performed to determine the skin irritation potential of dolutegravir using the SkinEthic Reconstituted Human Epidermal model [Report ED2010/00004, m ]. Tissue samples (n=3) were treated with 25 mg of dolutegravir and incubated at 37 C for treatment periods of 4 and 24 hours. Viability of the dolutegravir-treated tissue was assessed. A summary of the study design and the results from this study are presented in m2.6.7, Table The results showed the relative mean viability (relative to negative control) of dolutegravir-treated tissue was 100.7% after 4 hours exposure and 107% after 24 hours exposure. Dolutegravir was considered to be a non-irritant Rabbit Dolutegravir was administered percutaneously to male Japanese white rabbits (n=3) as 0.5 g applied to the intact and abraded skin (applied area: 2.5 x 2.5 cm) for 24 hours under occluded conditions [Report RD2010/00201, m ]. The application sites were observed macroscopically for dermal reactions 24, 48, and 72 hours after the start of application according to the evaluation criteria of the Draize method. Body weights and clinical observations were also monitored. A summary of the study design and the results from this study are presented in m2.6.7, Table Slight erythema was noted in the abraded skin in 2 of 3 animals, 24 hours after the start of application, but the erythema disappeared by 48 hours after the start of application. No dermal reactions were noted in the intact skin at any time of observation. The primary irritation index was No abnormalities were noted in general signs of body weight changes in any animal during the observation period. From these results, dolutegravir was classified as a substance which resulted in mild irritation under the conditions of this study Ocular Irritancy In vitro An ocular irritancy study was performed in vitro using the SkinEthic Reconstituted Human Corneal model and the irritancy potential for dolutegravir was assessed [Report ED2010/00005, m ]. Tissue samples (n=3) were treated with 30 mg of dolutegravir and incubated at 37 C for treatment periods of 10 and 60 minutes. Viability of the dolutegravir-treated tissue was assessed. A summary of the study design and the results from this study are presented in m2.6.7, Table

55 The results showed the relative mean viability (relative to negative control) of dolutegravir-treated tissue was 98.7% after a 10 minute exposure period and 103.1% after a 60 minute exposure period. Dolutegravir was considered not to be a significant ocular irritant Rabbit Dolutegravir was applied to the left eye of 6 male Japanese white rabbits once, and the ocular irritation potential was assessed [Report RD2010/00202, m ]. The dose was 0.1 g of dolutegravir per eye. The left eye of each animal in the eye-unwashed group (n=3) was left as it was after application of dolutegravir, and the left eye of the animals in the eye-washed group (n=3) was washed for 20 to 30 seconds after application. The right eyes of all animals were untreated and served as the untreated controls. The eyes were observed macroscopically for reaction at 1, 24, 48, and 72 hours and 4 and 7 days after application and evaluated according to the Draize method. Body weights and general signs were also monitored. A summary of the study design and the results from this study are presented in m2.6.7, Table In the eye-unwashed group, slight redness, chemosis, and discharge were noted in the conjunctivae. In the eye-washed group, slight redness was noted in the conjunctivae. The ocular reactions noted in the eye-unwashed group disappeared by 72 hours after application and those noted in the eye-washed group disappeared by 24 hours after application. The index of acute ocular irritation (I.A.O.I.) was 4.00 in the eye-unwashed group and 2.00 in the eye-washed group. No abnormalities were noted in general signs or body weight changes in any animal during the observation period. Dolutegravir was classified as slightly irritating according to the results of this study. Alleviating effects of eye washing were confirmed in the eye-washed group, since the mean score 1 hour after application was lower than in the eye-unwashed group, and ocular irritation disappeared earlier than in the eye-unwashed group Skin Sensitisation Potential Mouse Local Lymph Node Assay The skin sensitization potential of dolutegravir was determined in the CBA/Ca mouse following topical application to the dorsal surface of the ear [Report ED2009/00019, m ]. Dolutegravir (25%w/w) or vehicle (dimethylformamide) was topically administered to the ear (25 L/ear) of female mice (n=5/group) once a day for 3 consecutive days. On Day 6, the mice were given a single intravenous injection of [ 3 H]-methyl thymidine (20 Ci/mouse) and 5 hours later the auricular lymph nodes were removed, single cell suspensions prepared and the lymphocyte stimulation index (SI) determined. A summary of the study design and the results from this study are presented in m2.6.7, Table

56 No clinical observations or changes in body weights were noted. Dolutegravir was considered to be a non-sensitizer under the conditions of this assay with a SI of A second study was conducted to assess the skin sensitization potential of dolutegravir in the CBA/Ca mouse, using the same methodology [Report 2010N109153, m ]. A summary of the study design and the results from this study are presented in m2.6.7, Table No clinical observations or changes in body weights were noted. Dolutegravir was considered to be a non-sensitizer under the conditions of this assay with a SI of

57 Table 7.2 Batch Numbers of Dolutegravir and Formulations Used in Local Tolerance Studies Batch or Lot Number(s) Study Type Species (Strain) or Test System Duration of Dosing Report No. (Study No.) R09002B Skin irritation In vitro NA ED2010/00004 (1127/1884) A7Z002 Skin irritation Rabbit (Japanese white) Single RD2010/00201 (S TF-080-N) R09002B Eye irritation In vitro NA ED2010/00005 (1127/1885) A7Z002 Eye irritation Rabbit (Japanese white) A7Z002 R09002B Note: Skin sensitisation potential Skin sensitisation potential Single RD2010/00202 (S TF-079-N) Mouse (CBA/Ca) Single ED2009/00019 (1127/1834) Mouse (CBA/Ca) Single 2010N (1127/1886) Concentration (mg/ml) Formulation 25 mg NA 0.5 g /2.5x2.5cm NA 30 mg NA 0.1 g / eye NA 25% w/w 6 25% w/w 6 The synthetic route used to prepare the nonclinical batches was the same as the route which is proposed for the marketed product. Studies were performed with dolutegravir sodium salt form, unless otherwise noted. Key: NA = not applicable Formulation Code: 1 = polyethylene glycol = polysorbate 20 3 = mannitol 4 = sodium carboxymethylcellulose 5 = Sesame oil 6 = Dimethyl formamide 57

58 8. OTHER TOXICITY STUDIES 8.1. Introduction In order to characterise more fully the toxicological profile of dolutegravir, another toxicity study has been performed and is detailed in this section. A study was conducted in rats to assess the effects of oral administration of dolutegravir on T-cell dependent antibody response. The study was performed in full compliance with Good Laboratory Practice (GLP) regulations. The study was performed using the sodium salt of dolutegravir, which is the form proposed for use in humans; however, all concentrations and dosages quoted in this summary are expressed in terms of the parent compound (referred to simply as dolutegravir). The impurity profile for the batch of test material used in this investigation was consistent with that used in the clinical evaluation of dolutegravir and that proposed for use in the marketed product. A table showing the batch of dolutegravir used in this study, together with information on the method of formulation, is presented in Table 8.2. A list showing the study performed and the GLP status, together with the location of the report within this submission, is provided in Table

59 Table 8.1 List of Other Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Immunotoxicity Rat (Sprague Dawley) 10M/10F Oral (gavage) 10, 100, weeks Yes RD2009/00751 (S TB-064-L) m Note: All studies were performed with GSK A, the sodium salt form. No observed adverse effect levels (NOAEL) are bolded. Testing Facility: = 59

60 8.2. Immunotoxicity T-cell dependent antibody response The effects of dolutegravir on T-cell dependent antibody response were evaluated in rats (n=10/sex/group) after oral administration of doses at 10, 100, and 1000 mg/kg/day for one month [Report RD2009/00751, m ]. Data that were collected include: clinical observations, body weights, antibody response, select organ weights and gross pathology (spleen, adrenal glands, and thymus), and histopathology for select spleens (those which showed gross lesions). A summary of the study design and the results from this study are presented in m2.6.7, Table There were no treatment-related signs and no effects on body weights for any study animals. An increase of mean absolute and relative spleen weight was noted in males at 1000 mg/kg/day and 2 males had notable enlargement/swelling in the spleen. However, these changes in the spleen were not considered toxicologically significant due to a lack of significant histopathological changes in the spleen. No dolutegravir-related effects were noted in anti-hemocyanin, Keyhole limpet antibody titers in both males and females from any group, thus, dolutegravir had no effects on T- cell dependent antibody formation in this study. 60

61 Table 8.2 Batch Numbers of Dolutegravir and Formulations Used in Other Toxicity Studies Batch or Lot Study Type Species (Strain) or Duration of Report No. Concentration Formulation Number(s) Test System Dosing (Study No.) (mg/ml) B86001 Immunotoxicity Rat (Sprague Dawley) 1 month RD2009/00751 (S TB-064-L) 0.1, 1, 10% w/v 1 Note: The synthetic route used to prepare this nonclinical batch was the same as the route which is proposed for the marketed product. The study was performed with dolutegravir sodium salt form. Formulation Code: 1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 80 61

62 9. DISCUSSION AND CONCLUSIONS Dolutegravir has been evaluated in single and repeat oral dose studies of up to 26 weeks in duration in Sprague Dawley rats, 38 weeks in cynomolgus monkeys, 14 days in Japanese white rabbits, and 13 weeks in CD-1 mice. Fertility studies have been conducted in male and female Sprague Dawley rats, and embryofetal development studies have been conducted in pregnant Sprague Dawley rats and Japanese white rabbits. Juvenile toxicity studies have been conducted in juvenile Sprague Dawley rats. Oral safety pharmacology studies assessing effects on the respiratory, cardiovascular, central and peripheral nervous systems, and in vitro effects on the cardiac potassium ion channel (herg), have also been conducted. In addition, a battery of 3 assays were conducted to determine the mutagenic and clastogenic potential of dolutegravir. A tabular summary of the principal treatment-related findings and effect/no effect doses in repeat dose studies in rats and monkeys is presented in Table 9.1, and comparisons of systemic exposure to dolutegravir is presented in Table 9.2. Also, a comparison of dolutegravir animal to human exposure ratios (AUC 0-24, mg/kg and mg/m 2 ) in the definitive rat and monkey studies is presented in Table 9.3 and Table 9.4, respectively. Mortality/morbidity: In a 14 day study, one female monkey given 1000 mg/kg/day died on Day 13 after experiencing daily emesis and diarrhea. This animal s condition deteriorated over the dosing phase and the moribund condition was considered secondary to treatment-related effects on the digestive tract (emesis, diarrhea, ulcer in colon) and resultant changes in blood electrolytes. This animal s systemic exposure (AUC 0-24 ) on Day 1 was 277 g.h/ml. Gender-mean Day 14 exposure (AUC 0-24 ) at 1000 mg/kg/day was 360 g.h/ml, which corresponds to ~7X or ~5X above the anticipated human exposure for a 50 mg QD or BID dose, respectively. In a 38 week monkey toxicity study, two males in the high dose group (50 mg/kg/day) died or were euthanized on Days 59/55 after signs of gastrointestinal intolerance which consisted of diarrhea and emesis and subsequent body weight loss. These effects and appropriate safety metrics are described further in the discussion of gastrointestinal effects. Gastrointestinal effects: The primary finding from repeat dose toxicity studies with dolutegravir up to 26 weeks in rats and 38 weeks in monkeys was gastrointestinal (GI) toxicity. In monkeys, the most sensitive species, GI toxicity was characterized primarily by vomiting, diarrhea, and associated mortality as well as gastrointestinal lesions, and by gastric lesions in the rat. In both species, these effects were observed at progressively lower doses with increased study duration. The GI toxicity is believed to be the result of local drug administration at the mucosal surface of the gut following oral dosing, rather than systemic toxicity. The fact that affected animals had comparable exposures to animals at dose levels which were not affected is supportive of the conclusion that the GI toxicity is due to the larger local exposure in the GI tract in those dose groups. Therefore, mg/kg or mg/m 2 metrics are appropriate determinates of safety cover for this toxicity because it is not caused by systemic exposure. These estimates are provided below in addition to animal:human exposure comparisons based on AUC. Dermal and ocular irritancy studies in rabbits indicate dolutegravir is a mild irritant, and GI toxicity may be 62

63 a class effect of integrase inhibitors, as raltegravir (a marketed integrase inhibitor) caused irritation to GI mucosal surfaces in rodents [Merck Research Laboratories, 2007]. In rats, hemorrhage was observed in the lamina propria of the mucosa at 1000 mg/kg/day in the 4 week toxicity study and was reversible following a 4 week recovery period. The NOAEL was 100 mg/kg/day. Exposure (end of study, gender mean) at 100 mg/kg/day was 752 g.h/ml, which corresponds to ~14X or ~10X above the anticipated human exposure for a 50 mg QD or BID dose, respectively. The NOAEL (100 mg/kg/day) is 100X and 50X the human mg/kg equivalent dose (based on 50 kg human), and 18X and 9X the human mg/m 2 equivalent dose for a total daily clinical dose of 50 mg QD or BID, respectively. In the 26 week toxicity study, hemorrhage in the glandular stomach mucosa occurred in 1 male at the end of the 17 week dosing period and 1 male at the end of the 26 week dosing period in the 500 mg/kg/day group. No adverse findings were observed at the end of a 4 week recovery period. The NOAEL was 50 mg/kg/day. Exposure (end of study, gender mean) at 50 mg/kg/day was 765 g.h/ml, which corresponds to ~14X or ~10X above the anticipated human exposure for a 50 mg QD or BID dose, respectively. The NOAEL (50 mg/kg/day) is 50X and 25X the human mg/kg equivalent dose (based on 50 kg human), and 9X and 4X the human mg/m 2 equivalent dose for a total daily clinical dose of 50 mg QD and BID, respectively. Irritation of the gastrointestinal tract consisting of epithelial atrophy and mucosal hemorrhage in the stomach and lower GI tract (cecum, colon and/or rectum) was noted in monkeys given 300 mg/kg/day in the 14 day toxicity study. The NOAEL was 100 mg/kg/day. Exposure (end of study, gender mean) at 100 mg/kg/day was 190 g.h/ml, which corresponds to ~4X or 3X above the anticipated human exposure for a 50 mg QD or BID dose, respectively. The NOAEL (100 mg/kg/day) is 100X and 50X the human mg/kg equivalent dose (based on 50 kg human), and 35X and 18X the human mg/m 2 equivalent dose for a clinical dose of 50 mg QD and 50 mg BID, respectively. In the 4 week monkey toxicity study, histopathological changes of the GI tract occurred at 100 mg/kg/day and consisted of slight inflammatory cell infiltration in the lamina propria of the cecum, colon and rectum in both sexes; slight cell debris from the crypts of the cecum and colon in males; and atrophy of the mucosal epithelium of the cecum and colon. This dose was associated with clinical signs of vomiting, diarrhea, and body weight loss. The NOAEL was 50 mg/kg/day. Exposure (end of study, gender mean) at 50 mg/kg/day was 132 g.h/ml, which corresponds to ~2X above the anticipated human exposure for a 50 mg QD or BID dose. The NOAEL (50 mg/kg/day) is 50X and 25X the human mg/kg equivalent dose (based on 50 kg human), and 18X and 9X the human mg/m 2 equivalent dose for a clinical dose of 50 mg QD and BID, respectively. In the 38 week monkey toxicity study, the 50 mg/kg/day dose was reduced to 30 mg/kg/day on Day 70 for the remainder of the study due to GI intolerance. In the 17 week evaluation of the 38 week monkey study, slight mononuclear cell infiltration and hemorrhage in the lamina propria in the cecum and colon were noted in the animal that was euthanized on Day 55. Abnormal feces (observed through Day 131) associated with decreased food consumption and decreased body weight was noted in the 50/30 mg/kg/day group. At the end of the 38 week dosing period, 1 female in the 50/30 mg/kg/day group had adverse findings in the stomach consisting of multifocal 63

64 mononuclear cell infiltration and slight hemorrhage in the lamina propria, very slight multifocal erosions, and multifocal epithelial regeneration. At the end of a 4 week recovery period, multifocal mononuclear cell infiltration and very slight hemorrhage in the lamina propria and multifocal epithelial regeneration in the stomach were observed in one female. However, the changes in this animal were of lesser severity and there were no active erosions, suggesting recovery of changes upon cessation of treatment. Both animals with stomach lesions had diarrhea/vomiting prior to the dose reduction (50/30 mg/kg/day), but did not have clinical observations of toxicity following the dose reduction. Exposures at the end of study for the two affected females were lower compared to the other animals in this dose group (AUC 0-24 = 43.5 to 48.8 g.h/ml versus gender mean for 50/30 mg/kg/day group of 61.7 g.h/ml) and overlapped with exposures at 15 mg/kg/day (AUC 0-24 range = 25.8 to 54.0 g.h/ml). This observation is consistent with a local GI toxicity as opposed to a systemic effect. The NOAEL for the 38 week dosing period was 15 mg/kg/day (Day 270 gender mean AUC 0-24 and C max of 39 g.h/ml and 5.1 g/ml, respectively), which corresponds to 0.7X and 1.4X the human AUC and C max exposure, respectively, for a 50 mg QD dose) and corresponds to 0.5X and 1.2 X the human AUC and C max exposure, respectively, for a 50 mg BID dose. The NOAEL for the 38 week dosing period (15 mg/kg/day) is 15X and 8X the human mg/kg equivalent dose (based on 50 kg human), and 5X and 3X the human mg/m 2 equivalent dose for a 50 mg QD and BID dose, respectively. The NOAEL for the 17 week interim evaluation was also 15 mg/kg/day; thus, there was not a decrease in the NOAEL from 17 weeks of dosing to 38 weeks of dosing. Non-clinical evidence for GI toxicity with dolutegravir (including vomiting, diarrhea and gastric/colonic erosions), did not translate into significant findings for dolutegravir in double blinded randomized clinical trials, with a similar rate and nature of events reported for dolutegravir compared to raltegravir and efavirenz/tenofovir/emtricitabine (Atripla). Similar rates of nausea, vomiting and diarrhea were reported in studies of subjects with integrase resistance, who received dolutegravir 50 mg BID compared to studies of subjects who were integrase naive, and received dolutegravir 50 mg once daily. Therefore, there does not appear to be an increased risk for GI events with this higher dose of dolutegravir. Hepatic effects: Hepatocellular single cell necrosis and diffuse hepatocellular hypertrophy and/or vacuolation occurred in male monkeys given 1000 mg/kg/day in the 14 day study. Additional changes included transient ALT increases at 300 mg/kg/day, increased AST, bilirubin, GTP, and triglycerides at 1000 mg/kg/day and decreased total cholesterol at 1000 mg/kg/day. The NOAEL was 100 mg/kg/day. Exposure (end of study, gender mean) at 100 mg/kg/day was 190 g.h/ml, which corresponds to ~4X or ~3X above the anticipated human exposure for a 50 mg QD or BID dose, respectively. In the 38 week monkey toxicity study, liver findings were restricted to increased AST (2.5X) and bilirubin (2.8X) in the moribund animal in the 50 mg/kg/day group (euthanized on Day 55). The findings in the 38 week study were considered secondary to the moribund condition. Exposure (end of study, gender mean) at the NOAEL (15 mg/kg/day) was 39 g.h/ml, which corresponds to ~0.7X or ~0.5X the anticipated human exposure for a 50 mg QD or BID dose, respectively. No treatment related adverse effects on liver were observed in rats in studies up to 26 weeks. 64

65 Human subjects were carefully monitored for liver effects and cumulative data to date suggests a hepatic safety profile for dolutegravir that is comparable to raltegravir and efavirenz, the comparators used in the Phase III studies. Renal effects: In the 14 day rat study, there were statistically significant increases in urine specific gravity in males given 500 mg/kg/day and in females given 50 mg/kg/day. Because no treatment-related microscopic findings were observed in the kidneys, the change was not considered toxicologically significant. In the 4 week rat study there was an increased incidence of urine protein and increased urine specific gravity in animals given 1000 mg/kg/day, however, there were no related changes in blood chemistry or microscopic findings, and none of these changes occurred in the rat 26 week study at up to 500 mg/kg/day (Day 180 AUC at 500 mg/kg/day = 1558 g.h/ml, which corresponds to ~29X or ~21X above the anticipated human exposure for a 50 mg QD or BID dose, respectively). Renal tubule dilatation occurred in monkeys given 1000 mg/kg/day in the 14 day study. BUN and creatinine were increased while serum sodium and chloride were decreased in these monkeys. In the 38 week monkey toxicity study, renal findings were restricted to increased BUN (12.5X) and creatinine (3.7X), and slight kidney dilatation of distal renal tubules and cellular and hyaline casts in the moribund animal in the 50 mg/kg/day group (euthanized on Day 55). These findings (in both the 14 day and 38 week monkey toxicity studies) were considered secondary to the moribund condition related to GI toxicity. The NOAEL in the 38 week toxicity study was 15 mg/kg/day. Exposure (end of study, gender mean) at 15 mg/kg/day was 39 g.h/ml, which corresponds to ~0.7X or ~0.5X the anticipated human exposure for a 50 mg QD or BID dose, respectively. Analyses of adverse events from the Renal Systems Organ Class in clinical studies do not suggest that dolutegravir has an adverse effect on renal function. There was a low incidence of renal impairment or failure, and these events were more likely a consequence of underlying disease, co-morbid conditions, and concurrent drugs, and were not thought to be related to dolutegravir treatment. Mild elevations of creatinine are expected for dolutegravir. These are related to a likely benign effect on creatinine secretion with blockade of the OCT2 receptor, and do not progress on continued treatment with dolutegravir. A higher incidence of dipstick proteinuria was noted in efavirenz-controlled studies but not in a raltegravir-controlled study. However, quantitative measures of proteinuria showed no difference between dolutegravir and either efavirenz or raltegravir based combination antiretroviral therapy. Bone marrow and lymph node changes: In the 14 day monkey study, hypocellular and/or gelatinous bone marrow and atrophy of the white pulp in the spleen occurred in monkeys given 1000 mg/kg/day and a decrease in the paracortical lymphocytes of the submandibular and/or mesenteric lymph nodes and decreased reticulocytes occurred in monkeys given 300 mg/kg/day. Decreased reticulocytes, RBCs, and platelets and increased APTT occurred in monkeys given 1000 mg/kg/day and are believed to correlate with the microscopic bone marrow changes. The NOAEL was 100 mg/kg/day. Exposure (end of study, gender mean) at 100 mg/kg/day was 190 g.h/ml, which corresponds to ~4X or ~3X above the anticipated human exposure for a 50 mg QD or BID dose, 65

66 respectively. In the 4 week monkey study, decreased RBCs (0.91X) were observed in females given 100 mg/kg/day, with no correlating histopathology findings. The NOAEL was 50 mg/kg/day. Exposure (end of study, gender mean) at 50 mg/kg/day was 132 g.h/ml, which corresponds to ~2X above the anticipated human exposure for a 50 mg QD or BID dose. No treatment related adverse effects on bone marrow and lymph nodes were observed in non-moribund animals in the 9 month monkey toxicity study at doses 50/30 mg/kg/day. No treatment related adverse effects on bone marrow or lymph nodes were observed in rats in studies up to 26 weeks. A review of data from clinical trials revealed no signal for bone marrow or lymph node toxicity caused by dolutegravir. Genetic toxicology: Dolutegravir did not cause gene mutation or chromosomal damage in two in vitro tests (bacterial mutation assay and mouse lymphoma L5178Y cell assay) or in an in vivo oral rat micronucleus test. Data from the genotoxicity assessments suggest that dolutegravir does not present a genotoxic hazard to humans. Carcinogenicity: Dolutegravir was not carcinogenic to mice at doses up to 500 mg/kg/day or rats at doses up to 50 mg/kg/day following oral administration for 104 consecutive weeks. In both species, dolutegravir administration had no effect on survival, there were no treatment related clinical signs, and there were no neoplastic or non-neoplastic findings attributed to dolutegravir. The NOAEL for non-neoplastic findings after chronic oral administration was the high dose of 500 mg/kg/day for mice and 50 mg/kg/day for rats. When compared to the anticipated human exposure for a 50 mg QD or BID dose, the systemic exposures were ~20X or ~14X and ~17X or ~12X higher for mice and rats, respectively. Reproductive toxicology: Dolutegravir had no effects on male or female fertility in rats and no effect on embryofetal development in pregnant rats or rabbits at 1000 mg/kg/day. Therefore, based on animal data, dolutegravir is not anticipated to increase the risk of adverse developmental (or reproductive) outcomes in humans when used in accordance with dosing information in the product label. Dolutegravir administration resulted in suppressed body weight gain and decreased food consumption in a pre- and postnatal development study in rat dams (F0) receiving 1000 mg/k/g/day. Decreased body weights were noted in the subsequent generation (F1) in the 1000 mg/kg group from pre-weaning until adolescence. The NOAELs were 50 mg/kg/day for maternal general toxicity (F0) and for development of the subsequent generation (F1) and 1000 mg/kg/day for maternal reproductive functions (F0) such as maintenance of pregnancy, delivery and nursing. Based on the fact that effects on offspring body weights were noted at doses where maternal toxicity was observed, and the presence of considerable safety margins expected at the proposed clinical doses, there is minimal risk for adverse effects on postnatal development in offspring of mothers receiving dolutegravir. Juvenile toxicity: A juvenile toxicity study in rats was conducted with dolutegravir at oral doses of 0.5, 2 or 75 mg/kg/day from Day 4 to 66 postpartum (pp). Two preweanling deaths were considered test article related at 75 mg/kg/day. Over the 66

67 preweaning treatment period (Day 4 and 21 pp), mean body weight gain was decreased (0.86X control mean gain) for males and females in the 75 mg/kg/day group and the decrease persisted throughout the entire study for females during the postweaning period. There were no test article-related differences among the groups for the age at which offspring attained physical signs of sexual maturation (vaginal opening or balanopreputial skinfold separation). There were no changes considered related to dolutegravir administration in stage-dependent evaluation of spermatogenesis. There were no test article-related effects on T cell dependent antibody response (TDAR) measured on Day 67, and no effects on lymphocyte subsets (T cells, both CD4 and CD8 subsets, and B cells) and CD4 or CD8 T cell receptor V usage in peripheral blood. Therefore, the NOAEL in juvenile rats was 2 mg/kg/day (Day 32 pp gender mean AUC 0-24 = 90 g.h/ml and C max = 7.6 g/ml). Clinical studies in pediatric patients conducted to date have not revealed any safety issues specific to this population. Immunotoxicity: Oral administration of dolutegravir at doses up to 1000 mg/kg/day for 4 weeks had no effect on T cell dependent antibody response in male and female rats. Additionally, there were no signs of immunotoxicity from general toxicology studies findings or clinical safety data. Therefore, there is negligible risk of immunotoxicity potential to adult patient populations. Conclusion: The primary finding from repeat dose toxicity studies up to 38 weeks in monkeys and 26 weeks in rats with dolutegravir was gastrointestinal (GI) intolerance. In monkeys, the most sensitive species, GI intolerance was characterized primarily by vomiting, diarrhea, and associated mortality as well as gastrointestinal lesions, and by gastric lesions in the rat. In both species, these effects were observed at progressively lower doses with increased study duration. GI intolerance leading to mortality and morbidity in two animals occurred at 50 mg/kg/day in the 38 week monkey toxicity study. Subsequently (Day 70), the 50 mg/kg/day dose was reduced to 30 mg/kg/day for the duration of the study. At the end of the 38 week dosing period, 1 female in the 50/30 mg/kg/day group had adverse findings in the stomach consisting of multifocal mononuclear cell infiltration and slight hemorrhage in the lamina propria, very slight multifocal erosions, and multifocal epithelial regeneration. There was evidence of recovery at the end of the 4 week recovery period as 1 female in the 50/30 mg/kg/day group had similar but less severe lesions in the stomach with no active erosions. The GI intolerance is believed to be the result of local drug administration and not systemic toxicity. The NOAEL for the 38 week dosing period was 15 mg/kg/day (Day 270 gender mean AUC 0-24 and C max of 39 g.h/ml and 5.1 g/ml, respectively), which corresponds to 0.7X and 1.4X the human AUC and C max exposure, respectively, at a 50 mg once daily dose (AUC 0-24 of 53.6 g.h/ml and C max of 3.7 g/ml) and corresponds to 0.5X and 1.2X the human AUC and C max exposure, respectively, at a 50 mg twice daily (AUC 0-24 of 75.1 g.h/ml and C max of 4.2 g/ml). Because GI intolerance is considered to be due to local drug administration, mg/kg or mg/m 2 metrics are appropriate determinates of safety cover for this toxicity. The NOAEL for the 38 week dosing period (15 mg/kg/day) is 15X and 8X the human mg/kg equivalent dose (based on 50 kg human), and 5X and 3X the human mg/m 2 equivalent dose for a total daily clinical dose of 50 mg and 100 mg, respectively. Dermal and ocular irritancy studies in rabbits indicate dolutegravir is a mild irritant, and GI toxicity may be a class effect of integrase inhibitors, as raltegravir (a 67

68 marketed integrase inhibitor) caused irritation to GI mucosal surfaces in rodents [Merck Research Laboratories, 2007]. Table 9.1 Principal Toxicological Findings in Rats and Monkeys Following Oral Administration of Dolutegravir Effect Dose (mg/kg/day) Rat No Effect Dose (mg/kg/day) Effect Dose (mg/kg/day) Monkey No Effect Dose (mg/kg/day) Finding Mortality/Morbidity: Adult animals: Death preceded by repeated emesis, NO NO diarrhea with significant weight loss Juvenile animals: Mortality preceded by decreased 75 2 NA NA body weight gain Clinical Observation: Emesis, diarrhea NO NO a Body Weight Loss NO NO Gastrointestinal Effects: Stomach: Gastric mucosal hemorrhage, mononuclear cell infiltration, and/or multifocal epithelial regeneration Stomach: Multifocal erosions Cecum, colon, rectum: mucosal atrophy and/or hemorrhage Hepatic Effects: Hepatocellular single cell necrosis and vacuolation with AST, and GTP elevations and/or increased bilirubin and triglycerides 500 NO NO NO 50 NO NO NO 50/30 50/ ALT elevations without corresponding anatomic pathology changes AST & bilirubin elevations secondary to moribundity Renal Effects considered associated with NO NO moribund condition: Renal tubule dilatation, increased BUN and CRE, and/or decreased serum sodium and chloride Bone Marrow and Lymphoid changes considered NO NO associated with moribund condition and/or stress: Gelatinous or hypocellular bone marrow, thymic atrophy, spleenic lymphoid atrophy, decreased lymphocytes of the submandibular and mesesenteric lymph nodes, adrenal hypertrophy/increased weight, decreased retics, platelets, increased fibrinogen and/or prolonged APTT Decrease in RBCs (females) NO NO Findings Considered to be Associated with malnutrition: Acinar cell atrophy in the pancreas and/or parotid gland NO NO a. One male monkey in the 15 mg/kg/day group had non-adverse transient diarrhea with no effect on body weight that recovered during the dosing period. ALT = Alanine aminotransferase. APTT = Activated partial thromboplastin time. AST = Aspartate aminotransferase. BUN = Blood urea nitrogen. BW = Body weight. CRE = Creatinine. FC = Food consumption. NO = Not observed. NA = Not Applicable. RBC = Red blood cell. 68

69 Table 9.2 Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir Species (Duration) Rat d (14 days) Rat d (4 weeks) Dose (mg/kg/day) Sex Cmax ( g/ml) AUC0-24 ( g.h/ml) Animal to Day 1 End of Day 1 End of Human AUC Study Study Ratio a,b (50mg QD) Animal to Human AUC Ratio a,c (50mg BID) 50 M F M F (NOAEL) M F M F M F M (NOAEL) F M F Rat 5 M (26 weeks) d F M (NOAEL) F M F Monkey d (14 days) Monkey d (4 weeks) 100 M (NOAEL) F M F M F M F M (NOAEL) F M F Monkey 3 M (38 weeks) e F M F M (NOAEL) F /30 M F

70 Table 9.2 (Continued) Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir Species (Duration) Dose (mg/kg/day) Sex Cmax ( g/ml) AUC0-24 ( g.h/ml) Animal to Day 1 End of Study Day 1 End of Study Human AUC Ratio a,b (50mg QD) Animal to Human AUC Ratio a,c (50mg BID) Mouse 10 M (14 day) f F M F M F (NOAEL) M F Mouse 10 M (13-week) f F M F M F M (NOAEL) F Rat (embryofetal development) Rabbit (embryofetal development) 100 F F (NOAEL) g F F h F h F Rat 0.5 M (Juvenile) i F M (NOAEL) F M F Mouse (Carcinogenicity) f, j Rat (Carcinogenicity) k 7.5 M F M F M (NOAEL) F M F M F (NOAEL) M F

71 Table 9.2 (Continued) Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir Species (Duration) Dose (mg/kg/day) Sex Cmax ( g/ml) AUC0-24 ( g.h/ml) Animal to Day 1 End of Day 1 End of Human AUC Study Study Ratio a,b (50mg QD) Animal to Human AUC Ratio a,c (50mg BID) Human b 50 mg M/F NA NA Human c 100 mg M/F NA NA Key: The systemic exposure margins within the main body text of m2.4 are presented as gender averaged means. a. Calculated for AUC based on end of treatment values b. Based on the geometric mean of systemic human exposure AUC and Cmax values at a total daily dose of 50 mg from pooled data of Spring-1 and Spring-2 c. Based on the geometric mean of systemic human exposure AUC and Cmax values at a total daily dose of 100 mg (50 mg BID) from pooled data of Viking and SAILING. d. Values are the mean of n=3 to 5. e. Values are the mean of n= 7 to 9. f. Composite plasma toxicokinetic parameters from mice, n=3/sex/group/time point. g. The NOAEL was 1000 mg/kg/day for dams and embryo-fetal development. h. The NOAEL was 200 mg/kg/day for maternal general toxicity and 1000 mg/kg/day for maternal reproductive function and embryofetal development. i. Composite plasma toxicokinetic parameters for juvenile rats were examined on Day 13pp and Day 32pp. Composite parameters were derived from mean plasma concentration data. n=3/timepoint/dose, with the exception of the 8 hour timepoint on Day 13pp following 75 mg/kg/day, for which n=4. j. Toxicokinetics conducted on Day 26 and Day 182 instead of Day 1 and End of Study, respectively. k. Values are the mean of n=4/sex/group. Toxicokinetics conducted on Day 28 and Day 182 instead of Day 1 and End of Study, respectively. Note: No observed adverse effect levels (NOAEL) are bolded. QD = once daily. BID = twice daily. 71

72 Table 9.3 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M 2 ) Following Oral Administration of Dolutegravir in the 4 and 26 week Rat Toxicology Studies Species (Duration) Rat (4 weeks) Dose (mg/kg/day) Sex Day 1 Cmax ( g/ml) End of Study AUC0-24 ( g.h/ml) Day 1 End of Study 2 M F M F M (NOAEL) F M AUC (50mg QD) AUC (50mg BID) ANIMAL TO HUMAN RATIOS Based on AUC, MG/KG and MG/M 2 (gender averaged means) Mg/Kg (50mg QD) Mg/Kg (50mg BID) Mg/M 2 (50mg QD) Mg/M 2 (50mg BID) F Rat 5 M (26 weeks) F M (NOAEL) F M F Human 50 mg M/F NA NA NA NA NA NA Human 100 mg M/F NA NA NA NA NA NA Key: Calculations are based on 50 kg human. For conversion of animal doses in mg/kg to dose in mg/m 2, multiply by Km (Km rat = 6; Km human = 34). NA = Not applicable. 72

73 Table 9.4 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M 2 ) Following Oral Administration of Dolutegravir in the 14 Day and 4 and 38 Week Monkey Toxicology Studies Species (Duration) Dose (mg/kg/day) Sex Cmax ( g/ml) AUC0-24 ( g.h/ml) ANIMAL TO HUMAN RATIOS Based on AUC, MG/KG and MG/M 2 (gender averaged means) Day 1 End of Study Day 1 End of Study AUC (50mg QD) AUC (50mg BID) Mg/Kg (50mg QD) Mg/Kg (50mg BID) Mg/M 2 (50mg QD) Monkey 100 M (14 days) (NOAEL) F M F M F Monkey 25 M (4 weeks) F M (NOAEL) F M F Monkey 3 M (38 weeks) F M F M (NOAEL) F /30 M F /30 25/ / /5.3 Human 50 mg M/F NA NA NA NA NA NA Human 100 mg M/F NA NA NA NA NA NA Key: Calculations are based on 50 kg human. For conversion of animal doses in mg/kg to dose in mg/m 2, multiply by Km (Km monkey = 12; Km human = 34). NA = Not applicable. Mg/M 2 (50mg BID) 73

74 10. REFERENCES Key Literature References Skopek TR, Liber HL, Kaden DA and Thilly WG. Relative sensitivities of forward and reverse mutation assays in Salmonella typhimurium. Proc. Natl. Acad. Sci. (USA). 1978; 75: References (Available Upon Request) NTP CAS Registry Number: : Merck Research Laboratories. Raltegravir_ACM_BGPK_1.doc, Version 9.2, August FDA Advisory Committee, September 5, Guidelines (not provided) ICH Q3A(R2) (CPMP/ICH/2737/99): Impurities in New Drug Substances. October ICH S2(R1) (CHMP/ICH/126642/08): Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use (2008). ICH M3(R2) (CPMP/ICH/286/95): Note for guidance on non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. EMEA/CHMP/QWP/251344/2006 (CHMP/SWP/5199/02): Guideline on the limits of genotoxic impurities. London: EMEA (European Medicines Agency)

75 APPENDIX 1 CARCINOGENICITY TABLES 75

76 Table.1 List of Tissues Studied in Carcinogenicity Studies Tissue Rat (Report No ) Mouse (Report No ) Gross lesion Tissue masses Adrenals Aorta (thoracic) Bone (sternum, distal femur) Bone marrow (sternum,distal femur) Brain Cecum Colon Epididymides Esophagus Eyes Gallbladder X Heart Kidneys Lacrimal glands/harderian glands Liver Lungs (with mainstem bronchi) Lymph nodes (mesenteric,mediastinal) Mammary gland Muscle (rectus femoris) Nerve (sciatic) Ovaries Pancreas Pituitary Prostate Salivary glands (submandibular) Seminal vesicles Skin (dorsal base of tail) Small intestines (duodenum, ileum, jejunum and Peyer s patches/galt) Spinal cord (cervical) Spleen Stomach Testes Thymus Thyroid (with parathyroid) Trachea Urinary bladder Uterus (horns/body/cervix) Vagina Key: GALT = Gut Associated Lymphoid Tissue 76

77 Table.2 Carcinogenicity Study in Mice - Tumor Incidence Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Adipose Tissue Adrenal Glands Identification of Tumor Invasive Neoplasm (N) Medulla Benign Pheochromocytoma (B) Cortex Carcinoma (M) Cortex Adenoma (B) Invasive Neoplasm (N) Aorta Invasive Neoplasm (N) Bone (other) Osteoma (B) Sarcoma, NOS (M) Invasive Neoplasm (N) Brain Invasive Neoplasm (N) Cecum Invasive Neoplasm (N) Cervical Spinal Cord Invasive Neoplasm (N) Colon Invasive Neoplasm (N) Diaphragm Invasive Neoplasm (N) Distal Femur Invasive Neoplasm (N) Osteoma (B) Duodenum Invasive Neoplasm (N) Ears Invasive Neoplasm (N) Epididymides Invasive Neoplasm (N) Esophagus Invasive Neoplasm (N) Eyes Invasive Neoplasm (N)

78 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Femoral Marrow Gallbladder Harderian Glands Heart Invasive Neoplasm (N) Hemangioma (B) Invasive Neoplasm (N) Papilloma (B) Cystadenoma/Adenoma (B) Invasive Neoplasm (N) Adenoma (B) Carcinoma (M) Invasive Neoplasm (N) Endocardial Schwannoma (M) Hemangiosarcoma (M) Ileum Invasive Neoplasm (N) Intercostal Muscle Invasive Neoplasm (N) Jejunum Invasive Neoplasm (N) Kidneys Invasive Neoplasm (N) Lacrimal Gland Liver Invasive Neoplasm (N) Adenoma (B) Invasive Neoplasm (N) Hepatocellular Carcinoma (M) Hepatocellular Adenoma (B) Hemangiosarcoma (M) Hemangioma (B)

79 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Invasive Neoplasm (N) Lungs Bronchiolo/Alveolar Adenoma (B) Bronchiolo/Alveolar Carcinoma (M) Lymph Node (other) Invasive Neoplasm (N) Lymph/ Retic System Malignant Lymphoma (M) Histiocytic Sarcoma (M) Mammary Invasive Neoplasm (N) (protocol) Adenocarcinoma (M) Mandibular Lymph Nodes Invasive Neoplasm (N) Mediastinal Lymph Nodes Invasive Neoplasm (N) Mesenteric Lymph Invasive Neoplasm (N) Nodes Hemangiosarcoma (M) Mesentery/ Perito Invasive Neoplasm (N) Malignant Mesothelioma (M) Mesovarian Ligament Invasive Neoplasm (N) Muscle (Other) Invasive Neoplasm (N) Hemangiosarcoma (M) Rhabdomyosarcoma (M)

80 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Muscle (Protocol) Invasive Neoplasm (N) Hemangiosarcoma (M) Nerve Sciatic Invasive Neoplasm (N) Invasive Neoplasm (N) Tubulostromal Adenoma (B) Ovaries Adenocarcinoma (M) Cystadenoma (B) Benign Granulosa Cell Tumor (B) P. Patches/ GALT Invasive Neoplasm (N) Pancreas Invasive Neoplasm (N) Islet Cell Adenoma (B) Parotid Gland Invasive Neoplasm (N) Pericardium Invasive Neoplasm (N) Pituitary Invasive Neoplasm (N) Pars Distalis Adenoma (B) Prostate Invasive Neoplasm (N) Carcinoma (M) Rectum/ Low Colon Invasive Neoplasm (N) Salivary Gland Invasive Neoplasm (N) Seminal Vesicles Invasive Neoplasm (N)

81 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Carcinoma (M) Invasive Neoplasm (N) Skin (other) Hemangiosarcoma (M) Sarcoma NOS (M) Sebaceous Cell Adenoma (B) Histiocytoma (B) Skin (protocol) Invasive Neoplasm (N) Schwannoma (B) Fibrosarcoma (M) Invasive Neoplasm (N) Spleen Hemangioma (B) Hemangiosarcoma (M) Sternal Marrow Invasive Neoplasm (N) Sternum Stomach Testes Invasive Neoplasm (N) Osteogenic Sarcoma (M) Invasive Neoplasm (N) Glandular Mucosa: Adenoma (B) Forestomach: Squamous Cell Carcinoma (M) Invasive Neoplasm (N) Benign Interstitial Cell Tumor (B) Thymus Invasive Neoplasm (N)

82 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) (1) Water Control Frequency according to dose and sex (n) (2) Vehicle Control (3) Low 7.5 mg/kg/day (4) Inter. 25 mg/kg/day (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Thyroid Identification of Tumor Invasive Neoplasm (N) Follicular Cell Adenoma (B) Trachea Invasive Neoplasm (N) Ureters Invasive Neoplasm (N) Urinary Bladder Invasive Neoplasm (N) Leiomyosarcoma (N) Invasive Neoplasm (N) Endometrial Carcinoma (M) Endometrial Stromal Polyp (B) Leiomyosarcoma (M) Uterus w/ Cervix Hemangioma (B) Benign Granular Cell Tumor (B) Endometrial Stromal Sarcoma (M) Leiomyoma (B) Granular Cell Tumor (M) Hemagiosarcoma (M) Vagina Invasive Neoplasm (N) Vessel Hemangiosarcoma (C) Hemangioma (C) Zymbal s Gland Carcinoma (M)

83 Sex M/F M F M F M F M F M F Number of Animals Evaluated Number of Tissues Examined A). Macroscopically Abnormal Adipose tissue Bone (other) Bulbourethral Gland Coagulating Gland Diaphragm Ear(s) Extremity Intercostal Musc Lymph Node (other) Mandibular Lymph Nodes Mesentery/Perito Mesovarian Ligament Muscle (other) Parotid Gland Penis Pericardium Preput/Clit Gland Rectum/Low Colon Skin (other) Soft Tissue Tail Tongue Ureters Zymbal s Gland

84 Sex M/F M F M F M F M F M F Number of Animals Evaluated Number of Tissues Examined B). Protocol Tissue of Which Some Were Not Available For Histological Examination Adrenal Glands Aorta Cecum Colon Distal Femur Duodenum Esophagus Femoral Marrow Gallbladder Harderian Glands Ileum Jejunum Kidneys Lacrimal Gland Mammary (protocol) Mediastinal Lymph Node Mesenteric Lymph Node Nerve Sciatic Ovaries Peyer s Patches/GALT Parathyroid Pituitary Prostate Skin (protocol) Spleen Sternal Marrow Sternum Stomach

85 Sex M/F M F M F M F M F M F Number of Animals Evaluated Number of Tissues Examined Thymus Thyroid Trachea Urinary Bladder Vagina Key: - = Not examined (M) = Malignant (B) = Benign (N) = Metastatic NOS = Not otherwise specified (C) = Multicentric GALT = Gut Related Lymphoid Tissue P. Patches = Peyer s Patches Lymph/Retic = Lymphoreticular Mesentery/Perito = Mesentery/Peritoneum Preput/Clit = Preputial/Clitoral 85

86 Table.3 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence Males Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Water Control) 1041 D Muscle (other) Hemangioscarcoma Y (Water Control) 1029 D Skin (other) Carcinoma Y (Vehicle Control) 2048 D Tail [Skin (protocol)] Schwannoma N D Ear(s) [Skin (protocol)] Fibrosarcoma Y D Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N S Tail Keratin cyst N D Salivary Gland Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N S Ear(s) [Skin (protocol)] Schwannoma N 86

87 Table.4 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence Females Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Water Control) 1522 D Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N (Water Control) 1526 D Skin (other) Sebaceous Cell Adenoma N (Water Control) 1531 D Lymph Node (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N (Water Control) 1501 D Skin (other) Sarcoma Y Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) 0 (Water Control) 1523 D 101 Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N N 87

88 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Water Control) 1542 S Tail [Skin (protocol)] Fibrosarcoma Y (Vehicle Control) 2511 D Salivary Gland Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N (Vehicle Control) 2517 S Skin (other) Sarcoma Y D Skin (other) Carcinoma Y D Skin (other) [Mammary (protocol)] Adenocarcinoma Y D Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N D Skin (other) Hemangiosarcoma Y Key: D = Death S = Sacrificed Lymph/Retic = Lymphoreticular 88

89 Table.5 Carcinogenicity Study in Mice - Summary of Survival and Fate of Animals Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

90 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

91 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

92 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

93 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

94 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP Key: E = Number entering week. D = Deaths. K = Killed moribund/accidentally/for humane reasons. N = Necropsied completely. NP = Necropsied to some extent. 94

95 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

96 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

97 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

98 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

99 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

100 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP Key: E = Number entering week. D = Deaths. K = Killed moribund/accidentally/for humane reasons. N = Necropsied completely. NP = Necropsied to some extent. 100

101 Table.6 Carcinogenicity Study in Rats - Tumor Incidence Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Adipose Tissue Adrenal Glands Lipoma (B) Hibernoma (M) Invasive Neoplasm (N) Medulla Pheochromocytoma (B) Medulla Pheochromocytoma (M) Cortex Carcinoma (M) Cortex Adenoma (B) Invasive Neoplasm (N) Aorta Invasive Neoplasm (N) Bone (other) Brain Cecum Histiocytoma, fibrous (M) Fibrosarcoma (B) Astrocytoma (M) Granular Cell Tumor (B) Oligodendroglioma (M) Meningioma (B) Invasive Neoplasm (N) Fibroma (B) Invasive Neoplasm (N) Colon Invasive Neoplasm (N) Diaphragm Invasive Neoplasm (N) Duodenum Invasive Neoplasm (N) Ears Fibrosarcoma (M) Epididymides Invasive Neoplasm (N) Esophagus Invasive Neoplasm (N)

102 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Extremity Keratoacanthoma (B) ] ] Invasive Neoplasm (N) Eyes Invasive Neoplasm (N) Femoral Marrow Invasive Neoplasm (N) Harderian Gland Invasive Neoplasm (N) Heart Invasive Neoplasm (N) Ileum Jejunum Kidneys Lacrimal Adenocarcinoma (M) Invasive Neoplasm (N) Leiomyoma (B) Neuroendocrine Neoplasm (M) Leiomyosarcoma (M) Invasive Neoplasm (N) Carcinoma, Tubular (M) Carcinoma, Transitional Cells (M) Liposarcoma (M) Invasive Neoplasm (N) Gland Invasive Neoplasm (N) Liver Hepatocellular Adenoma (B) Hepatocellular Carcinoma (M) Hemangiosarcoma (M) Invasive Neoplasm (N)

103 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Lungs Carcinoma Nos (M) Bronchiolo/Alveolar Carcinoma (M) Hemangiosarcoma (M) Invasive Neoplasm (N) Lymph Node (other) Invasive Neoplasm (N) Lymph/ Retic System Mammary (protocol) Mediastinal Histiocytic Sarcoma (M) Malignant Lymphoma (M) Granulocytic Leukemia (M) Adenocarcinoma (M) Fibroadenoma (B) Adenoma (B) Invasive Neoplasm (N) Lymph Nodes Invasive Neoplasm (N) Meninges Mesenteric Malignant Meningioma (M) Lymph Nodes Invasive Neoplasm (N) Mesentery/ Perito Liposarcoma (M) Malignant Mesothelioma (M) Lipoma (B)

104 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Muscle (Other) Carcinoma (Nos) (M) Invasive Neoplasm (N) [ Muscle (Protocol) Invasive Neoplasm (N) Nerve Sciatic Invasive Neoplasm (N) Ovaries Invasive Neoplasm (N) P. Patches/ GALT Invasive Neoplasm (N) Pancreas Islet Cell Adenoma (B) Islet Cell Carcinoma (M) Hemangioma (B) Acinar Cell Adenoma (B) Invasive Neoplasm (N) Parathyroid Adenoma (B) Pericardium Invasive Neoplasm (N) Pituitary Pars Distalis Adenoma (B) Pars Distalis Carcinoma (M) Pars Intermedia Adenoma (B) Invasive Neoplasm (N) Prostate Invasive Neoplasm (N) Rectum/ Low Colon Fibroma (B) Salivary Gland Leiomyoma (B) Invasive Neoplasm (N)

105 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Seminal Vesicles Invasive Neoplasm (N) Skin (other) Skin (protocol) Malignant Basal Cell Tumor (M) Lipoma (B) Keratoacanthoma (B) Benign Basal Cell Tumor (B) Fibrosarcoma (M) Fibroma (B) Histiocytoma Fibrous (M) Squamous Cell Papilloma (B) Squamous Cell Carcinoma (M) Myxosarcoma (M) Malignant Schwannoma (M) Invasive Neoplasm (N) Keratoacanthoma (B) Histiocytoma Fibrous (M) Soft Tissue Invasive Neoplasm (N) Spleen Hemangioma (B) Invasive Neoplasm (N) Sternal Marrow Invasive Neoplasm (N) Stomach Carcinoma Squamous Cell (M) Invasive Neoplasm (N)

106 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Tail Testes Thymus Thyroid Keratoacanthoma (B) Basal Cell Tumor (B) Invasive Neoplasm (N) Benign Interstitial Cell Tumor (B) Invasive Neoplasm (N) Malignant Thymoma (M) Invasive Neoplasm (N) Carcinoma Follicular Cell (M) Follicular Cell Adenoma (B) C Cell Adenoma (B) C Cell Carcinoma (M) Invasive Neoplasm (N) Trachea Invasive Neoplasm (N) Urinary Bladder Invasive Neoplasm (N) Uterus w/ Cervix Vagina Vascular Tissue Endometrial Stromal Polyp (B) Benign Schwannoma (B) Leiomyoma (B) Invasive Neoplasm (N) Granular Cell Tumor (M) Invasive Neoplasm (N) Hemangiosarcoma (C) Hemangioma (C)

107 Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance) Frequency according to dose and sex (n) (2) (3) (4) Vehicle Low Inter. Control 2 mg/kg/day 10 mg/kg/day (1) Water Control (5) High 500 mg/kg/day Biometric Analysis: No M F M F M F M F M F Number of Animals Evaluated Organ Identification of Tumor Zymbal s Gland Adenoma (B) Carcinoma (M)

108 Sex M/F M F M F M F M F M F Number of Animals Evaluated Number of Tissues Examined A). Macroscopically Abnormal Adipose tissue Bone (other) Cavity Coagulating Gland Diaphragm Ear(s) Extremity Lymph Node (other) Meninges Mesentery/Perito Muscle (other) Penis Pericardium Preput/Clit Gland Rectum/Low Colon Skin (other) Soft Tissue Tail Ureters Urethra Vascular Tissue Vessel Zymbal s Gland

109 Sex M/F M F M F M F M F M F Number of Animals Evaluated Number of Tissues Examined B). Protocol Tissue of Which Some Were Not Available For Histological Examination Heart Mediastinal Lymph Node Mesenteric Lymph Node Mammary (protocol) Nerve Sciatic Parathyroid Salivary Gland Thymus Urinary Bladder Key: - = Not examined (M) = Malignant (B) = Benign (N) = Metastatic (C) = Multicentric GALT = Gut Related Lymphoid Tissue P. Patches = Peyer s Patches Lymph/Retic = Lymphoreticular Mesentery/Perito = Mesentery/Peritoneum Preput/Clit = Preputial/Clitoral 109

110 Table.7 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence Males Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Water Control) 1034 D Skin (other) Fibrosarcoma Y (Water Control) 1062 D Zymbal s Gland Carcinoma Y (Water Control) 1031 D Skin (other) Basal cell tumor Y (Water Control) 1020 D Skin (other) Cyst, keratinaceous N (Water Control) 1004 D Skin (other) Metastatic invasive neoplasm from Lymph/Retic System N (Water Control) 1007 D Skin (other) Keratoacanthoma N (Water Control) 1036 D Skin (other) Fibrosarcoma Y Squamous cell papilloma N 0 (Water Control) 1037 S Skin (other) 92 Keratoacanthoma N 110

111 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Myxosarcoma Y 0 (Water Control) 1035 S Skin (other) 104 Basal cell tumor N (Water Control) 1047 S Tail Basal cell tumor N (Vehicle Control) 2018 D Skin (other) Fibrosarcoma Y (Vehicle Control) 2020 D Skin (other) Squamous cell carcinoma Y (Vehicle Control) 2051 D Skin (other) Basal cell tumor N (Vehicle Control) 2037 D Skin (other) Histiocytoma, fibrous Y (Vehicle Control) 2043 D Skin (other) Keratoacanthoma N (Vehicle Control) 2004 D Skin (other) Keratoacanthoma N (Vehicle Control) 2057 D Mammary (protocol) Fibroadenoma N (Vehicle Control) 2028 D Skin (other) Fibrosarcoma Y (Vehicle Control) 2036 D Skin (other) Keratoacanthoma N (Vehicle Control) 2014 D Skin (other) Fibroma N (Vehicle Control) 2013 S Skin (other) Lipoma N (Vehicle Control) 2009 S Skin (other) Fibrosarcoma Y 111

112 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Vehicle Control) 2048 S Lymph/Retic System Histiocytic sarcoma Y D Skin (other) Histiocytoma, fibrous Y D Skin (other) Keratoacanthoma N Skin (other) Fibrosarcoma Y D 83 Skin (other) Myxosarcoma Y 52 Skin (other) Keratoacanthoma N D 71 Skin (other) Keratoacanthoma N D Skin (other) Keratoacanthoma N D Muscle (protocol) Metastatic invasive neoplasm from Lymph/Retic System N D Skin (other) Fibroma N D Skin (other) Squamous cell carcinoma Y S Skin (other) Lipoma N 112

113 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Skin (other) Keratoacanthoma N D 73 Skin (other) Fibrosarcoma Y D Skin (other) Keratoacanthoma N D Skin (other) Squamous cell papilloma N D Skin (other) Keratoacanthoma N S Skin (other) Fibroma N S Skin (other) Squamous cell papilloma N S Skin (other) Fibrosarcoma Y S Skin (other) Basal cell tumor N S Skin (other) Squamous cell papilloma N D Skin (other) Squamous cell papilloma N D Skin (other) Lipoma N D Skin (other) Keratoacanthoma N D Skin (other) Metastatic/invasive neoplasm from Lymph/Retic System N 113

114 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy D Skin (other) Histiocytoma, fibrous Y S Skin (other) Lipoma N 104 Key: D = Death S = Sacrificed Lymph/Retic = Lymphoreticular 93 Skin (other) Cyst, keratinaceous N S 97 Skin (other) Basal cell tumor N 114

115 Table.8 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence Females Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy 34 Mammary (protocol) Adenocarcinoma Y 34 Mammary (protocol) Adenoma N 41 Mammary (protocol) Adenoma N (Water Control) 1505 D Mammary (protocol) Fibroadenoma N 43 Mammary (protocol) Adenoma N 49 Mammary (protocol) Adenoma N 51 Mammary (protocol) Adenoma N (Water Control) 1560 D Mammary (protocol) Adenocarcinoma Y (Water Control) 1527 D Mammary (protocol) Adenocarcinoma Y (Water Control) 1506 D Mammary (protocol) Fibroadenoma N (Water Control) 1525 D Skin (other) Metastatic/invasive neoplasm (metastatic from Lymph/Retic System) N 115

116 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N 0 (Water Control) 1559 D 67 Mammary (protocol) Adenocarcinoma Y (Water Control) 1535 D Skin (other) Fibroma N (Water Control) 1563 D Mammary (protocol) Adenocarcinoma Y (Water Control) 1517 D Mammary (protocol) Hyperplasia, mammary, cytistic N (Water Control) 1503 D Mammary (protocol) Fibroadenoma N Mammary (protocol) Adenocarcinoma Y 0 (Water Control) 1554 D 65 Mammary (protocol) Fibroadenoma N (Water Control) 1550 D Mammary (protocol) Adenocarcinoma Y (Water Control) 1564 D Mammary (protocol) Adenocarcinoma Y 73 Mammary (protocol) Fibroadenoma N (Water Control) 1524 D Mammary (protocol) Hyperplasia, mammary N 80 Mammary (protocol) Fibroadenoma N (Water Control) 1555 D Mammary (protocol) Adenocarcinoma Y (Water Control) 1551 D Mammary (protocol) Fibroadenoma N 116

117 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N 0 (Water Control) 1512 D 80 Mammary (protocol) Fibroadenoma N 77 Mammary (protocol) Adenocarcinoma Y 0 (Water Control) 1547 D 77 Mammary (protocol) Fibroadenoma N 80 Mammary (protocol) Fibroadenoma N (Water Control) 1502 D Mammary (protocol) Fibroadenoma N 89 Mammary (protocol) Fibroadenoma N (Water Control) 1513 S Mammary (protocol) Fibroadenoma N 48 Mammary (protocol) Adenocarcinoma Y 69 Mammary (protocol) Adenocarcinoma Y 95 0 (Water Control) 1537 S 77 Mammary (protocol) Hyperplasia, Mammary, cystic N 77 Mammary (protocol) Adenocarcinoma Y 117

118 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy 51 Mammary (protocol) Fibroadenoma N Mammary (protocol) Adenocarcinoma Y 0 (Water Control) 1556 S 77 Mammary (protocol) Fibroadenoma N 81 Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N 0 (Water Control) 1501 S 85 Mammary (protocol) Adenocarcinoma Y (Water Control) 1536 S Mammary (protocol) Fibroadenoma N (Water Control) 1504 S Mammary (protocol) Fibroadenoma N Mammary (protocol) Hyperplasia, mammary, N cystic 89 0 (Water Control) 1528 S Mammary (protocol) Fibroadenoma N 93 Mammary (protocol) Fibroadenoma N (Water Control) 1565 S Skin (other) Keratoacanthoma N (Water Control) 1514 S Mammary (protocol) Fibroadenoma N 118

119 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Adenocarcinoma Y 0 (Water Control) 1529 S 93 Mammary (protocol) Adenocarcinoma Y 87 Mammary (protocol) Fibroadenoma N 0 (Water Control) 1540 S 92 Mammary (protocol) Fibroadenoma N 32 Mammary (protocol) Adenocarcinoma Y (Vehicle Control) 2539 D Mammary (protocol) Adenocarcinoma Y 60 Mammary (protocol) Adenocarcinoma Y (Vehicle Control) 2526 D Mammary (protocol) Fibroadenoma N 38 Mammary (protocol) Fibroadenoma N (Vehicle Control) 2564 D Mammary (protocol) Fibroadenoma N 65 Skin (other) Keratoacanthoma N (Vehicle Control) 2541 D Mammary (protocol) Hyperplasia, mammary, cystic N (Vehicle Control) 2548 D Mammary (protocol) Adenocarcinoma Y Skin (other) Keratoacanthoma N 0 (Vehicle Control) 2559 D 65 Mammary (protocol) Adenocarcinoma Y 119

120 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Vehicle Control) 2521 D Mammary (protocol) Adenocarcinoma Y (Vehicle Control) 2516 D Mammary (protocol) Fibroadenoma N 46 Mammary (protocol) Fibroadenoma N (Vehicle Control) 2547 D Mammary (protocol) Fibroadenoma N 59 Mammary (protocol) Fibroadenoma N (Vehicle Control) 2552 D Mammary (protocol) Fibroadenoma N (Vehicle Control) 2531 D Mammary (protocol) Fibroadenoma N (Vehicle Control) 2514 D Mammary (protocol) Fibroadenoma N (Vehicle Control) 2527 D Mammary (protocol) Hyperplasia, mammary N (Vehicle Control) 2535 D Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N 81 0 (Vehicle Control) 2536 D Mammary (protocol) Hyperplasia, mammary, cystic N (Vehicle Control) 2565 D Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N 0 (Vehicle Control) 2537 D 82 Mammary (protocol) Fibroadenoma N 120

121 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Vehicle Control) 2523 S Mammary (protocol) Adenocarcinoma Y 69 Mammary (protocol) Fibroadenoma N 95 0 (Vehicle Control) 2562 S 95 Mammary (protocol) Fibroadenoma N 71 Mammary (protocol) Fibroadenoma N 95 0 (Vehicle Control) 2504 S 92 Mammary (protocol) Fibroadenoma N 71 Mammary (protocol) Fibroadenoma N 95 0 (Vehicle Control) 2506 S 89 Skin (other) Squamous cell papilloma N 73 Mammary (protocol) Adenocarcinoma Y 95 0 (Vehicle Control) 2502 S 85 Mammary (protocol) Adenocarcinoma Y 121

122 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy 75 Mammary (protocol) Adenocarcinoma Y (Vehicle Control) 2540 S Mammary (protocol) Fibroadenoma N 89 Mammary (protocol) Fibroadenoma N 79 Mammary (protocol) Fibroadenoma N Mammary (protocol) Adenocarcinoma Y 88 Mammary (protocol) Adenoma N 95 0 (Vehicle Control) 2530 S 92 Mammary (protocol) Fibroadenoma N Mammary (protocol) Adenocarcinoma Y 92 Mammary (protocol) Adenoma N Mammary (protocol) Fibroadenoma N 0 (Vehicle Control) 2513 S 95 Mammary (protocol) Fibroadenoma N (Vehicle Control) 2558 S Mammary (protocol) Fibroadenoma N 122

123 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy (Vehicle Control) 2543 S Mammary (protocol) Fibroadenoma N (Vehicle Control) 2505 S Skin (other) Squamous cell papilloma N (Vehicle Control) 2524 S Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenoma N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N 123

124 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N D 82 Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Skin (other) Squamous cell papilloma N 124

125 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N D 90 Mammary (protocol) Fibroadenoma N 50 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N 77 Mammary (protocol) Fibroadenoma N S Tail Keratoacanthoma N 69 Mammary (protocol) Fibroadenoma N 73 Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Fibroadenoma N 87 Mammary (protocol) Fibroadenoma N 87 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N S Mammary (protocol) Adenoma N 125

126 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy S Mammary (protocol) Fibroadenoma N D Thymus Malignant thymoma Y D Mammary (protocol) Fibroadenoma N 41 Mammary (protocol) Fibroadenoma N Mammary (protocol) D Hyperplasia, mammary, cystic N 54 Mammary (protocol) Hyperplasia, mammary, cystic N Mammary (protocol) Adenocarcinoma Y D 37 Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N 126

127 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Hyperplasia mammay, cystic 66 Mammary (protocol) Fibroadenoma N N 63 Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N D 77 Mammary (protocol) Adenocarcinoma Y 82 Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y Mammary (protocol) Fibroadenoma N D 65 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y Mammary (protocol) Fibroadenoma N D 79 Mammary (protocol) Adenocarcinoma Y 127

128 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy 68 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y 82 Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N D 82 Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N D 82 Mammary (protocol) Fibroadenoma N 60 Mammary (protocol) Adenocarcinoma Y Mammary (protocol) D Fibroadenoma Adenoma N N 79 Mammary (protocol) Adenocarcinoma Y 85 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y 128

129 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy D 93 Mammary (protocol) Mammary (protocol) Fibroadenoma N Hyperplasia mammay, cystic 63 Mammary (protocol) Fibroadenoma N S 91 Mammary (protocol) Adenocarcinoma Y N S Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Fibroadenoma N 71 Mammary (protocol) Fibroadenoma N 82 Mammary (protocol) Adenocarcinoma Y S 85 Mammary (protocol) Adenocarcinoma Y 95 Mammary (protocol) Adenocarcinoma Y 129

130 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy S Mammary (protocol) Fibroadenoma Adenoma N N 95 Mammary (protocol) Adenoma N S Mammary (protocol) Fibroadenoma N Mammary (protocol) Fibroadenoma N S 87 Mammary (protocol) Fibroadenoma N 90 Mammary (protocol) Adenocarcinoma Y S 93 Mammary (protocol) Fibroadenoma N S Mammary (protocol) Fibroadenoma N S Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Adenocarcinoma Y S Skin (other) Benign basal cell tumor N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y 130

131 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N D 42 Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N D Mammary (protocol) Mammary (protocol) Hyperplasia, mammary, cystic Hyperplasia, mammary, cystic N N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y Mammary (protocol) Fibroadenoma N D 37 Mammary (protocol) Fibroadenoma N 48 Mammary (protocol) Fibroadenoma N D 65 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Adenocarcinoma Y 131

132 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N D 78 Mammary (protocol) Fibroadenoma N D Mammary (protocol) Adenocarcinoma Y D Mammary (protocol) Fibroadenoma N 62 Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Adenocarcinoma Y 87 Mammary (protocol) Fibroadenoma N S Mammary (protocol) Adenocarcinoma Y 68 Mammary (protocol) Fibroadenoma N S Mammary (protocol) Fibroadenoma N 91 Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Fibroadenoma N S Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Fibroadenoma N 132

133 Week of Necropsy Week Tumor Discovered Dose Group (mg/kg/day) Animal Number Mode of Death Tissue Tumor Type Malignancy Mammary (protocol) Fibroadenoma N S 90 Mammary (protocol) Fibroadenoma N S Mammary (protocol) Adenocarcinoma Y S Mammary (protocol) Fibroadenoma N 133

134 Table.9 Carcinogenicity Study in Rats - Summary of Survival and Fate of Animals Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

135 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

136 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

137 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

138 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

139 Week Group and Sex of 1M 2M 3M 4M 5M Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP Key: E = Number entering week. D = Deaths. K = Killed moribund/for humane reasons. N = Necropsied completely. NP = Necropsied to some extent. 139

140 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

141 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

142 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

143 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

144 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP

145 Week Group and Sex of 1F 2F 3F 4F 5F Study Water Control Vehicle Control Low Intermediate High E D K N NP E D K N NP E D K N NP E D K N NP E D K N NP Key: E = Number entering week. D = Deaths. K = Killed moribund/for humane reasons. N = Necropsied completely. NP = Necropsied to some extent. 145

146 APPENDIX 2 ADDITIONAL INFORMATION The following reports have been reviewed within GlaxoSmithKline (GSK) and the information is considered to have no bearing on safety. These reports are not included in the study listing tables because they were performed with intermediates in the synthesis of dolutegravir as part of routine investigations for worker health and safety during manufacturing, or they are either considered to be superseded. Report No. (Study No.) 2010N109154_00 (1127/1877) 2010N109158_00 (1127/1892) 2010N109159_00 (1127/1870) 2010N109160_00 (1127/1878) 2010N109161_00 (1127/1881) 2010N109162_00 (1127/1893) 2010N109274_00 (1127/1861) 2010N109275_00 (1127/1864) 2010N109276_00 (1127/1890) 2010N109277_00 (1127/1867) 2010N109278_00 (1127/1891) 2010N104267_00 (1127/1860) 2010N104313_00 (1127/1874) 2010N104314_00 (1127/1872) 2010N104299_00 (1127/1865) 2010N104269_00 (1127/1879) 2010N104268_00 (1127/1880) Title gsk021*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk022* RABBIT ENUCLEATED EYE TEST gsk022*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk023*: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD gsk023*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk023*: RABBIT ENUCLEATED EYE TEST gsk024*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk025*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk025*: RABBIT ENUCLEATED EYE TEST gsk026*: LOCAL LYMPH NODE ASSAY IN THE MOUSE gsk026*: RABBIT ENUCLEATED EYE TEST gsk024*: determination of eye irritation potential using an in vitro test strategy gsk027*: Determination of eye irritation potential using an in vitro test strategy gsk027*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model gsk026*: Determination of eye irritation potential using an in vitro test strategy gsk023*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model gsk023*: Determination of eye irritation potential using an in vitro test strategy * 新薬承認情報提供時に置き換え 146

147 Report No. (Study No.) 2010N104318_00 (1127/1876) 2010N104305_00 (1127/1869) 2010N104295_00 (1127/1863) 2010N104317_00 (1127/1875) 2010N104306_00 (1127/1868) 2010N104296_00 (1127/1862) 2010N104300_00 WD2007/01596/00 Title gsk021*: Determination of eye irritation potential using an in vitro test strategy gsk022*: Determination of eye irritation potential using an in vitro test strategy gsk025*: Determination of eye irritation potential using an in vitro test strategy gsk021*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model gsk022*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model gsk025*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model gsk026*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model GSK A: High Throughput Fluctuation Test (Screen) with Salmonella typhimurium * 新薬承認情報提供時に置き換え 147

148 MODULE TOXICOLOGY TABULATED SUMMARY

149 m Toxicology Tabulated Summary 2012N153953_00 TABLE OF CONTENTS PAGE 1. TOXICOLOGY: OVERVIEW FOR DOLUTEGRAVIR (also known as GSK or S ) TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS STUDIES TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS DATA TOXICOLOGY: DRUG SUBSTANCE SINGLE DOSE TOXICITY REPEAT DOSE TOXICITY: NON-PIVOTAL STUDIES REPEAT DOSE TOXICITY: PIVOTAL STUDIES GENOTOXICITY: IN VITRO GENOTOXICITY: IN VIVO CARCINOGENICITY REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: NON-PIVOTAL STUDIES REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: FERTILITY AND EARLY EMBRYONIC DEVELOPMENT TO IMPLANTATION (PIVOTAL) REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON EMBRYOFETAL DEVELOPMENT (PIVOTAL) REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT INCLUDING MATERNAL FUNCTION (PIVOTAL) STUDIES IN JUVENILE ANIMALS LOCAL TOLERANCE OTHER TOXICITY STUDIES

150 m Toxicology Tabulated Summary 2012N153953_00 LIST OF TABLES PAGE Table 1.1 List of Single Dose Toxicity Studies Performed with Dolutegravir...5 Table 1.2 List of Repeat Dose Toxicity Studies Performed with Dolutegravir...6 Table 1.3 List of Genotoxicity Studies Performed with Dolutegravir...7 Table 1.4 Table 1.5 Table 1.6 List of Carcinogenicity Studies Performed with Dolutegravir...8 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir...9 List of Local Tolerance Studies Performed with Dolutegravir...11 Table 1.7 List of Other Toxicity Studies Performed with Dolutegravir...12 Table 2.1 Table 3.1 Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir...13 Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC 0-24 ( g.h/ml)]...15 Table 4.1 Toxicology: Drug Substance for Dolutegravir...17 Table 5.1 Single Dose Toxicity for Dolutegravir...19 Table 6.1 Repeat Dose Toxicity: Non-Pivotal Studies for Dolutegravir...24 Table 7.1 Table 7.2 Table 7.3 Table 7.4 Table Week Oral Toxicity Study of GSK A (S ) in Mice Day Oral Toxicity Study of GSK A (ERC Sodium) in Rats Week Oral Toxicity Study of GSK A (ERC Sodium) in Rats Week Oral Toxicity Study of S Sodium in Rats Day Oral Toxicity Study of GSK A (ERC Sodium) in Monkeys...35 Table Month Oral Toxicity Study of ERC Sodium in Monkeys...37 Table Week Oral Toxicity Study of S Sodium in Monkeys...40 Table 8.1 GSK A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli

151 m Toxicology Tabulated Summary 2012N153953_00 Table 8.2 Table 8.3 GSK In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus...50 GSK A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus...51 Table 9.1 GSK A: Oral Bone Marrow Micronucleus Assay in Rats...53 Table 10.1 Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks...54 Table 10.2 Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks...62 Table 11.1 Table 11.2 Table 11.3 Table 12.1 Table 13.1 Table 13.2 Table 14.1 Table 15.1 Table 16.1 Table 17.1 Reproductive and Developmental Toxicity: Non-Pivotal Studies...71 Two week Oral toxicity Study of S Sodium in Non Pregnant Rabbits: Non-Pivotal Studies...73 Dose Range Finding Oral Study for Effects of S sodium on Embryofetal Development in Rabbits: Non-Pivotal Studies...75 Oral Study for Effects of S Sodium on Fertility and Early Embryonic Development to Implantation in Rats...77 Oral Study for Effects of S Sodium on Embryofetal Development in Rats...80 Oral Study for Effects of S Sodium on Embryofetal Development in Rabbits...85 Oral Study for Effects of S Sodium on Pre- and Post- Natal Development, Including Maternal Function, in Rats...91 GSK A: Oral Juvenile Toxicity Study in Rats...96 Local Tolerance Studies...99 Immunotoxicity Study of S Sodium in Rats: Determination of Specific Antibody Formation Against T-Cell- Dependent Antigen

152 m Toxicology Tabulated Summary 2012N153953_00 1. TOXICOLOGY: OVERVIEW FOR DOLUTEGRAVIR (also known as GSK or S ) Table 1.1 List of Single Dose Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Single Dose Rat (Sprague Dawley) 6M 6M Subcutaneous Intramuscular 2.5 Single No GSK RD2009/00921 (R42470) m Single Dose Rat (Sprague Dawley) 3M 3M Subcutaneous Intramuscular 2.5, 5 Single No GSK RD2009/00959 (R42475) m Single Dose Rat (Sprague Dawley) 3M Intramuscular 4, 7.3, 8.7 Single No GSK 2011N (R42826) m Single Dose Rat (Sprague Dawley) 3M Intramuscular 10 Single No GSK 2012N m Single Dose * Dog (beagle) 1F Oral 30, 100, 150, 250, 500 Single No RD2009/00963 m Single Dose Monkey (cynomolgus) 4F 4F 4F Oral Subcutaneous Intramuscular Single No GSK CD2009/00647 (D09113) m Single Dose Monkey (cynomolgus) 1F Oral (gavage) 50, 125, 250, 500 Single No RD2007/01184 (E TF-008-R) m Single Dose Monkey (cynomolgus) 3F Oral (gavage) 1, 3, 10, 50 Single No RD2008/01762 (S TB-44-R) m Note: All single dose oral studies were performed with dolutegravir, sodium salt form, while the subcutaneous and intramuscular studies were performed with the parent form of dolutegravir. * Dolutegravir is referred to in this report as MTS B. Testing Facility: GSK = GlaxoSmithKline = 5

153 m Toxicology Tabulated Summary 2012N153953_00 Table 1.2 List of Repeat Dose Toxicity Studies Performed with Dolutegravir Type of Study Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Repeat Dose Species (Strain)/ Test System Mouse (CD-1) Mouse (CD-1) Rat (Sprague Dawley) Rat (Sprague Dawley) Rat (Sprague Dawley) Monkey (cynomolgus) Monkey (cynomolgus) Monkey No./Sex/ Group 10M/10F 10M/10F 10M/10F a 10M/10F a,d 12M/12F f 3M/3F 3M/3F e 7M/7F Method of Administration Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral Dose (mg/kg/day) or Concentration Duration of Dosing (cynomolgus) (gavage) Note: All studies were performed with GSK A, the sodium salt form No observed adverse effect levels (NOAEL) are bolded. Key: a = Additional 4M/4F per group included for toxicokinetic parameters only. d = An additional 5M/5F were included at 1000 mg/kg/day for a 4 week recovery investigation. e = An additional 2M/2F were included at 100 mg/kg/day for a 4 week recovery investigation. f = An additional 6M/6F per group included for toxicokinetic parameters only. g = An additional 10M/10F per group were killed after 17 weeks of treatment for interim examinations. h = An additional 6M/6F were added at 500 mg/kg/day group for a 4 week recovery period. i = An additional 2 to 3M/2 to 3F per group were killed after 17 weeks of treatment for interim examinations. j = An additional 2M/2F were added at 15 and 50/30 mg/kg/day group for a 4 week recovery period. GLP Testing Facility Report No. (Study No.) 10, 100, 500, days No RD2009/01546 (S TF-066-R) 10, 50, 500, weeks Yes RD2009/00028 (S TF-068-L) 50, 150, days Yes RD2007/01140 (E TB-012-L) 2, 10, 100, weeks Yes RD2008/01628 (E TB-043-L) 5, 50, Yes RD2009/00410 weeks g,h (SBL ) 100, 300, days Yes RD2007/01142 (E TF-029-L) 25, 50, weeks Yes RD2008/00107 ((E TF-036-L) 3, 10, 15, 50/30 38 weeks i,j Yes RD2009/00036 Testing Facility: = = = = (SBL ) Location in CTD m m m m m m m m

154 m Toxicology Tabulated Summary 2012N153953_00 Table 1.3 List of Genotoxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Ames test NA NA In vitro 5 to 849 g/plate b NA Yes WD2007/00514 (V27467) m Screening mouse lymphoma assay Mouse lymphoma assay NA NA In vitro Up to 80 g/ml c NA No GSK WD2007/01581 (MLA-580) NA NA In vitro 5 to 85 g/ml b NA Yes WD2007/00515 (V27468) m m Rat micronucleus Rat (Sprague Dawley) 6M Oral (gavage) 50, 150, days Yes WD2007/00513 (R27469) m Note: All studies were performed with GSK A, the sodium salt form. Key: b = Highest concentration limited by solubility. c = Highest concentration limited by either solubility or toxicity. NA = Not applicable. Testing Facility: = = GSK = GlaxoSmithKline. 7

155 m Toxicology Tabulated Summary 2012N153953_00 Table 1.4 List of Carcinogenicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Carcinogenicity Mouse (CD-1) 65 Oral 45 TK (gavage) 7.5, 25, years Yes 2012N m Carcinogenicity Rat (Sprague Dawley) 65 Oral 12 TK (gavage) 2, 10, 50 2 years Yes 2012N m Key: TK = Satellite groups for toxicokinetics analysis. Testing Facility: = 8

156 m Toxicology Tabulated Summary 2012N153953_00 Table 1.5 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Fertility and early embryonic development Rat (Sprague Dawley) 20M/20F Oral (gavage) 100, 300, 1000 Up to 66 days (M) Up to 42 days (F) Yes XD2009/00368 (S TF-063-L) m Dose range embryofetal development study Rat (pregnant) (Sprague Dawley) 7F Oral (gavage) 100, 300, days (gestation Days 6 to 17) Yes RD2008/01761 (S TB-051-L) m Embryofetal development study Rat (pregnant) (Sprague Dawley) 20F Oral (gavage) 100, 300, days (gestation Days 6 to 17) Yes XD2009/00367 (S TB-062-L) m Dose range study Rabbit (non-pregnant) (Japanese white) 3F Oral (gavage) 30, 100, 300, days No RD2008/01760 (S TF-052-L) m Dose range embryofetal development study Rabbit (pregnant) (Japanese white) 4F Oral (gavage) 100, 300, days (gestation Days 6 to 18) Yes RD2009/00186 (S TF-060-L) m Embryofetal development study Rabbit (pregnant) (Japanese white) 18 to 20F Oral (gavage) 40, 200, days (gestation Days 6 to 18) Yes XD2009/0366 (S TF-065-L) m

157 m Toxicology Tabulated Summary 2012N153953_00 Table 1.5 (Continued) List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Pre- and postnatal development Rat (Sprague Dawley) 22F Oral (gavage) 5, 50, 1000 Gestation Day 6 to Lactation Day 20 Yes 2011N (SG10306) m Juvenile tolerability Rat (Sprague Dawley) (juvenile) 8M/8F Oral (gavage) 5, 50, 100, 500, days (Day 4 to Day 21 pp) No GSK CD2009/00409 (D09072) m Juvenile dose range Rat (Sprague Dawley) (juvenile) 20M/20F Oral (gavage) 2, 25, 75, days (Day 4 to Day 31 pp) No GSK CD2009/00770 (D09126) m Juvenile toxicity Rat (Sprague Dawley) (juvenile) 10M/10F Oral (gavage) 0.5, 2, 75 d 63 days (Days 4 to 66 pp) Yes GSK CD2010/00023 (G09229) m Note: All studies were performed with GSK A, the sodium salt form. No observed adverse effect levels (NOAEL) are bolded. d = Additional 30/sex/group were added for toxicokinetic analysis and 10/sex/group were added for T-cell-dependent antibody response assessments. pp = Post partum. Testing Facility: GSK = GlaxoSmithKline. = = 10

158 m Toxicology Tabulated Summary 2012N153953_00 Table 1.6 List of Local Tolerance Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Skin irritancy study Reconstituted human skin NA In vitro 25 mg/site NA Yes ED2010/00004 (1127/1884) m Skin irritancy study Rabbit (Japanese white) 3M Topical 500 mg/site Single No RD2010/00201 (S TF-080-N) m Ocular irritancy study Reconstituted human corneal model NA In vitro 30 mg/site NA Yes ED2010/00005 (1127/1885) m Ocular irritancy study Rabbit (Japanese white) 6M Topical 100 mg/eye Single No RD2010/00202 (S TF-079-N) m Local lymph node assay Mouse (CBA/Ca) 5F Topical 25 L/site (25% w/w) (sodium salt) 3 days Yes ED2009/00019 (1127/1834) m Local lymph node assay Mouse (CBA/Ca) 5F Topical 25 L/site (25% w/w) (parent) 3 days Yes 2010N (1127/1886) m Note: All studies were performed with GSK A, the sodium salt form. Testing Facility: = = GSK = GlaxoSmithKline. 11

159 m Toxicology Tabulated Summary 2012N153953_00 Table 1.7 List of Other Toxicity Studies Performed with Dolutegravir Type of Study Species (Strain)/ Test System No./Sex/ Group Method of Administration Dose (mg/kg/day) or Concentration Duration of Dosing GLP Testing Facility Report No. (Study No.) Location in CTD Immunotoxicity Rat (Sprague Dawley) 10M/10F Oral (gavage) 10, 100, weeks Yes RD2009/00751 (S TB-064-L) m Note: All studies were performed with GSK A, the sodium salt form. No observed adverse effect levels (NOAEL) are bolded. Testing Facility: = 12

160 m Toxicology Tabulated Summary 2012N153953_00 2. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS STUDIES Table 2.1 Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir Type of Study Test System Method of Administration Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Single dose Toxicokinetics Repeat dose Toxicokinetics Repeat dose Toxicokinetics Repeat dose Toxicokinetics Repeat dose Toxicokinetics Repeat dose Rat (Sprague Dawley) Rat (Sprague Dawley) Rat (Sprague Dawley) Rat (Sprague Dawley) Dog (beagle) Monkey (cynomolgus) Monkey (cynomolgus) Monkey (cynomolgus) Mouse (CD-1) Mouse (CD-1) Rat (Sprague Dawley) Rat (Sprague Dawley) Rat (Sprague Dawley) Subcutaneous Intramuscular Subcutaneous Intramuscular Doses (mg/kg) GLP Compliance Report (Study Number) 2.5 No RD2009/00921 (R42470) 2.5, 5 No RD2009/00959 (R42475) Intramuscular 4, 7.3, 8.7 No 2011N (R42826) Location (Module) m m m Intramuscular 10 No 2012N m Oral (gavage) Oral (gavage) Oral (gavage) 30, 100, 150, 250, , 125, 250, 500 No RD2009/00963 m No RD2007/01184 m , 3, 10, 50 No RD2008/01762 (S TB-44-R) Oral, SC, IM 3, 1, 1 No CD2009/00647 (D09113) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) Oral (gavage) 10, 100, , 50, 500, 1500 No Yes RD2009/01546 (S TF-066-R) RD2009/00028 (S TF-068-L) 50, 150, 500 Yes RD2007/01140 (E TB-012-L) 2, 10, 100, 1000 Yes RD2008/01628 (E TB-043-L) 5, 50, 500 Yes RD2009/00410 (S TF-055-L) m m m m m m m

161 m Toxicology Tabulated Summary 2012N153953_00 Table 2.1 (Continued) Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir Type of Study Test System Method of Administration Doses (mg/kg) GLP Compliance Report (Study Number) Location (Module) Toxicokinetics Reproductive and Developmental Toxicity Rat (pregnant) (Sprague Dawley) Oral (gavage) 100, 300, 1000 Yes XD2009/00367 (S TB-062-L) m Toxicokinetics Reproductive and Developmental Toxicity Rat (juvenile) (Sprague Dawley) Oral (gavage) 5, 50, 100, 500, 1000 No CD2009/00409 (D09072) m Toxicokinetics Reproductive and Developmental Toxicity Rat (juvenile) (Sprague Dawley) Oral (gavage) 2, 25, 75, 300 No CD2009/00770 (D09126) m Toxicokinetics Reproductive and Developmental Toxicity Rat (juvenile) (Sprague Dawley) Oral (gavage) 0.5, 2, 75 Yes CD2010/00023 (G09229) m Toxicokinetics Reproductive and Developmental Toxicity Rabbit (non-pregnant) (Japanese white) Oral (gavage) 30, 100, 300, 1000 Yes RD2008/01760 (S TF-052-L) m Toxicokinetics Reproductive and Developmental Toxicity Rabbit (pregnant) (Japanese white) Oral (gavage) 40, 200, 1000 Yes XD2009/00366 (S TF-065-L) m Toxicokinetics Repeat dose toxicity Monkey (cynomolgus) Oral (gavage) 100, 300, 1000 Yes RD2007/01142 (E TB-012-L) m Toxicokinetics Repeat dose toxicity Monkey (cynomolgus) Oral (gavage) 25, 50, 100 Yes RD2008/00107 (E TF-036-L) m Toxicokinetics Repeat dose toxicity Monkey (cynomolgus) Oral (gavage) 3, 10, 15, Yes RD2009/ /30 d (S TF-047-L) m Toxickinetics Local Tolerance Rat (Sprague Dawley) Intramuscular injection 4, 7.3, 8.7 No 2011N (R42826) m

162 m Toxicology Tabulated Summary 2012N153953_00 3. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS DATA Table 3.1 Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC 0-24 ( g.h/ml)] Daily Dose (mg/kg/day) Mouse Rat Rat (Juvenile) Rabbit Monkey Human Male Female Male Female Male Female Female Male Female g 27.1 g c 81.7 c 85.7 g 93.3 g d 15.5 d e 290 e a 256 a 274 c 378 c 32.3 d 37.7 d d 40.9 d c 83.9 c f 50/ d 61.7 d a 740 a 1040 b 1610 b 111 c 153 c 53.6 h e 922 e 75.1 i g 1044 g 15

163 m Toxicology Tabulated Summary 2012N153953_00 Table 3.1 (Continued) Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC 0-24 ( g.h/ml)] Daily Dose (mg/kg/day) Mouse Rat Rat (Juvenile) Rabbit Monkey Human Male Female Male Female Male Female Female Male Female c 781 c 148 c 92 c b 187 b b 1740 b f b 271 b a 1300 a 1710 b 1950 b e 1777 e c 1737 c 30.1 f 364 b 354 b a 1350 a Key: a = 13 weeks dosing in mice (Report RD2009/00028). AUC data from the end of the study. b = 14 days dosing in rats and monkeys (Reports RD2007/01140 and RD2007/01142, respectively). AUC data from the end of the study. c = 4 weeks dosing in rats and monkeys (Reports RD2008/01628 and RD2008/00107, respectively). AUC data from the end of the study. d = 38 weeks dosing in monkeys (Report RD2009/00036). AUC data from the end of the study. Note: the high dose of 50 was lowered to 30 during the study. e = 26 weeks dosing in rats (Report RD2009/00410). AUC data from the end of the study. f = 13 days dosing (Day 6 to Day 17 gestation) in pregnant rabbits (Report XD2009/00366). AUC data from the end of the study. g = 63 days dosing (Day 4 to Day 66 postpartum) in juvenile rats (Report CD2010/00023). AUC data from the end of the study. h = Human data for a 50 mg/day dose given once daily. i = Human data for a 50 mg dose given twice daily. 16

164 m Toxicology Tabulated Summary 2012N153953_00 4. TOXICOLOGY: DRUG SUBSTANCE Table 4.1 Toxicology: Drug Substance for Dolutegravir Batch Number Purity Specified Impurities (% w/w) Report Number Type of Study PROPOSED SPECIFICATION (% w/w) 452B 454B 451B 399A 453B 983A 009B Not more than 0.15 for each impurity A7Z <0.05 <0.05 <0.05 <0.05 a <0.05 <0.05 <0.05 RD2008/01628 Repeat Dose Toxicity RD2009/00410 RD2009/00036 Repeat Dose Toxicity Repeat Dose Toxicity R <0.05 <0.05 <0.05 <0.05 a <0.05 <0.05 <0.05 WD2007/00513 Genotoxicity WD2007/00514 WD2007/00515 RD2007/01039 RD2007/01141 Genotoxicity Genotoxicity Safety Pharmacology Safety Pharmacology <0.05 <0.05 <0.05 <0.05 a <0.05 <0.05 <0.05 RD2008/00107 Repeat Dose Toxicity MTS B <0.05 <0.05 <0.05 <0.05 a < NT RD2007/01038 Safety Pharmacology RD2007/01037 Safety Pharmacology B <0.05 <0.05 <0.05 <0.05 a 0.08 <0.05 <0.05 RD2009/00036 Repeat Dose Toxicity 2012N N RD2009/00751 XD2009/00367 XD2009/00368 Carcinogenicity Carcinogenicity Immunotoxicity Reproductive Toxicity Reproductive Toxicity 17

165 m Toxicology Tabulated Summary 2012N153953_00 Table 4.1 (Continued) Toxicology: Drug Substance for Dolutegravir Batch Number Purity (%) Specified Impurities (% w/w) Report Number Type of Study PROPOSED SPECIFICATION 452B 454B 451B 399A 453B 983A 009B Not more than 0.15 for each impurity <0.05 <0.05 <0.05 <0.05 a <0.05 <0.05 < N Carcinogenicity 2012N N Carcinogenicity Reproductive Toxicity B <0.05 <0.05 <0.05 <0.05 a 0.08 <0.05 <0.05 CD2010/00023 Juvenile Toxicity Key: a =.Calculated results without the response factor 2.5 for. NT = Not tested. 452B = ; 454B = ; 451B = ; 399A = ; 453B = ; 983A =, the enantiomer; and 009B = total of and, diastereomers. * 新薬承認情報提供時に置き換え 18

166 m Toxicology Tabulated Summary 2012N153953_00 5. SINGLE DOSE TOXICITY Table 5.1 Single Dose Toxicity for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Doses (mg/kg) Number of Animals/ Sex Noteworthy Findings Report/ Study No. (Module) Rat (Sprague Dawley) Subcutaneous and/or Intramuscular (Aqueous 2.0% (w/w) pluronic F127/0.2% (w/v) polysorbate 80/ 0.18 % (w/v) methylparaben)/ (0.02% (w/v) propylparaben/0.004 M NaH 2PO 4 H 20/ M Na 2HPO 4 with NaCl, ph 6.8) 2.5 a 6M The dose administered by both routes was well tolerated. Plasma concentrations of GSK were quantifiable up to Day 15 following the SC dose and up to Day 8 following the IM dose. Mean C max, AUC 0-24, and AUC 0-t values for GSK were similar between the SC and IM routes of administration. RD2009/00921 R42470 (m ) Rat (Sprague Dawley) Subcutaneous and/or Intramuscular (2% pluronic F127(w/v), 0.2% polysorbate 80 (w/v), 0.18% (w/v) methylparaben, 0.02% (w/v) propylparaben, 0.004M NaH 2PO 4, 0.006M Na 2HPO 4.H 2O, and 0.83% NaCl; approximate ph 6.8) 2.5 b 3M 5 b 3M Both doses were well tolerated. Mean GSK C max, AUC 0-24, and AUC 0-t values for the 2.5 or 5 mg/kg doses were similar between SC and IM administration. Following an increase in dose from 2.5 to 5 mg/kg, the mean AUC 0-t increased 2.3- to 3.2-fold after SC or IM administration. The duration that GSK plasma concentrations remained above the PAIC90 was up to Day 15 SC or Day 22 IM for the 2.5 mg/kg dose and up to Day 43 SC or IM for the 5 mg/kg dose. RD2009/00959 R42475 (m ) 19

167 m Toxicology Tabulated Summary 2012N153953_00 Table 5.1 (Continued) Single Dose Toxicity for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Doses (mg/kg) Number of Animals/ Sex Noteworthy Findings Report/ Study No. (Module) Rat (Sprague Dawley) Intramuscular injection No injection site irritation was noted with any formulation. The rats given the vehicle 20 mg/ml polysorbate 20, 20 mg/ml polyethylene glycol 3350, 10 mg/ml CMC, and 45 mg/ml mannitol in sterile water for injection (Formulation 2) with or without GSK B was associated with repeated soft feces and brown/orange staining of the anal area between Days 1 (day of dosing) and 4. None of these three formulations provided sustained concentrations of GSK >60 ng/ml for at least 43 days. The highest dose-normalized systemic exposure (C max and AUC 0- ) and the least amount of variability tended to be provided by the sesame oil formulation (Formulation 3). The results suggest that the rate of GSK absorption was lower when dosed in 20 mg/ml polysorbate 20, 20 mg/ml polyethylene glycol 3350, 10 mg/ml CMC, and 45 mg/ml mannitol in sterile water for injection (Formulation 2) and similar when dosing in sesame oil (Formulation 3), compared to that following dosing in 20 mg/ml polysorbate 20, 20 mg/ml polyethylene glycol 3350, and 45 mg/ml mannitol in sterile water for injection (Formulation 1). 2011N R42826 (m ) 20

168 m Toxicology Tabulated Summary 2012N153953_00 Table 5.1 (Continued) Single Dose Toxicity for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Doses (mg/kg) Number of Animals/ Sex Noteworthy Findings Report/ Study No. (Module) Dog (beagle) Oral (gavage) 30, 100, 150, 250, 500 1F Vomiting or vomitus was observed in the animals given 150 mg/kg between thirty minutes and two hours after dosing. It was speculated that vomiting or vomitus had influenced exposure level of the animals given 150 mg/kg, and thus it was difficult to achieve an appropriate dose relationship. These results indicate the difficulty to evaluate the toxicity of MTS B in dogs. RD2009/00963 (m ) Monkey (cynomolgus) Oral (gavage) (0.5% hydroxypropylmethyl-cellulose (HPMC)/ 0.1% Tween 80 TM (suspension)) Subcutaneous or Intramuscular (2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH 2PO 4H 2O, 0.006M Na 2HPO 4 and 0.83% NaCl (suspension)) 3 1 4F c 4F c All doses were well-tolerated and there were no clinical observations noted at the injection sites. Mean dose normalized C max following oral administration was higher than that following SC administration; however, the mean dose-normalized AUC 0-24 following oral administration was similar to the dose-normalized AUC 0-t following SC administration. There were no marked differences ( 2-fold) in the mean C max, AUC 0-24 or AUC 0-t values following SC or IM administration. Plasma AUC 0-t values following SC or IM administration were on average 3.2- or 3.9-fold higher, respectively, than corresponding AUC 0-24 values. CD2009/00647 (D09113) (m ) 21

169 m Toxicology Tabulated Summary 2012N153953_00 Table 5.1 (Continued) Single Dose Toxicity for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Doses (mg/kg) Number of Animals/ Sex Noteworthy Findings Report/ Study No. (Module) Monkey (cynomolgus) Oral (gavage) (0.5 w/w% hydroxypropylmethylcellulose (HPMC)/0.1 w/w% Tween 80 (aqueous solution)) 50, 125, 250, 500 1F ERC was detected in plasma at all dose levels. Plasma concentrationtime profiles were similar at all dose levels. No dose relationship was noted in the C max and AUC 0-24h values, although the reason was unclear. There were no apparent differences in the C max values between 125 and 500 mg/kg and the AUC 0-24h value at 500 mg/kg was only approximately 1.4-fold the AUC 0-24h value at 125 mg/kg. Therefore, the systemic exposure was considered to have attained the steady state at 125 mg/kg. RD2007/01184 (E TF-008-R) (m ) No treatment-related toxicity was evident in the animals physical condition, body weights, food consumption, hematology or clinical chemistry. 22

170 m Toxicology Tabulated Summary 2012N153953_00 Table 5.1 (Continued) Single Dose Toxicity for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Doses (mg/kg) Number of Animals/ Sex Noteworthy Findings Report/ Study No. (Module) Monkey (cynomolgus) Oral (gavage) (0.5 w/w% hydroxypropyl methylcellulose(hmpc) with 0.1 w/w% Tween 80 (aqueous solution)) 1, 3, 10, 50 3F When the monkeys were administered S orally with dose escalating manners from 1 to 50 mg/kg, the exposure of S was dose-related in nature. However, large inter-individual differences were noted at 10 mg/kg. RD2008/01762 (S TB-44-R) (m ) Key: a = One group of 6 rats was given a SC injection and another group of 6 rats was given an IM injection. b = One group of 3 rats was given a SC injection and another group of 3 rats was given an IM injection. c = On Day 1, a single oral dose of 0 or 3 mg/kg GSK A was given to monkeys in Groups 1 and 3, respectively. On Day 16, a single subcutaneous dose of 0 or 1 mg/kg GSK B was given to monkeys in Groups 1 and 3, respectively, and a single intramuscular dose of 0 or 1 mg/kg GSK B was given to monkeys in Groups 2 and 4, respectively. Toxicokinetic evaluation was performed on samples collected from monkeys in Groups 3 and 4 only. 23

171 m Toxicology Tabulated Summary 2012N153953_00 6. REPEAT DOSE TOXICITY: NON-PIVOTAL STUDIES Table 6.1 Repeat Dose Toxicity: Non-Pivotal Studies for Dolutegravir Species/ Strain Route (Vehicle/ Formulation) Duration of Dosing Doses (mg/kg) Number of Animals/Sex Noteworthy Findings Report/ Study No. (Module) Mouse (CD-1) Oral (gavage) (0.5 w/w% hydroxypropyl methylcellulose(hmpc) with 0.1 w/w% Tween 80 (aqueous solution)) 14 days 10, 100, 500, M/10F The no observed adverse effect level (NOAEL) for S was 1500 mg/kg/day because no adverse effects were noted at any dose level. RD2009/01546 (S TF-066-R) (m ) 24

172 m Toxicology Tabulated Summary 2012N153953_00 7. REPEAT DOSE TOXICITY: PIVOTAL STUDIES Table Week Oral Toxicity Study of GSK A (S ) in Mice Repeat Dose Toxicity Species/Strain: Albino mice (outbred) VAF/Plus Crl: CD-1 (ICR) BR Initial Age: Approximately 4 weeks Date of First Dose: 14 May 2009 Special Features: None No Observed Adverse Effect Level: 1500 mg/kg Report Title: Preliminary Carcinogenicity Study (Gavage) of S Sodium in Mice for 13 Weeks Duration of Dosing: 7 days per week for 13 weeks Duration of Post Dose: None Method of Administration: Oral gavage Vehicle/Formulation: 0.5% HPMC/0.1% Tween 80 Test Article: S sodium Report No.: RD2009/00028 CTD Module: m Study No.: [S TF-068-L] GLP Compliance: Yes Daily Dose (mg/kg) 0 (Control) M F M F M F M F M F Number of Animals Toxicokinetics AUC 0-24h ( g.h/ml), Day 1 NS NS AUC 0-24h ( g.h/ml), Day 28 NS NS AUC 0-24h ( g.h/ml), Day 85 NS NS C max ( g/ml), Day 1 NS NS C max ( g/ml), Day 28 NS NS C max ( g/ml), Day 85 NS NS

173 m Toxicology Tabulated Summary 2012N153953_00 Table 7.1 (Continued) 13 Week Oral Toxicity Study of GSK A (S ) in Mice Repeat-Dose Toxicity Study No. (Continued): Daily Dose (mg/kg) 0 (Control) M F M F M F M F M F Number of Animals: Toxicity Animals: Noteworthy Findings: Died or Sacrificed Moribund: 1 Body Weight: Feed Consumption: Clinical Observations: Hematology: Serum Chemistry: Total bilirubin (mg/dl) * Alkaline phosphatase (U/L) ** Aspartate aminotransferase (U/L) * Potassium (meq/l) * Organ Weights: Gross Pathology: Histopathology: Stomach Increased mucous neck cells NE NE NE NE NE NE 8 9 Key: = Glandular mucosa with occasional mucosal and/or submucosal eosinophilic and lymphocytic infiltrates. * = p<0.05. ** = p<0.01. = No noteworthy findings. NS = Not sampled. 26

174 m Toxicology Tabulated Summary 2012N153953_00 Table Day Oral Toxicity Study of GSK A (ERC Sodium) in Rats Repeat Dose Toxicity Test Article: ERC sodium Initial Age: 11 weeks Duration of Post Dose: None Report Title: 2 Week Oral Toxicity Study of ERC Sodium in Rats Date of First Dose: M: 22 Nov 06. F: 23 Nov 06 Method of Administration: Gavage No Observed Adverse-Effect Level: 500 mg/kg/day Special Features: None GLP Compliance: Yes Species/Strain: Rat/Sprague Dawley Duration of Dosing: 2 weeks Report No.: RD2007/01140 CTD Module: m Study No.: E TB-012-L Vehicle/Formulation: Aqueous 0.5 w/w% hydroxypropyl methylcellulose (HPMC) with 0.1 w/w% Tween 80/ Suspension Daily Dose (mg/kg) 0 (Control) Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Toxicokinetics Number of animals Cmax ( g/ml) Day 1 NC NC Day 14 NC NC AUC 0-24 h ( g.h/ml) Day 1 NC NC Day 14 NC NC Noteworthy Findings Died or Sacrificed Moribund Clinical Observations Body Weight Food Consumption Water Consumption Auditory Examination Ophthalmoscopy Key: No noteworthy findings. The first day of dosing is Day 1 NC: Not calculated because of insufficient data; control animals were sampled only at 1 hour after administration, and plasma ERC concentrations were alll below the lower limit of quantification (<0.03 g/ml). 27

175 m Toxicology Tabulated Summary 2012N153953_00 Table 7.2 (Continued) 14 Day Oral Toxicity Study of GSK A (ERC Sodium) in Rats Repeat Dose Toxicity Study No. E TB-012-L Daily Dose (mg/kg) 0 (Control) Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Urinalysis Number examined a) Specific gravity * ** 1.046** Hematology Number examined a, b) Eosinophil ( 10 3 /µl) * Blood Chemistry Number examined a, b) Creatinine (mg/dl) ** * - Total bilirubin (mg/dl) * Sodium (mmol/l) * Organ Weight c) (%) Gross Pathology Histopathology Stomach: eosinophil infiltration, glandular stomach Mild 4 0 N.E. N.E Stomach: increased mucous cell, glandular stomach Mild 3 0 N.E. N.E Stomach: edema, glandular stomach Mild 0 2 N.E. N.E Key: No noteworthy findings. The first day of dosing is Day 1 Cumulative Chi-squares test with Dunn-Sidak adj.: # - p<0.05 N.E.: Not examined Dunnett's parametric multi. t-test: * - p<0.05, ** - p<0.01 a) One male in the control group was excluded from statistical analysis due to abnormally high levels in AST, ALT and LD with incidental liver lesions. One male in the 50 mg/kg group was excluded from statistical analysis due to accidental loss of teeth. b) Samples of another male in the 50 mg/kg group were not collected due to a technical error. 28

176 m Toxicology Tabulated Summary 2012N153953_00 Table Week Oral Toxicity Study of GSK A (ERC Sodium) in Rats Report No.: RD2008/01628 CTD Module: m

177 m Toxicology Tabulated Summary 2012N153953_00 Table 7.3 (Continued) 4 Week Oral Toxicity Study of GSK A (ERC Sodium) in Rats 30

178 m Toxicology Tabulated Summary 2012N153953_00 Table 7.3 (Continued) 4 Week Oral Toxicity Study of GSK A (ERC Sodium) in Rats 31

179 m Toxicology Tabulated Summary 2012N153953_00 Table Week Oral Toxicity Study of S Sodium in Rats Repeat Dose Toxicity Report Title: 6 Month Oral Toxicity Study of S Sodium in Rats Species/Strain: Rat/Sprague Dawley Initial Age: 6 weeks Duration of Dosing: 4 or 6 months Duration of Post Dose: -1 month Date of First Dose: M: 30 July 2008; F: 31 July 2008 Method of Administration: Oral gavage Test Article: S sodium Report No.: RD2009/00410 CTD Module: m Study No.: SBL Vehicle/Formulation: 0.5% w/w hydroxypropylmethylcellulose (HPMC) aqueous solution with 0.1% w/w Tween 80/suspension No Observed Adverse Effect Level: 50 mg/kg/day Special Features: None GLP Compliance: Yes Daily Dose (mg/kg/day) 0 (Control) Toxicokinetics: Number of Animals # : M: 6 F: 6 M: 6 F: 6 M: 6 F: 6 M: 6 F: 6 C max ( g/ml): Day 1 of dosing NE NE Day 30 of dosing NE NE Day 120 of dosing NE NE Day 180 of dosing NE NE AUC 0-24h ( g.h/ml): Day 1 of dosing NE NE Day 30 of dosing NE NE Day 120 of dosing NE NE Day 180 of dosing NE NE Number of Animals: 4 Month Dosing Period: M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 6 Month Dosing Period (After 4 Months of Dosing): M: 12 F: 12 M: 12 F: 12 M: 12 F: 12 M: 12 F: 12 Recovery Period: M: 6 F: 6 M: 0 F: 0 M: 0 F: 0 M: 6 F: 6 Noteworthy Findings: Died or Sacrificed Moribund: Clinical Signs: Body Weight: Food Consumption: Ophthalmology: Urinalysis: Hematology:

180 m Toxicology Tabulated Summary 2012N153953_00 Table 7.4 (Continued) 26 Week Oral Toxicity Study of S Sodium in Rats Daily Dose (mg/kg/day) 0 (Control) Number of Animals: 4 Month Dosing Period: M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 6 Month Dosing Period (After 4 Months of M: 12 F: 12 M: 12 F: 12 M: 12 F: 12 M: 12 F: 12 Dosing): Recovery Period: M: 6 F: 6 M: 0 F: 0 M: 0 F: 0 M: 6 F: 6 Blood Chemistry: Gross Pathology*: End of the 4 Month Dosing Period: Stomach (glandular mucosa) White focus End of the 6 Month Dosing Period: Stomach (glandular mucosa) White focus Red focus End of the Recovery Period: Organ Weight: Histopathology*: End of the 4 Month Dosing Period Stomach (forestomach) Thickening, mucosa, limiting ridge, very slight Stomach (glandular stomach) Cellular infiltration, eosinophil, submucosa, very slight Cellular infiltration, eosinophil, submucosa, slight Hemorrhage, mucosa, very slight Increase, globule leukocyte, mucosa, very slight Increase, globule leukocyte, mucosa, slight

181 m Toxicology Tabulated Summary 2012N153953_00 Table 7.4 (Continued) 26 Week Oral Toxicity Study of S Sodium in Rats Daily Dose (mg/kg/day) 0 (Control) Stomach (glandular stomach) (Continued) Increase, mucous neck cell, very slight End of the 6 Month Dosing Period: Stomach (forestomach) Thickening, mucosa, limiting ridge, very slight Stomach (glandular stomach) Cellular infiltration, eosinophil, submucosa, very slight Cellular infiltration, eosinophil, submucosa, slight Hemorrhage, mucosa, very slight Increase, globule leukocyte, mucosa, very slight Increase, globule leukocyte, mucosa, slight Increase, mucous neck cell, very slight End of the Recovery Period: Stomach (forestomach) Thickening, mucosa, limiting ridge, very slight Stomach (glandular stomach) Increase, globule leukocyte, mucosa, very slight Key: # = 4 animals/sampling point. (Spare animals [2 males and 2 females] were not used for blood sampling.) * = Number of animals affected. NE = Not estimated. The first day of dosing is Day 1 of dosing. - = No noteworthy findings

182 m Toxicology Tabulated Summary 2012N153953_00 Table Day Oral Toxicity Study of GSK A (ERC Sodium) in Monkeys Repeat-Dose Toxicity Species/Strain: Cynomolgus Monkey Initial Age: 2 years and 6-9 months Date of First Dose: March 22, 2007 Report Title: Two-Week Oral Toxicity Study of ERC sodium in Monkeys Duration of Dosing: 2 Weeks Duration of Postdose: - Method of Administration: Gavage Vehicle/Formulation: Hydroxypropyl methylcellulose (0.5%), Tween 80 (0.1%) / Suspension Test Article: ERC sodium Report No.: RD2007/01142 CTD Module: m Study No.: SG07030 GLP Compliance: Yes Special Features: None No Observed Adverse Effect Level: 100 mg/kg/day as ERC Daily Dose as ERC (mg/kg) *0 (Control) * 100* 300 * 1000* Number of Animals M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 Toxicokinetics: AUC ( g.h/ml): ERC Day Day Noteworthy Findings Died or Sacrificed Moribund (Day 13) Clinical Signs Vomiting/Vomitus [1] Diarrhea (loose, muddy, watery stool) [1] Abnormal position (lateral, crouching) [1] Decrease in movement [1] Moribund condition a) [1] Key: The first day of dosing is Day 1 [ ]: Animal No. 72 (found dead on Day 13) a) Including paleness of oral mucosa and subnormal body surface temperature 35

183 m Toxicology Tabulated Summary 2012N153953_00 Table 7.5 (Continued) Repeat-Dose Toxicity 14 Day Oral Toxicity Study of GSK A (ERC Sodium) in Monkeys Study No. SG07030 Daily Dose as ERC (mg/kg) * 0 (Control)* *100* * 300* *1000* Number of Animals M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 Noteworthy Findings (Continued) Body Weight b) (%) 2.67 kg 2.38 kg Food Consumption b) (%) 100 g/day 100 g/day Ophthalmology Electrocardiography Hematology c) RBC (10 6 /µl) Week * Reticulocyte (%) Week ** Reticulocyte (10 9 /L) Week * ** 28.7 Platelet (10 3 /µl) Week * 260 APTT (s) Week * Fibrinogen (mg/dl) Week [442.8] Clinical Chemistry c) AST (U/L) Week {93} 40[87] ALT (U/L) Week * 167[442] γ-gtp (U/L) Week T-BIL (mg/dl) Week {0.35} 0.23[0.34] UN (mg/dl) Week {56.0} 28.0[54.4] CRE (mg/dl) Week * 0.63[0.87] CHO (mg/dl) Week * TG (mg/dl) Week * Na (meq/l) Week {124.6} 140.5[131.9] Cl (meq/l) Week {79.2} 96.0[84.4] A/G Week [0.51] Key: : No noteworthy findings [ ]: Animal No. 72 (dead animal) { }: Animal No. 32 Dunnett s test or mean rank test of Dunnett type: * p < 0.05 ** p < 0.01 b) At the Day 12 (body weight and food consumption). For controls, group means are shown. For treated groups, percent differences from controls are shown. c) Group means are shown. 36

184 m Toxicology Tabulated Summary 2012N153953_00 Table Month Oral Toxicity Study of ERC Sodium in Monkeys Repeat Dose Toxicity Species/Strain: Monkeys/Cynomolgus Initial Age: 2-3 years Date of First Dose: August 19, 2007 Report Title: 1 Month Oral Toxicity Study of ERC sodium in Monkeys Duration of Dosing: 1 Month (30 days) Duration of Postdose: 1 Month (30 days) Method of Administration: Gavage Vehicle/Formulation: 0.5%HPMC/0.1% Tween80 / Suspension Test Article: ERC sodium Report No.: RD2008/00107 CTD Module: m Study No. SG07224 GLP Compliance: Yes Special Features: None No Observed Adverse Effect Level: 50 mg/kg/day for males and females Daily Dose (mg/kg, as ERC ) * 0 (Control) * * 25 * * 50 * * 100 * Number of Animals M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Toxicokinetics: AUC ( g.h/ml): Day 1 NE NE Day 15 NE NE Day 30 NE NE C max ( g/ml): Day 1 NE NE Day 15 NE NE Day 30 NE NE Noteworthy Findings Died or Sacrificed Moribund Clinical Observations Vomiting/Vomitus a) Diarrhea (muddy or watery stools) b) 4 4 Crouching position Body Weight c) Day kg 2.63 kg d) -7 d) Key: The first day of dosing is Day 1. NE: Not estimated a) Only for 1 day b) Only for 3 days c) For controls, group means are shown. For treated groups, percent differences from controls are shown. d) Individual body weight of a male (Animal No. 35) decreased from 2.35 kg on Day 1 to 1.85 kg on Day 30 and that of a female (Animal No. 73) decreased from 2.55 kg on Day 1 to 2.00 kg on Day

185 m Toxicology Tabulated Summary 2012N153953_00 Table 7.6 (Continued) 1 Month Oral Toxicity Study of ERC Sodium in Monkeys Daily Dose (mg/kg) * 0 (Control) * * 25 * * 50 * * 100 * Number of Animals M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Noteworthy Findings (Continued) Food Consumption a) Day g 100 g b) -14 b) Ophthalmology Electrocardiography Hematology c) Week 2 RBC (106/μL) Platelet (103/μL) [221] Week 4 RBC (106/μL) * Reticulocyte (109/L) [7.4] 39.1 [9.6] Neutrophil (103/μL) [10.85] Fibrinogen (mg/dl) [381.1] Clinical Chemistry c) Week 4 Urea nitrogen (mg/dl) [34.0] Triglyceride (mg/dl) [151] Calcium (mg/dl) [10.75] Sodium (meq/l) [131.5] Potassium (meq/l) [5.43] Chloride (meq/l) [90.4] Total protein (g/dl) [8.65] Total bilirubin (mg/dl) [0.45] Key: : No noteworthy findings. Dunnett s test: * p < a) For controls, group means are shown. For treated groups, percent differences from controls are shown. b) Individual food consumption of a male (Animal No. 35) was -82% and that of a female (Animal No. 73) was -50% on Day 25. c) Group means are shown. [ ]: value of Animal Nos. 35 (male) or 73 (female) 38

186 m Toxicology Tabulated Summary 2012N153953_00 Table 7.6 (Continued) 1 Month Oral Toxicity Study of ERC Sodium in Monkeys Daily Dose (mg/kg) * 0 (Control) * * 25 * * 50 * * 100 * Number of Animals M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Noteworthy Findings (Continued) Urinalysis a) Week 4 Chloride (meq/day) ** 2.09 Gross Pathology Organ Weights Bone Marrow (nucleated cell counts) Histopathology Cecum: Inflammatory cell infiltration in the lamina propria Cecum: Cell debris from the cripts Cecum: Atrophy of the mucosal epithelium Colon: Inflammatory cell infiltration in the lamina propria Colon: Cell debris from the cripts Colon: Atrophy of the mucosal epithelium Rectum: Inflammatory cell infiltration in the lamina propria Thymus: Atrophy Pancreas: Atrophy of the acinar cells Postdose Evaluation: Number Evaluated Died or Sacrificed Moribund Noteworthy Findings Key: : No noteworthy findings. Dunnett s test: ** p < a) Group means are shown. 39

187 m Toxicology Tabulated Summary 2012N153953_00 Table Week Oral Toxicity Study of S Sodium in Monkeys Repeat Dose Toxicity Report Title: 9 Month Oral Toxicity Study of S Test Article: S sodium Sodium in Monkeys Species: Cynomolgus monkey Duration of Dosing: 4 or 9 month (120 or 270 days) Report No.: RD2009/00036 CTD Module: m Study No.: SBL (S TF-047-L) Initial Age: 4 to 5 years Duration of Post Dose: 1 month GLP Compliance: Yes Date of First Dose: M: June 6, 2008 F: June 13, 2008 Method of Administration: Oral Vehicle/Formulation: 0.5% w/w hydroxypropylmethylcellulose (HPMC)/aqueous solution No Observed Adverse Effect Level: 15 mg/kg/day Special Features: None with 0.1% w/w Tween 80/suspension Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Toxicokinetics: Number of Animals: 4 month dosing period month dosing period Cmax (μg/ml) Day 1 NC NC NE NE Day 30 NC NC NE NE Day 69 NC NC NE NE 9.54 c) 8.80 Day 120 NC NC NE NE Day 180 NC NC NE NE Day 270 NC NC NE NE AUC0-24 (μg.h/ml): Day 1 NC NC NE NE Day 30 NC NC NE NE Day 69 NC NC NE NE Day 120 NC NC NE NE Day 180 NC NC NE NE Day 270 NC NC NE NE Tmax (h): Day 1 NC NC NE NE Day 30 NC NC NE NE Day 69 NC NC NE NE Day 120 NC NC NE NE Day 180 NC NC NE NE Day 270 NC NC NE NE 40

188 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Noteworthy Findings: Died or Sacrificed Animals: [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [2] (#s. 64, 66) [0] Clinical Signs: Abnormal feces (diarrhea/soft stool) [0] [0] [0] [0] [0] [0] [1] (# 47) [0] [3] (#s. 61, 63, 68) [2] (#s. 72, 76) [4] (#s. 63, 64, 66, 68) [2] (#s. 74, 76) Absence of stool (no stool) [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [2] (#s. 64, 66) [1] (# 74) Salivation [0] [0] [0] [0] [0] [0] [1] (# 53) [0] [4] (#s. 61, 62, 68, 69) [7] (#s. 70, 71, 72, 73, 74, 76, 78) [4] (#s 64, 66, 68, 69) [6] (#s. 70, 71, 73, 74, 77, 78) Vomiting (transient) [0] [0] [0] [0] [0] [0] [1] [0] [0] [0] [2] [0] (# 48) (#s. 64, 69) Decreased activity [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [2] (#s. 64, 66) [1] (# 74) Abnormal position [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [2] [0] (#s. 64, 66) Emaciation [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [1] (# 74) Decreased body weight [0] [0] [0] [0] [0] [0] [0] [0] [1] (# 68) [0] [2] (#s. 64, 66) [1] (# 74) Decreased Food consumption [0] [0] [0] [0] [0] [0] [0] [0] [0] [0] [3] (#s. 64, 66, [1] (# 74) 68) Ophthalmology: Electrocardiography: Urinalysis:

189 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Hematology d) - Day 30 (Individual) e) : Neutrophils (x10 3 /mm 3 ) NE NE (# 64), (# 66) Neutrophils (%) NE NE (# 64) 69.5 (# 66) Monocytes (x10 3 /mm 3 ) NE NE (# 64), 0.93 (# 69) Leukocyte (x10 3 /mm 3 ) NE NE (# 64) Platelet (x10 3 /mm 3 ) NE NE (# 64) Hematology - Day 30 (Mean) Monocytes (x10 3 /mm 3 ) NE NE 0.541* - Neutrophils (x10 3 /mm 3 ) NE NE 6.822* - Day 69 (Individual) f) Neutrophils (x10 3 /mm 3 ) NE NE (# 68), (# 69) Neutrophils (%) NE NE 76.0 (# 68), 60.9 (# 69) Leukocyte (x10 3 /mm 3 ) NE NE (# 68) (# 74) (# 74) 42

190 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Monocytes (x10 3 /mm 3 ) NE NE 1.04 (# 69) 0.77 (# 76) APTT(s) 24.7 (# 61) 24.8 (# 74) Fibrinogen (mg/dl) NE NE (# 74) Erythrocyte (x10 6 /mm 3 ) NE NE (# 74) Hemoglobin (g/dl) NE NE (# 74) Hematocrit (%) NE NE (# 74) Hematology - Day 69 (Mean) Monocytes (x10 3 /mm 3 ) NE NE 0.541* - Neutrophils (x10 3 /mm 3 ) NE NE 6.822* - APTT(s) NE NE ** Day NE NE Day NE NE Day NE NE Blood Chemistry g) : Day NE NE - - Day 69 (Individual) h) Total protein (g/dl) NE NE (# 74) Albumin (g/dl) NE NE (# 74) Inorganic phosphorus (mg/dl) NE NE 8.05 (# - 68) Triglycerides (mg/dl) NE NE (# 74) 43

191 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Day 69 (Individual) i) Glucose (mg/dl) NE NE (# 68) (# 74) Chloride (meq/l) NE NE (# 68) (# 74) Day 69 (Mean) Inorganic phosphorus (mg/dl) NE NE 6.710* - Glucose (mg/dl) NE NE * Day 120 (Individual) j) Blood urea nitrogen (mg/dl) (# 76) Day NE NE Day NE NE Number of Animals Evaluated: 4 month dosing period M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 2 F: 2 M: 2 F: 3 M: 2 F: 0 9 month dosing period M: 4 F: 4 M: 4 F: 4 M: 4 F: 4 M: 3 F: 3 M: 3 F: 4 M: 0 F: 0 Gross Pathology: Moribund Animals: Enlargement of adrenals NE NE NE NE NE NE NE NE NE NE [2] (#s. 64, 66) Small thymus NE NE NE NE NE NE NE NE NE NE [2] NE (#s. 64, 66) Survived Animals: 4 month dosing period NE NE 9 month dosing period Stomach (Body) Red and recessed focus, mucosa [1] (# 76) Organ Weight: Moribund Animals: Increased absolute and relative adrenal weight NE NE NE NE NE NE NE NE NE NE [2] NE (#s. 64, 66) Survived Animals: NE NE NE NE NE NE NE 44

192 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Histopathology: Moribund Animals k) : Cecum and Colon Mononuclear cell infiltration and hemorrhage, lamina propria Esophagus and Tongue Inflammatory cell infiltration, epithelium NE NE NE NE NE NE NE NE NE NE [1] (# 64) NE NE NE NE NE NE NE NE NE NE [1] (# 64) Survived Animals: 4 month dosing period NE NE 9 month dosing period Stomach (Body) Mononuclear cell infiltration, and hemorrhage, lamina propria, multifocal [1] (# 76) Regeneration, epithelium, multifocal [1] (# 76) Erosion, multifocal [1] (# 76) Post Dose Evaluation: Number Evaluated: Died or Sacrificed Moribund: Noteworthy Findings: Gross Pathology: Stomach (body) Red focus, mucosa [1] (# 70) NE NE NE NE NE NE NE NE 45

193 m Toxicology Tabulated Summary 2012N153953_00 Table 7.7 (Continued) 38 Week Oral Toxicity Study of S Sodium in Monkeys Daily Dose (mg/kg) a) 0 (Control) b) 50 b) Number of Animals: M: 9 F: 9 M: 7 F: 7 M: 7 F: 7 M: 7 F: 7 M: 7 F: 9 M: 9 F: 9 Histopathology: Stomach (body) Cell infiltration, hemorrhage, and regeneration, multifocal [1] (# 70) Key: - = No noteworthy findings. NE = Not examined. The first day of dosing is Day 1 of dosing. [ ] = Number of animals. ( ) = Animal number. a) All doses are expressed as the parent entity. b) From Day 70, the dose of 50 mg/kg/day was reduced to 30 mg/kg/day because 2 males (#s. 64 and 66) were moribund/dead. In # 68 (male) and # 74 (female), dosing was stopped for several days (18 days for # 68 and 14 days for # 74) after Day 70 because of abnormal feces accompanied by body weight loss. c) n=7. d) In hematology, changes in the following parameters considered indicative of moribundity were observed in # 64 of the 50 mg/kg/day group: high value for platelet count and low values for reticulocytes, eosinophils, lymphocytes, and prolongation of PT and APTT. e) The values were compared with the pre-dosing values or background data. Pre-dosing values are as follows: # 64: neutrophils 6.25x10 3 /mm 3 and 44.7%, monocytes 0.50x10 3 /mm 3, leukocytes 13.98x10 3 /mm 3, platelets 545x10 3 /mm 3, # 66: neutrophils 0.87x10 3 /mm 3 and 11.2%; # 69: monocytes 0.44x10 3 /mm 3. f) The values were compared to the pre-dosing values or background data. Pre-dosing values are as follows: #61: APTT: 21.9 s; # 68: neutrophils 5.92x10 3 /mm 3 and 50.9%, leukocytes 11.64x10 3 /mm 3 ; # 69: neutrophils 4.02x10 3 /mm 3 and 34.9%, monocytes 0.44x10 3 /mm 3 ; # 74: erythrocytes 5.22x10 6 /mm 3, hemoglobin 13.1 g/dl, hematocrit 41.4%, neutrophils 5.82x10 3 /mm 3, leukocytes 11.07x10 3 /mm 3, fibrinogen mg/dl, APTT 18.4 s; #76: monocytes 0.22x10 3 /mm 3. g) In blood chemistry, changes in the following parameters considered due to secondary effects of moribundity were observed in # 64 of the 50 mg/kg/day group: high values for aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, and inorganic phosphorus, and low values for albumin, albumin/globulin ratio, total cholesterol, glucose, calcium, sodium and chloride. h) The values were compared with the pre-dosing values or background data. Pre-dosing values are as follows: # 68: inorganic phosphorus 5.91 mg/dl; # 74: total protein 7.4 g/dl, albumin 3.9 g/dl triglycerides 27 mg/dl. i) The values were compared with the pre-dosing values or background data. Pre-dosing values are as follows: # 68: glucose 112 mg/dl; # 74: glucose 84 mg/dl, chloride 108 meq/l. j) The value was compared with the pre-dosing value or background data. Pre-dosing value is as follows: # 76 blood urea nitrogen 26.2 mg/dl. k) In histopathology, the following findings considered due to secondary effects of moribundity were observed in #s 64 and 66 of the 50 mg/kg/day group: decreased lipid, hypertrophy of the fascicular zone, mononuclear cell infiltration in the adrenal, atrophy in the hepatocytes, acinar cells of the submandibular gland, parotid gland, and pancreas, adipose tissue of the aorta and heart, atrophy in the epithelium of the duodenum, dilatation of the distal tubule, cellular cast, or decrease in cellularity in the sternal bone marrow. NC = Not calculated, since all individual samples were BLQ (below the lower limit of quantification, <10 ng/ml). * = p<0.05: Significantly different from control mean. 46

194 m Toxicology Tabulated Summary 2012N153953_00 8. GENOTOXICITY: IN VITRO Table 8.1 GSK A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli Genotoxicity: In Vitro Report Title: GSK A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli. Test Compound: GSK A (sodium salt) Batch Number: R06001 Test for Induction of: Reverse mutation in bacterial cells Strain: S. typhimurium and E. coli No. of Independent Assays: 2 Covance Study No 2990/120 GSK Reference No: V27467 GSK Document Number: WD2007/00514 CTD Module: m No. of Replicate Plates: 3 (6 vehicle control, 3 positive control) Metabolising System: Aroclor-induced rat liver S9-mix containing 50 L S9-fraction/plate (final) Vehicles Test Article: Dimethyl sulphoxide (DMSO) Positive Controls: DMSO or water GLP Compliance: Yes Treatment: Plate incorporation for 3 days Date of Treatment: April 2007 Cytotoxic Effects: Reduction in background bacterial lawn and/or revertant colonies at 500 g per plate and/or 849 g per plate in strains TA100, TA1535 and TA1537 in the absence and presence of S9-mix. Genotoxic Effects: Negative 47

195 m Toxicology Tabulated Summary 2012N153953_00 Table 8.1 (Continued) GSK A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli Metabolic Activation Test Compound Concentration ( g/plate) 1 Main Plate Incorporation (Ames) Test 1 Mean Number of Revertant Colonies per Plate TA 98 TA 100 TA 1535 TA 1537 WP2 uvra pkm101 Without Activation DMSO 100 L/plate GSK GSK GSK GSK GSK GSK Nitrofluorene NT NT NT NT Sodium azide 2 NT NT NT 9-Aminoacridine 50 NT NT NT NT 4-Nitroquinoline-1-oxide 2 NT NT NT NT With Activation DMSO 100 L/plate GSK GSK GSK GSK GSK GSK Benzo[a]pyrene NT NT NT NT 2-Aminoanthracene 5 NT NT 2-Aminoanthracene 10 NT NT NT NT Key: 1. All concentrations are expressed in terms of parent compound 2. two fold increase in revertants for TA98, TA100 and WP2uvrA(pKM101) and three fold increase for TA1535 and TA Slight thinning of background bacterial lawn NT Not tested 48

196 m Toxicology Tabulated Summary 2012N153953_00 Table 8.1 (Continued) GSK A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli Metabolic Activation Test Compound Concentration ( g/plate) 1 Main Plate Incorporation (Ames) Test 2 Mean Number of Revertant Colonies per Plate TA 98 TA 100 TA 1535 TA 1537 WP2 uvra pkm101 Without Activation DMSO 100 L/plate GSK GSK GSK GSK GSK GSK Nitrofluorene NT NT NT NT Sodium Azide 2 NT NT NT 9-Aminoacridine 50 NT NT NT NT 4-Nitroquinoline-1-Oxide 2 NT NT NT NT With Activation DMSO 100 L/plate GSK GSK GSK GSK GSK GSK Benzo[a]pyrene NT NT NT NT 2-Aminoanthracene 5 NT NT 2-Aminoanthracene 10 NT NT NT NT Key: 1. All concentrations are expressed in terms of parent compound 2. two fold increase in revertants for TA98, TA100 and WP2uvrA(pKM101) and three fold increase for TA1535 and TA Slight thinning of background bacterial lawn NT Not tested 49

197 m Toxicology Tabulated Summary 2012N153953_00 Table 8.2 GSK In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus Study Type: Genetic Toxicology In Vitro Test for Induction of: Forward mutation at the TK +/- locus Cell Type: L5178Y Mouse Lymphoma Cells Report Title: GSK In Vitro Mutation Assay Test Article: GSK with L5178Y Mouse Lymphoma Cells at the TK Locus Batch Number: U23359/4/1 No. of Independent Tests: 2 Report No.: WD2007/01581 CTD Module: m Study No: MLA-580 No. of Replicate Cultures: 2 (vehicle); 1 (treatment and positives) Metabolising System: Phenobarbital 5,6-Benzoflavone -induced rat liver S9-mix containing 1% v/v S9-fraction (final) Vehicles Test Article: DMSO Positive Controls: DMSO GLP Compliance: No Treatment: 3 hr treatment with S9-mix; treatment for 24 hr without S9-mix Date of Treatment(s): May, 2006 Cytotoxic Effects: Reduction in RTG Genotoxic Effects: Positive Test Article Dose Level 1 mcg/ml Mean Relative Total Growth (%) 3 hr Treatment +S9-mix 24 hr Treatment -S9-mix Mean Relative Total Mean Mutant Frequency (x10-6 Growth ) (%) Mean Mutant Frequency (x10-6 ) DMSO GSK GSK GSK GSK GSK GSK GSK Methyl methane sulphonate Dimethylbenzanthracene Key: 1 = Mean mutant frequency exceeds the sum of the mean control mutant frequency plus Global Evaluation Factor (126x10-6 ) 50

198 m Toxicology Tabulated Summary 2012N153953_00 Table 8.3 GSK A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus Genotoxicity: In Vitro Test for Induction of: Forward mutation at the TK +/- locus Report Title: GSK A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus Test Article: GSK A (sodium salt) Batch Number: R06001 No. of Independent Tests: 3 Covance Study No: 2990/121 GSK Ref No: V27468 Report No.: WD2007/00515 CTD Module: m No. of Replicate Cultures: 4 (vehicle);2 (treatment and positive controls) Cell Type: L5178Y Mouse Lymphoma Cells Metabolising System: Aroclor-induced rat liver S9-mix. Final concentration of S9-fraction in cultures = 2% v/v Vehicles: Test Article: Dimethyl sulphoxide (DMSO) Positive Controls: DMSO GLP Compliance: Yes Treatment: 3 hr treatment with and without S9-mix; treatment for 24 hr without S9-mix Date of Treatment(s): April to May 2007 Cytotoxic Effects: Relative total growth (RTG) was reduced to 53% and 81% at 85 g/ml for 3 hour treatments in the absence and presence of S9-mix, respectively, and 26% at 85 g/ml for 24 hour treatment in the absence of S9-mix. Genotoxic Effects: Negative Test Article Dose Level 1 g/ml 3 hr Treatment -S9-mix 3 hr Treatment +S9-mix 24 hr Treatment -S9-mix Mean Mutant Mean Relative Mean Mutant Mean Relative Frequency Total Growth Frequency Total Growth (x10-6 ) (%) (x10-6 ) (%) Mean Relative Total Growth (%) Mean Mutant Frequency (x10-6 ) DMSO GSK GSK GSK NT NT NT NT GSK GSK GSK GSK GSK GSK GSK NT NT GSK Methyl methane sulphonate NT NT NT NT 51

199 m Toxicology Tabulated Summary 2012N153953_00 Table 8.3 (Continued) GSK A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus Test Article Dose Level 1 g/ml 3 hr Treatment -S9-mix 3 hr Treatment +S9-mix 24 hr Treatment -S9-mix Mean Mutant Mean Relative Mean Mutant Mean Relative Frequency Total Growth Frequency Total Growth (x10-6 ) (%) (x10-6 ) (%) Mean Relative Total Growth (%) Mean Mutant Frequency (x10-6 ) Methyl methane sulphonate 5 NT NT NT NT Benzo[a]pyrene 2 NT NT NT NT Key: 1 = All concentrations are expressed in terms of parent compound. 2 = Precipitation, observed by eye at the end of treatment (3 hour treatment only). NT = Not tested. 52

200 m Toxicology Tabulated Summary 2012N153953_00 9. GENOTOXICITY: IN VIVO Table 9.1 GSK A: Oral Bone Marrow Micronucleus Assay in Rats Genetic Toxicology: In Vivo Test for Induction of: Structural chromosomal damage and/or aneuploidy Species/Strain: Rat (Hsd:SD) Approximate Age: 8 weeks at time of dosing Cells Evaluated: Polychromatic erythrocytes (PCE) No. of Cells Analyzed/Animal: 2000 PCE Report Title: GSK A: Oral Bone Marrow Micronucleus Assay in Rats Treatment Schedule: Two doses 24 hours apart (vehicle & test compound). Positive control dosed once only. Sampling Time: 24 hours after final dose Method of Administration: Oral Dose volume: 10 ml/kg Vehicle/Formulation: 0.5% Hydroxypropylmethylcellulose (HPMC, K15M Premium) (w/v) with 0.1% Tween 80 (w/v) in reverse osmosis water (deionized water) Test Article: GSK A (sodium salt) Batch Number: R06001 PCS-MTL Study No.: GSK Reference No.: R27469 Report No.: WD2007/00513 CTD Module: m GLP Compliance: Yes Date of Dosing: April 2007 Special Features: None Toxic/Cytotoxic Effects: None Genotoxic Effects: Negative Evidence of Exposure: Exposure previously demonstrated in a 14-day toxicity study in SD rats [Sponsor Study No. E TB-012-L] Test Compound Dose 1 (mg/kg/day) Number of Animals Analyzed 2 Group Mean % PCE Vehicle 0 6M GSK M GSK M GSK M Cyclophosphamide 20 3M Key: 1 = All doses/concentrations are expressed in terms of parent compound. 2 = M = Male. 3 = Group mean number of micronucleated polychromatic erythrocytes (MPCE) per 2000 PCE analyzed. Group Mean MPCE 3 53

201 m Toxicology Tabulated Summary 2012N153953_ CARCINOGENICITY Table 10.1 Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Species/Strain: Mice/Crl:CD-1 (ICR)BR Duration of Dosing: Males: 104 weeks Study No.: Females: Group 5: 101 weeks Groups 1-4: weeks Report No.: 2012N Initial Age: 7 weeks Method of Administration: Oral gavage Location in CTD: m Date of First Dose: 25 March 2010 Vehicle/Formulation: Aqueous 0.5% w/w hydroxypropyl methylcellulose (HPMC) solution with 0.1% w/w Tween 80 (0.5% HPMC/0.1% Tween 80)/solution Conclusion: There were no test article-related Treatment of Controls: Group 1 Sterile Water for Injection, USP GLP Compliance: Yes neoplastic lesions 1Group 2 - Aqueous 0.5% w/w hydroxypropyl methylcellulose (HPMC) solution with 0.1% w/w Tween 80 (0.5% HPMC/0.1% Tween 80) Basis for High-Dose Selection: The high dose, 500 mg/kg/day, was chosen based on saturation of absorption between 500 mg/kg/day and 1500 mg/kg/day, and concern for gastrointestinal intolerance (observed in rats and monkeys) which may have manifested over time. Special Features: None Daily Dose (mg/kg/day) 0 (Water Control) 0 (Vehicle Control) Gender M F M F M F M F M F Toxicokinetics Number of Animals AUC0-24 ( g.h/ml) Day 26 BLQ BLQ BLQ BLQ AUC0-24 ( g.h/ml) Day 182 BLQ BLQ BLQ BLQ Cmax ( g/ml) Day 26 BLQ BLQ BLQ BLQ Cmax ( g/ml) Day 182 BLQ BLQ BLQ BLQ Number of Animals At Start Died/Sacrificed Moribund Terminal Sacrifice Survival (%) 46.2% 30.8% 44.6% 41.5% 56.9% 35.4% 40.0% 35.4% 40.0% 29.2% 54

202 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Drug-Related a Neoplastic Lesions - Incidental/Spontaneous Adipose Tissue b N-Metastatic/Invasive Neoplasm 0[3] 3[4] 1[1] 0[1] NE 1[1] 1[1] 0[3] 2[3] 2[2] Adrenal Glands c N-Metastatic/Invasive Neoplasm (64) 6(64) 4(64) 6 5(63) 3(64) 3(64) 8 B-Cortex: Adenoma 0 0 1(64) 2(64) 1(64) 0 1(63) 1(64) 1(64) 0 B-Medulla Benign Pheochromocytoma 1 0 0(64) 0(64) 0(64) 0 0(64) 0(64) 0(64) 0 M-Cortex: Carcinoma 0 0 0(64) 0(64) 0(64) 0 0(64) 1(64) 0(64) 0 Aorta c N-Metastatic/Invasive Neoplasm 4 6(64) 3 6 2(64) Bone (other) b B-Osteoma 1[7] 0[3] 1[3] 3[3] 0[6] NE 0[4] 0[3] 0[2]] 0[1] M-Sarcoma, NOS 0[7] 0[3] 0[3] 0[3] 0[6] NE 1[4] 0[3] 0[2] 0[1] N-Metastatic/Invasive Neoplasm 0[7] 0[3] 1[3] 0[3] 0[6] NE 0[4] 0[3] 0[2] 0[1] Brain N-Metastatic/Invasive Neoplasm Cecum c N-Metastatic/Invasive Neoplasm 5[63] 5[64] 6[64] [63] 2 0 1[63] Cervical, Spinal Cord N-Metastatic/Invasive Neoplasm Colon c N-Metastatic/Invasive Neoplasm (64) Diaphragm b N-Metastatic/Invasive Neoplasm NE 1[1] 1[1] NE NE NE 1[1] NE NE NE Distal femur N-Metastatic/Invasive Neoplasm 0 0(64) 0 1(64) B-Osteoma 0 0(64) 0 1(64) Duodenum c N-Metastatic/Invasive Neoplasm 1 3(64) 2(64) 1(64) 0(62) 0 1(61) 1 0 0(64) 55

203 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Ear(s) b N-Metastatic/Invasive Neoplasm 0[7] 0[3] 0[8] 0[5] 0[9] 0[5] 0[8] 0[6] 1[10] 0[2] Epididymides N-Metastatic/Invasive Neoplasm 8 NE 7 NE 5 NE 3 NE 3 NE Esophagus c N-Metastatic/Invasive Neoplasm (64) 3 3 2(64) Eyes N-Metastatic/Invasive Neoplasm Femoral Marrow N-Metastatic/Invasive Neoplasm (64) B-Hemangioma (64) Gallbladder c N-Metastatic/Invasive Neoplasm 1(55) 3(57) 5(58) 2(58) 2(60) 1(60) 1(53) 0(60) 0(59) 1(58) B-Papilloma 0(55) 0(57) 0(58) 1(58) 0(60) 0(60) 0(53) 0(60) 2(59) 0(58) B-Cystadenoma/Adenoma 0(55) 0(57) 0(58) 0(58) 0(60) 1(60) 2(53) 0(60) 0(59) 0(58) Harderian Gland c N-Metastatic/Invasive Neoplasm (64) B-Adenoma (64) M-Carcinoma (64) Heart N-Metastatic/Invasive Neoplasm M-Endocardial Schwannoma M-Hemangiosarcoma Ileum c N-Metastatic/Invasive Neoplasm 4(64) 5 3(64) 0(64) 0 1 1(64) 0 1 0(64) Intercostal Musc b N-Metastatic/Invasive Neoplasm NE NE 1[1] NE NE NE NE NE NE NE Jejunum c N-Metastatic/Invasive Neoplasm (64) 0(64) 0 0(62) 1 0(64) 0 Kidneys c N-Metastatic/Invasive Neoplasm 11 17(64)

204 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Lacrimal Gland c N-Metastatic/Invasive Neoplasm 3 13(63) (64) B-Adenoma 0 0(63) (64) Liver N-Metastatic/Invasive Neoplasm M-Hepatocellular Carcinoma B-Hepatocellular Adenoma M-Hemangiosarcoma B-Hemangioma Lungs N-Metastatic/Invasive Neoplasm B-Bronchiolo/Alveolar Adenoma M-Bronchiolo/Alveolar Carcinoma Lymph Node (other) b N-Metastatic/Invasive Neoplasm 12[27] 23[31] 16[31] 9[23] 3[10] 6[17] 8[21] 10[23] 5[13] 15[23] Lymphoreticular System M-Malignant Lymphoma M-Histiocytic Sarcoma Mammary (protocol) c N-Metastatic/Invasive Neoplasm 4(63) 18 4(60) 4(64) 3(63) (62) 7(64) M-Adenocarcinoma 0(63) 0 0(60) 0(64) 0(63) (62) 1(64) Mandibular Lymph Node b N-Metastatic/Invasive Neoplasm NE NE NE 1[1] NE NE NE NE NE NE Mediastinal Lymph Node c N-Metastatic/Invasive Neoplasm 12(61) 24 16(63) 13 4(62) 8 8(61) 15(63) 8(57) 17 Mesenteric Lymph Node c N-Metastatic/Invasive Neoplasm 12 24(64) 14(63) 12(64) 4(64) 6(64) 5(64) 9(62) 6(61) 12 M-Hemangiosarcoma 0 0(64) 0(63) 0(64) 0(64) 0(64) 1(64) 0(62) 0(61) 0 Mesentery/Peritoneum b N-Metastatic/Invasive Neoplasm 1[1] 0[2] 0[1] 2[4] NE 0[1] NE 1[2] 0[1] 1[2] M-Malignant Mesothelioma 0[1] 0[2] 0[1] 0[4] NE 1[1] NE 0[2] 0[1] 0[2] 57

205 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Mesovarian Ligament b N-Metastatic/Invasive Neoplasm NE 1[1] NE NE NE NE NE NE NE NE Muscle (other) b N-Metastatic/Invasive Neoplasm 0[1] 1[1] NE NE NE 0[1] 1[3] NE NE 1[2] M-Hemangiosarcoma 1[1] 0[1] NE NE NE 0[1] 0[3] NE NE 0[2] M-Rhabdomyosarcoma 0[1] 0[1] NE NE NE 0[1] 1[3] NE NE 0[2] Muscle (protocol) N-Metastatic/Invasive Neoplasm M-Hemangiosarcoma Nerve - Sciatic c N-Metastatic/Invasive Neoplasm (64) 1(64) (63) Ovaries c N-Metastatic/Invasive Neoplasm NE 19(64) NE 9 NE 4 NE 9 NE 9(64) B-Tubulostromal Adenoma NE 3(64) NE 0 NE 1 NE 0 NE 1(64) M-Adenocarcinoma NE 0(64) NE 1 NE 0 NE 0 NE 0(64) B-Cystadenoma NE 0(64) NE 2 NE 0 NE 0 NE 1(64) B-Benign Granulosa Cell Tumor NE 1(64) NE 0 NE 0 NE 0 NE 0(64) Peyer s Patches/GALT c N-Metastatic/Invasive Neoplasm 4(63) 6(64) 5(55) 2(59) 1(62) 1(58) 1(55) 1(61) 1(58) 1(58) Pancreas N-Metastatic/Invasive Neoplasm B-Islet Cell Adenoma Parotid Gland b N-Metastatic/Invasive Neoplasm NE 2[2] NE NE 1[2] NE 0[1] NE NE NE Pericardium b N-Metastatic/Invasive Neoplasm NE NE 1[1] 1[1] 0[1] 1[1] NE NE NE 1[1] Pituitary c N-Metastatic/Invasive Neoplasm 1 2(64) 0(64) 1 0(64) 0(64) 1(62) 0(64) 0(64) 1 B-Pars Distalis-Adenoma 0 0(64) 0(64) 0 0(64) 2(64) 0(62) 0(64) 0(64) 1 B-Pars Intermedia: Adenoma 0 0(64) 0(64) 0 0(64) 0(64) 0(62) 0(64) 0(64) 0 58

206 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Prostate c N-Metastatic/Invasive Neoplasm 6 NE 8 NE 6(63) NE 4 NE 4 NE M-Carcinoma 0 NE 0 NE 0(63) NE 1 NE 0 NE Rectum/Low Colon b N-Metastatic/Invasive Neoplasm NE 1[1] 0[1] NE NE NE NE 0[1] NE NE Salivary Gland N-Metastatic/Invasive Neoplasm Seminal Vesicles N-Metastatic/Invasive Neoplasm 3 NE 7 NE 2 NE 1 NE 2 NE Skin (other) b M-Carcinoma 1[17] 0[19] 0[21] 1[10] 1[14] 1[21] 0[12] 0[17] 1[18] 0[12] N-Metastatic/Invasive Neoplasm 1[17] 6[19] 1[21] 0[10] 1[14] 0[21] 1[12] 0[17] 2[18] 2[12] M-Hemangiosarcoma 0[17] 0[19] 0[21] 0[10] 0[14] 0[21] 0[12] 0[17] 0[18] 1[12] M-Sarcoma, NOS 0[17] 1[19] 0[21] 1[10] 0[14] 0[21] 0[12] 0[17] 0[18] 0[12] B-Sebaceous Cell Adenoma 0[17] 1[19] 0[21] 0[10] 0[14] 0[21] 0[12] 0[17] 0[18] 0[12] B-Histiocytoma 0[17] 0[19] 0[21] 0[10] 0[14] 0[21] 1[12] 0[17] 0[18] 0[12] Skin (protocol) c N-Metastatic/Invasive Neoplasm (64) B-Schwannoma (64) M-Fibrosarcoma (64) Spleen c N-Metastatic/Invasive Neoplasm 10(64) (64) B-Hemangioma 0(64) (64) M-Hemangiosarcoma 0(64) (64) Sternal Marrow c N-Metastatic/Invasive Neoplasm (64) (64) 3 Sternum c N-Metastatic/Invasive Neoplasm (64) (64) 8 M-Osteogenic Sarcoma (64) (64) 0 59

207 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Stomach c N-Metastatic/Invasive Neoplasm (64) 1 2(64) 5 2(64) 6 B-Glandular Mucosa: Adenoma (64) 0 0(64) 1 0(64) 1 M-Forestomach: Squamous Cell Carcinoma (64) 0 0(64) 1 0(64) 0 Testes N-Metastatic/Invasive Neoplasm 0 NE 1 NE 1 NE 0 NE 0 NE B-Benign Interstitial Cell Tumor 1 NE 1 NE 0 NE 2 NE 1 NE Thymus c N-Metastatic/Invasive Neoplasm 11(63) 24 17(60) 12(62) 7(60) 10 7(63) 16(64) 9(63) 15 Thyroid c N-Metastatic/Invasive Neoplasm (64) (61) 1(63) B-Follicular Cell Adenoma (64) (61) 1(63) Trachea c N-Metastatic/Invasive Neoplasm (64) Ureters b N-Metastatic/Invasive Neoplasm 0[2] 3[4] 2[3] 0[1] 1[1] 1[1] 0[1] NE 1[4] NE Urinary Bladder c N-Metastatic/Invasive Neoplasm 6 10(63) 6 4 1(64) 3 0(64) 4 3 8(63) N-Leiomyosarcoma 0 0(63) 0 0 0(64) 0 0(64) 0 0 1(63) Uterus w/cervix N-Metastatic/Invasive Neoplasm NE 11 NE 4 NE 6 NE 7 NE 4 M-Endometrial Carcinoma NE 0 NE 1 NE 0 NE 1 NE 0 B-Endometrial Stromal Polyp NE 2 NE 5 NE 4 NE 9 NE 7 M-Leiomyosarcoma NE 1 NE 0 NE 0 NE 3 NE 1 B-Hemangioma NE 0 NE 1 NE 3 NE 1 NE 1 B-Benign Granular Cell Tumor NE 0 NE 0 NE 1 NE 0 NE 0 M-Endometrial Stromal Sarcoma NE 2 NE 1 NE 0 NE 0 NE 0 B-Leiomyoma NE 1 NE 0 NE 2 NE 4 NE 1 M-Granular Cell Tumor NE 1 NE 0 NE 0 NE 0 NE 0 M-Hemangiosarcoma NE 0 NE 0 NE 0 NE 1 NE 0 60

208 m Toxicology Tabulated Summary 2012N153953_00 Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Vagina c N-Metastatic/Invasive Neoplasm NE 10(64) NE 1(63) NE 2 NE 2(64) NE 3(64) Vessel C-Hemangiosarcoma C-Hemangioma Zymbal s Gland b M-Carcinoma NE NE NE NE 1[1] NE NE NE NE NE Key: B = Benign; BLQ = Below lower limit of quantitation (<0.500 µg/ml) C = Multicentric; GALT = Gut-associate lymphoid tissue; M = Malignant; N = Metastatic; NE = Not examined; NOS = Not otherwise specified a There were no test article-related neoplastic lesions. b Not a protocol required organ/tissue. Organs/Tissues were examined grossly; number in [ ] indicates number of organs/tissues examined microscopically. c Missing protocol-required organs/tissues in some groups; number in ( ) indicates number of organs/tissues examined microscopically. 61

209 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Species/Strain: Rats/Sprague Dawley Duration of Dosing: Males: 24 months Study No.: Females: Group 5: 88 weeks Group 3: 90 weeks Group 4: 94 weeks Report No.: 2012N Groups 1 and 2: 95 weeks Initial Age: 7 weeks Method of Administration: Oral gavage Location in CTD: m Date of First Dose: 19 March 2010 Vehicle/Formulation: Aqueous 0.5 w/w% hydroxypropyl methylcellulose (HPMC) solution with 0.1 w/w% Tween 80 (0.5% HPMC/0.1% Tween 80)/solution Conclusion: There were no test article-related Treatment of Controls: Group 1 Sterile Water for GLP Compliance: Yes neoplastic lesions. Injection, USP Group 2 - Aqueous 0.5 w/w% hydroxypropyl methylcellulose (HPMC) solution with 0.1 w/w% Tween 80 (0.5% HPMC/0.1% Tween 80) Basis for High-Dose Selection: Maximum tolerated dose per ICH guidance (ICH Guidance, S1C (R2) Dose Selection For Carcinogenicity Studies of Pharmaceuticals) based on dosing period of 104 weeks Special Features: None Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Toxicokinetics Number of Animals AUC0-24 ( g.h/ml) Day 28 BLQ BLQ BLQ BLQ AUC0-24 ( g.h/ml) Day 182 BLQ BLQ BLQ BLQ Cmax ( g/ml) Day 28 BLQ BLQ BLQ BLQ Cmax ( g/ml) Day 182 BLQ BLQ BLQ BLQ Number of Animals At Start Died/Sacrificed Moribund Terminal Sacrifice Survival (%) 38.5% % 41.5% 33.8% 23.1% 38.5% 30.8% 26.2% 26.2% BLQ = Below lower limit of quantitation (<0.500 g/ml) 62

210 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Drug-Related a Neoplastic Lesions - Incidental/Spontaneous Adipose Tissue b N-Metastatic/Invasive Neoplasm 0[3] NE 0[2] 0[1] 0[1] 0[3] 0[1] 0[3] 2[3] 0[2] B Lipoma 0[3] NE 1[2] 0[1] 0[1] 0[3] 0[1] 0[3] 0[3] 0[2] M-Malignant Hibernoma 0[3] NE 0[2] 0[1] 0[1] 0[3] 1[1] 0[3] 0[3] 0[2] Adrenal Glands N-Metastatic/Invasive Neoplasm B-Medulla Benign Pheochromocytoma M-Cortex: Carcinoma B-Cortex: Adenoma M-Medulla: Malignant Pheochromocytoma Aorta N-Metastatic/Invasive Neoplasm Bone (other) b M-Histiocytoma, fibrous NE 0[2] NE NE 1[1] NE 0[2] NE 0[1]] NE B-Fibrosarcoma NE 0[2] NE NE 0[1] NE 0[2] NE 1[1] NE Brain N-Metastatic/Invasive Neoplasm M-Astrocytoma B-Granular Cell Tumor M-Oligodendroglioma B-Benign Meningioma Cecum N-Metastatic/Invasive Neoplasm B-Fibroma Colon N-Metastatic/Invasive Neoplasm Diaphragm b N-Metastatic/Invasive Neoplasm NE 0[1] NE NE NE NE 1[1] NE NE 1[1] 63

211 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Duodenum N-Metastatic/Invasive Neoplasm Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Ear(s) b M-Fibrosarcoma NE NE 0[1] NE NE NE NE NE 1[1] NE Epididymides N-Metastatic/Invasive Neoplasm 5 NE 1 NE 0 NE 0 NE 2 NE Esophagus N-Metastatic/Invasive Neoplasm Extremity b N-Metastatic/Invasive Neoplasm 1[34] 0[14] 0[40] 0[12] 0[26] 0[11] 0[27] 0[7] 0[16] 0[6] B-Keratoacanthoma 1[34] 0[14] 0[40] 0[12] 0[26] 0[11] 0[27] 0[7] 0[16] 0[6] Eyes N-Metastatic/Invasive Neoplasm Femoral Marrow N-Metastatic/Invasive Neoplasm Harderian Gland N-Metastatic/Invasive Neoplasm Heart c N-Metastatic/Invasive Neoplasm 2 0(64) (64) Ileum N-Metastatic/Invasive Neoplasm M-Adenocarcinoma Jejunum N-Metastatic/Invasive Neoplasm B-Leiomyoma M-Neuroendocrine Neoplasm M-Leiomyosarcoma

212 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Kidneys N-Metastatic/Invasive Neoplasm M-Carcinoma, Tubular M-Carcinoma, Transitional Cell M-Liposarcoma Lacrimal Gland N-Metastatic/Invasive Neoplasm Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Liver N-Metastatic/Invasive Neoplasm B-Hepatocellular Adenoma M-Hepatolcellular Carcinoma M-Hemangiosarcoma Lungs N-Metastatic/Invasive Neoplasm M-Carcinoma Nos M-Bronchiolo/Alveolar Carcinoma M-Hemangiosarcoma Lymph Node (other) b N-Metastatic/Invasive Neoplasm 5[20] 1[10] 1[20] 1[8] 1[13] 1[8] 1[16] 0[5] 1[15] 0[5] Lymph/Retic System M-Histiocytic Sarcoma M-Malignant Lymphoma M-Granulocytic Leukemia Mammary (protocol) c N-Metastatic/Invasive Neoplasm 2(56) 0 0(59) 0 0(54) 1 0(54) 0 0(52) 0 M-Adenocarcinoma 0(56) 19 0(59) 11 0(54) 14 0(54) 22 0(52) 17 B-Fibroadenoma 0(56) 20 1(59) 28 0(54) 17 0(54) 29 0(52) 15 B-Adenoma 0(56) 1 0(59) 1 0(54) 4 0(54) 3 0(52) 2 65

213 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Mediastinal Lymph Node N-Metastatic/Invasive Neoplasm Meninges b M-Malignant Meningioma NE NE 1[1] NE NE NE NE NE NE NE Mesenteric Lymph Node c N-Metastatic/Invasive Neoplasm 4 0(64) Mesentery/Perito b M-Liposarcoma NE 0[1] NE 0[1] NE 1[1] 0[1] 0[1] 0[2] NE M-Malignant Mesothelioma NE 0[1] NE 0[1] NE 0[1] 0[1] 0[1] 1[2] NE B-Lipoma NE 1[1] NE 0[1] NE 0[1] 0[1] 0[1] 0[2] NE Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Muscle (other) b N-Metastatic/Invasive Neoplasm 1[1] NE 0[1] NE NE 1[2] 0[2] NE 0[1] NE M-Carcinoma (Nos) 0[1] NE 0[1] NE NE 1[2] 0[2] NE 0[1] NE Muscle (protocol) N-Metastatic/Invasive Neoplasm Nerve - Sciatic N-Metastatic/Invasive Neoplasm 4 0(64) (64) 0 Ovaries N-Metastatic/Invasive Neoplasm NE 0 NE 1 NE 0 NE 0 NE 0 Peyer s Patches/GALT N-Metastatic/Invasive Neoplasm Pancreas N-Metastatic/Invasive Neoplasm B-Islet Cell Adenoma M-Islet Cell Carcinoma B-Hemangioma B-Acinar Cell Adenoma

214 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Parathyroid B-Adenoma 0(61) 0(62) 1(64) 0(63) 0(61) 0(59) 0(63) 0(64) 0(63) 0 Pericardium b N-Metastatic/Invasive Neoplasm 0[1] 0[2] 0[1] 0[1] NE NE 1[1] NE NE 1[1] Pituitary B-Pars Distalis-Adenoma M-Pars Distalis: Carcinoma N-Metastatic/Invasive Neoplasm B-Pars Intermedia: Adenoma Prostate N-Metastatic/Invasive Neoplasm 4 NE 1 NE 0 NE 0 NE 1 NE Rectum/Low Colon b B-Fibroma NE 1[1] NE NE NE NE NE NE NE NE Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous Salivary Gland c N-Metastatic/Invasive Neoplasm 2 1 0(64) B-Leiomyoma 0 0 0(64) Seminal Vesicles N-Metastatic/Invasive Neoplasm 1 NE 0 NE 0 NE 0 NE 1 NE 67

215 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Skin (other) b N-Metastatic/Invasive Neoplasm 2[18] 1[22] 0[20] 0[22] 0[18] 1[24] 0[20] 0[17] 1[17] 0[25] M-Malignant Basal Cell Tumor 1[18] 0[22] 0[20] 0[22] 0[18] 0[24] 0[20] 0[17] 0[17] 0[25] B-Lipoma 1[18] 0[22] 1[20] 0[22] 1[18] 0[24] 0[20] 0[17] 2[17] 0[25] B-Keratoacanthoma 2[18] 1[22] 4[20] 1[22] 3[18] 0[24] 4[20] 0[17] 3[17] 0[25] B-Benign Basal Cell Tumor 1[18] 0[22] 1[20] 0[22] 0[18] 0[24] 2[20] 1[17] 2[17] 0[25] M-Fibrosarcoma 2[18] 0[22] 3[20] 0[22] 1[18] 0[24] 2[20] 0[17] 0[17] 0[25] B-Fibroma 0[18] 1[22] 1[20] 0[22] 2[18] 1[24] 1[20] 0[17] 0[17] 0[25] M-Histiocytoma, Fibrous 0[18] 0[22] 1[20] 0[22] 1[18] 0[24] 0[20] 0[17] 1[17] 0[25] B-Squamous Cell Papilloma 1[18] 0[22] 0[20] 2[22] 0[18] 1[24] 3[20] 0[17] 1[17] 0[25] M-Squamous Cell Carcinoma 0[18] 0[22] 1[20] 0[22] 1[18] 0[24] 0[20] 0[17] 0[17] 0[25] M-Myxosarcoma 1[18] 0[22] 0[20] 0[22] 1[18] 0[24] 0[20] 0[17] 0[17] 0[25] M-Malignant Schwannoma 0[18] 0[22] 0[20] 0[22] 0[18] 0[24] 1[20] 0[17] 0[17] 0[25] Skin (protocol) B-Keratoacanthoma M-Histiocytoma, Fibrous Soft Tissue b N-Metastatic/Invasive Neoplasm 1[1] 0[1] NE NE NE 1[1] NE NE NE NE Spleen N-Metastatic/Invasive Neoplasm B-Hemangioma Sternal Marrow N-Metastatic/Invasive Neoplasm Stomach N-Metastatic/Invasive Neoplasm M-Carcinoma, Squamous Cell Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous 68

216 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Tail b N-Metastatic/Invasive Neoplasm 2[17] 0[1] 0[16] 0[5] 0[18] 0[7] 0[17] 0[6] 0[12] 0[10] B-Keratoacanthoma 0[17] 0[1] 0[16] 0[5] 0[18] 1[7] 0[17] 0[6] 0[12] 0[10] B-Basal Cell Tumor 1[17] 0[1] 0[16] 0[5] 0[18] 0[7] 0[17] 0[6] 0[12] 0[10] Testes N-Metastatic/Invasive Neoplasm 2 NE 0 NE 0 NE 0 NE 0 NE B-Benign Interstitial Cell Tumor 0 NE 1 NE 0 NE 2 NE 1 NE Thymus c N-Metastatic/Invasive Neoplasm 4[62] 1[64] 0[64] 1 0 1[63] 0[64] 0[62] 1 2 M-Malignant Thymoma 0[62] 0[64] 0[64] 0 0 0[63] 1[64] 1[62] 0 0 Thyroid M-Carcinoma, Follicular Cell B-Follicular, Cell Adenoma N-Metastatic/Invasive Neoplasm B-C-Cell Adenoma M-C-Cell Carcinoma Trachea N-Metastatic/Invasive Neoplasm Urinary Bladder N-Metastatic/Invasive Neoplasm 0 0(64) (64) (64) Uterus w/cervix B-Endometrial Stromal Polyp NE 2 NE 3 NE 1 NE 2 NE 2 N-Metastatic/Invasive Neoplasm NE 0 NE 1 NE 1 NE 0 NE 0 B-Benign Schwannoma NE 0 NE 0 NE 0 NE 0 NE 1 B-Leiomyoma NE 0 NE 1 NE 0 NE 0 NE 0 Vagina M-Granular Cell Tumor NE 0 NE 0 NE 0 NE 1 NE 0 N-Metastatic/Invasive Neoplasm NE 0 NE 0 NE 1 NE 0 NE 0 Gender M F M F M F M F M F Number of Evaluated Neoplastic Lesions - Incidental/Spontaneous 69

217 m Toxicology Tabulated Summary 2012N153953_00 Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks Daily Dose (mg/kg) 0 0 (Water Control) (Vehicle Control) Vascular Tissue b C-Hemangiosarcoma NE NE NE NE 0[2] NE NE NE NE 1[1] C-Hemangioma NE NE NE NE 2[2] NE NE NE NE 0[1] Zymbal s Gland b B-Adenoma 0[1] NE NE NE 1[1] NE NE NE NE NE M-Carcinoma 1[1] NE NE NE 0[1] NE NE NE NE NE Key: NE = Not examined a. There were no test article-related neoplastic lesions. b. Not a protocol required organ/tissue. Organs/Tissues were examined grossly; number in [ ] indicates number of organs/tissues examined microscopically. c. Missing protocol-required organs/tissues in some groups; number in ( ) indicates number of organs/tissues examined microscopically. 70

218 m Toxicology Tabulated Summary 2012N153953_ REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: NON-PIVOTAL STUDIES Table 11.1 Reproductive and Developmental Toxicity: Non-Pivotal Studies Species/ Strain Route (Vehicle/ Formulation) Duration of Dosing Doses (mg/kg) Number of Animals/Sex Noteworthy Findings Report/ Study No. (Module) Rat (pregnant) (Sprague Dawley) Oral (gavage) 12 days (gestation days 6 to 17) F 7F 7F There were no deaths and no dolutegravir-related clinical signs in any treatment group. No test substance-related changes on body weight, food consumption, maintenance of pregnancy and gross findings in dams were noted in any S sodium dosing groups. No test substance-related changes were noted on fetal viability, sex ratio, fetal body weight, placental weight, external malformations and external morphology of placenta of live fetuses. RD2008/01761 S TB-051-L (m ) Based on these results, 1000 mg/kg/day, which is the limit of daily dose on reproductive and developmental toxicity studies in the Guidelines for Toxicity Studies of Drugs, is considered to be suitable as the high dose level of the subsequent oral study for effects of S sodium on embryo-fetal development in rats. To assess the dose-response relationship, 300 and 100 mg/kg/day are considered to be suitable for the middle and low dose levels, respectively. 71

219 m Toxicology Tabulated Summary 2012N153953_00 Table 11.1 (Continued) Reproductive and Developmental Toxicity: Nonpivotal Studies Species/ Strain Rat (Sprague Dawley) Route (Vehicle/ Formulation) Oral (0.5% hydroxypropylmethylcellulose (HPMC) K15M / 0.1% polyoxyethylene sorbitan monooleate (Tween 80)./ suspension Duration of Dosing Day 4 to 21 postpartum (pp) inclusive Doses (mg/kg) Number of Animals/Sex 8/sex Noteworthy Findings Dose levels 500 mg/kg/day were not tolerated due to deaths starting on Day 14 pp. Necropsy observations included bright yellow discoloration of intestines, liver and skin and small adrenals. Clinical observations at 500 mg/kg/day included alopecia and loss of skin elasticity. There was growth retardation at 500 and 1000 mg/kg/day, evidenced as marked decreases in body weight and body length. Report/ Study No. (Module) CD2009/00409 D09072 (m ) Rats (Sprague Dawley) Oral (0.5% hydroxypropylmethylcellulose (HPMC) K15M / 0.1% polyoxyethylene sorbitan monooleate (Tween 80)./ solution or suspension Day 4 to 31 postpartum (pp) inclusive /sex A dose level of 300 mg/kg/day was not tolerated in juvenile rats; the entire group was euthanized one week early due to mortality. Clinical observations at 300 mg/kg/day included slightly decreased activity, various degrees of alopecia (hair loss) and moderate lack of skin elasticity in a few animals. The only macroscopic observation at 300 mg/kg/day was one male with a small thymus. The major histological effect at 300 mg/kg/day was decreased lymphocytes in the spleen, lymph nodes and thymus. Dose levels 75 mg/kg/day were tolerated, but caused reductions in body weight gain at 25 mg/kg/day. There were no macroscopic findings at 75 mg/kg/day, microscopic finding were minimal decreased lymphocytes in a few animals isolated to the spleen and lymph nodes. There was a trend of a slight decrease in the number of peripheral blood T cells in both male and female rats given > 25 mg/kg/day, with no effect on total B cells. CD2009/00770 D09126 (m ) 72

220 m Toxicology Tabulated Summary 2012N153953_00 Table 11.2 Two week Oral toxicity Study of S Sodium in Non Pregnant Rabbits: Non-Pivotal Studies Report No.: RD2008/01760 CTD Module: m

221 m Toxicology Tabulated Summary 2012N153953_00 Table 11.2 (Continued) Two week Oral toxicity Study of S Sodium in Non Pregnant Rabbits: Non-Pivotal Studies 74

222 m Toxicology Tabulated Summary 2012N153953_00 Table 11.3 Dose Range Finding Oral Study for Effects of S sodium on Embryofetal Development in Rabbits: Non-Pivotal Studies Report No.: RD2009/00186 CTD Module: m