module 2.6.6. toxicology written summary · confidential m2.6.6. toxicology written summary...

MODULE 2.6.6. TOXICOLOGY WRITTEN SUMMARY

CONFIDENTIALm2.6.6. Toxicology Written Summary 2012N154365_00

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TABLE OF CONTENTS

PAGE

1. BRIEF SUMMARY ...................................................................................................6

2. SINGLE DOSE TOXICITY......................................................................................102.1. Introduction.................................................................................................102.2. Rat..............................................................................................................12

2.2.1. Intramuscular and subcutaneous administration ..........................122.3. Dog.............................................................................................................13

2.3.1. Oral administration.......................................................................132.4. Monkey.......................................................................................................13

2.4.1. Oral administration.......................................................................132.4.2. Intramuscular and subcutaneous administration ..........................14

3. REPEAT DOSE TOXICITY ....................................................................................173.1. Introduction.................................................................................................173.2. Mouse.........................................................................................................19

3.2.1. Oral administration.......................................................................193.2.1.1. 14 day study...............................................................193.2.1.2. 13 week study ............................................................19

3.3. Rat..............................................................................................................203.3.1. Oral administration.......................................................................20

3.3.1.1. 14 day study...............................................................203.3.1.2. 4 week toxicity study...................................................213.3.1.3. 26 week toxicity study.................................................21

3.4. Monkey.......................................................................................................223.4.1. Oral administration.......................................................................22

3.4.1.1. 14 day study...............................................................223.4.1.2. 4 week toxicity study...................................................243.4.1.3. 38 week toxicity study.................................................25

4. GENOTOXICITY ....................................................................................................284.1. Introduction.................................................................................................284.2. In Vitro Studies (Dolutegravir) .....................................................................30

4.2.1. Non-mammalian cell systems ......................................................304.2.1.1. Ames assay................................................................30

4.2.2. Mammalian cell systems..............................................................304.2.2.1. Mutation test at the thymidine kinase locus in

mouse lymphoma L5178Y cells ..................................304.3. In Vivo Studies (Dolutegravir) .....................................................................31

4.3.1. Oral micronucleus test in the rat ..................................................314.4. Potential Impurities .....................................................................................32

4.4.1. gsk001* ........................................................................................324.4.1.1. Standard Ames test ....................................................324.4.1.2. Mouse lymphoma assay.............................................32

4.4.2. gsk002*........................................................................................324.4.2.1. Standard Ames test ....................................................32

4.4.3. gsk003* ........................................................................................334.4.3.1. Standard Ames test ....................................................33

* 新薬承認情報提供時に置き換え


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4.5. Route Assessment for Genotoxic Impurities ...............................................33

5. CARCINOGENICITY..............................................................................................365.1. Introduction.................................................................................................365.2. Mouse.........................................................................................................38

5.2.1. Oral administration.......................................................................385.2.1.1. Carcinogenicity study .................................................38

5.3. Rat..............................................................................................................395.3.1. Oral administration.......................................................................39

5.3.1.1. Carcinogenicity study .................................................39

6. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY .........................................416.1. Introduction.................................................................................................416.2. Fertility and Early Embryonic Development.................................................44

6.2.1. Rat...............................................................................................446.3. Embryofetal Development Studies..............................................................44

6.3.1. Rat...............................................................................................446.3.1.1. Oral administration .....................................................44

6.3.2. Rabbit ..........................................................................................456.3.2.1. Oral administration .....................................................45

6.4. Pre-Natal and Post-Natal Development, Including Maternal Function .........466.4.1. Rat...............................................................................................46

6.5. Juvenile Toxicity Studies.............................................................................476.5.1. Rat...............................................................................................47

6.5.1.1. Tolerability..................................................................476.5.1.2. Dose range study .......................................................486.5.1.3. Definitive Study ..........................................................49

7. LOCAL TOLERANCE.............................................................................................527.1. Introduction.................................................................................................527.2. Dermal Irritancy ..........................................................................................54

7.2.1. In vitro..........................................................................................547.2.2. Rabbit ..........................................................................................54

7.3. Ocular Irritancy ...........................................................................................547.3.1. In vitro..........................................................................................547.3.2. Rabbit ..........................................................................................55

7.4. Skin Sensitisation Potential.........................................................................557.4.1. Mouse..........................................................................................55

8. OTHER TOXICITY STUDIES.................................................................................588.1. Introduction.................................................................................................588.2. Immunotoxicity............................................................................................60

8.2.1. T-cell dependent antibody response ............................................60

9. DISCUSSION AND CONCLUSIONS......................................................................62

10. REFERENCES.......................................................................................................74

APPENDIX 1 CARCINOGENICITY TABLES.....................................................75

APPENDIX 2 ADDITIONAL INFORMATION ...................................................146


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LIST OF TABLES

PAGE

Table 2.1 List of Single Dose Toxicity Studies Performed with Dolutegravir...........11

Table 2.2 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 Batch Numbers of Dolutegravir and Formulations Used in Repeat Dose Toxicity Studies.............................................................................27

Table 4.1 List of Genotoxicity Studies Performed with Dolutegravir and Potential Impurities.................................................................................29

Table 4.2 Batch Numbers of Dolutegravir and Formulations Used in Genotoxicity Studies ..............................................................................35

Table 5.1 List of Carcinogenicity Studies Performed with Dolutegravir...................37

Table 5.2 Batch Numbers of Dolutegravir and Formulations Used in Carcinogenicity Studies..........................................................................40

Table 6.1 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir ...................................................................42

Table 6.2 Batch Numbers of Dolutegravir and Formulations Used in Reproductive and Developmental Toxicity Studies.................................51

Table 7.1 List of Local Tolerance Studies Performed with Dolutegravir..................53

Table 7.2 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 Batch Numbers of Dolutegravir and Formulations Used in Other Toxicity Studies......................................................................................61

Table 9.1 Principal Toxicological Findings in Rats and Monkeys Following Oral Administration of Dolutegravir.........................................................68

Table 9.2 Comparative Assessment of Mean Systemic Exposure FollowingOral Administration of Dolutegravir.........................................................69

Table 9.3 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M2) Following Oral Administration of Dolutegravir in the 4 and 26 week Rat Toxicology Studies .....................72


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Table 9.4 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M2) Following Oral Administration of Dolutegravir in the 14 Day and 4 and 38 Week Monkey Toxicology Studies .................................................................................73

Table.1 List of Tissues Studied in Carcinogenicity Studies..................................76

Table.2 Carcinogenicity Study in Mice - Tumor Incidence...................................77

Table.3 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence ............................................................................................86

Table.4 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence ............................................................................................87

Table.5 Carcinogenicity Study in Mice - Summary of Survival and Fate of Animals ..................................................................................................89

Table.6 Carcinogenicity Study in Rats - Tumor Incidence .................................101

Table.7 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence ..........................................................................................110

Table.8 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence ..........................................................................................115

Table.9 Carcinogenicity Study in Rats - Summary of Survival and Fate of Animals ................................................................................................134


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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 Route of Administration

Species

Repeat Dose ToxicityUp to 26 weeksUp to 38 weeks

OralOral

RatMonkey

GenotoxicityAmes testMouse lymphoma assayMicronucleus

In vitroIn vitro

Oral

Bacterial cellsL5178Y cells

Rat

Carcinogenicity Oral Mouse and Rat

Reproductive and DevelopmentalFertility/early embryonic developmentEmbryofetal developmentPre- and post-natal developmentJuvenile toxicity

OralOralOralOral

RatRat, Rabbit

RatRat

Immunotoxicity Oral Rat

Local ToleranceDermal irritancy

Ocular irritancy

Local lymph node assay

In vitroIn vivoIn vitroIn vivoTopical

Human cell modelRabbit

Human corneaRabbitMouse

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


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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 inAppendix 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 17.1. 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


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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.


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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.


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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 themonkey) and are discussed in Section 3.

2.1. 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 2.1.


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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.)

Locationin CTD

Single Dose Rat(Sprague Dawley)

6M6M

SubcutaneousIntramuscular

2.5 Single No GSK RD2009/00921(R42470)

m4.2.3.1


3M3M


2.5, 5 Single No GSK RD2009/00959(R42475)

m4.2.3.1


3M Intramuscular 4, 7.3, 8.7 Single No GSK 2011N123574(R42826)

m4.2.3.1


3M Intramuscular 10 Single No GSK 2012N136936 m4.2.3.1

Single Dose * Dog (beagle) 1F Oral 30, 100, 150, 250, 500 Single No RD2009/00963 m4.2.3.1

Single Dose Monkey(cynomolgus)

4F4F4F

OralSubcutaneousIntramuscular

311

Single No GSK CD2009/00647(D09113)

m4.2.3.1


1F Oral(gavage)

50, 125, 250, 500 Single No RD2007/01184(E-34572-TF-008-R)

m4.2.3.1


3F Oral(gavage)

1, 3, 10, 50 Single No RD2008/01762(S-349572-TB-44-R)

m4.2.3.1

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-0297994B.

Testing Facility:GSK = GlaxoSmithKline

=


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2.2. Rat

2.2.1. Intramuscular and subcutaneous administration

The toxicokinetics and potential toxicity of dolutegravir (2.5 mg/kg, parent form) wereinvestigated in male rats (n=6/group) following a single intramuscular or subcutaneousinjection [Report RD2009/00921, m4.2.3.1]. Dolutegravir was formulated as a long-acting parenteral wet bead-milled suspension in 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH2PO4H2O, 0.006M Na2HPO4

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 Cmax, AUC0-24 and AUC0-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, m4.2.3.1]. 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 NaH2PO4H2O, 0.006M Na2HPO4 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 Cmax, AUC0-24, and AUC0-t values for the 2.5 or 5 mg/kg doses were similar between subcutaneous and intramuscular administration. The mean AUC0-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, m4.2.3.1]. 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 5.1.


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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, m4.2.3.1]. 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 0.064 μg/mL (PA-IC90 [PA is protein adjusted]). The higher systemic exposure (mean Cmax and AUC0-t) values were achieved with the in situ gel with fast release, the in situ gel with slow release, or micronized free acid suspension.

2.3. Dog

2.3.1. 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, m4.2.3.1]. 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.

2.4. Monkey


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, m4.2.3.1]. Dolutegravir was formulated in 0.5% HPMC with0.1% Tween 80. The following were evaluated: clinical observations, body weights, food consumption, hematology, clinical chemistry and toxicokinetic analysis.


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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 Cmax or AUC0-24 values. There were no apparent differences in the Cmax values between 125 and 500 mg/kg and the AUC0-24 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, m4.2.3.1]. Dolutegravir was formulated in 0.5% HPMC with 0.1% Tween 80. Clinical observations, body weights, food consumption and toxicokinetics were evaluated. Atabulated 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, m4.2.3.1]. 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 2.4.2 below for discussion of other routes of administration).

Dolutegravir was well tolerated after a single oral dose of 3 mg/kg.

2.4.2. 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, m4.2.3.1]. Dolutegravir was formulated as a suspension in 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH2PO4H2O, 0.006M Na2HPO4 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 5.1.


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Mean dose-normalized Cmax 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 AUC0-24 following oral administration was similar to the dose-normalized AUC0-t following subcutaneous administration. There were no marked differences (2-fold) in the mean Cmax, AUC0-24 or AUC0-t values following subcutaneous or intramuscular administration.

Dolutegravir was well tolerated and no clinical observations were noted at the injection sites.


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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


Concentration (mg/mL)

Formulation

N10564-67-1 a IM, SC Rat (Sprague Dawley)

Single RD2009/00921

(R42470)

20 2

N10564-67-1 b IM, SC Rat (Sprague Dawley)

Single RD2009/00959

(R42475)

20, 40 2

eE537241-A, eE537241-B, and

eE537241-C

IM Rat (Sprague Dawley)

Single 2011N123574(R42826)

10, 20, 45 Multiple (refer to report)

091235661 IM Rat (Sprague Dawley)

Single 2012N136936 25.8 to 33 Multiple (refer to report)

05 Oral Monkey

(cynomolgus)

Single RD2007/01184

(E-34572-TF-008-R)

10, 25, 50, 100 1

A7Z001 Oral Monkey

(cynomolgus)

Single RD2008/01762

(S-349572-TB-044-R)

0.01, 0.03, 0.1,

0.5% w/v

1

A7Z002

N10564-67-1

Oral

IM, SC

Monkey

(cynomolgus)

Single CD2009/00647

(D09113)

0.3

20

1

2

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:IM = Intramuscular.SC = Subcutaneous.a = Long-acting parenteral wet bead-milled suspension. b = Homogenized suspension, at a larger particle size.

Formulation Code:1 = 0.5% w/w aqueous hydroxypropyl methylcellulose with 0.1% Tween 802 = 2% Pluronic F, 0.2% Polysorbate 80, 0.18% methylparaben, 0.02% propylparaben, 0.004M NaH2PO4H2O, 0.006M Na2HPO4 and 0.83%NaCl.


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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 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. Aninter-species comparison of dolutegravir plasma concentrations following repeated administration in the definitive toxicity studies is presented in Section 9. A tabularsummary of the repeat dose toxicokinetic data derived from the toxicity studies is presented in m2.6.7, Table 3.1.


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Table 3.1 List of Repeat Dose Toxicity Studies Performed with Dolutegravir

Type of Study

Species(Strain)/

Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Repeat Dose Mouse(CD-1)

10M/10F Oral(gavage)

10, 100, 500, 1500 14 days No RD2009/01546(S-349572-TF-066-R)

m4.2.3.2



10, 50, 500, 1500 13 weeks Yes RD2009/00028(S-349572-TF-068-L)

m4.2.3.2

Repeat Dose Rat(Sprague Dawley)

10M/10Fa Oral(gavage)

50, 150, 500 14 days Yes RD2007/01140(E-34572-TB-012-L)

m4.2.3.2


10M/10Fa,b Oral(gavage)

2, 10, 100, 1000 4 weeks Yes RD2008/01628(E-34572-TB-043-L)

m4.2.3.2


12M/12Fd Oral(gavage)

5, 50, 500 26 weekse,f Yes RD2009/00410(SBL055-082)

m4.2.3.2

Repeat Dose Monkey(cynomolgus)

3M/3F Oral(gavage)

100, 300, 1000 14 days Yes RD2007/01142(E-34572-TF-029-L)

m4.2.3.2


3M/3Fc Oral(gavage)

25, 50, 100 4 weeks Yes RD2008/00107((E-34572-TF-036-L)

m4.2.3.2


7M/7F Oral(gavage)

3, 10, 15, 50/30 38 weeksg,h

Yes RD2009/00036(SBL055-074)

m4.2.3.2

Note: All studies were performed with GSK1349572A, 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.

Testing Facility:= =

= =


19

3.2. Mouse


3.2.1.1. 14 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, m4.2.3.2]. 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 studyis 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.

3.2.1.2. 13 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, m4.2.3.2]. 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 studyis 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


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.

3.3. 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.


3.3.1.1. 14 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, m4.2.3.2]. 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 (Cmax and AUC0-24) to dolutegravir was generally less than dose-proportional 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 Cmax and AUC0-24 of 116 g/mL and 1830 g.h/mL, respectively on Day 14.


21

3.3.1.2. 4 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, m4.2.3.2]. 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 (Cmax and AUC0-24) increased with increasing dose although less thandose 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 Cmax and AUC0-24 of 55 g/mL and 751 g.h/mL, respectively, on Day 29.


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, m4.2.3.2]. 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 Cmax and AUC0-24 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


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 Cmax values of 764.5 g.h/mL and 47.3 g/mL, respectively).

3.4. 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.


3.4.1.1. 14 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, m4.2.3.2]. 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 (Cmax and AUC0-24) to dolutegravir was generally less than dose-proportional 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


23

the digestive tract (emesis, diarrhea, ulcer in colon) with significant change in bloodelectrolytes 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 animalfound 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 300


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 onemale 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 AUC0-24

(41 μg·h/mL) and Cmax (5.75 μg/mL) in the 100 mg/kg/day group was used to define the NOAEL exposure.


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, m4.2.3.2]. 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 thisstudy, 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, Cmax 17 g/mL).


25


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, m4.2.3.2]. 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 inm2.6.7, 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 prolongedAPTT (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


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 Cmax values of 38.8 g.h/mL and 5.05 g/mL, respectively).


27

Table 3.2 Batch Numbers of Dolutegravir and Formulations Used in Repeat Dose Toxicity Studies

Batch or Lot

Number(s)

Study Type Species Duration of

Dosing

Report No. Concentration

(mg/mL)

Formulation

B86001 Repeat Dose Mouse 14 days RD2009/01546 1, 10, 50, 150 1

B86001 Mouse 13 weeks RD2009/00028 1, 5, 50, 150 1

MTS-0297994B-07 Repeat Dose Rat 14 days RD2007/01140 5, 15 and 50 1

A7Z001 Rat 4 weeks RD2008/01628 0.2, 1, 10, 100 1

A7Z001 & B86001 Rat 26 weeks RD2009/00410 0.5, 5, 50 1

R06001 Repeat Dose Monkey 14 days RD2007/01142 10, 30, 100 1

02 Monkey 4 weeks RD2008/00107 2.5, 5, 10 1

A7Z001 & B86001 Monkey 38 weeks RD2009/00036 0.3, 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


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 standardAmes 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 DNA-reactive 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.


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 4.1.


29

Table 4.1 List of Genotoxicity Studies Performed with Dolutegravir and Potential Impurities


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Dolutegravir

Ames test NA NA In vitro 5 to 849 g/platea NA Yes WD2007/00514(V27467)

m4.2.3.3

Screening mouse lymphoma assay

NA NA In vitro Up to 80 g/mLb NA No GSK WD2007/01581(MLA-580)

m4.2.3.3

Mouse lymphoma assay

NA NA In vitro 5 to 85 g/mLa NA Yes WD2007/00515(V27468)

m4.2.3.3

Rat micronucleus Rat(Sprague Dawley)

6M Oral(gavage)

50, 150, 500 2 days Yes WD2007/00513(R27469)

m4.2.3.3

Impurities

gsk019* NA NA In vitro Up to 5000 g/plate

NA No GSK 2012N150346 m4.2.3.7.6

gsk002* NA NA In vitro Up to 500 g/plate NA No GSK 2010N105217 m4.2.3.7.6

gsk001* NA NA In vitro Up to 5000 g/plate

NA No GSK 2012N153013 m4.2.3.7.6

gsk001* NA NA In vitro Up to 1000 g/mL NA No GSK WD2010/00483 m4.2.3.7.6

Note: All studies using dolutegravir were performed with GSK1349572A, 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.



30

4.2. In Vitro Studies (Dolutegravir)

4.2.1. Non-mammalian cell systems

4.2.1.1. 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, m4.2.3.3.1]. 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.

4.2.2. 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 post-mitochondrial metabolising system (S9-mix). In both studies, the positive controls produced biologically significant increases in mutation frequency.

4.2.2.1. 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 alsowas tested for 24 hours in the absence of S9-mix [Report WD2007/01581, m4.2.3.3.1]. 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 inm2.6.7, 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.


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, m4.2.3.3.1]. 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.

4.3. 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 mouseand by the subcutaneous route in the rat.

4.3.1. Oral micronucleus test in the rat

Dolutegravir was tested in vivo in the rat bone marrow micronucleus assay following 2oral doses of 50, 150 or 500 mg/kg/day to male rats (n=6/group) given 24 hours apart[Report WD2007/00513, m4.2.3.3.2]. The highest dose tested was the dose producing maximum systemic exposure (Cmax 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.


32

4.4. Potential Impurities

4.4.1. gsk001*

4.4.1.1. 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, m4.2.3.7.6].

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.

4.4.1.2. 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, m4.2.3.7.6]. 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.

4.4.2. gsk002*



Single plate incorporation tests were conducted with gsk002* 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, gsk002* was mutagenic with



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).

4.4.3. gsk003*



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.

4.5. 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 mutagenicityand 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 4.4.1.1), gsk002* (see Section 4.4.2.1), gsk003* (see Section 4.4.3.1), gsk004* (NTP Reference), and gsk005* (Skopec, 1978). Additionally, gsk001* was considered genotoxic in a mouse lymphoma assay (see Section 4.4.1.2). 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].



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).


35

Table 4.2 Batch Numbers of Dolutegravir and Formulations Used in Genotoxicity Studies

Batch or Lot

Number(s)

Study Type Species (strain) or

Test System

Duration of

Dosing

Report No.

(Study No.)

Concentration

(mg/mL)

Formulation

R06001 Ames Test In vitro NA WD2007/00514

(V27467)

0.05 to 8.49 2

U23359/4/1 Mouse Lymphoma In vitro NA WD2007/01581

(MLA-580)

0.5 to 8.0 2

R06001 Mouse Lymphoma In vitro NA WD2007/00515

(V27468)

0.0332 to 8.50 2

R06001 Micronucleus Rat 2 days WD2007/00513

(R27469)

5, 15 and 50 1



Formulation Code:


2 = Dimethyl sulfoxide (DMSO)


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-1mouse 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 studieswere 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 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. Forthe definitive 104 week carcinogenicity studies in mice and rats, the electronic SAS data files are located with their corresponding reports in Module 4 (m4.2.3.4.1, rat SAS data and mouse SAS data).


37

Table 5.1 List of Carcinogenicity Studies Performed with Dolutegravir


Test System

No./Sex/Group


Dose (mg/kg/day)

Duration of Dosing



Location in CTD

Carcinogenicity Mouse(CD-1)

6545TK

Oral(gavage)

7.5, 25, 500 2 years Yes 2012N152419 m4.2.3.4.1

Carcinogenicity Rat(Sprague Dawley)

6512TK

Oral(gavage)

2, 10, 50 2 years Yes 2012N152418 m4.2.3.4.1

Key:TK = Satellite groups for toxicokinetics analysis.

Testing Facility:=


38

5.2. Mouse

Preliminary studies were performed to determine suitable dosages for use in a main oralcarcinogenicity study investigating the carcinogenic potential of dolutegravir in the CD-1 mouse. The preliminary studies determined that steady state systemic exposure based on AUC0-24 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 indose between 500 mg/kg/day and 1500 mg/kg/day.


5.2.1.1. 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, m4.2.3.4.1]. 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 10.1.

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 dolutegraviron Days 26 and 182. The systemic exposure (Cmax and AUC0-24) 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 orfemale 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


39

(NOAEL) for non-neoplastic findings after chronic oral administration was the high dose of 500 mg/kg/day.

5.3. Rat


5.3.1.1. 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, m4.2.3.4.1]. 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 10.2.

Surviving males were euthanized during Weeks 104 and 105 at the end of the 104 weekdosing 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 and182. No accumulation of dolutegravir was observed after repeated oral administration from Day 28 to Day 182. In general, the AUC0-24 and Cmax 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 signsand 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.


40

Table 5.2 Batch Numbers of Dolutegravir and Formulations Used in Carcinogenicity Studies

Batch or Lot

Number(s)

Study Type Species (strain)

or Test System

Duration of Dosing Report No.

(Study No.)

Concentration

(mg/mL)

Formulation

B86001 and

091001

Carcinogenicity Mouse 104 weeks 2012N152419 0.75, 2.5, 50 1

B86001 and

091001

Carcinogenicity Rat 104 weeks 2012N152418 0.2, 1, 5 1



Formulation Code:



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.




42

Table 6.1 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir

Type of Study

Species(Strain)/

Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Fertility and early embryonic development

Rat(Sprague Dawley)


100, 300, 1000 Up to 66 days (M)

Up to 42 days (F)

Yes XD2009/00368(S-349572-TF-063-L)

m4.2.3.5.1

Dose range embryofetal development study

Rat (pregnant)(Sprague Dawley)

7F Oral(gavage)

100, 300, 1000 12 days(gestation

Days 6 to 17)

Yes RD2008/01761(S-349572-TB-051-L)

m4.2.3.5.2

Embryofetal development study


20F Oral(gavage)


Days 6 to 17)

Yes XD2009/00367(S-349572-TB-062-L)

m4.2.3.5.2

Dose range study

Rabbit(non-pregnant)

(Japanese white)

3F Oral(gavage)

30, 100, 300, 1000 14 days No RD2008/01760(S-349572-TF-052-L)

m4.2.3.5.2


Rabbit (pregnant)(Japanese white)

4F Oral(gavage)


Days 6 to 18)

Yes RD2009/00186(S-349572-TF-060-L)

m4.2.3.5.2



18 to 20F

Oral(gavage)


Days 6 to 18)

Yes XD2009/0366(S-349572-TF-065-L)

m4.2.3.5.2


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



Duration of Dosing



Locationin CTD

Pre- and postnatal development

Rat(Sprague Dawley)

22F Oral(gavage)

5, 50, 1000 Gestation Day 6 to Lactation Day 20

Yes 2011N121663(SG10306)

m4.2.3.5.3

Juvenile tolerability

Rat(Sprague Dawley)

(juvenile)

8M/8F Oral(gavage)

5, 50, 100, 500, 1000

18 days(Day 4 to

Day 21 pp)

No GSK CD2009/00409(D09072)

m4.2.3.5.4

Juvenile dose range

Rat(Sprague Dawley)

(juvenile)


2, 25, 75, 300 28 days(Day 4 to

Day 31 pp)

No GSK CD2009/00770(D09126)

m4.2.3.5.4

Juvenile toxicity

Rat(Sprague Dawley)

(juvenile)


0.5, 2, 75a 63 days(Days 4 to

66 pp)

Yes GSK CD2010/00023(G09229)

m4.2.3.5.4

Note: All studies were performed with GSK1349572A, 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.

= =


44

6.2. Fertility and Early Embryonic Development

6.2.1. 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, m4.2.3.5.1]. 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 12.1.

There were no treatment-related effects and the NOAEL was considered to be 1000 mg/kg/day.

6.3. Embryofetal Development Studies

6.3.1. Rat

6.3.1.1. 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, m4.2.3.5.2]. 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 11.1.

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, m4.2.3.5.2]. Animals were observed for effects on clinical signs, body weights, and food consumption. Rats were killed on gestation Day


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 13.1.

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.

6.3.2. Rabbit

6.3.2.1. 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, m4.2.3.5.2]. The purpose of this study was to select dose levels for a dose range finding study to determinethe 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 11.2.

The mean Cmax 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 the300 mg/kg/day dose group. The mean AUC0-24 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, m4.2.3.5.2]. 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 11.3.


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 treatment-related 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, m4.2.3.5.2]. 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 13.2.

The mean Cmax values increased less than dose proportionally between 40 and 1000 mg/kg/day. The mean AUC0-24 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.

6.4. Pre-Natal and Post-Natal Development, Including Maternal Function

6.4.1. 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, m4.2.3.5.3]. Clinical observations, body weights, food consumption and gross pathology at termination of the lactation period were conducted on the dams (F0) to evaluate maternal toxicity. The following parameters


47

were evaluated for effects on the subsequent generation (F1): 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 (F2) on Day 13 of gestation. A summary of the study design and the results from this study are presented in m2.6.7, Table 14.1.

Suppressed body weight gain and decreased food consumption were noted in dams (F0) in the 1000 mg/kg/day group during the early stages of the lactation period. Decreased body weights were noted in the subsequent generation (F1) 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 (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.

6.5. Juvenile Toxicity Studies

6.5.1. Rat

6.5.1.1. 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, m4.2.3.5.4]. 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 11.1.

Due to unscheduled deaths, neither Cmax 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.


48

6.5.1.2. 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, m4.2.3.5.4]. 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 11.1.

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.


49

6.5.1.3. 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, m4.2.3.5.4]. 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 balano-preputial 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 15.1.

The systemic exposure to dolutegravir was generally lower on Day 32 pp compared to that on Day 13 pp. The Day 32 pp Cmax or AUC0-24 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


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 balano-preputial 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 AUC0-24 = 303 μg.h/mL and Cmax = 15.2 μg/mL and on Day 32 pp AUC0-24 = 85.7 μg.h/mL and Cmax

= 7.71 μg/mL, with no appreciable difference in gender.


51

Table 6.2 Batch Numbers of Dolutegravir and Formulations Used in Reproductive and Developmental Toxicity Studies

Batch or Lot Number(s)

Study Type Species (Strain) Duration of Dosing


Concentration (mg/mL)

Formulation

B86001 Fertility and early embryonic development

Rat (Sprague Dawley)

Up to 9 weeks XD2009/00368(S-349572-TF-063-L)

1, 3, 10% w/v 1

A7Z001 Dose range embryofetal development study


12 days RD2008/01761(S-349572-TB-051-L)

1, 3, 10% w/v 1

B86001 Embryofetal development study

Rat (SpragueDawley)

12 days XD2009/00367(S-349572-TB-062-L)

1, 3, 10% w/v 1

A7Z001 Dose range study Rabbit (Japanese White)

14 days RD2008/01760(S-349572-TF-052-L)

0.6, 2, 6, 20% w/v 1

A7Z002 Dose range embryofetal development study

Rabbit (Japanese White)

13 days RD2009/00186(S-349572-TF-060-L)

2, 6, 20% w/v 1

B86001 Embryofetal development study

Rabbit (Japanese White)

13 days XD2009/0366(S-349572-TF-065-L)

0.8, 4, 20% w/v 1

091001 Pre- and postnatal development


Day 6 gestation –Day 20 lactation

2011N121663(SG10306)

0.05, 0.5, 10% w/v 1

A7Z002 Juvenile tolerability Rat (Sprague Dawley)

18 days CD2009/00409(D09072)

1, 10, 20, 100, 200 1

A7Z002 Juvenile dose range Rat (Sprague Dawley)

Up to 4 weeks CD2009/00770(D09126)

0.4, 5, 15, 60 1

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:



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 sodiumsalt 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 (GSK1349572B) was used.




53

Table 7.1 List of Local Tolerance Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Skin irritancy study

Reconstituted human skin(SkinEthic

RHE)

NA In vitro 25 mg/site NA Yes ED2010/00004(1127/1884)

m4.2.3.6

Skin irritancy study *

Rabbit(Japanese

white)

3M Topical 500 mg/site Single No RD2010/00201(S-349572-TF-080-N)

m4.2.3.6

Ocular irritancy study

Reconstituted human corneal

model(SkinEthic

RHE)


m4.2.3.6

Ocular irritancy study *

Rabbit(Japanese

white)

6M Topical 100 mg/eye Single No RD2010/00202(S-349572-TF-079-N)

m4.2.3.6

Local lymph node assay *

Mouse(CBA/Ca)

5F Topical 25 L/site (25% w/w)(sodium salt)

3 days Yes ED2009/00019(1127/1834)

m4.2.3.6


Mouse(CBA/Ca)

5F Topical 25 L/site (25% w/w)(parent)

3 days Yes 2010N109153(1127/1886)

m4.2.3.6

Note: * These studies were performed with GSK1349572A, the sodium salt form, and the other studies used the free acid form, GSK1349572B.

Testing Facility: =

= GSK = GlaxoSmithKline.


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7.2. Dermal Irritancy

7.2.1. 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, m4.2.3.6]. Tissue samples (n=3) were treated with 25 mg of dolutegravir and incubated at 37C 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 16.1.

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.

7.2.2. 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, m4.2.3.6]. 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 16.1.

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 0.17.

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.

7.3. Ocular Irritancy

7.3.1. 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, m4.2.3.6]. Tissue samples (n=3) were treated with 30 mg of dolutegravir and incubated at 37C 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 16.1.


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.

7.3.2. 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, m4.2.3.6]. 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 16.1.

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 themean score 1 hour after application was lower than in the eye-unwashed group, and ocular irritation disappeared earlier than in the eye-unwashed group.

7.4. Skin Sensitisation Potential

7.4.1. 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, m4.2.3.6]. 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 [3H]-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 16.1.


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 1.27.

A second study was conducted to assess the skin sensitization potential of dolutegravir in the CBA/Ca mouse, using the same methodology [Report 2010N109153, m4.2.3.6]. A summary of the study design and the results from this study are presented in m2.6.7, Table 16.1.

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 0.83.


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.)

Concentration

(mg/mL)

Formulation

R09002B Skin irritation In vitro NA ED2010/00004(1127/1884)

25 mg NA

A7Z002 Skin irritation Rabbit (Japanese white)

Single RD2010/00201(S-349572-TF-080-N)

0.5 g /2.5x2.5cm NA

R09002B Eye irritation In vitro NA ED2010/00005(1127/1885)

30 mg NA

A7Z002 Eye irritation Rabbit (Japanese white)

Single RD2010/00202(S-349572-TF-079-N)

0.1 g / eye NA

A7Z002 Skin sensitisation potential

Mouse (CBA/Ca) Single ED2009/00019(1127/1834)

25% w/w 6

R09002B Skin sensitisation potential

Mouse (CBA/Ca) Single 2010N109153(1127/1886)

25% w/w 6

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.

Key:NA = not applicable

Formulation Code:1 = polyethylene glycol 33502 = polysorbate 203 = mannitol4 = sodium carboxymethylcellulose5 = Sesame oil6 = Dimethyl formamide


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 investigationwas 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 8.1.


59

Table 8.1 List of Other Toxicity Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Immunotoxicity Rat(Sprague Dawley)


10, 100, 1000 4 weeks Yes RD2009/00751(S-349572-TB-064-L)

m4.2.3.7.2

Note: All studies were performed with GSK1349572A, the sodium salt form.No observed adverse effect levels (NOAEL) are bolded.

Testing Facility: =


60

8.2. Immunotoxicity

8.2.1. 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, m4.2.3.7.2]. 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 17.1.

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.


61

Table 8.2 Batch Numbers of Dolutegravir and Formulations Used in Other Toxicity Studies

Batch or Lot

Number(s)

Study Type Species (Strain) or

Test System

Duration of

Dosing

Report No.

(Study No.)

Concentration

(mg/mL)

Formulation

B86001 Immunotoxicity Rat (Sprague Dawley) 1 month RD2009/00751

(S-349572-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:



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 (AUC0-24, mg/kg and mg/m2) 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 (AUC0-24) on Day 1 was 277 g.h/mL. Gender-mean Day 14 exposure (AUC0-24) 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 whichconsisted 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/m2 metrics are appropriate determinates of safety cover for this toxicitybecause 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


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/m2 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/m2 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 50Xthe human mg/kg equivalent dose (based on 50 kg human), and 35X and 18X the human mg/m2 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/m2 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


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 (AUC0-24 = 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 (AUC0-24 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 AUC0-24 and Cmax of 39 g.h/mL and 5.1 g/mL, respectively), which corresponds to 0.7X and 1.4X the human AUC and Cmax exposure, respectively, for a 50 mg QD dose) and corresponds to 0.5X and 1.2 X the human AUC and Cmax 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/m2 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.


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 ~0.5Xthe 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,


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


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 balano-preputial skinfold separation). There were no changes considered related to dolutegraviradministration 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 AUC0-24 = 90 g.h/mL and Cmax = 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 AUC0-24 and Cmax of 39 g.h/mL and 5.1 g/mL, respectively), which corresponds to 0.7X and 1.4X the human AUC and Cmax exposure, respectively, at a 50 mg once daily dose (AUC0-24 of 53.6 g.h/mL and Cmax of 3.7 g/mL) and corresponds to 0.5X and 1.2X the human AUC and Cmax exposure, respectively, at a 50 mg twice daily (AUC0-24 of 75.1 g.h/mL and Cmax of 4.2 g/mL). Because GI intolerance is considered to be due to local drug administration, mg/kg or mg/m2 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/m2 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


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

Finding

Rat MonkeyEffect Dose (mg/kg/day)

No Effect Dose (mg/kg/day)

Effect Dose (mg/kg/day)

No Effect Dose (mg/kg/day)

Mortality/Morbidity: Adult animals: Death preceded by repeated emesis, diarrhea with significant weight lossJuvenile animals: Mortality preceded by decreased body weight gain

NO

75

NO

2

50

NA

30

NA

Clinical Observation: Emesis, diarrhea NO NO 50 10a

Body Weight Loss NO NO 50 15Gastrointestinal Effects:

Stomach: Gastric mucosal hemorrhage, mononuclear cell infiltration, and/or multifocal epithelial regenerationStomach: Multifocal erosionsCecum, colon, rectum: mucosal atrophy and/or hemorrhage

500

NONO

50

NONO

50/30

50/3050

15

1515

Hepatic Effects: Hepatocellular single cell necrosis and vacuolation with AST, and GTP elevations and/or increased bilirubin and triglycerides

ALT elevations without corresponding anatomic pathology changes

AST & bilirubin elevations secondary to moribundity

NO NO

1000

300

50

300

100

15

Renal Effects considered associated with moribund condition: Renal tubule dilatation, increased BUN and CRE, and/or decreased serum sodium and chloride

NO NO 50 15

Bone Marrow and Lymphoid changes considered 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

NO NO 50 15

Decrease in RBCs (females) NO NO 100 50Findings Considered to be Associated with malnutrition: Acinar cell atrophy in the pancreas and/or parotid gland

NO NO 100 50

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.

s.kawakami

ノート注釈

s.kawakami : Marked


69

Table 9.2 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 Human AUC

Ratioa,b

(50mg QD)

Animal to Human AUC

Ratioa,c

(50mg BID)

Day 1 End of Study

Day 1 End of Study

Ratd

(14 days)50 M 58.5 65.7 881 1040 19.4 13.8

F 75.4 95.6 1110 1610 30.0 21.4

150 M 82.7 74.1 994 1150 21.5 15.3

F 83.3 106 1050 1740 32.5 23.2

500(NOAEL)

M 87.1 108 1360 1710 31.9 22.8

F 117 124 1350 1950 36.4 30.0

Ratd

(4 weeks)2 M 3.5 4.7 39.3 53.0 0.99 0.71

F 4.6 7.8 60.0 81.7 1.5 1.2

10 M 15.2 23.7 220 274 5.1 3.6

F 21.2 34.6 278 378 7.1 5.0

100(NOAEL)

M 43.7 49.2 693 722 13.5 9.6

F 54.4 61.6 775 781 14.6 10.4

1000 M 95.1 119 1678 1837 34.3 24.5

F 116 112 1615 1737 32.4 23.1

Rat(26 weeks)d

5 M 9.4 11.9 88.8 116 2.2 1.5

F 12.0 20.1 138 290 5.4 3.9

50(NOAEL)

M 47.3 38.0 637 607 11.3 8.1

F 56.5 56.6 731 922 17.2 12.3

500 M 95.3 85.1 1450 1338 30.0 17.8

F 103.5 107 1450 1777 33.2 23.7

Monkeyd

(14 days)100

(NOAEL)M 21.3 24.0 172 192 3.6 2.6

F 18.9 23.0 150 187 3.5 2.5

300 M 30.9 21.7 324 199 3.7 2.6

F 17.5 23.3 142 271 5.1 3.6

1000 M 27.5 26.2 358 364 6.8 4.8

F 20.9 30.3 237 354 6.6 4.7

Monkeyd

(4 weeks)25 M 8.7 13.9 60.3 108 2.0 1.4

F 11.2 15.4 67.9 83.9 1.6 1.1

50(NOAEL)

M 9.6 14.5 72.9 111 2.1 1.5

F 10.7 20.4 68.7 153 2.9 2.0

100 M 12.1 16.0 99.0 148 2.8 2.0

F 12.2 13.4 90.2 92.0 1.7 1.2

Monkey(38 weeks)e

3 M 2.9 3.0 15.2 18.9 0.35 0.25

F 3.1 2.3 15.3 15.5 0.29 0.21

10 M 4.7 4.4 30.7 32.3 0.60 0.43

F 6.1 5.1 34.5 37.7 0.70 0.50

15(NOAEL)

M 7.7 5.3 46.4 36.7 0.68 0.49

F 5.5 4.8 30.6 40.9 0.76 0.54

50/30 M 9.0 7.5 62.9 61.7 1.2 0.82

F 10.5 7.8 63.4 61.7 1.2 0.82


70

Table 9.2 (Continued) Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir

Species(Duration)

Dose (mg/kg/day)


Ratioa,b

(50mg QD)

Animal to Human AUC

Ratioa,c

(50mg BID)

Day 1 End of Study

Day 1 End of Study

Mouse(14 day)f

10 M 16.4 14.9 188 218 4.1 2.9

F 18.3 18.7 195 188 3.5 2.5

100 M 62.7 60.6 921 801 14.9 10.7

F 62.0 73.4 980 1170 21.8 15.6

500 M 96.5 77.0 1140 1090 20.3 14.5

F 93.2 90.7 1100 1190 22.2 15.8

1500(NOAEL)

M 106 104 1210 1240 23.1 16.5

F 115 123 1520 1630 30.4 21.7

Mouse(13-week)f

10 M 16.0 18.5 211 257 4.8 3.4

F 19.3 28.0 212 256 4.8 3.4

50 M 43.9 52.7 477 653 12.2 8.7

F 53.7 62.6 528 740 13.8 9.9

500 M 77.3 82.1 923 1010 18.8 13.4

F 88.3 109 1110 1300 24.3 17.3

1500(NOAEL)

M 109 103 1440 1320 24.6 17.6

F 114 118 1420 1350 25.2 18.0

Rat (embryofetal development)

100 F 64.1 78.0 949 1252 23.4 16.7

300 F 74.0 82.2 1096 1409 26.3 18.8

1000(NOAEL)g

F 139 109 1841 2032 37.9 27.1

Rabbit (embryofetal development)

40 F 0.8 1.3 2.1 2.6 0.049 0.035

200h F 1.5 1.7 15.6 14.5 0.27 0.19

1000h F 2.3 2.1 36.8 30.1 0.56 0.40

Rat(Juvenile)i

0.5 M 4.6 1.5 92.0 9.9 0.18 0.13

F 4.7 2.4 86.5 27.1 0.51 0.36

2(NOAEL)

M 15.2 7.7 303 85.7 1.6 1.1

F 16.4 7.5 316 93.3 1.7 1.2

75 M 88.0 69.9 1540 917 17.1 12.2

F 85.4 77.4 1549 1044 19.5 13.9

Mouse

(Carcino-genicity)f, j

7.5 M 13.4 14.5 176 148 2.8 2.0

F 20.3 16.6 235 157 2.9 2.1

25 M 40.8 27.4 579 327 6.1 4.4

F 44.4 43.3 565 494 9.2 6.6

500

(NOAEL)

M 77.7 71.9 1180 953 17.8 12.7

F 94.7 94.5 1300 1210 24.3 16.1

Rat

(Carcino-genicity)k

2 M 7.3 7.7 101 100 1.9 1.3

F 8.8 20.4 114 279 5.2 3.7

10 M 24.7 21.3 348 340 6.3 4.5

F 34.3 40.4 501 731 13.6 9.7

50

(NOAEL)

M 57.6 40.9 841 713 13.3 9.5

F 87.0 68.5 1150 1140 21.3 15.2


71

Table 9.2 (Continued) Comparative Assessment of Mean Systemic Exposure Following Oral Administration of Dolutegravir

Species(Duration)

Dose (mg/kg/day)


Ratioa,b

(50mg QD)

Animal to Human AUC

Ratioa,c

(50mg BID)

Day 1 End of Study

Day 1 End of Study

Humanb 50 mg M/F 3.7 53.6 NA NA

Humanc 100 mg M/F 4.2 75.1 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 valuesb. 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.


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Table 9.3 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M2) Following Oral Administration of Dolutegravir in the 4 and 26 week Rat Toxicology Studies

Species(Duration)

Dose (mg/kg/day)

SexCmax

(g/mL)AUC0-24

(g.h/mL)

ANIMAL TO HUMAN RATIOS Based on AUC, MG/KG and MG/M2

(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/M2

(50mg QD)Mg/M2

(50mg BID)

Rat(4 weeks)

2 M 3.5 4.7 39.3 53.01.25 0.96 2 1 0.35 0.18

F 4.6 7.8 60.0 81.7

10 M 15.2 23.7 220 2746.1 4.3 10 5 1.8 0.88

F 21.2 34.6 278 378

100(NOAEL)

M 43.7 49.2 693 72214.1 10.0 100 50 17.6 8.8

F 54.4 61.6 775 781

1000 M 95.1 119 1678 183733.4 23.8 1000 500 176 88

F 116 112 1615 1737

Rat(26 weeks)

5 M 9.4 11.9 88.8 1163.8 2.7 5 2.5 0.88 0.44

F 12.0 20.1 138 290

50(NOAEL)

M 47.3 38.0 637 60714.3 10.2 50 25 8.8 4.5

F 56.5 56.6 731 922

500 M 95.3 85.1 1450 133831.6 20.8 500 250 88 45

F 103.5 107 1450 1777

Human 50 mg M/F 3.7 53.6 NA NA NA NA NA NAHuman 100 mg M/F 4.2 75.1 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/m2 , multiply by Km (Km rat = 6; Km human = 34).NA = Not applicable.


73

Table 9.4 Comparative Assessment of Mean Animal to Human Exposure Ratios (AUC, Mg/Kg and Mg/M2) Following Oral Administration of Dolutegravir in the 14 Day and 4 and 38 Week Monkey Toxicology Studies

Species(Duration)

Dose (mg/kg/day)

SexCmax

(g/mL)AUC0-24(g.h/mL)

ANIMAL TO HUMAN RATIOS Based on AUC, MG/KG and MG/M2

(gender averaged means)Day 1 End of

StudyDay 1 End of

StudyAUC

(50mg QD)AUC

(50mg BID)Mg/Kg

(50mg QD)Mg/Kg

(50mg BID)Mg/M2

(50mg QD)Mg/M2

(50mg BID)Monkey

(14 days)100

(NOAEL)M 21.3 24.0 172 192

3.6 2.6 100 50 35 17.6F 18.9 23.0 150 187

300 M 30.9 21.7 324 1994.4 3.1 300 150 105 53

F 17.5 23.3 142 2711000 M 27.5 26.2 358 364

6.7 4.8 1000 500 353 176F 20.9 30.3 237 354

Monkey(4 weeks)

25 M 8.7 13.9 60.3 1081.8 1.3 25 12.5 8.8 4.4

F 11.2 15.4 67.9 83.950

(NOAEL)M 9.6 14.5 72.9 111

2.5 1.8 50 25 17.6 8.8F 10.7 20.4 68.7 153

100 M 12.1 16.0 99.0 1482.3 1.6 100 50 35 17.6

F 12.2 13.4 90.2 92.0Monkey

(38 weeks)3 M 2.9 3.0 15.2 18.9

0.32 0.23 3 1.5 1.1 0.53F 3.1 2.3 15.3 15.5

10 M 4.7 4.4 30.7 32.30.65 0.47 10 5 3.5 1.8

F 6.1 5.1 34.5 37.7

15(NOAEL)

M 7.7 5.3 46.4 36.70.72 0.52 15 7.5 5.3 2.6

F 5.5 4.8 30.6 40.9

50/30 M 9.0 7.5 62.9 61.71.2 0.82 50/30 25/15 17.6/10.6 8.8/5.3

F 10.5 7.8 63.4 61.7

Human 50 mg M/F 3.7 53.6 NA NA NA NA NA NAHuman 100 mg M/F 4.2 75.1 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/m2 , multiply by Km (Km monkey = 12; Km human = 34).NA = Not applicable.


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;75:4465-4469.

References (Available Upon Request)

NTP CAS Registry Number: 100-39-0: http://ntp.niehs.nih.gov/index.cfm?objectid=E87B62DC-BDB5-82F8-F5D7CACB96B43389

Merck Research Laboratories. Raltegravir_ACM_BGPK_1.doc, Version 9.2, August 2007.FDA Advisory Committee, September 5, 2007. http://www.fda.gov/ohrms/dockets/ac/07/briefing/2007-4314b1-00-index.htm.

Guidelines (not provided)

ICH Q3A(R2) (CPMP/ICH/2737/99): Impurities in New Drug Substances. October 2006.

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) 2006.


75

APPENDIX 1 CARCINOGENICITY TABLES


76

Table.1 List of Tissues Studied in Carcinogenicity Studies

Tissue Rat (Report No.

09-2178)

Mouse (Report No.

09-2177)

Gross lesion Tissue masses AdrenalsAorta (thoracic)

Bone (sternum, distal femur)Bone marrow (sternum,distal femur)BrainCecumColonEpididymidesEsophagusEyesGallbladderHeartKidneysLacrimal glands/Harderian glandsLiverLungs (with mainstem bronchi)Lymph nodes (mesenteric,mediastinal)Mammary glandMuscle (rectus femoris)Nerve (sciatic)OvariesPancreasPituitaryProstateSalivary glands (submandibular)Seminal vesiclesSkin (dorsal – base of tail)Small intestines (duodenum, ileum, jejunum and Peyer’s

patches/GALT)Spinal cord (cervical)SpleenStomachTestesThymusThyroid (with parathyroid)TracheaUrinary bladderUterus (horns/body/cervix)Vagina

X

Key: GALT = Gut Associated Lymphoid Tissue


77

Table.2 Carcinogenicity Study in Mice - Tumor Incidence

Number of Tumors in All Animals Which Were Evaluated (Without Consideration to Cause and Relevance)

Frequency according to dose and sex (n)

(1)Water

Control

(2)VehicleControl

(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 65 65 65 65 65 65 65 65 65 65

Organ Identification of Tumor

Adipose

TissueInvasive Neoplasm (N) 0 3 1 0 - 1 1 0 2 2

Adrenal

Glands

Medulla Benign

Pheochromocytoma (B) 1 0 0 0 0 0 0 0 0 0

Cortex Carcinoma (M) 0 0 0 0 0 0 0 1 0 0

Cortex Adenoma (B) 0 0 1 2 1 0 1 1 1 0

Invasive Neoplasm (N) 6 16 6 6 4 6 5 3 3 8

Aorta Invasive Neoplasm (N) 4 6 3 6 2 3 2 4 0 7

Bone

(other)

Osteoma (B) 1 0 1 3 0 - 0 0 0 0

Sarcoma, NOS (M) 0 0 0 0 0 - 1 0 0 0

Invasive Neoplasm (N) 0 0 1 0 0 - 0 0 0 0

Brain Invasive Neoplasm (N) 1 2 0 2 1 1 0 0 0 0

Cecum Invasive Neoplasm (N) 5 5 6 0 0 0 1 2 0 1

Cervical

Spinal Cord Invasive Neoplasm (N) 0 0 0 1 1 1 0 0 0 0

Colon Invasive Neoplasm (N) 0 3 3 0 0 0 0 0 0 0

Diaphragm Invasive Neoplasm (N) - 1 1 - - - 1 - - -

Distal

Femur


Osteoma (B) 0 0 0 1 0 0 0 0 0 0

Duodenum Invasive Neoplasm (N) 1 3 2 1 0 0 1 1 0 0

Ears Invasive Neoplasm (N) 0 0 0 0 0 0 0 0 1 0

Epididymides Invasive Neoplasm (N) 8 - 7 - 5 - 3 - 3 -

Esophagus Invasive Neoplasm (N) 2 3 2 2 3 3 2 5 2 5

Eyes Invasive Neoplasm (N) 3 3 1 0 1 1 0 0 1 3


78



(1)Water

Control

(2)VehicleControl

(3)Low

7.5 mg/kg/day

(4)Inter.

25 mg/kg/day

(5)High

500 mg/kg/day




Femoral

Marrow


Hemangioma (B) 0 1 0 0 1 0 0 0 0 0

Gallbladder


Papilloma (B) 0 0 0 1 0 0 0 0 2 0

Cystadenoma/Adenoma (B) 0 0 0 0 0 1 2 0 0 0

Harderian

Glands


Adenoma (B) 11 4 11 10 14 5 9 7 4 9

Carcinoma (M) 1 0 0 0 0 0 0 0 0 0

Heart


Endocardial

Schwannoma (M) 0 1 0 0 0 0 0 0 0 0

Hemangiosarcoma (M) 0 0 0 0 0 1 0 0 0 0

Ileum Invasive Neoplasm (N) 4 5 3 0 0 1 1 0 1 0

Intercostal

Muscle Invasive Neoplasm (N) - - 1 - - - - - - -

Jejunum Invasive Neoplasm (N) 2 1 1 0 0 0 0 1 0 0

Kidneys Invasive Neoplasm (N) 11 17 12 6 5 7 6 10 5 15

Lacrimal

Gland


Adenoma (B) 0 0 0 0 0 0 0 0 1 0

Liver


Hepatocellular

Carcinoma (M) 7 0 7 0 11 0 10 0 6 1

Hepatocellular

Adenoma (B) 11 0 13 0 15 0 16 0 10 1


Hemangioma (B) 0 0 0 0 1 0 0 0 0 0


79



(1)Water

Control

(2)VehicleControl

(3)Low

7.5 mg/kg/day

(4)Inter.

25 mg/kg/day

(5)High

500 mg/kg/day




Lungs


Bronchiolo/Alveolar

Adenoma (B) 12 8 8 0 15 4 5 8 6 5

Bronchiolo/Alveolar

Carcinoma (M) 10 2 3 4 8 5 9 5 11 2

Lymph

Node

(other) Invasive Neoplasm (N) 12 23 16 9 3 6 8 10 5 15

Lymph/

Retic

System

Malignant

Lymphoma (M) 14 27 17 16 8 9 6 14 10 19

Histiocytic Sarcoma (M) 3 2 1 3 2 3 3 6 1 4

Mammary

(protocol)


Adenocarcinoma (M) 0 0 0 0 0 0 0 1 0 1

Mandibular

Lymph

Nodes Invasive Neoplasm (N) - - - 1 - - - - - -

Mediastinal

Lymph

Nodes Invasive Neoplasm (N) 12 24 16 13 4 8 8 15 8 17

Mesenteric

Lymph

Nodes



Mesentery/

Perito

Invasive Neoplasm (N) 1 0 0 2 - 0 - 1 0 1

Malignant

Mesothelioma (M) 0 0 0 0 - 1 - 0 0 0

Mesovarian

Ligament Invasive Neoplasm (N) - 1 - - - - - - - -

Muscle

(Other)

Invasive Neoplasm (N) 0 1 - - - 0 1 - - 1

Hemangiosarcoma (M) 1 0 - - - 0 0 - - 0

Rhabdomyosarcoma (M) 0 0 - - - 0 1 - - 0


80



(1)Water

Control

(2)VehicleControl

(3)Low

7.5 mg/kg/day

(4)Inter.

25 mg/kg/day

(5)High

500 mg/kg/day




Muscle

(Protocol)



Nerve

Sciatic Invasive Neoplasm (N) 0 9 3 3 1 0 1 3 2 2

Ovaries

Invasive Neoplasm (N) - 19 - 9 - 4 - 9 - 9

Tubulostromal

Adenoma (B) - 3 - 0 - 1 - 0 - 1

Adenocarcinoma (M) - 0 - 1 - 0 - 0 - 0

Cystadenoma (B) - 0 - 2 - 0 - 0 - 1

Benign Granulosa

Cell Tumor (B) - 1 - 0 - 0 - 0 - 0

P. Patches/

GALT Invasive Neoplasm (N) 4 6 5 2 1 1 1 1 1 1

PancreasInvasive Neoplasm (N) 7 16 11 7 4 4 4 8 5 9

Islet Cell Adenoma (B) 1 0 1 0 0 0 1 1 0 0

Parotid

Gland Invasive Neoplasm (N) - 2 - - 1 - 0 - - -

Pericardium Invasive Neoplasm (N) - - 1 1 0 1 - - - 1

PituitaryInvasive Neoplasm (N) 1 2 0 1 0 0 1 0 0 1

Pars Distalis

Adenoma (B) 0 0 0 0 0 2 0 0 0 1

ProstateInvasive Neoplasm (N) 6 - 8 - 6 - 4 - 4 -

Carcinoma (M) 0 - 0 - 0 - 1 - 0 -

Rectum/

Low Colon Invasive Neoplasm (N) - 1 0 - - - - 0 - -

Salivary

Gland Invasive Neoplasm (N) 7 11 8 4 4 3 2 1 3 9

Seminal

Vesicles Invasive Neoplasm (N) 3 - 7 - 2 - 1 - 2 -


81



(1)Water

Control

(2)VehicleControl

(3)Low

7.5 mg/kg/day

(4)Inter.

25 mg/kg/day

(5)High

500 mg/kg/day




Skin

(other)

Carcinoma (M) 1 0 0 1 1 1 0 0 1 0



Sarcoma NOS (M) 0 1 0 1 0 0 0 0 0 0

Sebaceous Cell

Adenoma (B) 0 1 0 0 0 0 0 0 0 0

Histiocytoma (B) 0 0 0 0 0 0 1 0 0 0

Skin

(protocol)


Schwannoma (B) 0 0 1 0 0 0 0 1 1 0

Fibrosarcoma (M) 0 1 0 0 1 0 0 0 0 0

Spleen


Hemangioma (B) 0 0 1 0 0 0 0 0 0 0


Sternal

Marrow Invasive Neoplasm (N) 3 5 2 0 0 1 1 0 2 3

SternumInvasive Neoplasm (N) 4 12 3 2 2 2 5 4 6 8

Osteogenic Sarcoma (M) 0 1 0 0 0 0 0 0 0 0

Stomach


Glandular Mucosa:

Adenoma (B) 1 0 0 0 0 0 0 1 0 1

Forestomach:

Squamous Cell

Carcinoma (M) 0 0 0 0 0 0 0 1 0 0

TestesInvasive Neoplasm (N) 0 - 1 - 1 - 0 - 0 -

Benign Interstitial

Cell Tumor (B) 1 - 1 - 0 - 2 - 1 -

Thymus Invasive Neoplasm (N) 11 24 17 12 7 10 7 16 9 15


82



(1)Water

Control

(2)VehicleControl

(3)Low

7.5 mg/kg/day

(4)Inter.

25 mg/kg/day

(5)High

500 mg/kg/day




ThyroidInvasive Neoplasm (N) 0 3 1 1 1 1 1 1 1 1

Follicular Cell

Adenoma (B) 0 0 0 0 1 0 0 1 1 1

Trachea Invasive Neoplasm (N) 1 4 1 0 2 2 1 2 2 0

Ureters Invasive Neoplasm (N) 0 3 2 0 1 1 0 - 1 -

Urinary

Bladder


Leiomyosarcoma (N) 0 0 0 0 0 0 0 0 0 1

Uterus w/

Cervix


Endometrial

Carcinoma (M) - 0 - 1 - 0 - 1 - 0

Endometrial Stromal

Polyp (B) - 2 - 5 - 4 - 9 - 7

Leiomyosarcoma (M) - 1 - 0 - 0 - 3 - 1

Hemangioma (B) - 0 - 1 - 3 - 1 - 1

Benign Granular Cell

Tumor (B) - 0 - 0 - 1 - 0 - 0

Endometrial Stromal

Sarcoma (M) - 2 - 1 - 0 - 0 - 0

Leiomyoma (B) - 1 - 0 - 2 - 4 - 1

Granular Cell Tumor (M) - 1 - 0 - 0 - 0 - 0

Hemagiosarcoma (M) - 0 - 0 - 0 - 1 - 0

Vagina Invasive Neoplasm (N) - 10 - 1 - 2 - 2 - 3

VesselHemangiosarcoma (C) 2 3 2 1 3 2 3 1 1 2

Hemangioma (C) 1 1 1 1 2 2 1 0 0 1

Zymbal’s

Gland Carcinoma (M) - - - - 1 - - - - -


83

Sex M/F M F M F M F M F M F


Number of Tissues Examined 65 65 65 65 65 65 65 65 65 65

A). Macroscopically Abnormal

Adipose tissue 3 4 1 1 0 1 1 3 3 2

Bone (other) 7 3 3 3 6 0 4 3 2 1

Bulbourethral Gland 0 0 0 0 0 0 2 0 3 0

Coagulating Gland 2 0 2 0 5 0 1 0 0 0

Diaphragm 0 1 1 0 0 0 1 0 0 0

Ear(s) 7 3 8 5 9 5 8 6 10 2

Extremity 0 1 0 0 0 0 1 2 0 2

Intercostal Musc 0 0 1 0 0 0 0 0 0 0

Lymph Node (other) 27 31 31 23 10 17 21 23 13 23

Mandibular Lymph Nodes 0 0 0 1 0 0 0 0 0 0

Mesentery/Perito 1 2 1 4 0 1 0 2 1 2

Mesovarian Ligament 0 1 0 0 0 0 0 0 0 0

Muscle (other) 1 1 0 0 0 1 3 0 0 2

Parotid Gland 0 2 0 0 2 0 1 0 0 0

Penis 0 0 0 0 2 0 4 0 3 0

Pericardium 0 0 1 1 1 1 0 0 0 1

Preput/Clit Gland 1 0 1 0 0 0 2 1 2 0

Rectum/Low Colon 0 1 1 0 0 0 0 1 0 0

Skin (other) 17 19 21 10 14 21 12 17 18 12

Soft Tissue 0 0 0 0 0 0 0 0 1 0

Tail 1 0 1 0 0 0 3 0 1 0

Tongue 0 0 0 0 0 0 0 1 1 1

Ureters 2 4 3 1 1 1 1 0 4 0

Zymbal’s Gland 0 0 0 0 1 0 0 0 0 0


84




B). Protocol Tissue of Which Some Were Not Available For Histological Examination

Adrenal Glands 65 65 64 64 64 65 63 64 64 65

Aorta 65 64 65 65 64 65 65 65 65 65

Cecum 63 64 64 65 65 65 63 65 65 63

Colon 65 65 65 65 65 65 65 65 65 64

Distal Femur 65 64 65 64 65 65 65 65 65 65

Duodenum 65 64 64 64 62 65 61 65 65 64

Esophagus 65 65 65 64 65 65 64 65 65 65

Femoral Marrow 65 65 65 64 65 65 65 65 65 65

Gallbladder 55 57 58 58 60 60 53 60 59 58

Harderian Glands 65 65 65 65 65 65 64 65 65 65

Ileum 64 65 64 64 65 65 64 65 65 64

Jejunum 65 65 65 64 64 65 62 65 64 65

Kidneys 65 64 65 65 65 65 65 65 65 65

Lacrimal Gland 65 63 65 65 65 65 64 65 65 65

Mammary (protocol) 63 65 60 64 63 65 65 65 62 64

Mediastinal Lymph Node 61 65 63 65 62 65 61 63 57 65

Mesenteric Lymph Node 65 64 63 64 64 64 64 62 61 65

Nerve – Sciatic 65 65 65 64 64 65 65 65 65 63

Ovaries - 64 - 65 - 65 - 65 - 64

Peyer’s Patches/GALT 63 64 55 59 62 58 55 61 58 58

Parathyroid 49 41 44 42 51 36 55 48 47 41

Pituitary 65 64 64 65 64 64 62 64 64 65

Prostate 65 - 65 - 63 - 65 - 65 -

Skin (protocol) 65 65 65 65 64 65 65 65 65 65

Spleen 64 65 65 65 64 65 65 65 65 65

Sternal Marrow 65 65 65 65 64 65 65 65 64 65

Sternum 65 65 65 65 64 65 65 65 64 65

Stomach 65 65 65 65 64 65 64 65 64 65


85




Thymus 63 65 60 62 60 65 63 64 63 65

Thyroid 65 65 65 65 64 65 65 65 61 63

Trachea 65 65 65 65 65 65 64 65 65 65

Urinary Bladder 65 63 65 65 64 65 64 65 65 63

Vagina - 64 - 63 - 65 - 64 - 64

Key:- = Not examined(M) = Malignant(B) = Benign(N) = MetastaticNOS = Not otherwise specified(C) = MulticentricGALT = Gut Related Lymphoid TissueP. Patches = Peyer’s PatchesLymph/Retic = LymphoreticularMesentery/Perito = Mesentery/PeritoneumPreput/Clit = Preputial/Clitoral


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

94 94 0 (Water Control) 1041 D Muscle (other) Hemangioscarcoma Y

96 92 0 (Water Control) 1029 D Skin (other) Carcinoma Y

99 89 0 (Vehicle Control) 2048 D Tail [Skin (protocol)] Schwannoma N

103 97 7.5 3008 D Ear(s) [Skin (protocol)] Fibrosarcoma Y

93 90 25 4010 D Skin (other) Metastatic/invasive

neoplasm (metastatic from

Lymph/Retic System)

N

105 83 25 4023 S Tail Keratin cyst N

65 62 500 5062 D Salivary Gland Metastatic/invasive


Lymph/Retic System)

N

105 97 500 5041 S Ear(s) [Skin (protocol)] Schwannoma N


87

Table.4 Carcinogenicity Study in Mice - Chronological Listing of Tumor Occurrence

Females

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


34 33 0 (Water Control) 1522 D Skin (other) Metastatic/invasive


Lymph/Retic System)

N

87 86 0 (Water Control) 1526 D Skin (other) Sebaceous Cell Adenoma N

95 94 0 (Water Control) 1531 D Lymph Node (other) Metastatic/invasive


Lymph/Retic System)

N

100 89 0 (Water Control) 1501 D Skin (other) Sarcoma Y

102

101

0 (Water Control) 1523 D

Skin (other) Metastatic/invasive


Lymph/Retic System)

N

101 Skin (other) Metastatic/invasive


Lymph/Retic System)

N


88

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


103 100 0 (Water Control) 1542 S Tail [Skin (protocol)] Fibrosarcoma Y

94 87 0 (Vehicle Control) 2511 D Salivary Gland Metastatic/invasive


Lymph/Retic System)

N

102 97 0 (Vehicle Control) 2517 S Skin (other) Sarcoma Y

77 73 7.5 3523 D Skin (other) Carcinoma Y

80 68 500 5532 DSkin (other) [Mammary

(protocol)]

Adenocarcinoma Y



Lymph/Retic System)

N

88 87 500 5554 D Skin (other) Hemangiosarcoma Y

Key:D = DeathS = SacrificedLymph/Retic = Lymphoreticular


89

Table.5 Carcinogenicity Study in Mice - Summary of Survival and Fate of Animals

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M

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

1 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

2 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

3 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

4 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

5 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

6 65 - - - - 65 - 1 1 - 65 - - - - 65 - - - - 65 - - - -

7 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

8 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

9 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

10 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

11 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

12 65 - 1 1 - 64 - - - - 65 - - - - 65 1 - 1 - 65 - - - -

13 64 - - - - 64 - - - - 65 - - - - 64 - - - - 65 - - - -

14 64 - - - - 64 - - - - 65 - - - - 64 - - - - 65 - - - -

15 64 - - - - 64 - - - - 65 - - - - 64 - - - - 65 - - - -

16 64 - - - - 64 - - - - 65 - - - - 64 - - - - 65 - - - -

17 64 - - - - 64 - - - - 65 - - - - 64 - - - - 65 - 1 1 -

18 64 - 1 1 - 64 - - - - 65 - - - - 64 - - - - 64 - - - -


90

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



19 63 - - - - 64 - - - - 65 - - - - 64 - - - - 64 - - - -

20 63 - - - - 64 - - - - 65 - - - - 64 - - - - 64 - - - -

21 63 - - - - 64 - - - - 65 1 - 1 - 64 - - - - 64 - - - -

22 63 - - - - 64 - - - - 64 - - - - 64 - - - - 64 - - - -

23 63 - - - - 64 - - - - 64 - - - - 64 - - - - 64 - - - -

24 63 - - - - 64 - - - - 64 - - - - 64 1 - 1 - 64 - - - -

25 63 - - - - 64 - - - - 64 - - - - 63 - - - - 64 - - - -

26 63 - - - - 64 - - - - 64 - - - - 63 - - - - 64 - - - -

27 63 - - - - 64 - - - - 64 - - - - 63 - - - - 64 - - - -

28 63 - - - - 64 - - - - 64 - - - - 63 - - - - 64 - - - -

29 63 - - - - 64 1 - 1 - 64 - - - - 63 - - - - 64 - - - -

30 63 - - - - 63 - - - - 64 - 1 1 - 63 - - - - 64 - - - -

31 63 - - - - 63 - - - - 63 - - - - 63 - 1 1 - 64 - - - -

32 63 - - - - 63 - - - - 63 - - - - 62 - - - - 64 - - - -

33 63 - - - - 63 - - - - 63 - - - - 62 - - - - 64 - - - -

34 63 - - - - 63 - - - - 63 - - - - 62 - - - - 64 2 - 2 -

35 63 - - - - 63 - - - - 63 - - - - 62 - - - - 62 - - - -

36 63 - - - - 63 - - - - 63 - - - - 62 - - - - 62 - - - -

37 63 - - - - 63 - - - - 63 - - - - 62 - - - - 62 - - - -


91

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



38 63 - - - - 63 - - - - 63 - - - - 62 - 1 1 - 62 - - -

39 63 - - - - 63 - - - - 63 - - - - 61 - - - - 62 - - - -

40 63 - - - - 63 - 1 1 - 63 - - - - 61 - - - - 62 - - - -

41 63 - - - - 62 - - - - 63 - - - - 61 - - - - 62 - - - -

42 63 - - - - 62 - 1 1 - 63 - 1 1 - 61 - - - - 62 1 - 1 -

43 63 - - - - 61 - - - - 62 - - - - 61 - - - - 61 - - - -

44 63 - - - - 61 - - - - 62 - - - - 61 - - - - 61 - 1 1 -

45 63 1 - 1 - 61 - - - - 62 - - - - 61 - - - - 60 - - - -

46 62 - - - - 61 - - - - 62 - - - - 61 - - - - 60 - - - -

47 62 - - - - 61 - 1 1 - 62 - - - - 61 - - - - 60 - - - -

48 62 - - - - 60 - - - - 62 - - - - 61 - - - - 60 - - - -

49 62 - - - - 60 - - - - 62 - - - - 61 - - - - 60 - - - -

50 62 - - - - 60 2 - 2 - 62 - - - - 61 - - - - 60 - - - -

51 62 - - - - 58 - - - - 62 - - - - 61 - - - - 60 - - - -

52 62 - 1 1 - 58 - 1 1 - 62 - - - - 61 - - - - 60 - - - -

53 61 - - - - 57 - - - - 62 - - - - 61 - - - - 60 - - - -

54 61 - - - - 57 - 2 2 - 62 - - - - 61 - - - - 60 - 2 2 -

55 61 - - - - 55 - 1 1 - 62 - - - - 61 - - - - 58 - - - -

56 61 - - - - 54 - - - - 62 - - - - 61 - - - - 58 - - - -


92

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



57 61 - - - - 54 - - - - 62 - - - - 61 - - - - 58 - - - -

58 61 - - - - 54 - - - - 62 - - - - 61 - - - - 58 - - - -

59 61 - - - - 54 - 1 1 - 62 - - - - 61 - 1 1 - 58 1 - 1 -

60 61 - - - - 53 - - - - 62 - - - - 60 - - - - 57 - - - -

61 61 - - - - 53 - - - - 62 1 - 1 - 60 - - - - 57 - - - -

62 61 - - - - 53 - - - - 61 - - - - 60 1 - 1 - 57 - 1 1 -

63 61 - 1 1 - 53 - - - - 61 - - - - 59 - - - - 56 - - - -

64 60 - 2 2 - 53 - - - - 61 - 1 1 - 59 - - - - 56 - - - -

65 58 - - - - 53 - 1 1 - 60 - - - - 59 - - - - 56 - 1 1 -

66 58 - - - - 52 - - - - 60 - - - - 59 - - - - 55 - - - -

67 58 1 - 1 - 52 - - - - 60 - - - - 59 1 - 1 - 55 - 1 1 -

68 57 - - - - 52 1 - 1 - 60 - - - - 58 - - - - 54 - 1 1 -

69 57 - - - - 51 - - - - 60 - - - - 58 1 1 2 - 53 - - - -

70 57 - - - - 51 - 1 1 - 60 - - - - 56 - 1 1 - 53 - - - -

71 57 - 1 1 - 50 - - - - 60 - - - - 55 - - - - 53 - - - -

72 56 - - - - 50 - - - - 60 - 1 1 - 55 - - - - 53 - - - -

73 56 - - - - 50 - - - - 59 - - - - 55 1 - 1 - 53 - 1 1 -

74 56 - - - - 50 - - - - 59 1 - 1 - 54 - - - - 52 1 - 1 -

75 56 - - - - 50 - - - - 58 - - - - 54 - - - - 51 - - - -


93

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



76 56 1 - 1 - 50 - - - - 58 - - - - 54 - 1 1 - 51 - 2 2 -

77 55 - - - - 50 1 - 1 - 58 - 1 1 - 53 - - - - 49 - - - -

78 55 - - - - 49 - 1 1 - 57 - - - - 53 - 1 1 - 49 - - - -

79 55 - 1 1 - 48 - - - - 57 - - - - 52 - - - - 49 - 1 1 -

80 54 - - - - 48 1 2 3 - 57 - - - - 52 - 1 1 - 48 - - - -

81 54 - 1 1 - 45 - - - - 57 - 1 1 - 51 1 - 1 - 48 - 1 1 -

82 53 - - - - 45 - 1 1 - 56 - - - - 50 - 1 1 - 47 - 1 1 -

83 53 - - - - 44 - - - - 56 - - - - 49 - 1 1 - 46 - - - -

84 53 1 - 1 - 44 - - - - 56 - - - - 48 1 1 2 - 46 - - - -

85 52 2 - 2 - 44 2 - 2 - 56 - 1 1 - 46 - - - - 46 - 2 2 -

86 50 1 - 1 - 42 - - - - 55 2 - 2 - 46 - - - - 44 - - - -

87 49 1 1 2 - 42 - 2 2 - 53 - - - - 46 - 1 1 - 44 1 - 1 -

88 47 - - - - 40 - - - - 53 - - - - 45 1 - 1 - 43 - - - -

89 47 - 1 1 - 40 - - - - 53 - 1 1 - 44 1 - 1 - 43 - - - -

90 46 2 - 2 - 40 - 1 1 - 52 - 1 1 - 43 - - - - 43 - - - -

91 44 - - - - 39 - - - - 51 1 - 1 - 43 - - - - 43 - 1 1 -

92 44 - - - - 39 - - - - 50 - - - - 43 - 2 2 - 42 - 3 3 -

93 44 - - - - 39 1 - 1 - 50 - 1 1 - 41 1 - 1 - 39 - - - -

94 44 1 1 2 - 38 - 1 1 - 49 - 2 2 - 40 - - - - 39 - - - -


94

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



95 42 1 - 1 - 37 - 1 1 - 47 - 2 2 - 40 - - - - 39 - - - -

96 41 - 2 2 - 36 1 - 1 - 45 - - - - 40 - - - - 39 1 1 2 -

97 39 1 2 3 - 35 - - - - 45 - - - - 40 2 1 3 - 37 1 2 3 -

98 36 1 1 2 - 35 - 3 3 - 45 1 1 2 - 37 - 1 1 - 34 - - - -

99 34 - - - - 32 - 1 1 - 43 1 1 2 - 36 - 1 1 - 34 - - - -

100 34 - - - - 31 - - - - 41 1 - 1 - 35 - 1 1 - 34 - 2 2 -

101 34 - 1 1 - 31 - - - - 40 - - - - 34 1 1 2 - 32 - 1 1 -

102 33 1 1 2 - 31 - - - - 40 - 2 2 - 32 1 - 1 - 31 - - - -

103 31 - 1 1 - 31 - 1 1 - 38 1 - 1 - 31 1 1 2 - 31 - 2 2 -

104 30 - - - - 30 1 - 1 - 37 - - - - 29 3 - 3 - 29 2 1 3 -

Key:

E = Number entering week.

D = Deaths.

K = Killed moribund/accidentally/for humane reasons.

N = Necropsied completely.

NP = Necropsied to some extent.


95

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



1 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

2 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

3 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

4 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

5 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

6 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

7 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

8 65 - - - - 65 - - - - 65 - - - - 65 1 - 1 - 65 - 1 1 -

9 65 - - - - 65 - - - - 65 - - - - 64 - - - - 64 - - - -

10 65 - - - - 65 - - - - 65 - - - - 64 - - - - 64 - - - -

11 65 - - - - 65 - - - - 65 - - - - 64 - - - - 64 - 2 2 -

12 65 - - - - 65 - - - - 65 - - - - 64 - - - - 62 - - - -

13 65 - - - - 65 - - - - 65 - - - - 64 - - - - 62 - - - -

14 65 - - - - 65 - - - - 65 - - - - 64 - - - - 62 - - - -

15 65 - - - - 65 - - - - 65 - 1 1 - 64 - - - - 62 - - - -

16 65 - - - - 65 - 1 1 - 64 - - - - 64 - - - - 62 - 1 1 -

17 65 - - - - 64 - - - - 64 - - - - 64 - - - - 61 - - - -

18 65 - - - - 64 - - - - 64 - - - - 64 - - - - 61 - - - -

19 65 - - - - 64 - - - - 64 - - - - 64 - - - - 61 - - - -


96

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



20 65 - - - - 64 - - - - 64 - - - - 64 - - - - 61 - - - -

21 65 - - - - 64 - - - - 64 - - - - 64 - - - - 61 - - - -

22 65 - - - - 64 - - - - 64 - - - - 64 - 1 1 - 61 - - - -

23 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

24 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

25 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

26 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

27 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

28 65 - - - - 64 - - - - 64 - - - - 63 - - - - 61 - - - -

29 65 - - - - 64 - - - - 64 - - - - 63 - 1 1 - 61 - 1 1 -

30 65 - - - - 64 - 1 1 - 64 1 - 1 - 62 - - - - 60 - - - -

31 65 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

32 65 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

33 65 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

34 65 1 - 1 - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

35 64 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

36 64 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - - - -

37 64 - - - - 63 - - - - 63 - - - - 62 - - - - 60 - 1 1 -

38 64 - - - - 63 - - - - 63 - 2 2 - 62 - - - - 59 - - - -


97

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



39 64 1 - 1 - 63 - - - - 61 - - - - 62 - - - - 59 - - - -

40 63 - - - - 63 1 - 1 - 61 - - - - 62 - - - - 59 - 1 1 -

41 63 - 1 1 - 62 - - - - 61 - - - - 62 - - - - 58 - - - -

42 62 - - - - 62 - 1 1 - 61 - 1 1 - 62 - - - - 58 - - - -

43 62 - - - - 61 - - - - 60 - - - - 62 - - - - 58 - - - -

44 62 - - - - 61 1 - 1 - 60 - - - - 62 - - - - 58 - - - -

45 62 - - - - 60 - 1 1 - 60 - - - - 62 - - - - 58 1 - 1 -

46 62 - - - - 59 - - - - 60 1 - 1 - 62 - - - - 57 - - - -

47 62 1 - 1 - 59 - - - - 59 - - - - 62 - - - - 57 - - - -

48 61 - 1 1 - 59 - - - - 59 - 1 1 - 62 - - - - 57 - 1 1 -

49 60 - - - - 59 - - - - 58 - - - - 62 - - - - 56 1 - 1 -

50 60 - - - - 59 - - - - 58 - 1 1 - 62 - - - - 55 - 1 1 -

51 60 - 1 1 - 59 - - - - 57 - - - - 62 - - - - 54 - - - -

52 59 - - - - 59 - - - - 57 - - - - 62 - - - - 54 - 1 1 -

53 59 - - - - 59 - - - - 57 - - - - 62 - - - - 53 - - - -

54 59 - - - - 59 - - - - 57 - 1 1 - 62 - 1 1 - 53 - 2 2 -

55 59 - - - - 59 - - - - 56 - - - - 61 - - - - 51 - - - -

56 59 - - - - 59 - - - - 56 - 1 1 - 61 - 2 2 - 51 - 1 1 -

57 59 - 1 1 - 59 - - - - 55 - - - - 59 1 - 1 - 50 - - - -


98

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



58 58 - - - - 59 - - - - 55 - - - - 58 - - - - 50 - - - -

59 58 - - - - 59 - - - - 55 - - - - 58 - - - - 50 - 1 1 -

60 58 - - - - 59 - 1 1 - 55 - - - - 58 - - - - 49 - 1 1 -

61 58 - - - - 58 - - - - 55 - - - - 58 - 1 1 - 48 - - - -

62 58 1 1 2 - 58 - - - - 55 - 1 1 - 57 - 1 1 - 48 - - - -

63 56 1 - 1 - 58 - - - - 54 - - - - 56 - 1 1 - 48 - - - -

64 55 - - - - 58 - - - - 54 - 2 2 - 55 - 1 1 - 48 - - - -

65 55 - - - - 58 - - - - 52 - - - - 54 - 1 1 - 48 - - - -

66 55 - - - - 58 - - - - 52 - 1 1 - 53 - 1 1 - 48 - - - -

67 55 1 - 1 - 58 - - - - 51 - - - - 52 - - - - 48 - - - -

68 54 - - - - 58 - 2 2 - 51 - - - - 52 - 1 1 - 48 - 2 2 -

69 54 - 1 1 - 56 - - - - 51 - - - - 51 1 - 1 - 46 - 1 1 -

70 53 - - - - 56 - 1 1 - 51 - - - - 50 - - - - 45 - - - -

71 53 1 - 1 - 55 - - - - 51 - - - - 50 2 2 4 - 45 - - - -

72 52 - - - - 55 - - - - 51 1 - 1 - 46 - 1 1 - 45 - 1 1 -

73 52 - - - - 55 1 - 1 - 50 - - - - 45 - - - - 44 - 1 1 -

74 52 - - - - 54 - - - - 50 - - - - 45 - - - - 43 - 1 1 -

75 52 - 1 1 - 54 1 - 1 - 50 - - - - 45 - 2 2 - 42 1 - 1 -

76 51 1 - 1 - 53 - 1 1 - 50 - 1 1 - 43 - - - - 41 - - - -


99

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



77 50 - 1 1 - 52 - - - - 49 - 1 1 - 43 - 1 1 - 41 - 1 1 -

78 49 - 1 1 - 52 - 1 1 - 48 - 1 1 - 42 - 2 2 - 40 - 1 1 -

79 48 - - - - 51 - - - - 47 1 1 2 - 40 - 1 1 - 39 - 1 1 -

80 48 - 1 1 - 51 - - - - 45 - - - - 39 - - - - 38 - 2 2 -

81 47 - 3 3 - 51 1 - 1 - 45 - - - - 39 - - - - 36 1 - 1 -

82 44 1 1 2 - 50 - 2 2 - 45 - - - - 39 - - - - 35 1 1 2 -

83 42 - - - - 48 - 1 1 - 45 - 2 2 - 39 - 1 1 - 33 - - - -

84 42 - - - - 47 - 1 1 - 43 1 - 1 - 38 - - - - 33 - 1 1 -

85 42 - 1 1 - 46 1 - 1 - 42 - - - - 38 - - - - 32 - 1 1 -

86 41 - 2 2 - 45 1 2 3 - 42 - 1 1 - 38 - - - - 31 - - - -

87 39 3 - 3 - 42 1 - 1 - 41 - 1 1 - 38 - - - - 31 - - - -

88 36 - 1 1 - 41 - - - - 40 - 1 1 - 38 - 1 1 - 31 1 1 2 -

89 35 - 1 1 - 41 - - - - 39 1 - 1 - 37 - 2 2 - 29 - 1 1 -

90 34 1 1 2 - 41 - 1 1 - 38 - - - - 35 - 1 1 - 28 - - - -

91 32 1 - 1 - 40 - - - - 38 - 3 3 - 34 1 - 1 - 28 - - - -

92 31 2 2 4 - 40 - - - - 35 1 - 1 - 33 - - - - 28 2 1 3 -

93 27 - - - - 40 - - - - 34 - 1 1 - 33 - 1 1 - 25 - 1 1 -

94 27 - - - - 40 - 1 1 - 33 - - - - 32 - 1 1 - 24 - - - -

95 27 - 2 2 - 39 - 2 2 - 33 - 1 1 - 31 - 1 1 - 24 - - - -


100

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



96 25 1 - 1 - 37 2 2 4 - 32 - - - - 30 - - - - 24 - - - -

97 24 1 1 2 - 33 - 2 2 - 32 - 1 1 - 30 - 3 3 - 24 - - - -

98 22 - - - - 31 1 1 2 - 31 - 1 1 - 27 1 1 2 - 24 - - - -

99 22 - - - - 29 1 - 1 - 30 - - - - 25 - - - - 24 1 2 3 -

100 22 - 1 1 - 28 1 - 1 - 30 - 1 1 - 25 - - - - 21 - - - -

101 21 - - - - 27 - - - - 29 1 2 3 - 25 - - - - 21 - 2 2 -

102 21 - 1 1 - 27 - - - - 26 1 2 3 - 25 1 1 2 - 19 - - - -

103 20 - - - - 27 - 1 1 - 23 - - - - 23 - - - - 19 - - - -

104 20 - - - - 26 - - - - 23 - - - - 23 - - - - 19 - - - -

Key:


D = Deaths.

K = Killed moribund/accidentally/for humane reasons.




101

Table.6 Carcinogenicity Study in Rats - Tumor Incidence



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Adipose

Tissue

Lipoma (B) 0 - 1 0 0 0 0 0 0 0

Hibernoma (M) 0 - 0 0 0 0 1 0 0 0

Invasive Neoplasm (N) 0 - 0 0 0 0 0 0 2 0

Adrenal

Glands

Medulla

Pheochromocytoma (B) 4 0 5 0 3 1 4 1 4 1

Medulla

Pheochromocytoma (M) 0 0 1 0 0 0 0 0 0 0

Cortex Carcinoma (M) 0 2 0 1 0 0 0 2 0 0

Cortex Adenoma (B) 0 3 1 1 0 0 1 0 1 1


Aorta Invasive Neoplasm (N) 2 0 0 1 0 0 0 0 0 0

Bone

(other)

Histiocytoma, fibrous (M) - 0 - - 1 - 0 - 0 -

Fibrosarcoma (B) - 0 - - 0 - 0 - 1 -

Brain

Astrocytoma (M) 1 0 1 0 1 0 1 0 0 0

Granular Cell Tumor (B) 0 0 1 0 0 0 0 0 1 0

Oligodendroglioma (M) 0 0 1 0 0 0 1 2 0 0

Meningioma (B) 0 0 1 0 0 0 0 0 0 0


CecumFibroma (B) 0 1 0 0 0 0 0 0 0 0


Colon Invasive Neoplasm (N) 1 0 0 0 0 1 0 0 1 0

Diaphragm Invasive Neoplasm (N) - 0 - - - - 1 - - 1

Duodenum Invasive Neoplasm (N) 3 0 0 0 0 0 0 0 1 0

Ears Fibrosarcoma (M) - - 0 - - - - - 1 -

Epididymides Invasive Neoplasm (N) 5 - 1 - 0 - 0 - 2 -

Esophagus Invasive Neoplasm (N) 1 0 0 0 0 0 0 0 1 1


102



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




ExtremityKeratoacanthoma (B) 1 0 0 0 0 0] 0 0 0 0]


Eyes Invasive Neoplasm (N) 4 0 0 0 0 1 0 0 0 0

Femoral


Harderian


Heart Invasive Neoplasm (N) 2 0 0 0 0 0 1 0 0 1

IleumAdenocarcinoma (M) 0 0 0 1 0 0 0 0 0 0


Jejunum

Leiomyoma (B) 0 0 0 0 0 0 0 1 0 0

Neuroendocrine

Neoplasm (M) 0 0 0 0 0 0 1 0 0 0

Leiomyosarcoma (M) 0 0 0 1 0 0 0 0 0 0


Kidneys

Carcinoma, Tubular (M) 0 0 0 0 0 0 1 0 0 0

Carcinoma,

Transitional Cells (M) 0 0 0 0 0 0 0 0 1 0

Liposarcoma (M) 0 0 0 0 0 0 0 0 1 0


Lacrimal


Liver

Hepatocellular

Adenoma (B) 3 1 2 2 2 3 2 1 1 3

Hepatocellular

Carcinoma (M) 0 1 1 1 1 0 0 0 0 0




103



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Lungs

Carcinoma Nos (M) 0 0 0 1 0 0 0 0 0 0

Bronchiolo/Alveolar

Carcinoma (M) 0 0 0 0 0 0 0 0 0 1



Lymph

Node

(other) Invasive Neoplasm (N) 5 1 1 1 1 1 1 0 1 0

Lymph/

Retic

System

Histiocytic Sarcoma (M) 4 1 1 0 2 0 0 0 1 0

Malignant

Lymphoma (M) 3 2 0 1 0 1 1 0 0 1

Granulocytic

Leukemia (M) 1 0 0 0 0 0 0 0 0 0

Mammary

(protocol)

Adenocarcinoma (M) 0 19 0 11 0 14 0 22 0 17

Fibroadenoma (B) 0 20 1 28 0 17 0 29 0 15

Adenoma (B) 0 1 0 1 0 4 0 3 0 2


Mediastinal

Lymph


Meninges

Malignant

Meningioma (M) - - 1 - - - - - - -

Mesenteric

Lymph


Mesentery/

Perito

Liposarcoma (M) - 0 - 0 - 1 0 0 0 -

Malignant

Mesothelioma (M) - 0 - 0 - 0 0 0 1 -

Lipoma (B) - 1 - 0 - 0 0 0 0 -


104



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Muscle

(Other)

Carcinoma (Nos) (M) 0 - 0 - - 1 0 - 0 -

Invasive Neoplasm (N) 1 - 0 - - 1[ 0 - 0 -

Muscle

(Protocol) Invasive Neoplasm (N) 3 0 1 1 1 0 0 0 1 0

Nerve

Sciatic Invasive Neoplasm (N) 4 0 0 1 0 1 0 0 0 0

Ovaries Invasive Neoplasm (N) - 0 - 1 - 0 - 0 - 0

P. Patches/

GALT Invasive Neoplasm (N) 1 0 0 1 0 1 0 0 0 0

Pancreas

Islet Cell Adenoma (B) 2 2 3 1 1 1 7 1 4 0

Islet Cell Carcinoma (M) 2 1 3 2 1 0 3 2 5 2

Hemangioma (B) 0 0 0 0 1 0 0 0 0 0

Acinar Cell Adenoma (B) 1 0 0 0 0 0 1 0 1 0


Parathyroid Adenoma (B) 0 0 1 0 0 0 0 0 0 0

Pericardium Invasive Neoplasm (N) 0 0 0 0 - - 1 - - 1

Pituitary

Pars Distalis

Adenoma (B) 28 58 22 51 29 59 25 55 29 55

Pars Distalis

Carcinoma (M) 1 1 0 1 0 2 0 3 1 1

Pars Intermedia

Adenoma (B) 0 0 0 0 0 0 0 0 1 0


Prostate Invasive Neoplasm (N) 4 - 1 - 0 - 0 - 1 -

Rectum/

Low Colon Fibroma (B) - 1 - - - - - - - -

Salivary

Gland

Leiomyoma (B) 0 0 0 0 0 0 1 0 0 0



105



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Seminal

Vesicles Invasive Neoplasm (N) 1 - 0 - 0 - 0 - 1 -

Skin

(other)

Malignant Basal Cell

Tumor (M) 1 0 0 0 0 0 0 0 0 0

Lipoma (B) 1 0 1 0 1 0 0 0 2 0

Keratoacanthoma (B) 2 1 4 1 3 0 4 0 3 0

Benign Basal Cell

Tumor (B) 1 0 1 0 0 0 2 1 2 0

Fibrosarcoma (M) 2 0 3 0 1 0 2 0 0 0

Fibroma (B) 0 1 1 0 2 1 1 0 0 0

Histiocytoma Fibrous (M) 0 0 1 0 1 0 0 0 1 0

Squamous Cell

Papilloma (B) 1 0 0 2 0 1 3 0 1 0

Squamous Cell

Carcinoma (M) 0 0 1 0 1 0 0 0 0 0

Myxosarcoma (M) 1 0 0 0 1 0 0 0 0 0

Malignant

Schwannoma (M) 0 0 0 0 0 0 1 0 0 0


Skin

(protocol)


Histiocytoma Fibrous (M) 0 0 0 0 0 0 0 0 0 1

Soft Tissue Invasive Neoplasm (N) 1 0 - - - 1 - - - -

SpleenHemangioma (B) 0 0 0 0 1 0 0 0 0 0


Sternal


Stomach

Carcinoma

Squamous Cell (M) 0 0 1 0 0 0 0 0 0 0



106



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Tail


Basal Cell Tumor (B) 1 0 0 0 0 0 0 0 0 0


Testes

Benign Interstitial

Cell Tumor (B) 0 - 1 - 0 - 2 - 1 -

Invasive Neoplasm (N) 2 - 0 - 0 - 0 - 0 -

ThymusMalignant Thymoma (M) 0 0 0 0 0 0 1 1 0 0


Thyroid

Carcinoma Follicular

Cell (M) 2 1 0 0 1 1 0 1 0 0

Follicular Cell

Adenoma (B) 2 1 0 0 4 0 1 0 2 0

C Cell Adenoma (B) 4 1 4 1 2 4 5 3 1 1

C Cell Carcinoma (M) 2 0 0 2 1 0 1 1 2 1


Trachea Invasive Neoplasm (N) 2 0 0 0 0 0 0 0 0 0

Urinary

Bladder Invasive Neoplasm (N) 0 0 0 0 0 0 0 0 1 0

Uterus w/

Cervix

Endometrial Stromal

Polyp (B) - 2 - 3 - 1 - 2 - 2

Benign Schwannoma (B) - 0 - 0 - 0 - 0 - 1

Leiomyoma (B) - 0 - 1 - 0 - 0 - 0


VaginaGranular Cell Tumor (M) - 0 - 0 - 0 - 1 - 0


Vascular

Tissue

Hemangiosarcoma (C) - - - - 0 - - - - 1

Hemangioma (C) - - - - 2 - - - - 0


107



(1)Water

Control

(2)Vehicle

Control

(3)Low

2 mg/kg/day

(4)Inter.

10 mg/kg/day

(5)High

500 mg/kg/day




Zymbal’s

Gland

Adenoma (B) 0 - - - 1 - - - - -

Carcinoma (M) 1 - - - 0 - - - - -


108




A). Macroscopically AbnormalAdipose tissue 3 0 2 1 1 3 1 3 3 2

Bone (other) 0 2 0 0 1 0 2 0 1 0

Cavity 0 0 0 0 0 0 0 0 1 0

Coagulating Gland 0 - 0 - 0 - 1 - 0 -

Diaphragm 0 1 0 0 0 0 1 0 0 1

Ear(s) 0 0 1 0 0 0 0 0 1 0

Extremity 34 14 40 12 26 11 27 7 16 6

Lymph Node (other) 20 10 20 8 13 8 16 5 15 5

Meninges 0 0 1 0 0 0 0 0 0 0

Mesentery/Perito 0 1 0 1 0 1 1 1 2 0

Muscle (other) 1 0 1 0 0 2 2 0 1 0

Penis 0 - 0 - 0 - 0 - 1 -

Pericardium 1 2 1 1 0 0 1 0 0 1

Preput/Clit Gland 1 0 0 1 0 0 1 0 1 0

Rectum/Low Colon 0 1 0 0 0 0 0 0 0 0

Skin (other) 18 22 20 22 18 24 20 17 17 25

Soft Tissue 1 1 0 0 0 1 0 0 0 0

Tail 17 1 16 5 18 7 17 6 12 10

Ureters 1 0 0 1 1 0 1 0 1 1

Urethra 0 0 0 1 0 0 0 0 0 0

Vascular Tissue 0 0 0 0 2 0 0 0 0 1

Vessel 0 0 0 0 1 0 0 1 0 0

Zymbal’s Gland 1 0 0 0 1 0 0 0 0 0


109




B). Protocol Tissue of Which Some Were Not Available For Histological ExaminationHeart 65 64 65 65 65 64 65 65 65 65

Mediastinal Lymph Node 65 61 65 65 64 65 64 63 64 63

Mesenteric Lymph Node 65 64 65 65 65 65 65 65 65 65

Mammary (protocol) 56 65 59 65 54 65 54 65 52 65

Nerve Sciatic 65 64 65 65 65 65 65 65 64 65

Parathyroid 61 62 64 63 61 59 63 64 63 65

Salivary Gland 65 65 64 65 65 65 65 65 65 65

Thymus 62 64 64 65 65 63 64 62 65 65

Urinary Bladder 65 64 65 65 65 64 65 65 65 64

Key:- = Not examined(M) = Malignant(B) = Benign(N) = Metastatic(C) = MulticentricGALT = Gut Related Lymphoid TissueP. Patches = Peyer’s PatchesLymph/Retic = LymphoreticularMesentery/Perito = Mesentery/PeritoneumPreput/Clit = Preputial/Clitoral


110

Table.7 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence

Males

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


58 40 0 (Water Control) 1034 D Skin (other) Fibrosarcoma Y

59 39 0 (Water Control) 1062 D Zymbal’s Gland Carcinoma Y

79 57 0 (Water Control) 1031 D Skin (other) Basal cell tumor Y

83 73 0 (Water Control) 1020 D Skin (other) Cyst, keratinaceous N

87 83 0 (Water Control) 1004 D Skin (other) Metastatic invasive

neoplasm from Lymph/Retic

System

N

93 87 0 (Water Control) 1007 D Skin (other) Keratoacanthoma N

100 83 0 (Water Control) 1036 D Skin (other) Fibrosarcoma Y

10464

0 (Water Control) 1037 S Skin (other)Squamous cell papilloma N

92 Keratoacanthoma N


111

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


10494

0 (Water Control) 1035 S Skin (other)Myxosarcoma Y

104 Basal cell tumor N

104 101 0 (Water Control) 1047 S Tail Basal cell tumor N

78 64 0 (Vehicle Control) 2018 D Skin (other) Fibrosarcoma Y

91 55 0 (Vehicle Control) 2020 D Skin (other) Squamous cell carcinoma Y

91 88 0 (Vehicle Control) 2051 D Skin (other) Basal cell tumor N

92 89 0 (Vehicle Control) 2037 D Skin (other) Histiocytoma, fibrous Y

96 87 0 (Vehicle Control) 2043 D Skin (other) Keratoacanthoma N


99 71 0 (Vehicle Control) 2057 D Mammary (protocol) Fibroadenoma N

99 97 0 (Vehicle Control) 2028 D Skin (other) Fibrosarcoma Y


101 93 0 (Vehicle Control) 2014 D Skin (other) Fibroma N

104 76 0 (Vehicle Control) 2013 S Skin (other) Lipoma N

104 97 0 (Vehicle Control) 2009 S Skin (other) Fibrosarcoma Y


112

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


104 97 0 (Vehicle Control) 2048 S Lymph/Retic System Histiocytic sarcoma Y

76 69 2 3036 D Skin (other) Histiocytoma, fibrous Y

79 76 2 3010 D Skin (other) Keratoacanthoma N

9076

2 3017 DSkin (other) Fibrosarcoma Y

83 Skin (other) Myxosarcoma Y

9152

2 3005 DSkin (other) Keratoacanthoma N

71 Skin (other) Keratoacanthoma N


97 93 2 3026 D Muscle (protocol) Metastatic invasive


System

N

101 97 2 3035 D Skin (other) Fibroma N

103 61 2 3033 D Skin (other) Squamous cell carcinoma Y

104 95 2 3045 S Skin (other) Lipoma N


113

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


8171

10 4029 DSkin (other) Keratoacanthoma N

73 Skin (other) Fibrosarcoma Y


92 56 10 4045 D Skin (other) Squamous cell papilloma N


104 77 10 4028 S Skin (other) Fibroma N

104 87 10 4006 S Skin (other) Squamous cell papilloma N

104 91 10 4009 S Skin (other) Fibrosarcoma Y

104 99 10 4030 S Skin (other) Basal cell tumor N

105 94 10 4061 S Skin (other) Squamous cell papilloma N


80 60 50 5022 D Skin (other) Lipoma N




System

N


114

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


89 83 50 5062 D Skin (other) Histiocytoma, fibrous Y

104 67 50 5044 S Skin (other) Lipoma N

10493

50 5043 SSkin (other) Cyst, keratinaceous N

97 Skin (other) Basal cell tumor N

Key:D = DeathS = Sacrificed

Lymph/Retic = Lymphoreticular


115

Table.8 Carcinogenicity Study in Rats - Chronological Listing of Tumor Occurrence

Females

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


58

34


Mammary (protocol) Adenocarcinoma Y

34 Mammary (protocol) Adenoma N


42 Mammary (protocol) Fibroadenoma N




58 49 0 (Water Control) 1560 D Mammary (protocol) Adenocarcinoma Y


59 47 0 (Water Control) 1506 D Mammary (protocol) Fibroadenoma N

66 36 0 (Water Control) 1525 D Skin (other)

Metastatic/invasive


Lymph/Retic System)

N


116

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


7151

0 (Water Control) 1559 DMammary (protocol) Fibroadenoma N

67 Mammary (protocol) Adenocarcinoma Y

72 68 0 (Water Control) 1535 D Skin (other) Fibroma N


75 61 0 (Water Control) 1517 D Mammary (protocol)Hyperplasia, mammary,

cytisticN


7756

0 (Water Control) 1554 DMammary (protocol) Adenocarcinoma Y




80

73


Mammary (protocol) Fibroadenoma N

77 Mammary (protocol) Hyperplasia, mammary N





117

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


9055

0 (Water Control) 1512 DMammary (protocol) Fibroadenoma N


9077

0 (Water Control) 1547 DMammary (protocol) Adenocarcinoma Y


92

80





95 47 0 (Water Control) 1513 S Mammary (protocol) Fibroadenoma N

95

48

0 (Water Control) 1537 S



77 Mammary (protocol) Hyperplasia, Mammary,

cystic

N



118

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


95

51






9565

0 (Water Control) 1501 SMammary (protocol) Fibroadenoma N




95

80


Mammary (protocol) Hyperplasia, mammary,

cystic

N



95 81 0 (Water Control) 1565 S Skin (other) Keratoacanthoma N



119

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


9585

0 (Water Control) 1529 SMammary (protocol) Adenocarcinoma Y


9587

0 (Water Control) 1540 SMammary (protocol) Fibroadenoma N


65

32

0 (Vehicle Control) 2539 D





68

38




65 Skin (other) Keratoacanthoma N

68 54 0 (Vehicle Control) 2541 D Mammary (protocol)Hyperplasia, mammary,

cystic

N

75 64 0 (Vehicle Control) 2548 D Mammary (protocol) Adenocarcinoma Y

7565

0 (Vehicle Control) 2559 DSkin (other) Keratoacanthoma N



120

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


75 68 0 (Vehicle Control) 2521 D Mammary (protocol) Adenocarcinoma Y


77

46








81 55 0 (Vehicle Control) 2527 D Mammary (protocol) Hyperplasia, mammary N


86

81



81 Mammary (protocol) Hyperplasia, mammary,

cysticN


9354

0 (Vehicle Control) 2537 DMammary (protocol) Fibroadenoma N



121

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


95 65 0 (Vehicle Control) 2523 S Mammary (protocol) Adenocarcinoma Y

95

69

0 (Vehicle Control) 2562 S



95

71




95

71



89 Skin (other) Squamous cell papilloma N

95

73





122

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


95

75





95

79



88


Mammary (protocol) Adenoma N


92


Mammary (protocol) Adenoma N

9582

0 (Vehicle Control) 2513 SMammary (protocol) Fibroadenoma N


95 87 0 (Vehicle Control) 2558 S Mammary (protocol) Fibroadenoma N


123

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death



95 91 0 (Vehicle Control) 2505 S Skin (other) Squamous cell papilloma N


52 47 2 3552 D Mammary (protocol) Fibroadenoma N



68 51 2 3532 D Mammary (protocol) Adenoma N

73 63 2 3534 D Mammary (protocol) Adenocarcinoma Y









124

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death





9073

2 3501 DMammary (protocol) Fibroadenoma N






125

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


9374



95

50

2 3543 D




95 55 2 3563 S Tail Keratoacanthoma N

95

69

2 3505 S







95 79 2 3526 S Mammary (protocol) Adenoma N


126

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


95 85 2 3557 S Mammary (protocol) Fibroadenoma N

50 34 10 4517 D Thymus Malignant thymoma Y


55

41

10 4502 D


41 Mammary (protocol)Hyperplasia, mammary,

cysticN

54 Mammary (protocol)Hyperplasia, mammary,

cysticN

5734

10 4536 DMammary (protocol) Adenocarcinoma Y









127

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death





8360

10 4535 DMammary (protocol)

Hyperplasia mammay,

cysticN


83

63

10 4547 D






8654




8677




128

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


88

68

10 4556 D




8880




9047



90

60

10 4521 D


74 Mammary (protocol)Fibroadenoma

Adenoma

N

N





129

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


94

79

10 4537 D


93 Mammary (protocol)Hyperplasia mammay,

cysticN

9563

10 4555 SMammary (protocol) Fibroadenoma N


95 70 10 4559 S Mammary (protocol) Adenocarcinoma Y


95

71

10 4523 S






130

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


95

71

10 4533 S

Mammary (protocol)Fibroadenoma

Adenoma

N

N



9585



9590

10 4501 SMammary (protocol) Adenocarcinoma Y





95 93 10 4549 S Skin (other) Benign basal cell tumor N




131

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


4527




55

41

50 5555 D

Mammary (protocol)Hyperplasia, mammary,

cysticN

44Mammary (protocol)

Hyperplasia, mammary,

cysticN



8237



8248






132

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


8562





95

62

50 5510 S





95

68

50 5547 S








133

Week of Necropsy

Week Tumor

Discovered


Animal Number

Mode of Death


9589






134

Table.9 Carcinogenicity Study in Rats - Summary of Survival and Fate of Animals

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



1 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

2 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

3 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

4 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

5 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

6 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

7 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

8 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

9 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

10 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

11 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

12 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

13 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

14 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

15 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

16 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

17 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

18 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -


135

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



19 65 - - - - 65 1 - 1 - 65 - - - - 65 - - - - 65 - - - -

20 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

21 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

22 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

23 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

24 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

25 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

26 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

27 65 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

28 65 1 - 1 - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

29 64 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

30 64 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

31 64 - - - - 64 - - - - 65 - - - - 65 - - - - 65 - - - -

32 64 - - - - 64 - 1 1 - 65 - - - - 65 - - - - 65 - - - -

33 64 - - - - 63 - - - - 65 - - - - 65 - - - - 65 - - - -

34 64 - - - - 63 - - - - 65 - - - - 65 - - - - 65 - - - -

35 64 - - - - 63 - - - - 65 - - - - 65 - - - - 65 - - - -

36 64 - - - - 63 1 - 1 - 65 - - - - 65 - - - - 65 - - - -

37 64 - - - - 62 - - - - 65 - - - - 65 1 - 1 - 65 - - - -


136

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



38 64 - - - - 62 - - - - 65 - - - - 64 - - - - 65 - - - -

39 64 - - - - 62 - - - - 65 - - - - 64 - - - - 65 - 1 1 -

40 64 - - - - 62 - - - - 65 - - - - 64 - - - - 64 - - - -

41 64 - - - - 62 - - - - 65 - - - - 64 - - - - 64 1 - 1 -

42 64 1 - 1 - 62 - - - - 65 - - - - 64 - - - - 63 - - - -

43 63 - - - - 62 - - - - 65 - - - - 64 - - - - 63 - - - -

44 63 - - - - 62 - - - - 65 - - - - 64 - - - - 63 - - - -

45 63 - 1 1 - 62 - - - - 65 - - - - 64 - - - - 63 - - - -

46 62 - - - - 62 - - - - 65 - - - - 64 - - - - 63 - - - -

47 62 - - - - 62 - - - - 65 - - - - 64 1 - 1 - 63 - - - -

48 62 - - - - 62 - - - - 65 - - - - 63 1 - 1 - 63 - - - -

49 62 - - - - 62 - 1 1 - 65 - - - - 62 - - - - 63 - - - -

50 62 - - - - 61 - - - - 65 - - - - 62 - 1 1 - 63 - - - -

51 62 - - - - 61 - - - - 65 - - - - 61 - - - - 63 - - - -

52 62 - - - - 61 - - - - 65 1 1 2 - 61 - - - - 63 - - - -

53 62 - - - - 61 - - - - 63 - 1 1 - 61 - 1 1 - 63 - - - -

54 62 - - - - 61 - - - - 62 - - - - 60 - - - - 63 - - - -

55 62 - - - - 61 - - - - 62 1 - 1 - 60 - - - - 63 - - - -

56 62 - - - - 61 - - - - 61 - - - - 60 1 - 1 - 63 1 1 2 -


137

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



57 62 - - - - 61 - - - - 61 - - - - 59 - - - - 61 - - - -

58 62 - 1 1 - 61 - - - - 61 1 - 1 - 59 - - - - 61 2 1 3 -

59 61 1 1 2 - 61 - - - - 60 1 - 1 - 59 - - - - 58 - - - -

60 59 - - - - 61 - - - - 59 - - - - 59 - - - - 58 - - - -

61 59 - - - - 61 - - - - 59 - 1 1 - 59 - - - - 58 1 - 1 -

62 59 - - - - 61 - - - - 58 - - - - 59 - - - - 57 - 1 1 -

63 59 1 - 1 - 61 - - - - 58 - - - - 59 - - - - 56 - - - -

64 58 - - - - 61 - - - - 58 - - - - 59 - - - - 56 - 1 1 -

65 58 - - - - 61 1 - 1 - 58 - - - - 59 - - - - 55 - - - -

66 58 - - - - 60 - - - - 58 - - - - 59 - - - - 55 1 - 1 -

67 58 - - - - 60 - - - - 58 - - - - 59 - - - - 54 - 1 1 -

68 58 - - - - 60 - - - - 58 1 - 1 - 59 1 - 1 - 53 - - - -

69 58 - - - - 60 1 - 1 - 57 - - - - 58 - - - - 53 - - - -

70 58 - 1 1 - 59 - - - - 57 - - - - 58 - - - - 53 - 1 1 -

71 57 - - - - 59 - - - - 57 - - - - 58 1 - 1 - 52 - - - -

72 57 - - - - 59 1 - 1 - 57 - 1 1 - 57 1 3 4 - 52 - 1 1 -

73 57 - 1 1 - 58 - - - - 56 - - - - 53 - - - - 51 - 2 2 -

74 56 - - - - 58 1 1 2 - 56 - - - - 53 - - - - 49 1 - 1 -

75 56 1 - 1 - 56 - - - - 56 - 1 1 - 53 - 1 1 - 48 - - - -


138

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



76 55 - - - - 56 - 1 1 - 55 - 1 1 - 52 - - - - 48 - 1 1 -

77 55 1 - 1 - 55 - - - - 54 - - - - 52 - 1 1 - 47 - - - -

78 54 - - - - 55 - 1 1 - 54 - - - - 51 - - - - 47 - 1 1 -

79 54 - 1 1 - 54 1 - 1 - 54 2 1 3 - 51 1 1 2 - 46 2 - 2 -

80 53 1 - 1 - 53 - - - - 51 1 1 2 - 49 - 1 1 - 44 1 2 3 -

81 52 - 2 2 - 53 2 - 2 - 49 1 - 1 - 48 1 - 1 - 41 - - - -

82 50 1 2 3 - 51 - 1 1 - 48 1 1 2 - 47 - 1 1 - 41 - - - -

83 47 1 - 1 - 50 - 2 2 - 46 - 1 1 - 46 - - - - 41 - - - -

84 46 - - - - 48 1 - 1 - 45 1 - 1 - 46 2 1 3 - 41 1 1 2 -

85 46 2 - 2 - 47 - 1 1 - 44 - - - - 43 - - - - 39 - - - -

86 44 - 1 1 - 46 - - - - 44 - - - - 43 - 1 1 - 39 - - - -

87 43 1 - 1 - 46 - - - - 44 - - - - 42 - - - - 39 - 1 1 -

88 42 - - - - 46 - - - - 44 2 - 2 - 42 - - - - 38 2 1 3 -

89 42 1 1 2 - 46 - - - - 42 1 1 2 - 42 - - - - 35 1 1 2 -

90 40 - - - - 46 - 1 1 - 40 1 1 2 - 42 1 - 1 - 33 - - - -

91 40 1 3 4 - 45 - 4 4 - 38 1 1 2 - 41 - 2 2 - 33 - 2 2 -

92 36 - - - - 41 - 2 2 - 36 - - - - 39 2 1 3 - 31 - - - -

93 36 1 - 1 - 39 - - - - 36 1 - 1 - 36 - 1 1 - 31 1 - 1 -

94 35 1 - 1 - 39 - 1 1 - 35 - 2 2 - 35 - 2 2 - 30 1 - 1 -


139

Week

of

Study

Group and Sex

1M 2M 3M 4M 5M



95 34 3 - 3 - 38 - - - - 33 1 - 1 - 33 - - - - 29 1 - 1 -

96 31 1 1 2 - 38 - 3 3 - 32 1 2 3 - 33 - 1 1 - 28 - - - -

97 29 - 1 1 - 35 - - - - 29 - 2 2 - 32 - 1 1 - 28 1 1 2 -

98 28 - - - - 35 1 1 2 - 27 1 - 1 - 31 - - - - 26 - - - -

99 28 - - - - 33 2 - 2 - 26 - - - - 31 1 1 2 - 26 1 1 2 -

100 28 1 1 2 - 31 - 3 3 - 26 - - - - 29 2 1 3 - 24 1 1 2 -

101 26 - - - - 28 4 1 5 - 26 1 1 2 - 26 - - - - 22 1 - 1 -

102 26 - - - - 23 - - - - 24 - - - - 26 1 - 1 - 21 - - - -

103 26 1 - 1 - 23 1 - 1 - 24 1 1 2 - 25 - - - - 21 - 1 1 -

104 25 - - - - 22 - - - - 22 - - - - 25 - - - - 20 1 2 3 -

Key:


D = Deaths.

K = Killed moribund/for humane reasons.




140

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



1 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

2 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

3 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

4 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

5 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

6 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

7 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

8 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

9 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

10 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

11 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

12 65 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

13 65 - 1 1 - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

14 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

15 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

16 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

17 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

18 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -


141

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



19 64 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

20 64 - 1 1 - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

21 63 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

22 63 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

23 63 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

24 63 - 1 1 - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

25 62 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

26 62 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

27 62 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

28 62 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - - - -

29 62 - - - - 65 - - - - 65 - - - - 65 - - - - 65 - 1 1 -

30 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

31 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

32 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

33 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

34 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

35 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - - - -

36 62 - - - - 65 - - - - 65 - - - - 65 - - - - 64 - 1 1 -

37 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - - - -


142

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



38 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - - - -

39 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - - - -

40 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - - - -

41 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - - - -

42 62 - - - - 65 - - - - 65 - - - - 65 - - - - 63 - 1 1 -

43 62 - - - - 65 - - - - 65 - - - - 65 - - - - 62 - 1 1 -

44 62 - - - - 65 - - - - 65 - - - - 65 - - - - 61 - 1 1 -

45 62 - - - - 65 - - - - 65 - - - - 65 - - - - 60 - 1 1 -

46 62 - - - - 65 - 1 1 - 65 - 1 1 - 65 - - - - 59 - - - -

47 62 - - - - 64 - - - - 64 - - - - 65 - - - - 59 - - - -

48 62 - - - - 64 - - - - 64 - 1 1 - 65 - - - - 59 - - - -

49 62 - - - - 64 - - - - 63 - - - - 65 - - - - 59 - - - -

50 62 - - - - 64 - 1 1 - 63 1 1 2 - 65 - 1 1 - 59 - 1 1 -

51 62 - - - - 63 - - - - 61 - - - - 64 1 - 1 - 58 - - - -

52 62 - - - - 63 - - - - 61 - 1 1 - 63 - 1 1 - 58 - - - -

53 62 - - - - 63 - - - - 60 - 1 1 - 62 - - - - 58 - - - -

54 62 - 1 1 - 63 - - - - 59 - - - - 62 - - - - 58 - 1 1 -

55 61 2 - 2 - 63 - 1 1 - 59 - - - - 62 1 2 3 - 57 - 2 2 -

56 59 - - - - 62 - - - - 59 1 - 1 - 59 - - - - 55 - - - -


143

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



57 59 - - - - 62 - 1 1 - 58 - - - - 59 - 2 2 - 55 - - - -

58 59 1 1 2 - 61 - - - - 58 - 1 1 - 57 1 1 2 - 55 - 1 1 -

59 57 - 3 3 - 61 1 1 2 - 57 - - - - 55 - 1 1 - 54 1 1 2 -

60 54 - - - - 59 - - - - 57 - - - - 54 - 1 1 - 52 1 - 1 -

61 54 - - - - 59 - - - - 57 - 1 1 - 53 1 - 1 - 51 - 1 1 -

62 54 - 1 1 - 59 - 1 1 - 56 - 1 1 - 52 - 1 1 - 50 - - - -

63 53 - - - - 58 - - - - 55 - 1 1 - 51 - - - - 50 - - - -

64 53 - 1 1 - 58 - 1 1 - 54 - 1 1 - 51 - - - - 50 - - - -

65 52 - - - - 57 - 2 2 - 53 - - - - 51 - - - - 50 - - - -

66 52 1 - 1 - 55 - - - - 53 - 2 2 - 51 - 1 1 - 50 - - - -

67 51 - 1 1 - 55 - - - - 51 1 1 2 - 50 - - - - 50 1 - 1 -

68 50 - 2 2 - 55 - 2 2 - 49 1 1 2 - 50 - 1 1 - 49 - 2 2 -

69 48 1 - 1 - 53 - - - - 47 - - - - 49 - 1 1 - 47 - 2 2 -

70 47 - - - - 53 - 1 1 - 47 - - - - 48 - 1 1 - 45 - - - -

71 47 - 2 2 - 52 - - - - 47 - - - - 47 - - - - 45 - - - -

72 45 1 - 1 - 52 - 1 1 - 47 - 2 2 - 47 - 1 1 - 45 - 3 3 -

73 44 - 1 1 - 51 - 1 1 - 45 - 1 1 - 46 - - - - 42 - 1 1 -

74 43 - - - - 50 - - - - 44 1 - 1 - 46 - 1 1 - 41 1 - 1 -

75 43 - 3 3 - 50 - 5 5 - 43 1 2 3 - 45 1 - 1 - 40 - 2 2 -


144

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



76 40 1 - 1 - 45 - - - - 40 - 1 1 - 44 - - - - 38 - - - -

77 39 - 2 2 - 45 1 2 3 - 39 2 1 3 - 44 - - - - 38 1 - 1 -

78 37 - 1 1 - 42 1 2 3 - 36 - 1 1 - 44 1 - 1 - 37 - 4 4 -

79 36 - 2 2 - 39 - 1 1 - 35 - - - - 43 - 1 1 - 33 - 1 1 -

80 34 1 - 1 - 38 - - - - 35 - - - - 42 - - - - 32 - 3 3 -

81 33 - 1 1 - 38 - 1 1 - 35 - 4 4 - 42 - - - - 29 - - - -

82 32 1 - 1 - 37 - 1 1 - 31 - - - - 42 - 3 3 - 29 1 3 4 -

83 31 - - - - 36 1 - 1 - 31 - - - - 39 1 2 3 - 25 - 2 2 -

84 31 - - - - 35 - - - - 31 - 2 2 - 36 - 1 1 - 23 - - - -

85 31 - 2 2 - 35 - - - - 29 - 2 2 - 35 - 1 1 - 23 - 1 1 -

86 29 - - - - 35 1 2 3 - 27 - - - - 34 - 3 3 - 22 - - - -

87 29 - - - - 32 - - - - 27 - 1 1 - 31 1 - 1 - 22 - - - -

88 29 - - - - 32 - 2 2 - 26 - 1 1 - 30 2 3 5 - 22 1 1 2 -

89 29 - - - - 30 - 1 1 - 25 - 2 2 - 25 - - - - 20 - - - -

90 29 1 1 2 - 29 - 1 1 - 23 - 4 4 - 25 1 1 2 - 20 1 1 2 -

91 27 - - - - 28 - - - - 19 - 1 1 - 23 - 2 2 - 18 - - - -

92 27 1 - 1 - 28 - - - - 18 - 1 1 - 21 - - - - 18 - 1 1 -

93 26 - 3 3 - 28 - 1 1 - 17 1 1 2 - 21 - - - - 17 - - - -

94 23 2 - 2 - 27 - - - - 15 - - - - 21 1 - 1 - 17 - - - -


145

Week

of

Study

Group and Sex

1F 2F 3F 4F 5F



95 21 - 1 1 - 27 - - - - 15 - - - - 20 - - - - 17 - - - -

Key:


D = Deaths.

K = Killed moribund/for humane reasons.




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.)

Title

2010N109154_00 (1127/1877)

gsk021*: LOCAL LYMPH NODE ASSAY IN THE MOUSE

2010N109158_00 (1127/1892)

gsk022* RABBIT ENUCLEATED EYE TEST

2010N109159_00 (1127/1870)


2010N109160_00 (1127/1878)

gsk023*: ACUTE ORAL TOXICITY IN THE RAT - FIXED DOSE METHOD

2010N109161_00 (1127/1881)


2010N109162_00 (1127/1893)

gsk023*: RABBIT ENUCLEATED EYE TEST

2010N109274_00 (1127/1861)


2010N109275_00 (1127/1864)


2010N109276_00 (1127/1890)


2010N109277_00 (1127/1867)


2010N109278_00 (1127/1891)


2010N104267_00

(1127/1860)

gsk024*: determination of eye irritation potential using an in vitro test strategy

2010N104313_00

(1127/1874)

gsk027*: Determination of eye irritation potential using an in vitro test strategy

2010N104314_00

(1127/1872)

gsk027*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model

2010N104299_00

(1127/1865)


2010N104269_00

(1127/1879)


2010N104268_00

(1127/1880)




147

Report No.

(Study No.)

Title

2010N104318_00

(1127/1876)

gsk021*: Determination of eye irritation potential using anin vitro test strategy

2010N104305_00

(1127/1869)


2010N104295_00

(1127/1863)


2010N104317_00

(1127/1875)


2010N104306_00

(1127/1868)


2010N104296_00

(1127/1862)


2010N104300_00 gsk026*: Determination of skin irritation potential using the skinethic reconstituted human epidermal model

WD2007/01596/00 GSK1349572A: High Throughput Fluctuation Test (Screen) with Salmonella typhimurium


MODULE 2.6.7. TOXICOLOGY TABULATED SUMMARY

CONFIDENTIALm2.6.7. Toxicology Tabulated Summary 2012N153953_00

2

TABLE OF CONTENTS

PAGE

1. TOXICOLOGY: OVERVIEW FOR DOLUTEGRAVIR (also known as GSK1349572 or S-349572) ......................................................................................5

2. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS STUDIES ......................13

3. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS DATA............................15

4. TOXICOLOGY: DRUG SUBSTANCE....................................................................17

5. SINGLE DOSE TOXICITY......................................................................................19

6. REPEAT DOSE TOXICITY: NON-PIVOTAL STUDIES .........................................24

7. REPEAT DOSE TOXICITY: PIVOTAL STUDIES ..................................................25

8. GENOTOXICITY: IN VITRO..................................................................................47

9. GENOTOXICITY: IN VIVO ....................................................................................53

10. CARCINOGENICITY..............................................................................................54

11. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: NON-PIVOTAL STUDIES ...............................................................................................................71

12. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: FERTILITY AND EARLY EMBRYONIC DEVELOPMENT TO IMPLANTATION (PIVOTAL) ..............77

13. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON EMBRYOFETAL DEVELOPMENT (PIVOTAL).......................................................80

14. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT INCLUDING MATERNAL FUNCTION (PIVOTAL) ..........................................................................................91

15. STUDIES IN JUVENILE ANIMALS.........................................................................96

16. LOCAL TOLERANCE.............................................................................................99

17. OTHER TOXICITY STUDIES...............................................................................101


3

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 List of Carcinogenicity Studies Performed with Dolutegravir.....................8

Table 1.5 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir .....................................................................9

Table 1.6 List of Local Tolerance Studies Performed with Dolutegravir..................11

Table 1.7 List of Other Toxicity Studies Performed with Dolutegravir .....................12

Table 2.1 Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir............................................................................................13

Table 3.1 Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC0-24 (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 13 Week Oral Toxicity Study of GSK1349572A (S-349572) in Mice .......................................................................................................25

Table 7.2 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats......................................................................................27

Table 7.3 4 Week Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats......................................................................................29

Table 7.4 26 Week Oral Toxicity Study of S-349572 Sodium in Rats .....................32

Table 7.5 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Monkeys...............................................................................35

Table 7.6 1 Month Oral Toxicity Study of ERC-349572 Sodium in Monkeys ..........37

Table 7.7 38 Week Oral Toxicity Study of S-349572 Sodium in Monkeys ..............40

Table 8.1 GSK1349572A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli.........................................47


4

Table 8.2 GSK1349572 In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus...........................................................50

Table 8.3 GSK1349572A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus...........................................................51

Table 9.1 GSK1349572A: 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 Reproductive and Developmental Toxicity: Non-Pivotal Studies............71

Table 11.2 Two week Oral toxicity Study of S-349572 Sodium in Non Pregnant Rabbits: Non-Pivotal Studies .................................................73

Table 11.3 Dose Range Finding Oral Study for Effects of S-349572 sodium on Embryofetal Development in Rabbits: Non-Pivotal Studies...............75

Table 12.1 Oral Study for Effects of S-349572 Sodium on Fertility and Early Embryonic Development to Implantation in Rats ....................................77

Table 13.1 Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rats..............................................................................80

Table 13.2 Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rabbits .........................................................................85

Table 14.1 Oral Study for Effects of S-349572 Sodium on Pre- and Post-Natal Development, Including Maternal Function, in Rats ......................91

Table 15.1 GSK1349572A: Oral Juvenile Toxicity Study in Rats.............................96

Table 16.1 Local Tolerance Studies.........................................................................99

Table 17.1 Immunotoxicity Study of S-349572 Sodium in Rats: Determination of Specific Antibody Formation Against T-Cell-Dependent Antigen ..............................................................................101


5

1. TOXICOLOGY: OVERVIEW FOR DOLUTEGRAVIR (also known as GSK1349572 or S-349572)

Table 1.1 List of Single Dose Toxicity Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD


6M6M


2.5 Single No GSK RD2009/00921(R42470)

m4.2.3.1


3M3M


2.5, 5 Single No GSK RD2009/00959(R42475)

m4.2.3.1


3M Intramuscular 4, 7.3, 8.7 Single No GSK 2011N123574(R42826)

m4.2.3.1


3M Intramuscular 10 Single No GSK 2012N136936 m4.2.3.1

Single Dose * Dog (beagle) 1F Oral 30, 100, 150, 250, 500 Single No RD2009/00963 m4.2.3.1


4F4F4F

OralSubcutaneousIntramuscular

311

Single No GSK CD2009/00647(D09113)

m4.2.3.1


1F Oral(gavage)

50, 125, 250, 500 Single No RD2007/01184(E-34572-TF-008-R)

m4.2.3.1


3F Oral(gavage)

1, 3, 10, 50 Single No RD2008/01762(S-349572-TB-44-R)

m4.2.3.1

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-0297994B.

Testing Facility:GSK = GlaxoSmithKline

=


6

Table 1.2 List of Repeat Dose Toxicity Studies Performed with Dolutegravir

Type of Study

Species(Strain)/

Test System

No./Sex/Group



Duration of Dosing



Locationin CTD



10, 100, 500, 1500 14 days No RD2009/01546(S-349572-TF-066-R)

m4.2.3.2



10, 50, 500, 1500 13 weeks Yes RD2009/00028(S-349572-TF-068-L)

m4.2.3.2


10M/10Fa Oral(gavage)

50, 150, 500 14 days Yes RD2007/01140(E-34572-TB-012-L)

m4.2.3.2


10M/10Fa,d Oral(gavage)

2, 10, 100, 1000 4 weeks Yes RD2008/01628(E-34572-TB-043-L)

m4.2.3.2


12M/12Ff Oral(gavage)

5, 50, 500 26 weeksg,h

Yes RD2009/00410(SBL055-082)

m4.2.3.2


3M/3F Oral(gavage)

100, 300, 1000 14 days Yes RD2007/01142(E-34572-TF-029-L)

m4.2.3.2


3M/3Fe Oral(gavage)

25, 50, 100 4 weeks Yes RD2008/00107((E-34572-TF-036-L)

m4.2.3.2


7M/7F Oral(gavage)

3, 10, 15, 50/30 38 weeksi,j Yes RD2009/00036(SBL055-074)

m4.2.3.2

Note: All studies were performed with GSK1349572A, the sodium salt formNo 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.

Testing Facility: = =

= =


7

Table 1.3 List of Genotoxicity Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Ames test NA NA In vitro 5 to 849 g/plateb NA Yes WD2007/00514(V27467)

m4.2.3.3.1

Screening mouse lymphoma assay

NA NA In vitro Up to 80 g/mLc NA No GSK WD2007/01581(MLA-580)

m4.2.3.3.1

Mouse lymphoma assay

NA NA In vitro 5 to 85 g/mLb NA Yes WD2007/00515(V27468)

m4.2.3.3.1

Rat micronucleus Rat(Sprague Dawley)

6M Oral(gavage)

50, 150, 500 2 days Yes WD2007/00513(R27469)

m4.2.3.3.2

Note: All studies were performed with GSK1349572A, 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.


8

Table 1.4 List of Carcinogenicity Studies Performed with Dolutegravir


Test System

No./Sex/Group


Dose (mg/kg/day)

Duration of Dosing



Location in CTD

Carcinogenicity Mouse(CD-1)

6545TK

Oral(gavage)

7.5, 25, 500 2 years Yes 2012N152419 m4.2.3.4.1

Carcinogenicity Rat(Sprague Dawley)

6512TK

Oral(gavage)

2, 10, 50 2 years Yes 2012N152418 m4.2.3.4.1

Key:TK = Satellite groups for toxicokinetics analysis.

Testing Facility: =


9

Table 1.5 List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir

Type of Study

Species(Strain)/

Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Fertility and early embryonic development

Rat(Sprague Dawley)


100, 300, 1000 Up to 66 days (M)

Up to 42 days (F)

Yes XD2009/00368(S-349572-TF-063-L)

m4.2.3.5.1



7F Oral(gavage)


Days 6 to 17)

Yes RD2008/01761(S-349572-TB-051-L)

m4.2.3.5.2



20F Oral(gavage)


Days 6 to 17)

Yes XD2009/00367(S-349572-TB-062-L)

m4.2.3.5.2

Dose range study


(Japanese white)

3F Oral(gavage)

30, 100, 300, 1000 14 days No RD2008/01760(S-349572-TF-052-L)

m4.2.3.5.2



4F Oral(gavage)


Days 6 to 18)

Yes RD2009/00186(S-349572-TF-060-L)

m4.2.3.5.2



18 to 20F

Oral(gavage)


Days 6 to 18)

Yes XD2009/0366(S-349572-TF-065-L)

m4.2.3.5.2


10

Table 1.5 (Continued) List of Reproductive and Developmental Toxicity Studies Performed with Dolutegravir

Type of Study

Species(Strain)/

Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Pre- and postnatal development

Rat(Sprague Dawley)

22F Oral(gavage)

5, 50, 1000 Gestation Day 6 to Lactation Day 20

Yes 2011N121663(SG10306)

m4.2.3.5.3

Juvenile tolerability

Rat(Sprague Dawley)

(juvenile)

8M/8F Oral(gavage)

5, 50, 100, 500, 1000

18 days(Day 4 to

Day 21 pp)

No GSK CD2009/00409(D09072)

m4.2.3.5.4

Juvenile dose range

Rat(Sprague Dawley)

(juvenile)


2, 25, 75, 300 28 days(Day 4 to

Day 31 pp)

No GSK CD2009/00770(D09126)

m4.2.3.5.4

Juvenile toxicity

Rat(Sprague Dawley)

(juvenile)


0.5, 2, 75d 63 days(Days 4 to

66 pp)

Yes GSK CD2010/00023(G09229)

m4.2.3.5.4

Note: All studies were performed with GSK1349572A, 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.

= =


11

Table 1.6 List of Local Tolerance Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD


Reconstituted human skin


m4.2.3.6


Rabbit(Japanese

white)

3M Topical 500 mg/site Single No RD2010/00201(S-349572-TF-080-N)

m4.2.3.6



model


m4.2.3.6


Rabbit(Japanese

white)

6M Topical 100 mg/eye Single No RD2010/00202(S-349572-TF-079-N)

m4.2.3.6


Mouse(CBA/Ca)

5F Topical 25 L/site (25% w/w)(sodium salt)

3 days Yes ED2009/00019(1127/1834)

m4.2.3.6


Mouse(CBA/Ca)

5F Topical 25 L/site (25% w/w)(parent)

3 days Yes 2010N109153(1127/1886)

m4.2.3.6

Note: All studies were performed with GSK1349572A, the sodium salt form. Testing Facility: =

= GSK = GlaxoSmithKline.


12

Table 1.7 List of Other Toxicity Studies Performed with Dolutegravir


Test System

No./Sex/Group



Duration of Dosing



Locationin CTD

Immunotoxicity Rat(Sprague Dawley)


10, 100, 1000 4 weeks Yes RD2009/00751(S-349572-TB-064-L)

m4.2.3.7.2

Note: All studies were performed with GSK1349572A, the sodium salt form.No observed adverse effect levels (NOAEL) are bolded.

Testing Facility: =


13

2. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS STUDIES

Table 2.1 Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir

Type of Study Test System Method ofAdministration

Doses(mg/kg)

GLPCompliance

Report (StudyNumber)

Location(Module)

Toxicokinetics – Single dose Rat(Sprague Dawley)


2.5 No RD2009/00921(R42470)

m4.2.3.1



2.5, 5 No RD2009/00959(R42475)

m4.2.3.1


Intramuscular 4, 7.3, 8.7 No 2011N123574(R42826)

m4.2.3.1


Intramuscular 10 No 2012N136936 m4.2.3.1

Toxicokinetics – Single dose Dog(beagle)

Oral(gavage)

30, 100, 150, 250, 500

No RD2009/00963 m4.2.3.1

Toxicokinetics – Single dose Monkey(cynomolgus)

Oral(gavage)

50, 125, 250, 500

No RD2007/01184 m4.2.3.1


Oral(gavage)

1, 3, 10, 50 No RD2008/01762(S-349572-TB-44-R)

m4.2.3.1


Oral, SC, IM 3, 1, 1 No CD2009/00647(D09113)

m4.2.3.1

Toxicokinetics – Repeat dose Mouse(CD-1)

Oral(gavage)

10, 100, 500 1500

No RD2009/01546(S-349572-TF-066-R)

m4.2.3.2

Toxicokinetics – Repeat dose Mouse(CD-1)

Oral(gavage)

10, 50, 500, 1500

Yes RD2009/00028(S-349572-TF-068-L)

m4.2.3.2

Toxicokinetics – Repeat dose Rat(Sprague Dawley)

Oral(gavage)

50, 150, 500 Yes RD2007/01140(E-349572-TB-012-L)

m4.2.3.2

Toxicokinetics – Repeat dose Rat(Sprague Dawley)

Oral(gavage)

2, 10, 100, 1000

Yes RD2008/01628(E-34572-TB-043-L)

m4.2.3.2

Toxicokinetics –Repeat dose Rat(Sprague Dawley)

Oral(gavage)

5, 50, 500 Yes RD2009/00410(S-349572-TF-055-L)

m4.2.3.2


14

Table 2.1 (Continued) Toxicokinetics: Overview of Toxicokinetics Studies for Dolutegravir

Type of Study Test System Method ofAdministration

Doses(mg/kg)

GLPCompliance

Report (StudyNumber)

Location(Module)

Toxicokinetics – Reproductive and Developmental Toxicity


Oral(gavage)

100, 300, 1000

Yes XD2009/00367(S-349572-TB-062-L)

m4.2.3.5.2


Rat (juvenile)(Sprague Dawley)

Oral(gavage)

5, 50, 100, 500, 1000

No CD2009/00409(D09072)

m4.2.3.5.4



Oral(gavage)

2, 25, 75, 300 No CD2009/00770(D09126)

m4.2.3.5.4



Oral(gavage)

0.5, 2, 75 Yes CD2010/00023(G09229)

m4.2.3.5.4



(Japanese white)

Oral(gavage)

30, 100, 300, 1000

Yes RD2008/01760(S-349572-TF-052-L)

m4.2.3.5.2



Oral(gavage)

40, 200, 1000 Yes XD2009/00366(S-349572-TF-065-L)

m4.2.3.5.2

Toxicokinetics – Repeat dose toxicity Monkey(cynomolgus)

Oral(gavage)

100, 300, 1000

Yes RD2007/01142(E-349572-TB-012-L)

m4.2.3.2


Oral(gavage)

25, 50, 100 Yes RD2008/00107(E-349572-TF-036-L)

m4.2.3.2


Oral(gavage)

3, 10, 15, 50/30d

Yes RD2009/00036(S-349572-TF-047-L)

m4.2.3.2

Toxickinetics – Local Tolerance Rat(Sprague Dawley)

Intramuscular injection 4, 7.3, 8.7 No 2011N123574(R42826)

m4.2.3.6


15

3. TOXICOKINETICS: OVERVIEW OF TOXICOKINETICS DATA

Table 3.1 Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC0-24 (g.h/mL)]

Daily Dose (mg/kg/day)

Mouse Rat Rat (Juvenile) Rabbit Monkey Human

Male Female Male Female Male Female Female Male Female

0.5 9.9g 27.1g

2 53.0c 81.7c 85.7g 93.3g

3 18.9d 15.5d

5 116e 290e

10 257a 256a 274c 378c 32.3d 37.7d

15 36.7d 40.9d

25 108c 83.9c

40 2.6f

50/30 61.7d 61.7d

50 653a 740a 1040b 1610b 111c 153c 53.6h

50 607e 922e 75.1i

75 917g 1044g


16

Table 3.1 (Continued) Mean Toxicokinetics Data for Dolutegravir at Steady State [AUC0-24 (g.h/mL)]


Mouse Rat Rat (Juvenile) Rabbit Monkey Human

Male Female Male Female Male Female Female Male Female

100 722c 781c 148c 92c

100 192b 187b

150 1150b 1740b

200 14.5f

300 199b 271b

400

500 1010a 1300a 1710b 1950b

500 1338e 1777e

1000 1837c 1737c 30.1f 364b 354b

1500 1320a 1350a

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.


17

4. TOXICOLOGY: DRUG SUBSTANCE

Table 4.1 Toxicology: Drug Substance for Dolutegravir

Batch Number Purity Specified Impurities (% w/w) Report Number Type of Study

(% w/w) 452B 454B 451B 399A 453B 983A 009B

PROPOSED SPECIFICATION

Not more than 0.15 for each impurity

A7Z001 99.6 <0.05 <0.05 <0.05 <0.05a <0.05 <0.05 <0.05 RD2008/01628 Repeat Dose Toxicity

RD2009/00410 Repeat Dose Toxicity

RD2009/00036 Repeat Dose Toxicity

R06001 100.0 <0.05 <0.05 <0.05 <0.05a <0.05 <0.05 <0.05 WD2007/00513 Genotoxicity

WD2007/00514 Genotoxicity

WD2007/00515 Genotoxicity

RD2007/01039 Safety Pharmacology


02 99.6 <0.05 <0.05 <0.05 <0.05a <0.05 <0.05 <0.05 RD2008/00107 Repeat Dose Toxicity

MTS-0297994B-07 98.1 <0.05 <0.05 <0.05 <0.05a <0.05 0.10 NT RD2007/01038 Safety Pharmacology


B86001 99.8 <0.05 <0.05 <0.05 <0.05a 0.08 <0.05 <0.05 RD2009/00036 Repeat Dose Toxicity

2012N152419 Carcinogenicity


RD2009/00751 Immunotoxicity

XD2009/00367 Reproductive Toxicity

XD2009/00368 Reproductive Toxicity


18

Table 4.1 (Continued) Toxicology: Drug Substance for Dolutegravir

Batch Number Purity (%) Specified Impurities (% w/w) Report Number Type of Study

452B 454B 451B 399A 453B 983A 009B

PROPOSED SPECIFICATION

Not more than 0.15 for each impurity

091001 99.3 <0.05 <0.05 <0.05 <0.05a <0.05 <0.05 <0.05 2012N152419 Carcinogenicity


2011N121663 Reproductive Toxicity

B87002 99.6 <0.05 <0.05 <0.05 <0.05a 0.08 <0.05 <0.05 CD2010/00023 Juvenile Toxicity

Key:a =.Calculated results without the response factor ‘2.5’ for gsk009*.NT = Not tested.452B = gsk006*; 454B = gsk007*; 451B = gsk008*; 399A = gsk009*;453B = gsk010*; 983A = gsk011*, the enantiomer; and 009B = total of gsk012* and gsk028*, diastereomers.



19

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 NaH2PO4 H20/0.006 M Na2HPO4 with NaCl, pH 6.8)

2.5a 6M The dose administered by both routeswas well tolerated. Plasma concentrations of GSK1349572 were quantifiable up to Day 15 following the SC dose and up to Day 8 following the IM dose. Mean Cmax, AUC0-24, and AUC0-t

values for GSK1349572 were similar between the SC and IM routes of administration.

RD2009/00921R42470

(m4.2.3.1)

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 NaH2PO4, 0.006M Na2HPO4.H2O, and 0.83% NaCl; approximate pH 6.8)

2.5b

5b

3M3M

Both doses were well tolerated. Mean GSK1349572 Cmax, AUC0-24, and AUC0-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 AUC0-t increased 2.3- to 3.2-fold after SC or IM administration. The duration that GSK1349572 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.

RD2009/00959R42475

(m4.2.3.1)


20

Table 5.1 (Continued) Single Dose Toxicity for Dolutegravir

Species/ Strain


Number of Animals/

Sex


Rat(Sprague Dawley)

Intramuscular injection 0004

7.38.7

333333

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 GSK1349574B 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 GSK1349572 >60 ng/mL for at least 43 days. The highest dose-normalized systemic exposure (Cmax and AUC0-) and the least amount of variability tended to be provided by the sesame oil formulation (Formulation 3). The results suggest that the rate of GSK1349572 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).

2011N123574R42826

(m4.2.3.1)


21


Species/ Strain


Number of Animals/

Sex


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 hadinfluenced 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-0297994B in dogs.

RD2009/00963(m4.2.3.1)

Monkey(cynomolgus)

Oral (gavage)(0.5% hydroxypropylmethyl-cellulose (HPMC)/

0.1% Tween 80TM (suspension))

Subcutaneous or Intramuscular(2% Pluronic F, 0.2% Polysorbate 80, 0.18%

methylparaben, 0.02% propylparaben, 0.004M NaH2PO4H2O, 0.006M Na2HPO4 and 0.83%

NaCl (suspension))

3

1

4Fc

4Fc

All doses were well-tolerated and there were no clinical observations noted at the injection sites. Mean dose–normalized Cmax

following oral administration was higher than that following SC administration; however, the mean dose-normalized AUC0-

24 following oral administration was similar to the dose-normalized AUC0-t following SC administration. There were no marked differences ( 2-fold) in the mean Cmax, AUC0-24 or AUC0-t values following SC or IM administration. Plasma AUC0-t values following SC or IM administration were on average 3.2- or 3.9-fold higher, respectively, than corresponding AUC0-24

values.

CD2009/00647(D09113)(m4.2.3.1)


22


Species/ Strain


Number of Animals/

Sex


Monkey(cynomolgus)

Oral (gavage)(0.5 w/w% hydroxypropylmethylcellulose

(HPMC)/0.1 w/w% Tween 80(aqueous solution))

50, 125, 250, 500

1F ERC-349572 was detected in plasma at all dose levels. Plasma concentration-time profiles were similar at all dose levels. No dose relationship was noted in the Cmax and AUC0-24h values, although the reason was unclear. There were no apparent differences in the Cmax values between 125 and 500 mg/kg and the AUC0-24h value at 500 mg/kg was only approximately 1.4-fold the AUC0-24h value at 125 mg/kg. Therefore, the systemic exposure was considered to have attained the steady state at 125 mg/kg.

No treatment-related toxicity was evident in the animals’ physical condition, body weights, food consumption, hematology or clinical chemistry.

RD2007/01184(E-34572-TF-008-R)

(m4.2.3.1)


23


Species/ Strain


Number of Animals/

Sex


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-349572 orally with dose escalating manners from 1 to 50 mg/kg, the exposure of S-349572 was dose-related in nature. However, large inter-individual differences were noted at 10 mg/kg.

RD2008/01762(S-349572-TB-44-R)

(m4.2.3.1)

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 GSK1349572A was given to monkeys in Groups 1 and 3, respectively. On Day 16, a single

subcutaneous dose of 0 or 1 mg/kg GSK1349572B was given to monkeys in Groups 1 and 3, respectively, and a single intramuscular dose of 0 or1 mg/kg GSK1349572B was given to monkeys in Groups 2 and 4, respectively. Toxicokinetic evaluation was performed on samples collected frommonkeys in Groups 3 and 4 only.


24

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


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, 1500

10M/10F The no observed adverse effect level (NOAEL) for S-349572 was 1500 mg/kg/day because no adverse effects were noted at any dose level.

RD2009/01546(S-349572-TF-066-R)

(m4.2.3.2)


25

7. REPEAT DOSE TOXICITY: PIVOTAL STUDIES

Table 7.1 13 Week Oral Toxicity Study of GSK1349572A (S-349572) in Mice

Repeat Dose Toxicity Report Title: Preliminary Carcinogenicity Study (Gavage) of S-349572 Sodium in Mice for 13 Weeks

Test Article: S-349572 sodium

Species/Strain: Albino mice (outbred) VAF/Plus®

Crl: CD-1® (ICR) BRDuration of Dosing: 7 days per week for 13 weeks Report No.: RD2009/00028

CTD Module: m4.2.3.2Study No.: 09-2119 [S-349572-TF-068-L]

Initial Age: Approximately 4 weeks Duration of Post Dose: None

Date of First Dose: 14 May 2009 Method of Administration: Oral gavageVehicle/Formulation: 0.5% HPMC/0.1% Tween 80

GLP Compliance: Yes

Special Features: None

No Observed Adverse Effect Level: 1500 mg/kg

Daily Dose (mg/kg) 0 (Control) 10 50 500 1500M F M F M F M F M F

Number of Animals Toxicokinetics

27 27 54 54 54 54 54 54 54 54

AUC0-24h (g.h/mL), Day 1 NS NS 211 212 477 528 923 1110 1440 1420



Cmax (g/mL), Day 1 NS NS 16.0 19.3 43.9 53.7 77.3 88.3 109 114

Cmax (g/mL), Day 28 NS NS 19.7 24.6 51.0 65.3 83.4 89.4 89.3 116

Cmax (g/mL), Day 85 NS NS 18.5 28.0 52.7 62.6 82.1 109 103 118


26

Table 7.1 (Continued) 13 Week Oral Toxicity Study of GSK1349572A (S-349572) in Mice

Repeat-Dose Toxicity Study No. (Continued): 09-2119Daily Dose (mg/kg) 0 (Control) 10 50 500 1500

M F M F M F M F M FNumber of Animals: 10 10 10 10 10 10 10 10 10 10Toxicity Animals:Noteworthy Findings:Died or Sacrificed Moribund: – 1 – – – – – – – –Body Weight: – – – – – – – – – –Feed Consumption: – – – – – – – – – –Clinical Observations: – – – – – – – – – –Hematology: – – – – – – – – – –Serum Chemistry:Total bilirubin (mg/dL) 0.09 – 0.12 – 0.11 – 0.12 – 0.13* –Alkaline phosphatase (U/L) 38 – 37 – 37 – 39 – 56** –Aspartate aminotransferase (U/L) – 76 – 80 – 72 – 96 – 110*Potassium (mEq/L) – 4.6 – 4.6 – 5.0 – 4.8 – 5.1*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.


27

Table 7.2 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats

Repeat Dose ToxicityTest Article: ERC-349572 sodium

Report Title: 2 Week Oral Toxicity Study of ERC-349572 Sodium in Rats

Species/Strain: Rat/Sprague DawleyDuration of Dosing: 2 weeksReport No.: RD2007/01140CTD Module: m4.2.3.2Study No.: E-349572-TB-012-L

Initial Age: 11 weeksDuration of Post Dose: None

Date of First Dose: M: 22 Nov 06. F: 23 Nov 06Method of Administration: Gavage

Vehicle/Formulation: Aqueous 0.5 w/w% hydroxypropyl methylcellulose (HPMC) with 0.1 w/w% Tween 80/ Suspension

No Observed Adverse-Effect Level: 500 mg/kg/day Special Features: None GLP Compliance: Yes

Daily Dose (mg/kg) 0 (Control) 50 150 500Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10ToxicokineticsNumber of animals 3 3 3 3 3 3 3 3Cmax (g/mL)Day 1 NC NC 58.5 75.4 82.7 83.3 87.1 117Day 14 NC NC 65.7 95.6 74.1 106 108 124AUC0-24 h (g.h/mL)Day 1 NC NC 881 1110 994 1050 1360 1350Day 14 NC NC 1040 1610 1150 1740 1710 1950Noteworthy FindingsDied or Sacrificed Moribund 0 0 0 0 0 0 0 0Clinical Observations - - - - - - - -Body Weight - - - - - - - -Food Consumption - - - - - - - -Water Consumption - - - - - - - -Auditory Examination - - - - - - - -Ophthalmoscopy - - - - - - - -

Key: No noteworthy findings. The first day of dosing is Day 1NC: Not calculated because of insufficient data; control animals were sampled only at 1 hour after administration, and plasma ERC-349572 concentrations were alll below the lower limit of quantification (<0.03 g/mL).


28

Table 7.2 (Continued) 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats

Repeat Dose Toxicity Study No. E-349572-TB-012-L

Daily Dose (mg/kg) 0 (Control) 50 150 500Number of Animals M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10UrinalysisNumber examined a) 9 10 9 10 10 10 10 10Specific gravity 1.019 1.021 1.036 1.039* 1.032 1.036 1.047** 1.046**HematologyNumber examined a, b) 9 10 8 10 10 10 10 10Eosinophil (× 103/µL) - 0.05 - 0.07 - 0.09* - 0.07Blood ChemistryNumber examined a, b) 9 10 8 10 10 10 10 10Creatinine (mg/dL) 0.26 - 0.28 - 0.30** - 0.29* -Total bilirubin (mg/dL) - 0.06 - 0.05 - 0.05 - 0.05*Sodium (mmol/L) - 142 - 142 - 143 - 143*Organ Weight c) (%) - - - - - - - -Gross Pathology - - - - - - - -HistopathologyStomach: eosinophil infiltration, glandular stomach

Mild 4 0 N.E. N.E. 2 5 9 5Stomach: increased mucous cell, glandular stomach

Mild 3 0 N.E. N.E. 1 2 5 4Stomach: edema, glandular stomach

Mild 0 2 N.E. N.E. 3 2 7 4

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 examinedDunnett'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.


29

Table 7.3 4 Week Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats

Report No.: RD2008/01628 CTD Module: m4.2.3.2


30

Table 7.3 (Continued) 4 Week Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats


31

Table 7.3 (Continued) 4 Week Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Rats


32

Table 7.4 26 Week Oral Toxicity Study of S-349572 Sodium in Rats

Repeat Dose Toxicity Report Title: 6 Month Oral Toxicity Study of S-349572 Sodium in Rats

Test Article: S-349572 sodiumReport No.: RD2009/00410CTD Module: m4.2.3.2Study No.: SBL055-082

Species/Strain: Rat/Sprague DawleyInitial Age: 6 weeksDuration of Dosing: 4 or 6 monthsDuration of Post Dose: -1 month

Date of First Dose: M: 30 July 2008; F: 31 July 2008Method of Administration: Oral gavage

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: YesDaily Dose (mg/kg/day) 0 (Control) 5 50 500Toxicokinetics:Number of Animals#: M: 6 F: 6 M: 6 F: 6 M: 6 F: 6 M: 6 F: 6Cmax (g/mL): Day 1 of dosing NE NE 9.4 12.0 47.3 56.5 95.3 103.5

Day 30 of dosing NE NE 12.5 19.0 49.9 62.6 93.2 108.9Day 120 of dosing NE NE 11.2 20.8 37.7 59.6 84.6 101.5Day 180 of dosing NE NE 11.9 20.1 38.0 56.6 85.1 106.8

AUC0-24h (g.h/mL): Day 1 of dosing NE NE 88.8 138.4 636.7 731.0 1450.1 1450.2Day 30 of dosing NE NE 123.5 216.7 708.7 883.2 1300.1 1508.4

Day 120 of dosing NE NE 126.7 267.2 594.0 932.7 1274.3 1660.3Day 180 of dosing NE NE 115.7 289.7 606.9 921.6 1337.6 1777.1

Number of Animals:4 Month Dosing Period: M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 106 Month Dosing Period (After 4 Months of Dosing): M: 12 F: 12 M: 12 F: 12 M: 12 F: 12 M: 12 F: 12Recovery Period: M: 6 F: 6 M: 0 F: 0 M: 0 F: 0 M: 6 F: 6Noteworthy Findings:Died or Sacrificed Moribund: 0 0 0 0 0 0 0 0Clinical Signs: - - - - - - - -Body Weight: - - - - - - - -Food Consumption: - - - - - - - -Ophthalmology: - - - - - - - -Urinalysis: - - - - - - - -Hematology: - - - - - - - -


33

Table 7.4 (Continued) 26 Week Oral Toxicity Study of S-349572 Sodium in Rats

Daily Dose (mg/kg/day) 0 (Control) 5 50 500Number of Animals:4 Month Dosing Period: M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 106 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: 6Blood Chemistry: - - - - - - - -Gross Pathology*: - - - - - - - -End of the 4 Month Dosing Period:Stomach (glandular mucosa)

White focus 0 0 0 0 0 0 1 0End of the 6 Month Dosing Period:Stomach (glandular mucosa)

White focus 0 0 0 0 0 0 1 0Red focus 0 0 0 0 0 0 1 2

End of the Recovery Period: - - - -Organ Weight: - - - - - - - -Histopathology*:End of the 4 Month Dosing PeriodStomach (forestomach)

Thickening, mucosa, limiting ridge, very slight

0 0 0 0 0 0 5 5

Stomach (glandular stomach)Cellular infiltration, eosinophil, submucosa, very slight

2 1 2 1 4 4 8 7

Cellular infiltration, eosinophil, submucosa, slight

0 0 0 0 0 0 2 3

Hemorrhage, mucosa, very slight 0 0 0 0 0 0 1 0Increase, globule leukocyte, mucosa, very slight

0 0 0 1 3 4 0 0

Increase, globule leukocyte, mucosa, slight

0 0 0 0 6 6 10 10


34

Table 7.4 (Continued) 26 Week Oral Toxicity Study of S-349572 Sodium in Rats

Daily Dose (mg/kg/day) 0 (Control) 5 50 500Stomach (glandular stomach) (Continued)

Increase, mucous neck cell, very slight 0 0 0 0 2 3 10 10End of the 6 Month Dosing Period:Stomach (forestomach)


0 0 0 0 1 0 4 4

Stomach (glandular stomach)Cellular infiltration, eosinophil, submucosa, very slight

3 1 0 1 4 7 12 8

Cellular infiltration, eosinophil, submucosa, slight

0 0 0 0 0 0 0 3

Hemorrhage, mucosa, very slight 0 0 0 0 0 0 1 0Increase, globule leukocyte, mucosa, very slight

0 0 1 0 2 3 0 0

Increase, globule leukocyte, mucosa, slight

0 0 0 0 9 9 12 12

Increase, mucous neck cell, very slight 0 0 0 0 4 7 10 10End of the Recovery Period:Stomach (forestomach)


0 0 1 2

Stomach (glandular stomach)Increase, globule leukocyte, mucosa, very slight

0 0 2 1

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.


35

Table 7.5 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Monkeys

Repeat-Dose Toxicity Report Title: Two-Week Oral Toxicity Study of ERC-349572 sodium in Monkeys

Test Article: ERC-349572 sodium

Species/Strain: Cynomolgus MonkeyInitial Age: 2 years and 6-9 monthsDate of First Dose: March 22, 2007

Duration of Dosing: 2 WeeksDuration of Postdose: -Method of Administration: GavageVehicle/Formulation: Hydroxypropyl methylcellulose (0.5%), Tween 80 (0.1%) / Suspension

Report No.: RD2007/01142CTD Module: m4.2.3.2Study No.: SG07030GLP Compliance: Yes

Special Features: NoneNo Observed Adverse Effect Level: 100 mg/kg/day as ERC-349572Daily Dose as ERC-349572 (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: 3Toxicokinetics: AUC (g.h/mL):ERC-349572 Day 1 172 150 324 142 358 237

Day 14 192 187 199 271 364 354Noteworthy FindingsDied or Sacrificed Moribund 0 0 0 0 0 0 0 1 (Day 13)Clinical SignsVomiting/Vomitus 0 0 2 1 1 3 3 3[1]Diarrhea (loose, muddy, watery stool) 0 0 0 0 2 2 3 3[1]Abnormal position (lateral, crouching) 0 0 0 0 0 0 2 1[1]Decrease in movement 0 0 0 0 0 0 2 1[1]Moribund condition a) 0 0 0 0 0 0 0 1[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


36

Table 7.5 (Continued) 14 Day Oral Toxicity Study of GSK1349572A (ERC-349572 Sodium) in Monkeys

Repeat-Dose Toxicity Study No. SG07030

Daily Dose as ERC-349572 (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: 3Noteworthy Findings (Continued)Body Weight b) (%) 2.67 kg 2.38 kg -1 0 -3 -7 -13 -5Food Consumption b) (%) 100 g/day 100 g/day 0 0 0 -13 -29 -21Ophthalmology – – – – – – – –Electrocardiography – – – – – – – –Hematology c)

RBC (106/µL) Week 1 – 6.43 – 6.03 – 5.87 – 5.76*Reticulocyte (%) Week 1 1.3 – 0.7 – 0.6 – 0.2** –Reticulocyte (109/L) Week 1 76.5 60.8 38.6 66.0 34.8* 28.2 12.9** 28.7Platelet (103/µL) Week 2 431 372 320 316 294 319 227* 260APTT (s) Week 2 22.9 – 22.8 – 22.8 – 25.5* –Fibrinogen (mg/dL) Week 2 222.6 226.3 255.6 252.3 292.6 315.6 364.8 312.4[442.8]Clinical Chemistry c)

AST (U/L) Week 1 19 22 18 25 27 20 45{93} 40[87]ALT (U/L) Week 1 31 32 35 56 72 18 303* 167[442]γ-GTP (U/L) Week 1 70.5 – 62.1 – 56.3 – 94.2 –T-BIL (mg/dL) Week 2 0.06 0.11 0.10 0.07 0.10 0.30 0.20{0.35} 0.23[0.34]UN (mg/dL) Week 2 18.9 17.2 13.2 18.5 14.4 18.0 34.3{56.0} 28.0[54.4]CRE (mg/dL) Week 2 0.46 0.51 0.55 0.50 0.51 0.56 0.68* 0.63[0.87]CHO (mg/dL) Week 1 – 157 – 139 – 130 – 116*TG (mg/dL) Week 2 71 – 17* – 35 – 137 –Na (mEq/L) Week 2 145.9 149.8 146.2 152.0 144.1 145.9 139.6{124.6} 140.5[131.9]Cl (mEq/L) Week 2 108.3 108.8 106.4 109.8 106.0 106.5 94.7{79.2} 96.0[84.4]A/G Week 2 1.31 1.33 1.16 1.20 1.09 1.11 0.94 0.97[0.51]

Key:–: No noteworthy findings [ ]: Animal No. 72 (dead animal) { }: Animal No. 32Dunnett’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.


37

Table 7.6 1 Month Oral Toxicity Study of ERC-349572 Sodium in Monkeys

Repeat Dose Toxicity Report Title: 1 Month Oral Toxicity Study of ERC-349572 sodium in Monkeys

Test Article: ERC-349572 sodium

Species/Strain: Monkeys/CynomolgusInitial Age: 2-3 yearsDate of First Dose: August 19, 2007

Duration of Dosing: 1 Month (30 days)Duration of Postdose: 1 Month (30 days)Method of Administration: GavageVehicle/Formulation: 0.5%HPMC/0.1% Tween80 / Suspension

Report No.: RD2008/00107CTD Module: m4.2.3.2Study No. SG07224

GLP Compliance: YesSpecial Features: NoneNo Observed Adverse Effect Level: 50 mg/kg/day for males and femalesDaily Dose (mg/kg, as ERC-349572) * 0 (Control) * * 25 * * 50 * * 100 *Number of Animals M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5Toxicokinetics: AUC (g.h/mL): Day 1 NE NE 60.31 67.88 72.87 68.74 99.00 90.18

Day 15 NE NE 116.21 111.60 120.35 104.17 109.79 128.93Day 30 NE NE 108.30 83.93 111.32 153.17 148.41 92.04

Cmax (g/mL): Day 1 NE NE 8.70 11.15 9.64 10.66 12.11 12.15Day 15 NE NE 15.34 18.83 14.53 16.00 14.30 18.19Day 30 NE NE 13.92 15.38 14.45 20.38 15.95 13.39

Noteworthy FindingsDied or Sacrificed Moribund 0 0 0 0 0 0 0 0Clinical ObservationsVomiting/Vomitus 0 0 0 0 1 a) 0 5 5Diarrhea (muddy or watery stools) 0 0 0 0 0 1 b) 4 4Crouching position 0 0 0 0 0 0 1 1Body Weight c)

Day 30 2.68 kg 2.63 kg +1 +3 -5 -2 -7 d) -7 d)

Key:The first day of dosing is Day 1. NE: Not estimated a) Only for 1 day b) Only for 3 daysc) 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 30.


38

Table 7.6 (Continued) 1 Month Oral Toxicity Study of ERC-349572 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 25 100 g 100 g 0 0 0 0 -16 b) -14 b)

Ophthalmology – – – – – – – –Electrocardiography – – – – – – – –Hematology c)Week 2 RBC (106/μL) – 6.19 – 6.25 – 6.24 – 5.67

Platelet (103/μL) – 422 – 409 – 404 – 331 [221]Week 4 RBC (106/μL) – 6.04 – 6.17 – 6.03 – 5.48*

Reticulocyte (109/L) 28.3 42.1 46.6 53.4 42.5 60.5 44.3 [7.4] 39.1 [9.6]Neutrophil (103/μL) 1.78 – 1.98 – 3.75 – 5.70 [10.85] –Fibrinogen (mg/dL) 199.5 – 215.6 – 208.1 – 244.6 [381.1] –

Clinical Chemistry c)Week 4 Urea nitrogen (mg/dL) 17.0 – 15.7 – 18.3 – 21.2 [34.0] –

Triglyceride (mg/dL) 21 – 19 – 39 – 58 [151] –Calcium (mg/dL) 9.25 – 9.21 – 8.94 – 9.37 [10.75] –Sodium (mEq/L) 152.0 – 152.0 – 152.0 – 146.2 [131.5] –Potassium (mEq/L) 3.81 – 3.96 – 3.75 – 4.08 [5.43] –Chloride (mEq/L) 111.1 – 110.3 – 111.0 – 105.6 [90.4] –Total protein (g/dL) 7.33 – 7.26 – 7.28 – 7.52 [8.65] –Total bilirubin (mg/dL) – 0.12 – 0.11 – 0.08 – 0.15 [0.45]

Key:–: No noteworthy findings.Dunnett’s test: * –p < 0.05.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)


39

Table 7.6 (Continued) 1 Month Oral Toxicity Study of ERC-349572 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) 7.72 4.74 4.47 3.08 5.13 5.23 3.57** 2.09Gross Pathology – – – – – – – –Organ Weights – – – – – – – –Bone Marrow (nucleated cell counts) – – – – – – – –HistopathologyCecum: Inflammatory cell infiltration in the lamina propria

0 0 0 0 0 0 2 1

Cecum: Cell debris from the cripts 0 0 0 0 0 0 1 0Cecum: Atrophy of the mucosal epithelium 0 0 0 0 0 0 0 1Colon: Inflammatory cell infiltration in the lamina propria

0 0 0 0 0 0 1 2

Colon: Cell debris from the cripts 0 0 0 0 0 0 1 0Colon: Atrophy of the mucosal epithelium 0 0 0 0 0 0 0 1Rectum: Inflammatory cell infiltration in the lamina propria

0 0 0 0 0 0 1 1

Thymus: Atrophy 0 0 0 0 0 0 0 2Pancreas: Atrophy of the acinar cells 0 0 0 0 0 0 0 1

Postdose Evaluation:Number Evaluated 2 2 0 0 0 0 2 2Died or Sacrificed Moribund 0 0 0 0Noteworthy Findings – – – –

Key:–: No noteworthy findings.Dunnett’s test: ** –p < 0.01.a) Group means are shown.


40

Table 7.7 38 Week Oral Toxicity Study of S-349572 Sodium in Monkeys

Repeat Dose Toxicity Report Title: 9 Month Oral Toxicity Study of S-349572 Sodium in Monkeys


Species: Cynomolgus monkey Duration of Dosing: 4 or 9 month (120 or 270 days) Report No.: RD2009/00036CTD Module: m4.2.3.2Study No.: SBL055-074 (S-349572-TF-047-L)

Initial Age: 4 to 5 years Duration of Post Dose: 1 month GLP Compliance: YesDate of First Dose: M: June 6, 2008

F: June 13, 2008Method of Administration: Oral Vehicle/Formulation: 0.5% w/w

hydroxypropylmethylcellulose (HPMC)/aqueous solution with 0.1% w/w Tween 80/suspensionNo Observed Adverse Effect Level: 15 mg/kg/day Special Features: None

Daily Dose (mg/kg)a) 0 (Control) 3 10 15 30b) 50b)

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: 9Toxicokinetics:Number of Animals:4 month dosing period 9 9 7 7 7 7 7 7 7 9 9 99 month dosing period 6 6 4 4 4 4 5 5 5 6 0 0Cmax (μg/mL) Day 1 NC NC 2.89 3.08 4.71 6.14 7.67 5.53 NE NE 9.01 10.47

Day 30 NC NC 3.83 3.34 5.07 5.98 6.71 8.62 NE NE 9.92 9.86Day 69 NC NC 3.37 2.58 4.67 6.80 7.48 11.06 NE NE 9.54c) 8.80

Day 120 NC NC 3.27 3.19 5.54 7.86 7.64 10.63 8.42 10.23 NE NEDay 180 NC NC 2.75 2.51 4.19 6.26 6.32 6.38 7.75 6.83 NE NEDay 270 NC NC 3.03 2.27 4.44 5.13 5.31 4.80 7.52 7.77 NE NE

AUC0-24 (μg.h/mL):Day 1 NC NC 15.24 15.30 30.71 34.48 46.42 30.59 NE NE 62.86 63.42

Day 30 NC NC 19.12 17.24 32.01 39.06 42.62 51.32 NE NE 67.25 66.47Day 69 NC NC 17.88 15.62 31.34 44.85 46.23 63.81 NE NE 75.14 66.11


Tmax (h):Day 1 NC NC 1.4 1.0 1.7 1.6 1.7 1.6 NE NE 1.7 2.1

Day 30 NC NC 1.1 1.2 1.6 1.3 1.9 1.7 NE NE 1.9 1.7Day 69 NC NC 1.1 0.9 1.4 2.0 1.7 1.9 NE NE 2.0 1.7



41

Table 7.7 (Continued) 38 Week Oral Toxicity Study of S-349572 Sodium in Monkeys


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](# 48)

[0] [0] [0] [2](#s. 64, 69)

[0]

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](#s. 64, 66)

[0]

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,

68)

[1](# 74)

Ophthalmology: - - - - - - - - - - - -

Electrocardiography: - - - - - - - - - - - -

Urinalysis: - - - - - - - - - - - -


42



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: 9Hematologyd) - Day 30 (Individual)e):Neutrophils (x103/mm3) - - - - - - - - NE NE ↑14.40

(# 64),↑10.34(# 66)

-

Neutrophils (%) - - - - - - - - NE NE ↑62.9(# 64)↑69.5(# 66)

-

Monocytes (x103/mm3) - - - - - - - - NE NE ↑1.01(# 64),↑0.93(# 69)

-

Leukocyte (x103/mm3) - - - - - - - - NE NE ↑22.91(# 64)

-

Platelet (x103/mm3) - - - - - - - - NE NE ↑676(# 64)

-

Hematology - Day 30 (Mean)Monocytes (x103/mm3) 0.282 - 0.319 - 0.313 - 0.501 - NE NE 0.541* -Neutrophils (x103/mm3) 3.306 - 4.306 - 4.180 - 5.744 - NE NE 6.822* -Day 69 (Individual)f)

Neutrophils (x103/mm3) - - - - - - - - NE NE ↑16.88(# 68),↑11.97(# 69)

↑18.22(# 74)

Neutrophils (%) - - - - - - - - NE NE ↑76.0(# 68),↑60.9(# 69)

-

Leukocyte (x103/mm3) - - - - - - - - NE NE ↑22.20(# 68)

↑25.00(# 74)


43



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: 9Monocytes (x103/mm3) - - - - - - - - NE NE ↑1.04

(# 69)↑0.77(# 76)

APTT(s) ↑24.7(# 61)

↑24.8(# 74)

Fibrinogen (mg/dL) - - - - - - - - NE NE - ↑291.4(# 74)

Erythrocyte (x106/mm3) - - - - - - - - NE NE - ↓4.31(# 74)

Hemoglobin (g/dL) - - - - - - - - NE NE - ↓10.7(# 74)

Hematocrit (%) - - - - - - - - NE NE - ↓33.8(# 74)

Hematology - Day 69 (Mean)Monocytes (x103/mm3) 0.282 - 0.319 - 0.313 - 0.501 - NE NE 0.541* -Neutrophils (x103/mm3) 3.306 - 4.306 - 4.180 - 5.744 - NE NE 6.822* -APTT(s) - 17.82 - 19.59 - 18.16 - 21.23 NE NE - 21.21**Day 120 - - - - - - - - - - NE NEDay 180 - - - - - - - - - - NE NEDay 266 - - - - - - - - - - NE NEBlood Chemistryg):Day 30 - - - - - - - - NE NE - -Day 69 (Individual)h)

Total protein (g/dL) - - - - - - - - NE NE - ↓6.4(# 74)

Albumin (g/dL) - - - - - - - - NE NE - ↓3.2(# 74)

Inorganic phosphorus (mg/dL) - - - - - - - - NE NE ↑8.05 (# 68)

-

Triglycerides (mg/dL) - - - - - - - - NE NE - ↑143(# 74)


44



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: 9Day 69 (Individual)i)

Glucose (mg/dL) - - - - - - - - NE NE ↓53(# 68)

↓40(# 74)

Chloride (mEq/L) - - - - - - - - NE NE ↓103(# 68)

↓97(# 74)

Day 69 (Mean)Inorganic phosphorus (mg/dL) 5.680 - 5.820 - 6.049 - 5.977 - NE NE 6.710* -Glucose (mg/dL) - 79.1 - 82.7 - 80.0 - 73.9 NE NE - 63.7*Day 120 (Individual)j)

Blood urea nitrogen (mg/dL) - - - - - - - - - 31.4(# 76)

NE NE

Day 180 - - - - - - - - - - NE NEDay 270 - - - - - - - - - - NE NENumber 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: 09 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: 0Gross Pathology:Moribund Animals:Enlargement of adrenals NE NE NE NE NE NE NE NE NE NE [2]

(#s. 64, 66)NE

Small thymus NE NE NE NE NE NE NE NE NE NE [2](#s. 64, 66)

NE

Survived Animals:4 month dosing period - - - - - - - - - - NE NE9 month dosing periodStomach (Body)Red and recessed focus, mucosa - - - - - - - - - [1]

(# 76)NE NE

Organ Weight:Moribund Animals:Increased absolute and relative adrenal weight

NE NE NE NE NE NE NE NE NE NE [2](#s. 64, 66)

NE

Survived Animals: - - - - - - - - - - NE NE


45



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: 9Histopathology:Moribund Animalsk):Cecum and Colon

Mononuclear cell infiltration and hemorrhage, lamina propria

NE NE NE NE NE NE NE NE NE NE [1](# 64)

NE

Esophagus and TongueInflammatory cell infiltration, epithelium

NE NE NE NE NE NE NE NE NE NE [1](# 64)

NE

Survived Animals:4 month dosing period - - - - - - - - - - NE NE9 month dosing periodStomach (Body)

Mononuclear cell infiltration, and hemorrhage, lamina propria, multifocal

- - - - - - - - - [1](# 76)

NE NE

Regeneration, epithelium, multifocal

- - - - - - - - - [1](# 76)

NE NE

Erosion, multifocal - - - - - - - - - [1](# 76)

NE NE

Post Dose Evaluation:Number Evaluated: 2 2 2 2 2 2Died or Sacrificed Moribund: 0 0 0 0 0 0Noteworthy Findings:Gross Pathology:Stomach (body)

Red focus, mucosa - - - - - [1](# 70)


46



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: 9Histopathology: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 6.25x103/mm3 and 44.7%, monocytes

0.50x103/mm3, leukocytes 13.98x103/mm3, platelets 545x103/mm3, # 66: neutrophils 0.87x103/mm3 and 11.2%; # 69: monocytes 0.44x103/mm3.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 5.92x103/mm3 and 50.9%,

leukocytes 11.64x103/mm3; # 69: neutrophils 4.02x103/mm3 and 34.9%, monocytes 0.44x103/mm3; # 74: erythrocytes 5.22x106/mm3, hemoglobin 13.1 g/dL, hematocrit 41.4%,neutrophils 5.82x103/mm3, leukocytes 11.07x103/mm3, fibrinogen 145.8 mg/dL, APTT 18.4 s; #76: monocytes 0.22x103/mm3.

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 foraspartate 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, chloride108 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 tissueof 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.


47

8. GENOTOXICITY: IN VITRO

Table 8.1 GSK1349572A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli

Genotoxicity: In Vitro Report Title: GSK1349572A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli.

Test Compound: GSK1349572A (sodium salt)Batch Number: R06001

Test for Induction of: Reverse mutation in bacterial cells

No. of Independent Assays: 2 Covance Study No 2990/120GSK Reference No: V27467GSK Document Number: WD2007/00514CTD Module: m4.2.3.3.1

Strain: S. typhimurium and E. coli 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


48

Table 8.1 (Continued) GSK1349572A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli

Metabolic Activation

Test CompoundConcentration

(g/plate)1

Main Plate Incorporation (Ames) Test 1Mean Number of Revertant Colonies per Plate

TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA pKM101Without Activation DMSO 100 L/plate 26 100 15 16 110

GSK1349572 5 18 94 14 10 114GSK1349572 15 19 95 17 16 110GSK1349572 50 24 101 15 14 151GSK1349572 150 28 98 16 17 101GSK1349572 500 14 113 16 11 112GSK1349572 849 14 433 133 93 104

2-Nitrofluorene 5 13202 NT NT NT NTSodium azide 2 NT 11262 7892 NT NT

9-Aminoacridine 50 NT NT NT 1762 NT4-Nitroquinoline-1-oxide 2 NT NT NT NT 15402

With Activation DMSO 100 L/plate 30 97 16 13 149GSK1349572 5 33 126 19 14 163GSK1349572 15 41 95 17 17 170GSK1349572 50 33 97 14 17 168GSK1349572 150 27 92 20 23 177GSK1349572 500 27 623 173 15 167GSK1349572 849 25 133 163 83 146

Benzo[a]pyrene 10 3302 NT NT NT NT2-Aminoanthracene 5 NT 17612 4312 1592 NT2-Aminoanthracene 10 NT NT NT NT 10442

Key:1. All concentrations are expressed in terms of parent compound2. two fold increase in revertants for TA98, TA100 and WP2uvrA(pKM101) and three fold increase for TA1535 and TA15373. Slight thinning of background bacterial lawnNT Not tested


49

Table 8.1 (Continued) GSK1349572A: Bacterial Mutation Assay (Ames Test) with Salmonella typhimurium and Escherichia coli

Metabolic Activation

Test CompoundConcentration

(g/plate)1

Main Plate Incorporation (Ames) Test 2Mean Number of Revertant Colonies per Plate

TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA pKM101Without Activation DMSO 100 L/plate 22 110 16 14 130

GSK1349572 5 24 88 15 19 146GSK1349572 15 28 95 17 16 140GSK1349572 50 27 116 19 16 162GSK1349572 150 26 122 20 18 129GSK1349572 500 21 753 203 11 122GSK1349572 849 24 283 133 33 124

2-Nitrofluorene 5 15482 NT NT NT NTSodium Azide 2 NT 10602 7572 NT NT

9-Aminoacridine 50 NT NT NT 1822 NT4-Nitroquinoline-1-Oxide 2 NT NT NT NT 18352

With Activation DMSO 100 L/plate 40 117 14 21 198GSK1349572 5 36 120 22 15 187GSK1349572 15 38 108 15 18 209GSK1349572 50 34 111 19 19 204GSK1349572 150 38 124 19 23 221GSK1349572 500 29 98 11 183 188GSK1349572 849 25 333 163 153 151

Benzo[a]pyrene 10 4232 NT NT NT NT2-Aminoanthracene 5 NT 15962 2242 1252 NT2-Aminoanthracene 10 NT NT NT NT 12002

Key:1. All concentrations are expressed in terms of parent compound2. two fold increase in revertants for TA98, TA100 and WP2uvrA(pKM101) and three fold increase for TA1535 and TA15373. Slight thinning of background bacterial lawnNT Not tested


50

Table 8.2 GSK1349572 In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus

Study Type: Genetic Toxicology In VitroReport Title: GSK1349572 In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus

Test Article: GSK1349572 Batch Number: U23359/4/1

Test for Induction of: Forward mutation at the TK+/- locus

No. of Independent Tests: 2 Report No.: WD2007/01581CTD Module: m4.2.3.3.1Study No: MLA-580

Cell Type: L5178Y Mouse Lymphoma Cells 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: NoTreatment: 3 hr treatment with S9-mix; treatment for 24 hr without S9-mix Date of Treatment(s): May, 2006Cytotoxic Effects: Reduction in RTGGenotoxic Effects: Positive

Test ArticleDose Level1

mcg/mL

3 hr Treatment +S9-mix 24 hr Treatment -S9-mixMean Relative Total

Growth(%)

Mean MutantFrequency (x10-6)

Mean Relative Total Growth

(%)

Mean MutantFrequency (x10-6)

DMSO 0 100 86.69 100 79.2GSK1349572 5 81 113.21GSK1349572 10 49 102.58GSK1349572 20 15 279.041

GSK1349572 30 60 119.24GSK1349572 40 57 160.54GSK1349572 60 55 110.79GSK1349572 80 54 130.44

Methyl methane sulphonate 5 80 477.061

Dimethylbenzanthracene 1 21 1019.681

Key:1 = Mean mutant frequency exceeds the sum of the mean control mutant frequency plus Global Evaluation Factor (126x10-6)


51

Table 8.3 GSK1349572A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus

Genotoxicity: In Vitro Report Title: GSK1349572A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus

Test Article: GSK1349572A (sodium salt)Batch Number: R06001

Test for Induction of: Forward mutation at the TK+/-

locusNo. of Independent Tests: 3 Covance Study No: 2990/121

GSK Ref No: V27468Report No.: WD2007/00515CTD Module: m4.2.3.3.1

Cell Type: L5178Y Mouse Lymphoma Cells No. of Replicate Cultures: 4 (vehicle);2 (treatment and positive controls)

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


g/mL

3 hr Treatment -S9-mix 3 hr Treatment +S9-mix 24 hr Treatment -S9-mix

Mean RelativeTotal Growth

(%)

Mean MutantFrequency

(x10-6)


(%)


(x10-6)


(%)


(x10-6)

DMSO 0 100 84.06 100 78.30 100 74.05

GSK1349572 5 96 77.13 107 54.86 75 72.25

GSK1349572 10 71 62.86 104 54.60 68 75.78

GSK1349572 15 NT NT NT NT 54 93.75

GSK1349572 20 73 83.51 94 64.15 47 73.37

GSK1349572 30 74 81.87 92 56.25 38 94.69

GSK1349572 40 72 72.44 77 63.70 30 81.00

GSK1349572 50 65 78.07 78 76.68 28 92.05

GSK1349572 60 70 87.68 86 80.73 28 83.43

GSK1349572 70 56 75.22 83 62.32 29 97.31

GSK1349572 80 69 67.36 81 74.80 NT NT

GSK1349572 852 532 81.082 812 65.852 26 74.38

Methyl methane sulphonate 15 39 896.08 NT NT NT NT


52

Table 8.3 (Continued) GSK1349572A: In Vitro Mutation Assay with L5178Y Mouse Lymphoma Cells at the TK Locus


g/mL

3 hr Treatment -S9-mix 3 hr Treatment +S9-mix 24 hr Treatment -S9-mix


(%)


(x10-6)


(%)


(x10-6)


(%)


(x10-6)

Methyl methane sulphonate 5 NT NT NT NT 35 912.04

Benzo[a]pyrene 2 NT NT 50 526.43 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.


53

9. GENOTOXICITY: IN VIVO

Table 9.1 GSK1349572A: Oral Bone Marrow Micronucleus Assay in Rats

Genetic Toxicology: In Vivo Report Title: GSK1349572A: Oral Bone Marrow Micronucleus Assay in Rats

Test Article: GSK1349572A (sodium salt)Batch Number: R06001

Test for Induction of: Structural chromosomal damage and/or aneuploidy

Treatment Schedule: Two doses 24 hours apart (vehicle & test compound).Positive control dosed once only.

PCS-MTL Study No.: 961547GSK Reference No.: R27469Report No.: WD2007/00513CTD Module: m4.2.3.3.2

Species/Strain: Rat (Hsd:SD) Sampling Time: 24 hours after final doseApproximate Age: 8 weeks at time of dosing Method of Administration: Oral

Dose volume: 10 mL/kgGLP Compliance: Yes

Cells Evaluated: Polychromatic erythrocytes (PCE) Vehicle/Formulation: 0.5% Hydroxypropylmethylcellulose (HPMC, K15M Premium) (w/v) with 0.1% Tween 80 (w/v) in reverse osmosis water (deionized water)

Date of Dosing: April 2007No. of Cells Analyzed/Animal: 2000 PCE

Special Features: NoneToxic/Cytotoxic Effects: NoneGenotoxic Effects: NegativeEvidence of Exposure: Exposure previously demonstrated in a 14-day toxicity study in SD rats [Sponsor Study No. E-349572-TB-012-L]

Test Compound Dose1

(mg/kg/day)Number of Animals

Analyzed2

Group Mean% PCE

Group MeanMPCE3

Vehicle 0 6M 53.3 2.7GSK1349572 50 6M 52.7 2.3GSK1349572 150 6M 52.6 2.5GSK1349572 500 6M 53.9 1.5

Cyclophosphamide 20 3M 45.7 56.7

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.


54

10. 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

Females: Group 5: 101 weeks

Groups 1-4: 102 -103 weeks

Study No.: 09-2177Report No.: 2012N152419

Initial Age: 7 weeks Method of Administration: Oral gavage Location in CTD: m4.2.3.4.1

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 neoplastic lesions

Treatment of Controls: Group 1 – Sterile Water for Injection, USP

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)

GLP Compliance: Yes

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.



0

(Water Control)

0

(Vehicle Control) 7.5 25 500

Gender M F M F M F M F M F

Toxicokinetics

Number of Animals 20 20 20 20 45 45 45 45 45 45

AUC0-24 (g.h/mL) – Day 26 BLQ BLQ BLQ BLQ 176 235 579 565 1180 1300


Cmax (g/mL) – Day 26 BLQ BLQ BLQ BLQ 13.4 20.3 40.8 44.4 77.7 94.7

Cmax (g/mL) – Day 182 BLQ BLQ BLQ BLQ 14.5 16.6 27.4 43.3 71.9 94.5Number of Animals

At Start 65 65 65 65 65 65 65 65 65 65Died/Sacrificed Moribund 35 45 36 38 28 42 39 42 39 46

Terminal Sacrifice 30 20 29 27 37 23 26 23 26 19Survival (%) 46.2% 30.8% 44.6% 41.5% 56.9% 35.4% 40.0% 35.4% 40.0% 29.2%


55

Table 10.1 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Mice for 104 Weeks

Daily Dose (mg/kg) 0(Water Control)

0(Vehicle Control) 7.5 25 500

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Drug-Relateda - - - - - - - - -- -Neoplastic Lesions - Incidental/SpontaneousAdipose Tissueb

N-Metastatic/Invasive Neoplasm 0[3] 3[4] 1[1] 0[1] NE 1[1] 1[1] 0[3] 2[3] 2[2]Adrenal Glandsc

N-Metastatic/Invasive Neoplasm 6 16 6(64) 6(64) 4(64) 6 5(63) 3(64) 3(64) 8B-Cortex: Adenoma 0 0 1(64) 2(64) 1(64) 0 1(63) 1(64) 1(64) 0B-Medulla Benign Pheochromocytoma 1 0 0(64) 0(64) 0(64) 0 0(64) 0(64) 0(64) 0M-Cortex: Carcinoma 0 0 0(64) 0(64) 0(64) 0 0(64) 1(64) 0(64) 0

Aortac

N-Metastatic/Invasive Neoplasm 4 6(64) 3 6 2(64) 3 2 4 0 7Bone (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]

BrainN-Metastatic/Invasive Neoplasm 1 2 0 2 1 1 0 0 0 0

Cecumc

N-Metastatic/Invasive Neoplasm 5[63] 5[64] 6[64] 0 0 0 1[63] 2 0 1[63]Cervical, Spinal Cord

N-Metastatic/Invasive Neoplasm 0 0 0 1 1 1 0 0 0 0Colonc

N-Metastatic/Invasive Neoplasm 0 3 3 0 0 0 0 0 0 0(64)Diaphragmb

N-Metastatic/Invasive Neoplasm NE 1[1] 1[1] NE NE NE 1[1] NE NE NEDistal femur

N-Metastatic/Invasive Neoplasm 0 0(64) 0 1(64) 0 0 0 0 0 0B-Osteoma 0 0(64) 0 1(64) 0 0 0 0 0 0

Duodenumc

N-Metastatic/Invasive Neoplasm 1 3(64) 2(64) 1(64) 0(62) 0 1(61) 1 0 0(64)


56




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousEar(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 NEEsophagusc

N-Metastatic/Invasive Neoplasm 2 3 2 2(64) 3 3 2(64) 5 2 5Eyes

N-Metastatic/Invasive Neoplasm 3 3 1 0 1 1 0 0 1 3Femoral Marrow

N-Metastatic/Invasive Neoplasm 3 3 2 1(64) 0 0 1 1 0 3B-Hemangioma 0 1 0 0(64) 1 0 0 0 0 0

Gallbladderc

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 Glandc

N-Metastatic/Invasive Neoplasm 3 13 7 4 1 0 2(64) 2 3 6B-Adenoma 11 4 11 10 14 5 9(64) 7 4 9M-Carcinoma 1 0 0 0 0 0 0(64) 0 0 0

HeartN-Metastatic/Invasive Neoplasm 3 9 3 5 4 4 4 4 3 6M-Endocardial Schwannoma 0 1 0 0 0 0 0 0 0 0M-Hemangiosarcoma 0 0 0 0 0 1 0 0 0 0

Ileumc

N-Metastatic/Invasive Neoplasm 4(64) 5 3(64) 0(64) 0 1 1(64) 0 1 0(64)Intercostal Muscb

N-Metastatic/Invasive Neoplasm NE NE 1[1] NE NE NE NE NE NE NEJejunumc

N-Metastatic/Invasive Neoplasm 2 1 1 0(64) 0(64) 0 0(62) 1 0(64) 0Kidneysc

N-Metastatic/Invasive Neoplasm 11 17(64) 12 6 5 7 6 10 5 15


57




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousLacrimal Glandc

N-Metastatic/Invasive Neoplasm 3 13(63) 8 5 3 2 2(64) 2 4 6B-Adenoma 0 0(63) 0 0 0 0 0(64) 0 1 0

LiverN-Metastatic/Invasive Neoplasm 12 18 12 10 7 7 9 6 6 14M-Hepatocellular Carcinoma 7 0 7 0 11 0 10 0 6 1B-Hepatocellular Adenoma 11 0 13 0 15 0 16 0 10 1M-Hemangiosarcoma 3 1 2 0 5 1 4 0 1 0B-Hemangioma 0 0 0 0 1 0 0 0 0 0

LungsN-Metastatic/Invasive Neoplasm 6 15 6 7 3 4 5 9 2 7B-Bronchiolo/Alveolar Adenoma 12 8 8 0 15 4 5 8 6 5M-Bronchiolo/Alveolar Carcinoma 10 2 3 4 8 5 9 5 11 2

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 14 27 17 16 8 9 6 14 10 19M-Histiocytic Sarcoma 3 2 1 3 2 3 3 6 1 4

Mammary (protocol)c

N-Metastatic/Invasive Neoplasm 4(63) 18 4(60) 4(64) 3(63) 4 3 5 3(62) 7(64)M-Adenocarcinoma 0(63) 0 0(60) 0(64) 0(63) 0 0 1 0(62) 1(64)

Mandibular Lymph Nodeb

N-Metastatic/Invasive Neoplasm NE NE NE 1[1] NE NE NE NE NE NEMediastinal Lymph Nodec

N-Metastatic/Invasive Neoplasm 12(61) 24 16(63) 13 4(62) 8 8(61) 15(63) 8(57) 17Mesenteric Lymph Nodec

N-Metastatic/Invasive Neoplasm 12 24(64) 14(63) 12(64) 4(64) 6(64) 5(64) 9(62) 6(61) 12M-Hemangiosarcoma 0 0(64) 0(63) 0(64) 0(64) 0(64) 1(64) 0(62) 0(61) 0

Mesentery/Peritoneumb

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]


58




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousMesovarian Ligamentb

N-Metastatic/Invasive Neoplasm NE 1[1] NE NE NE NE NE NE NE NEMuscle (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 1 11 3 1 2 0 3 2 2 3M-Hemangiosarcoma 1 0 0 0 0 0 0 0 0 0

Nerve - Sciaticc

N-Metastatic/Invasive Neoplasm 0 9 3 3(64) 1(64) 0 1 3 2 2(63)Ovariesc

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/GALTc

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 7 16 11 7 4 4 4 8 5 9B-Islet Cell Adenoma 1 0 1 0 0 0 1 1 0 0

Parotid Glandb

N-Metastatic/Invasive Neoplasm NE 2[2] NE NE 1[2] NE 0[1] NE NE NEPericardiumb

N-Metastatic/Invasive Neoplasm NE NE 1[1] 1[1] 0[1] 1[1] NE NE NE 1[1]Pituitaryc

N-Metastatic/Invasive Neoplasm 1 2(64) 0(64) 1 0(64) 0(64) 1(62) 0(64) 0(64) 1B-Pars Distalis-Adenoma 0 0(64) 0(64) 0 0(64) 2(64) 0(62) 0(64) 0(64) 1B-Pars Intermedia: Adenoma 0 0(64) 0(64) 0 0(64) 0(64) 0(62) 0(64) 0(64) 0


59




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousProstatec

N-Metastatic/Invasive Neoplasm 6 NE 8 NE 6(63) NE 4 NE 4 NEM-Carcinoma 0 NE 0 NE 0(63) NE 1 NE 0 NE

Rectum/Low Colonb

N-Metastatic/Invasive Neoplasm NE 1[1] 0[1] NE NE NE NE 0[1] NE NESalivary Gland

N-Metastatic/Invasive Neoplasm 7 11 8 4 4 3 2 1 3 9Seminal Vesicles

N-Metastatic/Invasive Neoplasm 3 NE 7 NE 2 NE 1 NE 2 NESkin (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 1 6 2 0 1(64) 1 0 1 0 0B-Schwannoma 0 0 1 0 0(64) 0 0 1 1 0M-Fibrosarcoma 0 1 0 0 1(64) 0 0 0 0 0

Spleenc

N-Metastatic/Invasive Neoplasm 10(64) 21 14 12 5(64) 5 5 11 8 14B-Hemangioma 0(64) 0 1 0 0(64) 0 0 0 0 0M-Hemangiosarcoma 0(64) 2 0 1 0(64) 0 0 1 0 1

Sternal Marrowc

N-Metastatic/Invasive Neoplasm 3 5 2 0 0(64) 1 1 0 2(64) 3Sternumc

N-Metastatic/Invasive Neoplasm 4 12 3 2 2(64) 2 5 4 6(64) 8M-Osteogenic Sarcoma 0 1 0 0 0(64) 0 0 0 0(64) 0


60




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousStomachc

N-Metastatic/Invasive Neoplasm 3 10 7 0 2(64) 1 2(64) 5 2(64) 6B-Glandular Mucosa: Adenoma 1 0 0 0 0(64) 0 0(64) 1 0(64) 1M-Forestomach: Squamous Cell Carcinoma 0 0 0 0 0(64) 0 0(64) 1 0(64) 0

TestesN-Metastatic/Invasive Neoplasm 0 NE 1 NE 1 NE 0 NE 0 NEB-Benign Interstitial Cell Tumor 1 NE 1 NE 0 NE 2 NE 1 NE

Thymusc

N-Metastatic/Invasive Neoplasm 11(63) 24 17(60) 12(62) 7(60) 10 7(63) 16(64) 9(63) 15Thyroidc

N-Metastatic/Invasive Neoplasm 0 3 1 1 1(64) 1 1 1 1(61) 1(63)B-Follicular Cell Adenoma 0 0 0 0 1(64) 0 0 1 1(61) 1(63)

Tracheac

N-Metastatic/Invasive Neoplasm 1 4 1 0 2 2 1(64) 2 2 0Uretersb

N-Metastatic/Invasive Neoplasm 0[2] 3[4] 2[3] 0[1] 1[1] 1[1] 0[1] NE 1[4] NEUrinary Bladderc

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/CervixN-Metastatic/Invasive Neoplasm NE 11 NE 4 NE 6 NE 7 NE 4M-Endometrial Carcinoma NE 0 NE 1 NE 0 NE 1 NE 0B-Endometrial Stromal Polyp NE 2 NE 5 NE 4 NE 9 NE 7M-Leiomyosarcoma NE 1 NE 0 NE 0 NE 3 NE 1B-Hemangioma NE 0 NE 1 NE 3 NE 1 NE 1B-Benign Granular Cell Tumor NE 0 NE 0 NE 1 NE 0 NE 0M-Endometrial Stromal Sarcoma NE 2 NE 1 NE 0 NE 0 NE 0B-Leiomyoma NE 1 NE 0 NE 2 NE 4 NE 1M-Granular Cell Tumor NE 1 NE 0 NE 0 NE 0 NE 0M-Hemangiosarcoma NE 0 NE 0 NE 0 NE 1 NE 0


61




Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousVaginac

N-Metastatic/Invasive Neoplasm NE 10(64) NE 1(63) NE 2 NE 2(64) NE 3(64)Vessel

C-Hemangiosarcoma 2 3 2 1 3 2 3 1 1 2C-Hemangioma 1 1 1 1 2 2 1 0 0 1

Zymbal’s Glandb

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

aThere were no test article-related neoplastic lesions.bNot a protocol required organ/tissue. Organs/Tissues were examined grossly; number in [ ] indicates number of organs/tissues examined microscopically.cMissing protocol-required organs/tissues in some groups; number in ( ) indicates number of organs/tissues examined microscopically.


62

Table 10.2 Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks

Species/Strain: Rats/Sprague Dawley Duration of Dosing: Males: 24 monthsFemales: Group 5: 88 weeks

Group 3: 90 weeksGroup 4: 94 weeksGroups 1 and 2: 95 weeks

Study No.: 09-2178

Report No.: 2012N152418

Initial Age: 7 weeks Method of Administration: Oral gavage Location in CTD: m4.2.3.4.1Date 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 neoplastic lesions.

Treatment of Controls: Group 1 – Sterile Water for Injection, USPGroup 2 - Aqueous 0.5 w/w% hydroxypropyl methylcellulose (HPMC) solution with 0.1 w/w% Tween 80 (0.5% HPMC/0.1% Tween 80)

GLP Compliance: Yes

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: NoneDaily Dose (mg/kg) 0

(Water Control)0

(Vehicle Control) 2 10 50Gender M F M F M F M F M FToxicokineticsNumber of Animals 4 4 4 4 12 12 12 12 12 12



Cmax (g/mL) – Day 28 BLQ BLQ BLQ BLQ 7.29 8.78 24.7 34.3 57.6 87.0

Cmax (g/mL) – Day 182 BLQ BLQ BLQ BLQ 7.71 20.4 21.3 40.4 40.9 68.5Number of Animals

At Start 65 65 65 65 65 65 65 65 65 65Died/Sacrificed Moribund 40 45 43 38 43 50 40 45 48 48Terminal Sacrifice 25 20 22 27 22 15 25 20 17 17

Survival (%) 38.5% 30.8 33.8% 41.5% 33.8% 23.1% 38.5% 30.8% 26.2% 26.2%

BLQ = Below lower limit of quantitation (<0.500 g/mL)


63

Table 10.2 (Continued) Carcinogenicity Study of Dolutegravir Sodium in Rats for 104 Weeks


0(Vehicle Control) 2 10 50

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Drug-Relateda - - - - - - - - -- -Neoplastic Lesions - Incidental/SpontaneousAdipose Tissueb

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 5 0 0 1 0 0 0 0 1 0

B-Medulla Benign Pheochromocytoma 4 0 5 0 3 1 4 1 4 1M-Cortex: Carcinoma 0 2 0 1 0 0 0 2 0 0B-Cortex: Adenoma 0 3 1 1 0 0 1 0 1 1M-Medulla: Malignant Pheochromocytoma 0 0 1 0 0 0 0 0 0 0

AortaN-Metastatic/Invasive Neoplasm 2 0 0 1 0 0 0 0 0 0

Bone (other)b

M-Histiocytoma, fibrous NE 0[2] NE NE 1[1] NE 0[2] NE 0[1]] NEB-Fibrosarcoma NE 0[2] NE NE 0[1] NE 0[2] NE 1[1] NE

BrainN-Metastatic/Invasive Neoplasm 1 0 0 0 0 0 0 0 0 0M-Astrocytoma 1 0 1 0 1 0 1 0 0 0B-Granular Cell Tumor 0 0 1 0 0 0 0 0 1 0M-Oligodendroglioma 0 0 1 0 0 0 1 2 0 0B-Benign Meningioma 0 0 1 0 0 0 0 0 0 0

CecumN-Metastatic/Invasive Neoplasm 1 0 0 1 0 1 0 0 1 0B-Fibroma 0 1 0 0 0 0 0 0 0 0

ColonN-Metastatic/Invasive Neoplasm 1 0 0 0 0 1 0 0 1 0

Diaphragmb

N-Metastatic/Invasive Neoplasm NE 0[1] NE NE NE NE 1[1] NE NE 1[1]


64




DuodenumN-Metastatic/Invasive Neoplasm 3 0 0 0 0 0 0 0 1 0

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousEar(s)b

M-Fibrosarcoma NE NE 0[1] NE NE NE NE NE 1[1] NEEpididymides

N-Metastatic/Invasive Neoplasm 5 NE 1 NE 0 NE 0 NE 2 NEEsophagus

N-Metastatic/Invasive Neoplasm 1 0 0 0 0 0 0 0 1 1Extremityb

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]

EyesN-Metastatic/Invasive Neoplasm 4 0 0 0 0 1 0 0 0 0

Femoral MarrowN-Metastatic/Invasive Neoplasm 5 1 1 0 0 0 0 0 0 0

Harderian GlandN-Metastatic/Invasive Neoplasm 4 0 0 1 0 1 0 0 0 0

Heartc

N-Metastatic/Invasive Neoplasm 2 0(64) 0 0 0 0(64) 1 0 0 1Ileum

N-Metastatic/Invasive Neoplasm 0 0 0 1 0 1 0 0 0 0M-Adenocarcinoma 0 0 0 1 0 0 0 0 0 0

JejunumN-Metastatic/Invasive Neoplasm 0 0 0 0 0 1 0 0 0 0B-Leiomyoma 0 0 0 0 0 0 0 1 0 0M-Neuroendocrine Neoplasm 0 0 0 0 0 0 1 0 0 0M-Leiomyosarcoma 0 0 0 1 0 0 0 0 0 0


65




KidneysN-Metastatic/Invasive Neoplasm 6 0 0 1 0 2 0 0 2 0M-Carcinoma, Tubular 0 0 0 0 0 0 1 0 0 0M-Carcinoma, Transitional Cell 0 0 0 0 0 0 0 0 1 0M-Liposarcoma 0 0 0 0 0 0 0 0 1 0

Lacrimal GlandN-Metastatic/Invasive Neoplasm 3 0 0 0 0 1 0 0 1 0

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousLiver

N-Metastatic/Invasive Neoplasm 8 2 0 1 2 1 1 0 1 0B-Hepatocellular Adenoma 3 1 2 2 2 3 2 1 1 3M-Hepatolcellular Carcinoma 0 1 1 1 1 0 0 0 0 0M-Hemangiosarcoma 0 0 0 0 0 0 0 0 0 1

LungsN-Metastatic/Invasive Neoplasm 6 1 1 2 2 0 1 0 1 0M-Carcinoma Nos 0 0 0 1 0 0 0 0 0 0M-Bronchiolo/Alveolar Carcinoma 0 0 0 0 0 0 0 0 0 1M-Hemangiosarcoma 0 0 0 0 0 0 0 0 0 1

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 4 1 1 0 2 0 0 0 1 0M-Malignant Lymphoma 3 2 0 1 0 1 1 0 0 1M-Granulocytic Leukemia 1 0 0 0 0 0 0 0 0 0

Mammary (protocol)c

N-Metastatic/Invasive Neoplasm 2(56) 0 0(59) 0 0(54) 1 0(54) 0 0(52) 0M-Adenocarcinoma 0(56) 19 0(59) 11 0(54) 14 0(54) 22 0(52) 17B-Fibroadenoma 0(56) 20 1(59) 28 0(54) 17 0(54) 29 0(52) 15B-Adenoma 0(56) 1 0(59) 1 0(54) 4 0(54) 3 0(52) 2


66




Mediastinal Lymph NodeN-Metastatic/Invasive Neoplasm 6 3 0 1 0 1 1 0 1 1

Meningesb

M-Malignant Meningioma NE NE 1[1] NE NE NE NE NE NE NE

Mesenteric Lymph Nodec

N-Metastatic/Invasive Neoplasm 4 0(64) 0 1 0 1 0 0 1 0Mesentery/Peritob

M-Liposarcoma NE 0[1] NE 0[1] NE 1[1] 0[1] 0[1] 0[2] NEM-Malignant Mesothelioma NE 0[1] NE 0[1] NE 0[1] 0[1] 0[1] 1[2] NEB-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 FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousMuscle (other)b

N-Metastatic/Invasive Neoplasm 1[1] NE 0[1] NE NE 1[2] 0[2] NE 0[1] NEM-Carcinoma (Nos) 0[1] NE 0[1] NE NE 1[2] 0[2] NE 0[1] NE

Muscle (protocol)N-Metastatic/Invasive Neoplasm 3 0 1 1 1 0 0 0 1 0

Nerve - SciaticN-Metastatic/Invasive Neoplasm 4 0(64) 0 1 0 1 0 0 0(64) 0

OvariesN-Metastatic/Invasive Neoplasm NE 0 NE 1 NE 0 NE 0 NE 0

Peyer’s Patches/GALTN-Metastatic/Invasive Neoplasm 1 0 0 1 0 1 0 0 0 0

PancreasN-Metastatic/Invasive Neoplasm 3 0 0 1 0 1 0 0 1 0B-Islet Cell Adenoma 2 2 3 1 1 1 7 1 4 0M-Islet Cell Carcinoma 2 1 3 2 1 0 3 2 5 2B-Hemangioma 0 0 0 0 1 0 0 0 0 0B-Acinar Cell Adenoma 1 0 0 0 0 0 1 0 1 0


67




ParathyroidB-Adenoma 0(61) 0(62) 1(64) 0(63) 0(61) 0(59) 0(63) 0(64) 0(63) 0

Pericardiumb

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 28 58 22 51 29 59 25 55 29 55M-Pars Distalis: Carcinoma 1 1 0 1 0 2 0 3 1 1N-Metastatic/Invasive Neoplasm 3 0 0 1 0 0 0 0 0 0B-Pars Intermedia: Adenoma 0 0 0 0 0 0 0 0 1 0

ProstateN-Metastatic/Invasive Neoplasm 4 NE 1 NE 0 NE 0 NE 1 NE

Rectum/Low Colonb

B-Fibroma NE 1[1] NE NE NE NE NE NE NE NEGender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/SpontaneousSalivary Glandc

N-Metastatic/Invasive Neoplasm 2 1 0(64) 1 0 1 0 0 1 0B-Leiomyoma 0 0 0(64) 0 0 0 1 0 0 0

Seminal VesiclesN-Metastatic/Invasive Neoplasm 1 NE 0 NE 0 NE 0 NE 1 NE


68




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 0 0 1 1 0 0 0 0 0 0M-Histiocytoma, Fibrous 0 0 0 0 0 0 0 0 0 1

Soft Tissueb

N-Metastatic/Invasive Neoplasm 1[1] 0[1] NE NE NE 1[1] NE NE NE NESpleen

N-Metastatic/Invasive Neoplasm 5 2 0 1 1 1 1 0 0 0B-Hemangioma 0 0 0 0 1 0 0 0 0 0

Sternal MarrowN-Metastatic/Invasive Neoplasm 5 1 1 0 0 0 0 0 0 0

StomachN-Metastatic/Invasive Neoplasm 2 0 0 1 0 1 0 0 2 0M-Carcinoma, Squamous Cell 0 0 1 0 0 0 0 0 0 0

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/Spontaneous


69




Tailb

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]

TestesN-Metastatic/Invasive Neoplasm 2 NE 0 NE 0 NE 0 NE 0 NEB-Benign Interstitial Cell Tumor 0 NE 1 NE 0 NE 2 NE 1 NE

Thymusc

N-Metastatic/Invasive Neoplasm 4[62] 1[64] 0[64] 1 0 1[63] 0[64] 0[62] 1 2M-Malignant Thymoma 0[62] 0[64] 0[64] 0 0 0[63] 1[64] 1[62] 0 0

ThyroidM-Carcinoma, Follicular Cell 2 1 0 0 1 1 0 1 0 0B-Follicular, Cell Adenoma 2 1 0 0 4 0 1 0 2 0N-Metastatic/Invasive Neoplasm 2 0 0 1 0 0 0 0 0 0B-C-Cell Adenoma 4 1 4 1 2 4 5 3 1 1M-C-Cell Carcinoma 2 0 0 2 1 0 1 1 2 1

TracheaN-Metastatic/Invasive Neoplasm 2 0 0 0 0 0 0 0 0 0

Urinary BladderN-Metastatic/Invasive Neoplasm 0 0(64) 0 0 0 0(64) 0 0 1 0(64)

Uterus w/CervixB-Endometrial Stromal Polyp NE 2 NE 3 NE 1 NE 2 NE 2N-Metastatic/Invasive Neoplasm NE 0 NE 1 NE 1 NE 0 NE 0B-Benign Schwannoma NE 0 NE 0 NE 0 NE 0 NE 1B-Leiomyoma NE 0 NE 1 NE 0 NE 0 NE 0

VaginaM-Granular Cell Tumor NE 0 NE 0 NE 0 NE 1 NE 0N-Metastatic/Invasive Neoplasm NE 0 NE 0 NE 1 NE 0 NE 0

Gender M F M F M F M F M FNumber of Evaluated 65 65 65 65 65 65 65 65 65 65Neoplastic Lesions - Incidental/Spontaneous


70




Vascular Tissueb

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 Glandb

B-Adenoma 0[1] NE NE NE 1[1] NE NE NE NE NEM-Carcinoma 1[1] NE NE NE 0[1] NE NE NE NE NE

Key:NE = Not examineda. 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.


71

11. 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)




Oral (gavage) 12 days(gestation days 6 to

17)

100300

1000

7F7F7F

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-349572 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.

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-349572 sodium on embryo-fetal development in rats. To assess the dose-response relationship, 300 and 100 mg/kg/day are considered to be suitable forthe middle and low dose levels, respectively.

RD2008/01761S-349572-TB-051-L

(m4.2.3.5.2)


72

Table 11.1 (Continued) Reproductive and Developmental Toxicity: Nonpivotal Studies

Species/ Strain Route (Vehicle/ Formulation)

Duration of Dosing

Doses (mg/kg)



Rat(Sprague Dawley)

Oral (0.5% hydroxypropyl-methylcellulose (HPMC) K15M /

0.1% polyoxyethylene

sorbitan monooleate

(Tween™80)./ suspension

Day 4 to 21

postpartum (pp)

inclusive

550

100500

1000

8/sexDose 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.

CD2009/00409D09072

(m4.2.3.5.4)

Rats (Sprague Dawley)

Oral (0.5% hydroxypropyl-methylcellulose (HPMC) K15M /

0.1% polyoxyethylene

sorbitan monooleate

(Tween™80)./ solution or suspension

Day 4 to 31

postpartum (pp)

inclusive

02

2575

300

20/sexA 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 causedreductions 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.

CD2009/00770D09126

(m4.2.3.5.4)


73

Table 11.2 Two week Oral toxicity Study of S-349572 Sodium in Non Pregnant Rabbits: Non-Pivotal Studies

Report No.: RD2008/01760 CTD Module: m4.2.3.5.2


74

Table 11.2 (Continued) Two week Oral toxicity Study of S-349572 Sodium in Non Pregnant Rabbits: Non-Pivotal Studies


75

Table 11.3 Dose Range Finding Oral Study for Effects of S-349572 sodium on Embryofetal Development in Rabbits:Non-Pivotal Studies



76

Table 11.3 (Continued) Dose Range Finding Oral Study for Effects of S-349572 sodium on Embryofetal Development in Rabbits: Non-Pivotal Studies


77

12. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: FERTILITY AND EARLY EMBRYONIC DEVELOPMENT TO IMPLANTATION (PIVOTAL)

Table 12.1 Oral Study for Effects of S-349572 Sodium on Fertility and Early Embryonic Development to Implantation in Rats

Reproductive and Developmental Toxicology -Fertility and Early Embryonic Development to Implantation

Study Title: Oral Study for Effects of S-349572 Sodium on Fertility and Early Embryonic Development to Implantation in Rats


Design Similar to ICH 4.1.1? Yes Duration of Dosing: M: 4 weeks prior to mating (63 to 66 total days)F: 2 weeks prior to mating, through Day 7 of gestation (23 to 42 total days)

Report No.: XD2009/00368CTD Module: m4.2.3.5.1Study No.: SG08065

Species/Strain: Rats/Sprague Dawley Sponsor’s Study No.: S-349572-TF-063-L

Initial Age of Dosing: M: 9 weeksF: 10 weeks


Day of Mating: Day 0

Date of First Dose: M: 14 Oct 2008F: 28 Oct 2008

Day of C-Section: Day 13 of gestation GLP Compliance: Yes

Method of Administration: Oral Gavage

Vehicle/Formulation: 0.5% w/w hydroxypropylmethylcellulose in 0.1% w/w Tween 80 aqueous solution/Suspension

No Observed Adverse Effect Level: F0 Males: 1000 mg/kg/dayF0 Females: 1000 mg/kg/dayF1 Litters: 1000 mg/kg/day


78

Table 12.1 (Continued) Oral Study for Effects of S-349572 Sodium on Fertility and Early Embryonic Development to Implantation in Rats

Daily Dose (mg/kg) 0 (Control) 100 300 1000Males No. Evaluated 20 20 20 20

No. Died or Sacrificed Moribund 0 0 0 0Clinical Observations – – – –Necropsy Observations – – – –Body Weights (% a) a 438 g −1 −1 0Food Consumption (% a) a 27.6 g/day −2 −3 +2Genital Organ Weightsb – – – –Sperm Evaluationsc – – – –Mean No. Days Prior to Mating 2.7 2.9 3.2 3.0No. of Males that Mated 19 20 19 20No. of Fertile Males 19 20 19 20

Key:a = After 4 weeks of dosing. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical significance

is based on actual data (not on the percent differences).b = Testes, epididyumides, seminal vesicles (including coagulating glands) and (prostate ventral lobe).c = Sperm motility total sperm count and sperm malformations.- = No noteworthy findings.


79

Table 12.1 (Continued) Oral Study for Effects of S-349572 Sodium on Fertility and Early Embryonic Development to Implantation in Rats

Daily Dose (mg/kg) 0 (control) 100 300 1000Females No. Evaluated 20 20 20 20

No. Died or Sacrificed Moribund 0 0 0 0Clinical Observations – – – –Necropsy Observations – – – –Pre-mating Body Weights (%) d 268 g +1 +2 +2Gestation Body Weights (%) d 312 g +2 +5 * +4Pre-mating Food Consumption (%) d 20.4 g/day +1 0 +1Gestation Food Consumption (%) d 26.4 g/day –2 +6 +3Mean No. Estrous Cycles/15 days 3.3 3.3 3.0 3.2Mean No. Days Prior to Mating 2.7 2.9 3.2 3.0No. of Females Sperm-Positive e 19 20 20 20No. of Pregnant Females e 19 20 20 20Mean No. Corpora Lutea 16.9 16.2 16.8 16.2Mean No. Implantations 14.9 14.9 15.6 14.8Mean % Pre-implantation Loss 12.3 7.8 7.0 11.5Mean No. Live Embryos 14.2 14.2 14.9 13.8Mean % Post-implantation Loss 6.0 4.5 4.5 6.0

Key:d = At end of pre-mating or dosing period on Day 15 of dosing or dosing period on Day 8 of gestation. For controls, group means are shown. For

treated groups, percent differences from controls are shown. Statistical significance is based on actual data (not on the percent differences).e = Total of the 1st and 2nd mating periods.Dunnett’s Test * - p < 0.05.


80

13. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON EMBRYOFETAL DEVELOPMENT (PIVOTAL)

Table 13.1 Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rats

Report No.: XD2009/00367 CTD Module: m4.2.3.5.2


81

Table 13.1 (Continued) Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rats


82



83



84



85

Table 13.2 Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rabbits

Reproductive and Developmental Toxicology -Embryofetal Development Study

Study Title: Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rabbits


Design similar to ICH 4.1.3? Yes Duration of Dosing: G6-G18 Report No.: XD2009/00366CTD Module: m4.2.3.5.2Study No.: SG08064

Species/Strain/Sex: Rabbits/Kbl:JW/Female Day of C-Section: G29 Sponsor’s Study No.: S-349572-TF-065-L

Day of Mating: G0 Method of Administration: Oral GavageInitial Age of Dosing: 19 weeks Special Features: None GLP Compliance: YesDate of First Dose: Nov. 10, 2008Vehicle/Formulation: 0.5 w/w% hydroxypropyl methylcellulose in 0.1 w/w% Tween 80 aqueous solution/SuspensionNo Observed Adverse-Effect Level:F0 Females: 200 mg/kg/day for general toxicity and 1000 mg/kg/day for reproductive toxicityF1 Litters: 1000 mg/kg/dayDaily Dose (mg/kg) 0 (control) 40 200 1000Dams No. Pregnant Animals 20 20 18 19

Toxicokinetics: AUC0-24h (g.h/mL) G6 NE 2.1 15.6 36.8

AUC0-24h (g.h/mL) G18 NE 2.6 14.5 30.1

Cmax (g/mL) G6 NE 0.8 1.5 2.3

Cmax (g/mL) G18 NE 1.3 1.7 2.1No. Died or Sacrificed Moribund 0 0 0 0No. Aborted or with Total Resorption of Litter 0 1 a 0 0Clinical Observations: No. Scant Feces/Urine 2 5 5 7Necropsy Observations – – – –

Key:a = Abortion on G22.G = Gestational day.NE = Not estimated.– = No noteworthy findings.


86

Table 13.2 (Continued) Oral Study for Effects of S-349572 Sodium on Embryofetal Development in Rabbits

Daily Dose (mg/kg) 0 (control) 40 200 1000Dams Body Weights (%) b G7 3.65 kg 0 0 −2

G19 3.88 kg −1 −2 −4Body Weight Gain from G6 G7 −0.01 kg 0.00 kg −0.03 kg −0.10 kg **

G19 0.22 kg 0.21 kg 0.13 kg * 0.03 kg **Food Consumption (%) b G7 201 g/day +4 −14 −47 **

G19 199 g/day 0 −7 −18Mean No. Corpora Lutea 11.1 10.9 9.7 10.2Mean No. Implantation 9.3 9.5 8.3 8.6Mean % Pre-implantation Loss 14.9 12.1 15.3 15.2

Litters No. Litters Evaluated 20 19 18 19Mean No. Live Fetuses 8.2 8.8 7.9 7.4No. Dead Fetuses c 23 13 8 23Mean % Post-implantation Loss 11.2 7.3 5.7 14.7Mean Fetal Body Weight (g) Male 44.9 43.8 46.8 45.4

Female 44.0 43.0 45.4 43.6Mean Placental Weight (g) Male 3.55 3.39 3.89 3.67

Female 3.40 3.36 3.59 3.59Mean Fetal Sex Ratios (Males/Total) 0.51 0.54 0.58 0.53

Key:Dunnett’s Test: * - p < 0.05 ** - p < 0.01b = For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical significance is based on actual

data (not on the percent differences).c = Total of early deaths after implantation (implantation sites only), unformed fetuses and formed fetuses with placentae or macerated fetuses.


87


Daily Dose (mg/kg) 0 (control) 40 200 1000Litters Fetal Alterations:

External Malformations (%) d 1/163 (0.5) 1/167 (0.5) 0/142 0/141Cranioschisis

No. Fetuses (%) 0 1 (0.5) 0 0No. Litters (%) 0 1 (5.3) 0 0

OmphaloceleNo. Fetuses (%) 1 (0.5) 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Visceral Malformations (%) d 2/163 (1.1) 0/167 0/142 0/141Membranous ventricular septum defect

No. Fetuses (%) 1 (0.5) 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Small gallbladderNo. Fetuses (%) 1 (0.6) 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Key:d = Mean % of fetuses with malformations.


88


Daily Dose (mg/kg) 0 (control) 40 200 1000Litters Skeletal Malformations (%) d 5/163 (2.7) 3/167 (1.8) 4/142 (2.4) 2/141 (1.0)

Absent skullNo. Fetuses (%) 0 1 (0.5) 0 0No. Litters (%) 0 1 (5.3) 0 0

Split cervical archNo. Fetuses (%) 1 (0.5) e 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Absent thoracic archNo. Fetuses (%) 1 (0.7) f 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Fused thoracic archNo. Fetuses (%) 1 (0.7) f 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Absent thoracic centrumNo. Fetuses (%) 1 (0.7) f 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Fused thoracic centrumNo. Fetuses (%) 1 (0.5) g 0 1 (0.6) h 0No. Litters (%) 1 (5.0) 0 1 (5.6) 0

Hemicentric thoracic centrumNo. Fetuses (%) 1 (0.5) 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Key:d - Mean % of fetuses with malformations.e, f, g, h - The skeletal malformation with each shoulder note was observed in the same fetus.


89


Daily Dose (mg/kg) 0 (control) 40 200 1000Litters Thoracic hemivertebra

No. Fetuses (%) 1 (0.5) g 0 1 (0.6) h 0No. Litters (%) 1 (5.0) 0 1 (5.6) 0

Fused lumbar centrumNo. Fetuses (%) 1 (0.5) g 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Fused caudal centrumNo. Fetuses (%) 1 (0.5) 0 0 0No. Litters (%) 1 (5.0) 0 0 0

Fused sternebraNo. Fetuses (%) 1 (0.5) e 2 (1.2) 3 (1.9) 2 (1.0)No. Litters (%) 1 (5.0) 2 (10.5) 2 (11.1) 2 (10.5)

Absent ribNo. Fetuses (%) 1 (0.5) g 0 1 (0.6) h 0No. Litters (%) 1 (5.0) 0 1 (5.6) 0

Skeletal Variations (%) d 68/163 (43.6) 32/167 (19.3) * 34/142 (22.9) 29/141 (19.2) *Extra cranial ossification site

No. Fetuses (%) 4 (2.3) 1 (0.6) 1 (0.7) 3 (1.6)No. Litters (%) 3 (15.0) 1 (5.3) 1 (5.6) 2 (10.5)

Supernumerary lumbar vertebraNo. Fetuses (%) 10 (9.6) 6 (3.9) 4 (2.3) 1 (0.5)No. Litters (%) 5 (25.0) 5 (26.3) 2 (11.1) 1 (5.3)

Key:d - Mean % of fetuses with malformationsDunnett’s Test * - p < 0.05e, g, h - The skeletal malformation with each shoulder note was observed in the same fetus.


90


Daily Dose (mg/kg) 0 (control) 40 200 1000Litters Decreased lumbar vertebra

No. Fetuses (%) 0 0 1 (0.7) 1 (0.5)No. Litters (%) 0 0 1 (5.6) 1 (5.3)

LumbarizationNo. Fetuses (%) 1 (0.5) 1 (0.6) 0 1 (1.1)No. Litters (%) 1 (5.0) 1 (5.3) 0 1 (5.3)

Bipartite ossification of sternebraNo. Fetuses (%) 5 (2.7) 2 (1.2) 2 (1.2) 0No. Litters (%) 4 (20.0) 2 (10.5) 2 (11.1) 0

Misaligned sternebraNo. Fetuses (%) 1 (0.5) 0 0 2 (1.0)No. Litters (%) 1 (5.0) 0 0 2 (10.5)

Cervical ribNo. Fetuses (%) 12 (7.5) 15 (8.5) 9 (5.5) 6 (5.3)No. Litters (%) 6 (30.0) 8 (42.1) 5 (27.8) 5 (26.3)

Full supernumerary ribNo. Fetuses (%) 17 (9.5) 9 (5.6) 8 (4.8) 10 (6.0)No. Litters (%) 8 (40.0) 6 (31.6) 5 (27.8) 6 (31.6)

Short supernumerary ribNo. Fetuses (%) 32 (22.7) 8 (5.2) * 14 (10.7) 12 (7.1)No. Litters (%) 12 (60.0) 5 (26.3) 8 (44.4) 6 (31.6)

Degree of Skeletal Ossification – – – –

Key:– = No noteworthy findings.Dunnett’s Test * - p < 0.05


91

14. REPRODUCTIVE AND DEVELOPMENTAL TOXICITY: EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT INCLUDING MATERNAL FUNCTION (PIVOTAL)

Table 14.1 Oral Study for Effects of S-349572 Sodium on Pre- and Post-Natal Development, Including Maternal Function, in Rats

Reproductive and Developmental Toxicology - Effects on Pre- and Postnatal Development, Including Maternal Function

Study Title: Oral Study for Effects of S-349572 sodium on Pre- and Postnatal Development, Including Maternal Function, in Rats


Design similar to ICH 4.1.2.: Yes Duration of Dosing: G6-L20 Study No.: SG10306Day of Mating: G0 Method of Administration: Oral gavage Report No.: 2011N121663Species/Strain/Sex: Rats/Sprague Dawley/Female Initial Age: 10-12 weeks S-349572-TF-093-L CTD Module: m4.2.3.5.3Date of First Dose: 1 Nov 2010 Litters Culled/Not Culled: Culled to 4/sex/litter GLP Compliance: YesVehicle/Formulation: 0.5 w/w% hydroxypropyl methylcellulose in 0.1 w/w% Tween 80 aqueous solution/SuspensionSpecial Features: NoneNo Observed Adverse-Effect Level:

F0 : 50 mg/kg/day for general toxicity and 1000 mg/kg/day for reproductive functionF1 : 50 mg/kg/day

Daily Dose (mg/kg) 0 (control) 5 50 1000F0 Females: No. Pregnant 22 22 21 20

No. Died or Sacrificed Moribund 0 0 0 0Clinical Observations − − − −Necropsy Observations − − − −Gestation Body Weights (%a) G20 396 g +2 +2 −1Body Weight Gains from G6 G20 119 g 121 g 123 g 116 g

Key:−: No noteworthy findings G: Gestation day L: Lactation daya = For controls, group means are shown. For treated groups, percent differences from controls are shown.


92

Table 14.1 (Continued) Oral Study for Effects of S-349572 Sodium on Pre- and Post-Natal Development, Including Maternal Function, in Rats

Daily Dose (mg/kg) 0 (control) 5 50 1000F0 Females: Lactation Body Weights (%a) L4 310 g +4 +2 −3

Lactation Body Weights (%a) L7 322 g +2 +1 −2Lactation Body Weights (%a) L21 311 g +1 +1 +2Body Weight Gains from L0 L4 18 g 21 g 14 g −4 g **Body Weight Gains from L0 L7 29 g 30 g 25 g 10 g **Body Weight Gains from L0 L21 19 g 15 g 13 g 13 gGestation Food Consumption (%a) G20 24.1 g/day +2 +2 −3Lactation Food Consumption (%a) L4 28.7 g/day +5 +5 −14 *Lactation Food Consumption (%a) L7 45.3 g/day −4 −2 −8 *Lactation Food Consumption (%a) L21 67.9 g/day −5 −3 −6Mean Duration of Gestation (days) 21.8 21.9 21.8 22.0No. Delivery (Gestation Index, %) 22 (100) 22 (100) 21 (100) 20 (100)Abnormal Parturition − − − −

F1 Litters: No. Litters Evaluated 22 22 21 20(Pre-weaning) No. of Total Litter Losses 1 1 0 0

Mean No. Pups/Litter 14.3 14.0 14.5 14.3Mean No. Liveborn Pups/Litter 13.9 13.5 14.2 13.7External Malformations (No. of Pup) 0 0 0 1 b

Key:−: No noteworthy findings G: Gestation day L: Lactation day Dunnett’s Test * - p<0.05 ** - p<0.01a = For controls, group means are shown. For treated groups, percent differences from controls are shown.

Statistical significance is based on actual data (not on the percent differences).b = Multiple malformations (meningocele and absent eye bulge)


93


Daily Dose (mg/kg) 0 (control) 5 50 1000F1 Litters: No. Dead Newborns 9 11 6 11(Pre-weaning) Mean % Postnatal Survival to L4 94.0 94.5 99.4 88.4

Mean % Postnatal Survival to Weaning 100 100 100 100Mean Pup Body Weights (g) L0 Male 6.53 6.63 6.66 6.45Mean Pup Body Weights (g) L0 Female 6.14 6.30 6.24 6.05Mean Pup Body Weights (g) L11 Male 29.1 29.4 29.3 27.2Mean Pup Body Weights (g) L11 Female 28.0 28.3 28.1 26.4 *Mean Pup Body Weights (g) L14 Male 37.8 38.1 37.7 35.3Mean Pup Body Weights (g) L14 Female 36.5 36.6 36.3 34.2 **Mean Pup Body Weights (g) L18 Male 48.6 48.2 48.0 45.2Mean Pup Body Weights (g) L18 Female 47.0 46.3 46.1 44.0 **Mean Pup Body Weights (g) L21 Male 61.4 61.1 60.5 57.9Mean Pup Body Weights (g) L21 Female 59.1 58.7 57.9 56.0 *Mean Pup Sex Ratios (% Males) 48.1 54.0 49.7 52.6Pup Clinical Signs − − − −Physical Development c − − − −Early Behavior d − − − −Pup Necropsy Observations − − − −

Key−: No noteworthy findings L: Lactation day Dunnett’s Test * - p<0.05 ** - p<0.01c = Pinna unfolding, growth of abdominal hair, eruption of upper incisor and eyelid openingd = Back righting and negative geotaxis


94


Daily Dose (mg/kg) 0 (control) 5 50 1000F1 Males: No. Evaluated Post-weaning (R, B) 42 (21, 21) 42 (21, 21) 42 (21, 21) 40 (20, 20)(Post-weaning) No. Died or Sacrificed Moribund 0 0 0 0

Clinical Observations − − − −Necropsy Observations − − − −Body Weights (g) 28 Days of Age 103 104 102 98Body Weights (g) 35 Days of Age 163 166 163 156Body Weights (g) 42 Days of Age 232 237 233 222Body Weights (g) 70 Days of Age 442 451 449 432Sensory Function 4 Weeks of Age e − − − −Open Field Test 5 Weeks of Age − − − −Conditioned Avoidance Response f − − − −Preputial Separation − − − −Mean No. Days Prior to Mating 3.6 3.3 2.6 3.2No. Mated (% Copulation) g 20 (95.2) 20 (95.2) 21 (100) 19 (100)No. Fertile Males (% Fertility) g 20 (100) 19 (95.0) 21 (100) 19 (100)

F1 Females: No. Evaluated Post-weaning (R, B) 42 (21, 21) 42 (21, 21) 42 (21, 21) 38 (19, 19)(Post-weaning) No. Died or Sacrificed Moribund 0 0 0 0

Clinical Observations − − − −Necropsy Observations − − − −

Key:−: No noteworthy findings R: for reproductive function observations B: for behavioral evaluationse = Visual placing response, pupillary reflex, Preyer’s reflex and pain response f - 7-8 Weeks of Ageg = Results during the 1st (14 days) and 2nd (7 days) mating periods


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Daily Dose (mg/kg) 0 (control) 5 50 1000F1 Females: Body Weights (g) 28 Days of Age 93 93 92 89 *(Post-weaning) Body Weights (g) 35 Days of Age 139 139 137 133 *

Body Weights (g) 42 Days of Age 174 175 174 166 *Body Weights (g) 70 Days of Age 261 262 259 250Sensory Function 4 Weeks of Age e − − − −Open Field Test 5 Weeks of Age − − − −Conditioned Avoidance Response f − − − −Opening of Vagina − − − −Mean Days of Estrous Cycle 4.3 4.7 4.1 4.0No. Mated (% Copulation) g 20 (95.2) 20 (95.2) 21 (100) 19 (100)No. Pregnant Females (% Fertility) g 20 (100) 19 (95.0) 21 (100) 19 (100)Gestation Body Weights (g) G13 365 362 378 348Mean No. Corpora Lutea 16.4 16.4 18.0 16.8Mean No. Implantations 15.4 15.5 16.2 15.8Mean % Pre-implantation Loss 5.5 5.0 8.6 5.8

F2 Litters: Mean No. Live Conceptuses/Litter 14.6 14.7 15.5 14.8No. Dead Conceptuses h 16 15 15 18Mean % Post-implantation Loss 5.2 5.1 4.2 6.2

Key:−: No noteworthy findings G: Gestation day Dunnett’s Test * - p<0.05e = Visual placing response, pupillary reflex, Preyer’s reflex and pain response f = 7-8 Weeks of Ageg = Results during the 1st (14 days) and 2nd (7 days) mating periods h = Total of early deaths after implantation, unformed embryos and formed embryos with placenta


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15. STUDIES IN JUVENILE ANIMALS

Table 15.1 GSK1349572A: Oral Juvenile Toxicity Study in Rats

Study Type: Reproductive and Developmental Toxicology

Report Title: GSK1349572A: Oral Juvenile Toxicity Study in Rats Test Article: GSK1349572A, sodium saltBatch No.: B87002

Design Similar to ICH 4.1.2: Yes Duration of Dosing: Days 4 to 66 post partum (pp)a inclusive Study No.: G09229

Species/Strain: Rat/Sprague Dawley Method of Administration/Frequency: Oral gavage/once daily

Initial Age: 4 days of age (Day 4 pp) Vehicle/Formulation: 0.5% hydroxypropylmethylcellulose (K15M) and 0.1% Tween™ 80/suspension

GSK Document Number: CD2010/00023CTD Module: m4.2.3.5.4

Date of First Dose: 1 February 2010 Litter Handling: Culled to 5/sex/litter on Day 3 pp, weaned on Day 21 pp and single housed on Day 28 pp

GLP Compliance: Yes

Special Features: Immunology Parameters: T-cell-dependent antibody response and immunophenotyping of lymphocyte subsets (T- and B-cell enumeration). Also, T-cell receptor V beta usage in peripheral blood was assessed.Additional groups were included (Groups 5 and 6, n=10/sex) as a positive control TDAR experiment with cyclosporine to validate the method in juvenile rats.No Observed Adverse Effect Level: 2 mg/kg/day

Data Collected: Viability, clinical observations, body weight, food consumption (post-weaning), Day 13 pp and Day 32 pp toxicokinetics, physical development (including vaginal opening in females and balano-preputial skin fold separation in males), hematology, coagulation, clinical chemistry, urinalysis, macroscopic and microscopic pathology, stage-dependant evaluation of spermatogenesis, organ weights, femur length, T-cell-dependent antibody response (juvenile rats were immunized with KLH on Day 45 pp; anti-KLH IgM evaluation on Day 50 pp and anti-KLH IgG evaluation on Day 59 pp), immunophenotyping of lymphocyte subsets (T-cells, both CD4 and CD8 subsets, and B-cells) and CD4 or CD8 T-cell receptor V beta usage (Vβs 8.2/8.4, 8.5, 10 and 16) in peripheral blood.

Conclusion: There were pre-weanling deaths that occurred on Days 12 and 17 pp; both males that were found dead without a cause of death identified. There were statistically significant test article-related body weight effects at 75 mg/kg/day in males and females during the pre-weaning period and in females during the post-weaning period. With the exception of nasal degeneration/regeneration at all dose levels which was considered a local irritant effect secondary to the expelling of the gavaged material rather than a systemic effect, there were no other test article-related macroscopic or microscopic findings. There were no test article-related effects on immunologic competence as measured by T-cell-dependent antibody response, and no effects on lymphocyte subset counts (T-cells, both CD4 and CD8 subsets, and B-cells) and CD4 or CD8 T-cell receptor V beta usage at any dose level. Thus, 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 the AUC0-24 was 303 g.h/mL and the Cmax was 15.2 g/mL and on Day 32 pp the AUC0-24 was 85.7 g.h/mL and the Cmax was 7.71 g/mL, with no appreciable difference in gender.


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Table 15.1 (Continued) GSK1349572A: Oral Juvenile Toxicity Study in Rats

Study Type: Oral Toxicity Study in Juvenile Rats Study No. G09229GSK1349572 Daily Dose (mg/kg)a 0 (Control) 0.5 2 75

M F M F M F M FGroup Composition (10 litters/group each litter 5 pups/sex/litter) 50 50 50 50 50 50 50 50Juveniles Assigned to Study on Day 3 pp, treatment initiated on Day 4 pp for all subset groups:

Subset I - Main Study Necropsy Day 67 pp, nonlittermates 10 10 10 10 10 10 10 10Subset II -TDAR, Day 50 and 59 pp, nonlittermates 10 10 10 10 10 10 10 10Subset III – Toxicokinetic Evaluation on Day 13 pp 20 20 20 20 20 20 20 20Subset IV – Toxicokinetic Evaluation on Day 32 pp 10 10 10 10 10 10 10 10

Subset I: Main Study Necropsy (Day 67 pp) Mortalityb (found dead, one on Day 12 pp and one on Day 17 pp) 0 0 0 0 0 0 2b 0

Clinical Observations of early decedents noneMacroscopic Observations of early decedents none

Clinical Observations - - - - - - - -Body Weight Gain (g)c

Day 4 to 13 pp 19.30g 19.22g 1.04X 1.03X 1.06X 1.01X 1.01X 0.99XDay 13 to 21 pp 28.75g 26.46g 0.98X 1.03X 0.92X 1.03X 0.75X* 0.74X*Day 4 to 21 pp (preweaning period) 48.05g 45.67g 1.00X 1.03X 0.98X 1.03X 0.86X* 0.85XDay 21 to 66 pp (postweaning period) 400.7g 225.9g 1.00X 0.91X 1.00X 0.93X 1.05X 0.92XDay 4 to 66 pp (entire treatment period) 448.8g 271.5g 1.00X 0.93X 0.99X 0.95X 1.03X 0.91X

Body Weight (g)c

Day 15 pp 38.73g 37.13g 1.00X 1.04X 1.00X 1.01X 0.95X 0.93X*Day 16 pp 42.03g 40.21g 1.00X 1.03X 1.00X 1.01X 0.93X* 0.91X*Day 17 pp 44.89g 43.03g 1.01X 1.03X 0.99X 1.01X 0.93X* 0.90X*Day 18 pp 47.33g 45.39g 1.02X 1.04X 0.99X 1.01X 0.92X* 0.90X*Day 19 pp 50.01g 47.70g 1.02X 1.04X 0.98X 1.01X 0.90X* 0.90X*Day 20 pp 53.83g 51.03g 1.01X 1.04X 0.99X 1.02X 0.88X* 0.88X*

Day 21 pp 58.45g 55.24g 1.01X 1.04X 0.99X 1.03X 0.88X* 0.88X*Day 28 pp 102.7g 95.97g 1.01X 1.01X 1.02X 1.03X 0.95X 0.89X*Day 42 pp 242.2g 184.2g 1.0X 0.98X 0.98X 1.00X 0.98X 0.90X*Day 66 pp (end of treatment period) 459.6g 282.3g 1.00X 0.93X 0.99X 0.95X 1.03X 0.91X


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Table 15.1 (Continued) GSK1349572A: Oral Juvenile Toxicity Study in Rats

Study Type: Oral Toxicity Study in Juvenile Rats Study No. G09229GSK1349572 Daily Dose (mg/kg)a 0 (Control) 0.5 2 75

M F M F M F M FSubset I: Main Study Necropsy (Day 67 pp) continued

Physical Development (Sexual Maturity), day of occurrenceVaginal Opening 31.9 32.3 31.3 32.2Balano-preputial Skin Fold Separation 44.4 44.7 45.0 46.0

Femur Length (mm) 36.4 33.4 36.2 32.9 36.5 32.8 35.8 32.0Subset III: Toxicokinetic Evaluation on Day 13 pp

AUC0-24 (g.h/mL) NQ NQ 92.0 86.5 303 316 1540 1549

Cmax (g/mL) NQ NQ 4.55 4.69 15.2 16.4 88.0 85.4

Subset IV: Toxicokinetic Evaluation on Day 32 pp

AUC0-24 (g.h/mL) NQ NQ 9.90 27.1 85.7 93.3 917 1044

Cmax (g/mL) NQ NQ 1.50 2.41 7.71 7.46 69.9 77.4

Key:- No noteworthy findings.a. Dose levels for Groups 1 through 4 are expressed as parent compound, GSK1349572. b. One of the early decedents was not assigned to Main Study Necropsy Subset, however, for consistency purposes both are reported here. The animal that was

assigned to the Main Study Necropsy Subset was replaced with an animal from the Toxicokinetic Day 32 pp Subset so that 10 nonlittermates/sex/group completed Day 67 pp necropsy.

c. For controls, group means are shown. For treated groups, the magnitude of change relative to controls are shown. Body weight data for all animals was analyzed forpreweaning period (Day 4 to 21 pp; n=30/sex/group) and for main study animals only for postweaning period (Day 21 to 66 pp; n=10/sex/group).

* = Statistical significance (p 0.05) is based on raw data (not on the magnitude difference from control)NQ = Not Quantifiable (concentration below the lower limit of quantification (250 ng/mL)NA = Not Applicablepp = postpartum


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16. LOCAL TOLERANCE

Table 16.1 Local Tolerance Studies

Species/ Strain

Route (Vehicle/Formulation)

Doses (mg/kg)



Mouse(CBA/Ca)

Topical(dimethyl formamide

(emulsion))

25 L/site (25% w/w)

(sodium salt)

5F The test material was considered to be a non-sensitiser under the conditions of the test.

The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.

ED2009/000191127/1834(m4.2.3.6)

Mouse(CBA/Ca)

Topical(dimethyl formamide

(solution))

25 L/site (25% w/w)

(parent)

5F The test material was considered to be a non-sensitiser under the conditions of the test.

The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.

2010N1091531127/1886(m4.2.3.6)

Reconstituted human skin

In vitro(NA)

25 mg/site NA The test material was considered to be a Non-Irritant.

GHS: Considered not to be a significant irritant.

ED2010/000041127/1884(m4.2.3.6)


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Table 16.1 (Continued) Local Tolerance Studies

Species/ Strain Route (Vehicle/Formulation)

Doses (mg/kg)



Rabbit(Japanese white)

Topical(NA)

500 mg/site 3M The primary irritation index (P.I.I.) was 0.17. RD2010/00201S-349572-TF-080-N

(m4.2.3.6)


model

In vitro(NA)

30 mg/site NA Negative result, considered not to be a significant ocular irritant.

ED2010/000051127/1885(m4.2.3.6)

Rabbit(Japanese white)

Topical(NA)

100 mg/eye 6M Index of acute ocular irritation (I.A.O.I.): 4.00 (unwashed) and 2.00 (washed).

Evaluation: slightly irritating.

RD2010/00202S-349572-TF-079-N

(m4.2.3.6)


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17. OTHER TOXICITY STUDIES

Table 17.1 Immunotoxicity Study of S-349572 Sodium in Rats: Determination of Specific Antibody Formation Against T-Cell-Dependent Antigen



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Table 17.1 (Continued) Immunotoxicity Study of S-349572 Sodium in Rats: Determination of Specific Antibody Formation Against T-Cell-Dependent Antigen

module 2.6.6. toxicology written summary · confidential m2.6.6. toxicology written summary...

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