Property of the sanofi-aventis group - strictly confidential

2.6.7 NONCLINICAL TOXICOLOGY TABULATED SUMMARY

Lixisenatide (AVE0010)

Date: xx-xxx-20xx Document Number: -

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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LIST OF TABLES

1 PROGRAM OVERVIEW .................................................................................................................. 5 TS 2.6.7.1 - Toxicology tabulated summary – overview - study program ....................................................... 5

2 OVERVIEW OF TOXICOKINETICS STUDIES ............................................................................. 19 TS 2.6.7.2 – Overview of Toxicokinetics Studies .......................................................................................... 19

3 OVERVIEW OF TOXICOKINETICS DATA ................................................................................... 21 TS 2.6.7.3A – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) –

mouse/human ................................................................................................................................ 21 TS 2.6.7.3B – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – rat/human . 22 TS 2.6.7.3C – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – rat

(CAR)/human ................................................................................................................................. 23 TS 2.6.7.3D – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) –

dog/human ..................................................................................................................................... 24 TS 2.6.7.3E – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – pregnant

rat – rabbit /human ......................................................................................................................... 25

4 TOXICOLOGY DRUG SUBSTANCE ............................................................................................ 26 TS 2.6.7.4 – Toxicology Drug Substance ...................................................................................................... 26

5 SINGLE-DOSE TOXICITY ............................................................................................................. 37 TS 2.6.7.5 – Single-Dose Toxicity ................................................................................................................. 37

6 REPEAT-DOSE TOXICITY – NONPIVOTAL STUDIES ............................................................... 45 TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies ............................................................................. 45

7 REPEAT-DOSE TOXICITY ........................................................................................................... 51 TS 2.6.7.7A – Repeat-Dose Toxicity (Rat, 2 weeks) .................................................................................... 51 TS 2.6.7.7B – Repeat-Dose Toxicity (Rat, 13 weeks) ................................................................................... 59 TS 2.6.7.7C – Repeat-Dose Toxicity (Rat, 6 months) ................................................................................... 69 TS 2.6.7.7D – Repeat-Dose Toxicity (Rat, 4 weeks) .................................................................................... 76 TS 2.6.7.7E – Repeat-Dose Toxicity (Dog, 4 weeks) .................................................................................... 82 TS 2.6.7.7F – Repeat-Dose Toxicity (Dog, 13 weeks) .................................................................................. 89 TS 2.6.7.7G – Repeat-Dose Toxicity (Dog, 12 months)................................................................................ 97

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8 GENOTOXICITY: IN VITRO ........................................................................................................ 105 TS 2.6.7.8A - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxx ........................................................... 105 TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx .................................................. 108 TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx .................................................. 114 TS 2.6.7.8D - Genotoxicity: In vitro – Mammalian chromosome aberration test in human lymphocytes

batch xxxxxxxxx ........................................................................................................................... 120 TS 2.6.7.8E - Genotoxicity: In vitro – Mammalian chromosome aberration test in human lymphocytes

batch xxxxxxxxxxxxxx .................................................................................................................. 126 TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes

batch xxxxxxxxxxxxxx .................................................................................................................. 132

9 GENOTOXICITY: IN VIVO .......................................................................................................... 138 TS 2.6.7.9A - Genotoxicity: In vivo micronucleus test (mouse) .................................................................. 138

10 CARCINOGENICITY ................................................................................................................... 139 TS 2.6.7.10A – Dose Range Finding Toxicity - Nonpivotal Studies ............................................................ 139 TS 2.6.7.10B – Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) ............................................. 142 TS 2.6.7.10C - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks, toxicokinetics) ...................... 149 TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse ..................................................................... 152 TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat ............................................................................ 157 TS 2.6.7.10F – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in mice . 163 TS 2.6.7.10G – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in rats .. 164 TS 2.6.7.10H – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in knock

out mice ........................................................................................................................................ 165

11 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - NONPIVOTAL STUDIES ................. 166 TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies .......................................... 166

12 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - FERTILITY AND EARLY EMBRYONIC, DEVELOPMENT TO IMPLANTATION ............................................................... 173

TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation.............................................................................................................................. 173

13 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - EFFECTS ON EMBRYO-FETAL DEVELOPMENT .......................................................................................................................... 179

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats . 179 TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development,

Rabbits (1) .................................................................................................................................... 192 TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development,

Rabbits (2) .................................................................................................................................... 201

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14 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT, INCLUDING MATERNAL FUNCTION ............................................. 221

TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function ......................................................................................................... 221

15 STUDIES IN JUVENILE ANIMALS ............................................................................................. 240 TS 2.6.7.15A – Studies in juvenile animals – Exploratory study in male juvenile dogs .............................. 240 TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs .............................. 244

16 LOCAL TOLERANCE ................................................................................................................. 251 TS 2.6.7.16A - Local Tolerance AVE0010 batch xxxxxxxx ......................................................................... 251 TS 2.6.7.16B - Local Tolerance AVE0010 batch xxxxxxxx ......................................................................... 253 TS 2.6.7.16C - Local Tolerance Placebo of AVE0010 ................................................................................ 255

17 OTHER TOXICITY STUDIES ...................................................................................................... 257 TS 2.6.7.17A - Other Toxicity Studies – Antigenicity – B-cell response ..................................................... 257 TS 2.6.7.17B - Other Toxicity Studies – Antigenicity – T-cell response ...................................................... 258 TS 2.6.7.17C - Other Toxicity Studies – Degradation products – 2-week mouse study ............................. 259 TS 2.6.7.17D - Other Toxicity Studies – Batch qualification – 2-week rat study ......................................... 261 TS 2.6.7.17E - Other Toxicity Studies – Batch qualification – 13-week rat study ....................................... 264 TS 2.6.7.17F - Other Toxicity Studies – Toxicology Safety assessment AVE0010 - drug substance ........ 265 TS 2.6.7.17G - Other Toxicity Studies – Toxicology Safety assessment AVE0010 - xxxxxxxxxxxxx ........ 266

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1 PROGRAM OVERVIEW TS 2.6.7.1 - Toxicology tabulated summary – overview - study program

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg)a

GLP Compliance

Testing Facility

Study Number

Location

Single-Dose Toxicity Mouse (Crl:CD-1(ICR)BR)

Subcutaneous - 500 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20xx-1181] 4.2.3.1-1

Mouse (Crl:CD-1(ICR)BR)

Intravenous - 500 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 x-1180] 4.2.3.1-2

Rat (Crl:WI(G1x/BRL/Han)BR)

Subcutaneous - 5000 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 x-1179] 4.2.3.1-3

Rat (Crl:WI(G1x/BRL/Han)BR)

Subcutaneous - 50, 125, 500 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1186] 4.2.3.1-4

Rat (Crl:WI(G1x/BRL/Han)BR)

Intravenous - 5000 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1178] 4.2.3.1-5

Rat (Crl:WI(G1x/BRL/Han)BR)

Intravenous - 1, 2.5, 10 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1184] 4.2.3.1-6

Abbreviations: a Doses expressed as peptide content; the NOAEL (no-observed-adverse-effect-level) is underlined.

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TS 2.6.7.1 - Toxicology tabulated summary – overview- study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg)a

GLP Compliance

Testing Facility

Study Number

Location

Single-Dose Toxicity (continued) Dog (Beagle, Harlan) Subcutaneous - 10, 40, 200 Yes xxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxx

England

[20 xx-1187] 4.2.3.1-7

Dog (Beagle, Harlan) Intravenous - 5, 20, 100 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1185] 4.2.3.1-8

Abbreviations: a Doses expressed as peptide content, the NOAEL (no-observed-adverse-effect-level) is underlined.

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses a (μg/kg BID)

GLP Compliance

Testing Facility

Study Number

Location

Repeat-Dose Toxicity Rodents Rat (Hsd: Sprague Dawley SD)

Subcutaneous 5 days 70, 1000, 2000 No Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1276] 4.2.3.2-1

Rat (Hsd: Sprague Dawley SD, Crl: CD®(SD) BR)

Subcutaneous 5 days 2000 No Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1986] 4.2.3.2-2

Rat (Crl:WI(WU)BR)

Subcutaneous 5 days 2000 No Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1987] 4.2.3.2-3

Rat (Hsd: Sprague Dawley SD)

Subcutaneous 14 days 2, 20, 200 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1925] 4.2.3.2-4

Rat (Crl:CD®(SD)IGS BR)

Intravenous 4 weeks 3, 10, 30 Yes xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxx

England

[20 xx-2059] 4.2.3.2-10

Abbreviations: a: Doses expressed as peptide content, for GLP repeat-dose toxicity studies, the highest NOAEL (no-observed-adverse-effect-level) is underlined (not defined for exploratory non-GLP studies).

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses a (μg/kg BID)

GLP Compliance

Testing Facility

Study Number

Location

Repeat-Dose Toxicity Rodents (continued) Rat (Crl:CD®(SD)IGS BR)

Subcutaneous 13 weeks 4.1, 16.6, 165.6, 828.2, 1656.4

Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20xx-0063] 4.2.3.2-14 4.2.3.2-14a

Rat (Hsd: Sprague Dawley SD)

Subcutaneous 6 months 5, 100, 2000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0085] 4.2.3.2-15

Abbreviations: a: Doses expressed as peptide content, for GLP repeat-dose toxicity studies, the highest NOAEL (no-observed-adverse-effect-level) is underlined (not defined for exploratory non-GLP studies).

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses a (μg/kg BID)

GLP Compliance

Testing Facility

Study Number

Location

Repeat-Dose Toxicity Non-Rodents Dog (Beagle, Marshall) Subcutaneous 3 days Escalating: 2.5 to 500 No Sanofi-aventis

Drug Safety Evaluation Frankfurt Mainzer Landstrasse 500

65795 Hattersheim Germany

[20 xx-0233] 4.2.3.2-16

Dog (Beagle, Harlan) Subcutaneous 4 weeks 10, 40, 200 Yes xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxx

England

[20 xx-2060] 4.2.3.2-17

Dog (Beagle, Marshall) Subcutaneous 13 weeks 20, 300/100, 1000/400/250 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1926] 4.2.3.2-19 4.2.3.2-19a

Dog (Beagle, Marshall) Subcutaneous 12 months 2, 200, 1000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0064] 4.2.3.2-20 4.2.3.2-20a

Abbreviations: a Doses expressed as peptide content, for repeat-dose toxicity studies, the NOAEL (no-observed-adverse-effect-level) is underlined.

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide

Species (Strain)

Method of Administration

Duration of Dosing

Doses (μg/kg/day) or Concentration

GLP Compliance

Testing Facility

Study Number

Location

Genotoxicity In vitro Ames test Salmonella typhimurium TA98, TA100, TA1535, TA1537 E. coli - WP2 uvrA

in vitro - 1.6 - 5000 µg/mL Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-1183] 4.2.3.3.1-1

Salmonella typhimurium TA98, TA100, TA1535, TA1537 E. coli - WP2 uvrA

in vitro - 0.16 – 5000 µg/mL Yes Aventis Pharma ProTox

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1342] 4.2.3.3.1-2

Salmonella typhimurium TA98, TA100, TA1535, TA1537 E. coli - WP2 uvrA

in vitro - 0.16 – 5000 µg/mL Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0234] 4.2.3.3.1-3

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide

Species (Strain)

Method of Administration

Duration of Dosing

Doses (μg/kg/day) or Concentration

GLP Compliance

Testing Facility

Study Number

Location

Genotoxicity In vitro (continued) Chromosome aberration test Human lymphocytes in vitro 0.39 - 50 µM Yes xxxxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxxx England

[MAF0100] 4.2.3.3.1-4

Human lymphocytes in vitro 312.5 - 5000 µg/mL Yes xxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxx Germany

[20 xx-1343] 4.2.3.3.1-5

Human lymphocytes in vitro 312.5 - 5000 µg/mL Yes xxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxx Germany

[20 xx-0386] 4.2.3.3.1-6

Genotoxicity In vivo (Micronucleus test in bone marrow) Mice (CD-1) Intravenous single 1250, 2500, 5000 µg/mL Yes xxxxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxxx England

[MUT0212] 4.2.3.3.2-1

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TS 2.6.7.1 – Toxicology tabulated summary – overview – study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg) a BID

GLP Compliance

Testing Facility

Study Number

Location

Carcinogenicity Mouse (Hsd:ICR (CD-1)) Exploratory study

Subcutaneous 14 days 20, 200, 1000, 2000 No Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1952] 4.2.3.4.2-1

Mouse (Crl: CD-1) Dose-range finding study

Subcutaneous 3 months 16.6, 165.6, 828.2, 1656.4 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-0062] 4.2.3.4.2-2

Mouse (Crl: CD-1) Toxicokinetic study

Subcutaneous 3 months 200, 1000, 2000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0443] 4.2.3.4.2-3

Mouse (Crl: CD-1) Carcinogenicity study

Subcutaneous 2 year 40, 200, 1000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[CAR0085] 4.2.3.4.1-1

Rat (Crl: SD) Carcinogenicity study

Subcutaneous 2 year 40, 200, 1000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[CAR0084] 4.2.3.4.1-2

Abbreviations: a Doses expressed as peptide content; for repeat-dose toxicity studies, the NOAEL (no-observed-adverse-effect-level) is underlined (not defined for exploratory non-GLP studies and toxicokinetic studies).

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TS 2.6.7.1 – Toxicology tabulated summary – overview – study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg) a BID

GLP Compliance

Testing Facility

Study Number

Location

Carcinogenicity (Continued) Mouse (Crl: CD-1) Mechanistic study

Subcutaneous 1 Day 2000 QD / 2000 BID Yes Sanofi-aventis SCP Disposition, Safety &

Animal Research Operational Center Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[DIV1333] 4.2.3.4.3-8

Rat (Crl: SD) Mechanistic study

Subcutaneous 1 Day 2000 QD / 2000 BID Yes Sanofi-aventis R&D - SCP Disposition, Safety &

Animal Research Operational Center Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[DIV1332] 4.2.3.4.3-9

Mouse (Crl: CD-1 and GLP-1R (-/-) knockout mice) Mechanistic study

Subcutaneous 2 weeks 1000 No sanofi-aventis recherche & développement

Centre de Montpellier 371 rue du Pr. J. Blayac

34184 Montpellier Cedex 04 France

[TSA1481] 4.2.3.4.3-10

Abbreviations: a Doses expressed as peptide content; for repeat-dose toxicity studies, the NOAEL (no-observed-adverse-effect-level) is underlined (not defined for exploratory non-GLP studies and toxicokinetic studies).

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TS 2.6.7.1 – Toxicology tabulated summary – overview – study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg) a BID

GLP Compliance

Testing Facility

Study Number

Location

Reproductive and Developmental Toxicity Fertility and Early Embryonic Development Rat (Crl:CD®(SD)IGSBR)

Subcutaneous M: 4 weeks before pairing until study week 8

F: 2 weeks before pairing until day 6 of gestation

2, 29, 414 Yes xxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxx England

[20 xx-0550] 4.2.3.5.1-1 4.2.3.5.1-1a

Abbreviations: - a Doses expressed as peptide content; for repeat-dose toxicity studies, the NOAEL (no-observed-adverse-effect-level) for fertility and early embryonic development is underlined.

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg) a BID

GLP Compliance

Testing Facility

Study Number

Location

Reproductive and Developmental Toxicity (continued) Embryo-Fetal Development Rat (Hsd:SpragueDawley SD)

Subcutaneous 12 days 70, 1000, 2000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1923] 4.2.3.5.2-1

Rat (Hsd:SpragueDawley SD)

Subcutaneous 12 days 2.5, 35, 500 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0551] 4.2.3.5.2-2

Rabbit (Chbb:HM (SPF) Kleinrusse)

Subcutaneous 13 days 40, 200, 1000 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1924] 4.2.3.5.2-5

Rabbit (Chbb:HM (SPF) Kleinrusse)

Subcutaneous 13 days 2.5, 25, 250 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0552] 4.2.3.5.2-6

Rabbit (Chbb:HM (SPF) Kleinrusse)

Subcutaneous 13 days 0.15, 1, 2.5 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-1086] 4.2.3.5.2-9

Abbreviations: a Doses expressed as peptide content, unless otherwise specified, for repeat-dose toxicity studies, the highest NOAEL (no-observed-adverse-effect-level) for embryo-fetal effects is underlined.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (µg/kg) a BID

GLP Compliance

Testing Facility

Study Number

Location

Reproductive and Developmental Toxicity (continued) Prenatal and postnatal development, including maternal function

Rat (Hsd:SpragueDawley SD)

Subcutaneous gestation day 6 until lactation

day 3

2.5, 35, 500 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[DPP0025] 4.2.3.5.3-1

Rat (Hsd:SpragueDawley SD)

Subcutaneous gestation day 6 until lactation

day 20

2, 20, 200 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[DPN0327] 4.2.3.5.3-2

Juvenile Toxicity Studies

Dog (Beagle, Marshall) Subcutaneous 2 weeks 5, 20, 200 No Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[DIV1328] 4.2.3.5.4-1

Dog (Beagle, Marshall) Subcutaneous 8 months 5, 20, 200 Yes Sanofi-aventis R&D – SCP Disposition, Safety &

Animal Research Operational Center Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[TXC1462] 4.2.3.5.4-2

Abbreviations: a Doses expressed as peptide content, unless otherwise specified, for repeat-dose toxicity studies, the NOEL (no-observed-effect-level) for the F1-generation is underlined.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Dosesa

GLP Compliance

Testing Facility

Study Number

Location

Local Tolerance (Aqueous solution) Rabbit (New Zealand white)

IV, IA, SC, IM PV

single 0.5 mL/animal 0.1 mL/animal

Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0327]* 4.2.3.6-3

Rabbit (New Zealand white)

IV, IA, SC, IM PV

single 0.5 mL/animal 0.1 mL/animal

Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0519] 4.2.3.6-1

Rabbit (New Zealand white)

SC, IM PV

single 0.5 mL/animal 0.1 mL/animal

Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[20 xx-0771] 4.2.3.6-2 4.2.3.6-2a

Other Toxicity Studies Immunotoxicity/Antigenicity**

Mouse (BALB/cJ) Oral SC

5 times, each separated by 2

weeks

19950 µg/kg 1.995 µg/kg

No xxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxx

xxxxxxxxxxxxxxxx Denmark

[DIV1155] 4.2.3.7.1-1

Mouse (BALB/cJ) Injection in hindpaws and base

of tail

single 0.65 mM (≈ 65 nmol

peptide/animal)

No xxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxx

xxxxxxxxxxxxxxxx Denmark

[DIV1158] 4.2.3.7.1-2 4.2.3.7.1-2a

Abbreviations: IV = intravenous, PV = perivenous, IA = intraarterial, IM = intra muscular, SC = subcutaneous a Doses expressed as peptide content *Study performed with placebo of AVE0010 ** Antibody determination is in addition included in general toxicity studies 20xx-1925 (2-week rat), 20xx-2059 (4-week rat), 20xx-0063 (13-week rat), 20xx-0085 (6-month rat), 20xx-2060 (4-week dog), 20xx-1926 (13-week dog), 20xx-0064 (12-month dog) as well as in carcinogenicity range finding study 20xx-0443 (3-month mouse TK), and carcinogenicity studies CAR0084 and CAR0085.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 18

TS 2.6.7.1 - Toxicology tabulated summary – overview - study program (continued)

Test Article: Lixisenatide Species (Strain)

Method of Administration

Duration of Dosing

Doses (μg/kg a BID)

GLP Compliance

Testing Facility

Study Number

Location

Other Toxicity Studies (continued) Qualification of degradation products / clinical batches Mouse (Crl:CD-1(ICR)) SC 14 days 2, 20, 200 Yes xxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxx xxxxxxxxxxxxxxxxxx

UK

[TSA1242] 4.2.3.7.6-1

Rat (Hsd: Sprague Dawley SD)

SC 14 days 2, 20, 200 Yes Sanofi-aventis Drug Safety Evaluation Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[TSA1331] 4.2.3.7.6-2

Rat (CRL:SD)

SC 13 weeks 200 Yes Sanofi-aventis R&D – SCP Disposition, Safety &

Animal Research Operational Center Frankfurt

Mainzer Landstrasse 500 65795 Hattersheim

Germany

[TXC1482] 4.2.3.7.6-3

Safety assessment (cutaneous and ocular irritation and sensitization potential) of drug substance and intermediate synthesis product (xxxxxxxxxxxxx) Mouse (Balb/c) Rabbit (New Zealand White) In vitro

dermal dermal

3-day single

2.5 mgb 500 mgb

100 mgb

No Sanofi-aventis Drug Safety Evaluation Montpellier

371 rue du Pr. J. Blayac 34184 Montpellier Cedex 04

France

[IST0273] 4.2.3.7.7-1

Mouse (Balb/c) Rabbit (New Zealand White) In vitro

dermal dermal

3-day single

6.25 mgb 500 mgb

100 mgb

No Sanofi-aventis Drug Safety Evaluation Montpellier

371 rue du Pr. J. Blayac 34184 Montpellier Cedex 04

France

[IST0274] 4.2.3.7.7-2

Abbreviations: a: Doses expressed as peptide content, unless otherwise specified, for repeat-dose toxicity studies, the highest NOAEL (no-observed-adverse-effect-level) is underlined. b: unadjusted for salt, water, or purity content.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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2 OVERVIEW OF TOXICOKINETICS STUDIES TS 2.6.7.2 – Overview of Toxicokinetics Studies

Test Article: Lixisenatide Type of Study Test

System Method of

Administration Doses

(µg/kg BID) GLP

Compliance Study

Number Location

Repeat-Dose Toxicity Rat SC (14-day) 2, 20, 200 yes [20xx-1925] 4.2.3.2-4 Rat IV (4-week)* 3, 10, 30 yes [20 xx-2059] 4.2.3.2-10 Rat SC (13-week) 4.1, 16.6, 165.6,

828.2, 1656.4 yes [20 xx-0063] 4.2.3.2-14

Rat SC (6-month) 5, 100, 2000 yes [20 xx-0085] 4.2.3.2-15 Dog SC (13-week) 20, 300/100,

1000/400/250** yes [20 xx-1926] 4.2.3.2-19

Dog SC (12-month) 2, 200, 1000 yes [20 xx-0064] 4.2.3.2-20 4.2.3.2-20a

Carcinogenicity Mouse

SC (2-week) 20, 200, 1000, 2000 no [20 xx-1952] 4.2.3.4.2-1 Mouse

SC (13-week) 16.6, 165.6, 828.2, 1656.4

yes [20 xx-0062] 4.2.3.4.2-2

Mouse SC (3-month) 200, 1000, 2000 yes [20 xx-0443] 4.2.3.4.2-3 Mouse SC (2-year) 40, 200, 1000 yes [CAR0085] 4.2.3.4.1-1 Rat SC (2-year) 40, 200, 1000 yes [CAR0084] 4.2.3.4.1-2 Abbreviations: SC = subcutaneous, IV = intravenous * In study 20xx-2059 TK analysis was performed x years after sampling due to technical reasons, so that sample stability is not guranteed. Results are therefore not included in the subsequent toxicokinetics overview table. ** mid and high dose reduced during study course.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.2 – Overview of Toxicokinetics Studies (continued)

Test Article: Lixisenatide Type of Study Test

System Method of

Administration Doses

(µg/kg BID) GLP

Compliance Study

Number Location

Reproductive and Developmental Toxicity

Rat SC (GD 6 to 17) 2.5, 35, 500 yes [20 xx-0551] 4.2.3.5.2-2

Rabbit SC (GD 6 to 18) 2.5, 25, 250 yes [20 xx-0552] 4.2.3.5.2-6 Rabbit SC (GD 6 to 18) 0.15, 1, 2.5 yes [20 xx-1086] 4.2.3.5.2-9

Juvenile Toxicity Dog SC 5, 20, 200 yes [DIV1328] 4.2.3.5.4-1 Dog SC 5, 20, 200 and 200QD yes [TXC1462] 4.2.3.5.4-2 Abbreviations: GD=gestation day, SC = subcutaneous

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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3 OVERVIEW OF TOXICOKINETICS DATA TS 2.6.7.3A – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – mouse/human

Test Article: Lixisenatide Species/

Strain (Study No.)

Mouse/CD-1 [20xx-1952]

Mouse/CD-1 [20 xx-0062]

Mouse/CD-1 [20 xx-0443]

Mouse/CD-1 [CAR0085]

Human [ACT6011]

Daily Dose (µg/kg BID) Day 1 Day 14 Day 1 Day 1/2 Day 92/93 Day 176 Day 29

(AUC)a M F M F M F M F M F M F AB- AB+

16.6* 26.9 22.4 20* 15 13 27 21 40 67.3 51.2

165.6* 384.1 259.2 200* 150 98 289 133 200 192 145 351 337 1970 706

828.2* 2009.5 1613.6 1000* 952 621 1283 618 1000 927 787 9521 6980 20000 36700 1656* 2848 2387.7 2000* 1619 1141 2183 1928 2000 1590 1490 20351 13039

20 μg QD per subject

0.847 7.25

Abbreviations: M= male, F = female, AUC = area under the concentration curve, AB- = antibody negative subjects, AB+ = antibody positive subjects a All AUC and AUC0-24 values have been rounded to 3 significant figures or less. * initial TK assay not covering AVE0010 bound to antibodies

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TS 2.6.7.3B – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – rat/human

Test Article: Lixisenatide Species/

Strain (Study No.)

Rat/Sprague-Dawley [20 xx-1925]

Rat/Sprague Dawley [20 xx-0063]

Rat/Sprague Dawley [20 xx-0085]

Human [ACT6011]

Daily Dose (µg/kg BID)

Day 1 Day 14 Day 1 Day 29 Day 92 Day 1 Day 7 Day 183 Day 29 (AUC)a

M F M F M F M F M F M F M F M F AB- AB+ 2 1.49 0.38 1.22 0.76 5 8.77 7.74 10.5 11.7 15.7 12.8

20 31.78 24.5 24.09 11.52 200 89.3 298.95 154.48 326.61 4.1* 3.12 4.53 16.6* 21.31 19.63 100 143 110 146 132 2735 3914

165.6* 143.19 132.61 165.6 121 113 3223 3363 17642 20637 828.2* 293.61 345.91 828.2 173 218 6195 6613 39235 28916 1656.4 321.70 535.59 1656.4 255 429 1822 3464 14348 15144 2000 711 697 2815 2298 52597 35563

20 μg QD per subject

0.847 7.25

Abbreviations: M= male, F = female, AUC = area under the concentration curve, AB- = antibody negative subjects, AB+ = antibody positive subjects a : All AUC and AUC0-24 values have been rounded to 3 significant figures or less. * initial TK assay not covering AVE0010 bound to antibodies

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.3C – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – rat (CAR)/human

Test Article: Lixisenatide Species/

Strain (Study No.) Rat/Sprague-Dawley

[CAR0084] Human

[ACT6011]

Daily Dose (µg/kg BID) Day 4 Week 13 Week 52 Day 29

(AUC)a M F M F M F AB- AB+

40 66.7 69.5 15500 8340 8290 6620 200 242 268 53100 31200 39400 31700 1000 863 823 20400 14100 45700 32800

20 µg QD per subject 0.847 7.25 Abbreviations: M= male, F = female, AUC = area under the concentration curve, AB- = antibody negative subjects, AB+ = antibody positive subjects a : All AUC and AUC0-24 values have been rounded to 3 significant figures or less.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.3D – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – dog/human

Test Article: Lixisenatide Species/

Strain (Study No.)

Dog / Beagle [20 xx-1926]

Dog / Beagle

[20 xx-0064]

Human

[ACT6011]

Daily Dose (µg/kg BID) Day 1 Day 28 Day 91

Day 1/31/85 for 2/200/1000

µg/kg

Day 184/216/268

for 2/200/1000 µg/kg

Day 370/370/371 for

2/200/1000 µg/kg

Day 29 (AUC)a

Day 29 (AUC)a

M F M F M F M F M F M F AB- AB+ 2 19.3 18.6 150 24.7 126 21.7

20* 233 203 20 215 197 392 322 2700 957

200 2650 3230 33300 31000 25600 33800 300* 1230 1289

300/100** 1019 1395 4906 1749 21607 3252 1000* 4112 3528

1000/400/250*** 3578 3225 1438 3037 8289 11072 1000 120000 339000 247000 43700 161000 33900

20 µg QD per subject 0.847 7.25 Abbreviations: M= male, F = female, AUC = area under the concentration curve, AB- = antibody negative subjects, AB+ = antibody positive subjects a : All AUC and AUC0-24 values have been rounded to 3 significant figures or less. * initial TK assay not covering AVE0010 bound to antibodies ** dose reduced from Day 29 to 100 µg/kg BID, *** dose reduced from Day 15 to 400 µg/kg BID and from Day 29 to 250 µg/kg BID

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.3E – Toxicokinetics – Overview of toxicokinetics Data (mean AUC(0-24h) [ng.h/mL]) – pregnant rat – rabbit /human

Test Article: Lixisenatide

Species/ Strain (Study No.)

Rat/Sprague-Dawley [20 xx-0551]

Rabbit / Himalayan Kleinrusse [20 xx-0552]

Rabbit / Himalayan Kleinrusse [20 xx-1086]

Human [ACT6011]

Daily Dose (µg/kg BID) Day 7 Day 7 Day 7 Day 29 (AUC)a

Day 29 (AUC)a

F F F AB- AB+ 2.5 3.9 35 49.4

500 581.4 0.15 0.929

1 16.0 2.5 41.5 2.5 27.2 25 292.5

250 3899.2 20 µg QD per subject 0.847 7.25

Abbreviations: M= male, F = female, AUC = area under the concentration curve, AB- = antibody negative subjects, AB+ = antibody positive subjects a : All AUC and AUC0-24 values have been rounded to 3 significant figures or less.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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4 TOXICOLOGY DRUG SUBSTANCE TS 2.6.7.4 – Toxicology Drug Substance

Test Article: Lixisenatide

Batch No. Purity (%) Specified Impurities (%) Study No. Type of Study Location

xxx Proposed Specification

≤x

xxxxxxxxxxxxxxxxxxx xx (xxxxxxx xxxxxxx)

xx [20xx-1180] Single dose IV mouse

4.2.3.1-2

[20x-1181] Single dose SC mouse

4.2.3.1-1

[20x-1178] Single dose IV rat

4.2.3.1-5

[20x-1179] Single dose SC rat

4.2.3.1-3

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%) Specified Impurities (%) Study No. Type of Study Location

xxx xxxxxxxxxx xxxxxxxxx Proposed Specification

≤xxx ≤xxxxxx ≤xxxxxxxxxxxx

xxxxxxxxx xxxx (xxxxxxx xxxxxxx)*

xxxxx xxxxxx xxxxxxxxxxxxxxx

[20xx-1184] Single dose IV rat

4.2.3.1-6

[20 xx-1186] Single dose SC rat

4.2.3.1-4

[20 xx-1185] Single dose IV dog

4.2.3.1-8

[20 xx-1187] Single dose SC dog

4.2.3.1-7

[20 xx-2059] 4-week IV rat

4.2.3.2-10

[20 xx-2060] 4-week SC dog

4.2.3.2-17

[20 xx-1183] Ames Test 4.2.3.3.1-1 [MAF0100] CA human

lympho-cytes

4.2.3.3.1-4

[MUT0212] Mouse micro-

nucleus test

4.2.3.3.2-1

Abbreviations: RF = dose range finding study a xxxxxxxxxxxxxxx according to Certificate of Analysis * based on analytical certificate dated xx xxx 20xx and xx xxx 20xx ** xxxxxxxx: based on analytical certificates dated xx xxxxxxxx 20xx and xx xxxxxxxx 20xx

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%) a Specified Impurities (%) Study No. Type of Study Location

xxx xxxxxxxxxx xxxxxxxxx Proposed Specification

≤0.5 ≤200ppm ≤10 USP-EU/mg

xxxxxxxxx xxxxxxx xxxx% (xxxxxxx

xxxxxxx)**

xxxxx xxxxxxx xxxxxxxxxxxxxx

[20xx-1276] 5-day SC rat (Hsd:

SD)

4.2.3.2-1

[20 xx-1986] 5-day SC rat (Hsd:

SD and Crl: SD)

4.2.3.2-2

[20 xx-1987] 5-day SC rat (Wistar)

4.2.3.2-3

[20 xx-1925] 2-week SC rat

4.2.3.2-4

[20 xx-1952] 2-week SC mouse

4.2.3.4.2-1

[20 xx-1923] Embryo-fetal toxicity

rat RF

4.2.3.5.2-1

[20 xx-1924] Embryo-fetal toxicity rabbit RF

4.2.3.5.2-5

[20 xx-0551] Embryo-fetal toxicity

rat

4.2.3.5.2-2

Abbreviations: RF = dose range finding study a xxxxxxxxxxxxxxx according to Certificate of Analysis * based on analytical certificate dated xx xxx 20xx and xx xxx 20xx ** xxxxxxxx: based on analytical certificates dated xx xxxxxxxx 20xx and xx xxxxxxxx 20xx

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%)a Specified Impurities (%) Study No. Type of Study Location

xxx xxxxxxxxxx xxxxxxxxx Proposed Specification

≤xxx ≤xxxxxx ≤xxxxxxxxxxxx

xxxxxxxxx [20 xx-0552] Embryo-fetal toxicity

rabbit

4.2.3.5.2-6

[20 xx-1086] Embryo-fetal toxicity

rabbit

4.2.3.5.2-9

Abbreviations: RF = dose range finding study a xxxxxxxxxxxxxxx according to Certificate of Analysis * based on analytical certificate dated xx xxx 20xx and xx xxx 20xx ** xxxxxxxx: based on analytical certificates dated xx xxxxxxxx 20xx and xx xxxxxxxx 20xx

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%)a

Specified Impurities (%) Study No. Type of

Study Location xxx xxxxxxxxxx xxxxxxxxx xxxxxxxxx xxxxxxxxxxxxx

Proposed Specification

≤xxx ≤xxxxxx ≤xxxxxxxxxxxxx

≤xxxxxxxxxx

xxxxxxxxxxxxx xxxx (xxxxxxx xxxxxxx)

xxxx xxxxxx (xxxxx)

xxxxxxx-xxxxxx

xxxxx (xxxxxxxx) [20 xx-0062] 13-week SC mouse

4.2.3.4.2-2

[20 xx-0063] 13-week SC rat

4.2.3.2-14 4.2.3.2-14a

[20 xx-1926] 13-week dog

4.2.3.2-19 4.2.3.2-19a

[20 xx-0550] Fertility rat 4.2.3.5.1-1 xxxxxxxxxxxxxx xxxx (xxxxxxx

xxxxxxx) Xxxxxx (xxxx)

xxxxx (xxxxx)

Xxxxxxx xxxxx

xxxxx (xxxxxxxx) [20 xx-0062] 13-week SC mouse

4.2.3.4.2-2

[20 xx-0063] 13-week SC rat

4.2.3.2-14 4.2.3.2-14a

[20 xx-1926] 13-week dog

4.2.3.2-19 4.2.3.2-19a

[20 xx-1342] Ames test 4.2.3.3.1-2 [20 xx-1343] CA human

lympho-cytes

4.2.3.3.1-5

Abbreviations: LOQ=limit of quantification, LOD=limit of detection, * xxxxxxx dated xx xxx 20xx a xxxxxxxxxxxxxxx according to Certificate of Analysis

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%)a

Specified Impurities (%) Study No. Type of

Study Location xxx xxxxxxxxxx xxxxxxxxx xxxxxxxxx xxxxxxxxxxxxx

Proposed Specification

≤xxx ≤xxxxx ≤xxxxxxx xxxxx

≤xxxxxxxxx

xxxxxxxxxxxxxx xxxx (xxxxxxx xxxxxxx)

xxxxxx : xxxx%

(xxxxxxx xxxxxxx)*

xxxxxx (xxxxx)

Xxxxxx (xxxxx)

Xxxxxxx xxxxx

xxxxx (xxxxxxxx) [20 xx-0064] 12-month SC dog

4.2.3.2-20 4.2.3.2-20a

xxxxxxxxxxxxxx xxxx (xxxxxxx xxxxxxx) (xx xxxxx 20xx)

xxxxxx : xxx%

(xxxxxxx xxxxxxx)*

xxxxx (xxxx)

xxxxx (xxxxx)

Xxxxxxx xxxxx

xxxxx (xxxxxxxx) [20 xx-0443] 3-month SC mouse TK

4.2.3.4.2-3

[20 xx-0233] 3-day SC rising-dose

dog

4.2.3.2-16

[20 xx-0085] 6-month SC rat

4.2.3.2-15

[20 xx-0064] 12-month SC dog

4.2.3.2-20 4.2.3.2-20a

[20 xx-0234] Ames Test 4.2.3.3.1-3 xxxx%** [20 xx-0386] CA human

lympho-cytes

4.2.3.3.1-6

Abbreviations: LOQ=limit of quantification, LOD=limit of detection a xxxxxxxxxxxxxxx according to Certificate of Analysis * xxxxxxxx dated xx xxxxxxx 20xx, ** batch release xx xxxxxxx 20xx

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%)a

Specified Impurities (%) Study No. Type of

Study Location xxx xxxxxxxxxx xxxxxxxxx xxxxxxxxx xxxxxxxxxxxxxx

Proposed Specification

≤xxx ≤xxxxx ≤xxxxxxx xxxxx

≤xxxxxxxxx

xxxxxxxxxxxxxx xxx% (xxxxxxx xxxxxxx)

xxxxx (xxxx)

xxxxx Xxxxxxx xxxxx

xxxxx (xxxxxxxx) [TSA1331] 2-week rat 4.2.3.7.6-2

[DPP0025] Rat Pre/ postnatal

developm. range finding

4.2.3.5.3-1

[DPN0327] Rat Pre/ postnatal

developm. range finding

4.2.3.5.3-2

xxx% (xxxxxxx xxxxxxx)*

xxxxx (xxxxxxxx) [CAR0084] 2-year rat 4.2.3.4.1-2 [CAR0085] 2-year

mouse 4.2.3.4.1-1

Abbreviations: LOQ=limit of quantification, LOD=limit of detection a xxxxxxxxxxxxxxx according to Certificate of Analysis * xxxxxxx xx xxxxxxx 20xx

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide

Batch No. Purity (%)a Specified Impurities (%)

Study No. Type of Study Location xxx xxxxxxxxxx xxxxxxxxx xxxxxxxxx

xxxxxxxxxxxxx xxxxxxx xxxxx

xxxxxxxxx

Proposed Specification

≤xxx ≤xxxxx ≤xxxxxxx xxxxx

≤xxxxxxxxx ≤xxx ≤xxx

xxxx xxx% (xxxxxxx xxxxxxx)

xxxxx (xxxx)

xxxxx (xxxxx)

Xxxxxxxx xxxxx

xxxxxxxxxxx xxxx xxx [CAR0084] 2-year rat 4.2.3.4.1-2

[CAR0085] 2-year mouse

4.2.3.4.1-1

xxxx xxx% (xxxxxxx xxxxxxx)

xxx xxxxxx (xxxxx)

Xxxxxxxx xxxxx

xxxxxxxxxxx Xxxxx (xxxxx)

xxx [CAR0084] 2-year rat 4.2.3.4.1-2

[CAR0085] 2-year mouse

4.2.3.4.1-1

[DIV1332] 1 Day rat 4.2.3.4.3-9 [DIV1333] 1 Day

mouse 4.2.3.4.3-8

[TSA1481] 14-day knockout

mice

4.2.3.4.3-10

[DIV1328] 14-day juvenile dog

4.2.3.5.4-1

[TXC1462] 8-month juvenile dog

4.2.3.5.4-2

Xxxxxxxxxxxxxxx [TXC1482] 13-week rat 4.2.3.7.6-3 xxxxxxxxxxxxxxx [TXC1482] 13-week rat 4.2.3.7.6-3 Abbreviations: LOQ=limit of quantification, LOD=limit of detection a xxxxxxxxxxxxxxx according to Certificate of Analysis

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide (xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx)

Batch No. Purity (%) Specified Impurities (%)

Study No. Type of Study Location Xxx

xxxxxxx* xxxxxxxx

Proposed Specification

xxxx to xxxxx xxxxxx

<xxx xxxx xxxxx

xxxxxxxxxxxxxx* xxxxxxx xxxxxxxxx AVE0010

Xxxx xxxxxxx

Xxxxxxxx xxxxx

[TSA1242] 2-week mouse

4.2.3.7.6-1

Abbreviations: * stored under xxxxxxxxxxxxxxxxx at xxxxx for xxxxxxx, xxxxxxxxxxxxxxxxxxxxxx xxxx%, ** xxxxxxxxxxxxxxxxxxxxxxxxxx (used to xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide and Placebo of Lixisenatide (xxxxxxxxxxxxxxxxxxxxxxxxx)

Batch No. Purity (%) Specified Impurities (%) Study No. Type of Study Location

Proposed Specification

xxxxxxxx xxxxxxx AVE0010 (Placebo)

[20 xx-0327] Rabbit local tolerance

4.2.3.6-3

xxxxxxxx xxxxxxxxxxxx AVE0010

[20 xx-0519] Rabbit local tolerance

4.2.3.6-1

xxxxxxxx xxxxxxxxxxx AVE0010

[20 xx0771] Rabbit local tolerance

4.2.3.6-2 4.2.3.6-2a

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.4 – Toxicology Drug Substance (continued)

Test Article: Lixisenatide (xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx)

Batch No. Purity (%) Specified Impurities (%) Study No. Type of Study Location

Proposed Specification

xxxxxxxxxxxxxxxx and xxxxxxxxxx

Xxx [DIV1155] B-cell response

mouse

4.2.3.7.1-1

Xxxxxxxxxxxxxxxx xx [DIV1158] T-cell response

mouse

4.2.3.7.1-2 4.2.3.7.1-2a

xxxxxxxxxxx* Xxxx [IST0273] Cutaneous and ocular irritation,

sensitization

4.2.3.7.7-1

xxxxxxxxx** xxxx [IST0274] Cutaneous and ocular irritation,

sensitization

4.2.3.7.7-2

Abbreviations: * drug substance, ** xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

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5 SINGLE-DOSE TOXICITY TS 2.6.7.5 – Single-Dose Toxicity

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)a

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Mouse (Crl:CD-

1(ICR)BR)

SC (Phosphate

buffered saline)

500

1M/1F preliminary

study

5M/5F main study

500 >500 Preliminary study: Mortality: none Clinical signs: NAD Necropsy: NAD Main study: Mortality: none Clinical signs: NAD Body weight: NAD Necropsy: NAD

[20 xx-1181]

4.2.3.1-1

Abbreviations: M = male, F = female, NAD = No abnormality detected, p.a. = post administration, Dose expressed as peptide content

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Mouse (Crl:CD-

1(ICR)BR)

IV (Phosphate

buffered saline)

500

1M/1F preliminary

study

5M/5F main study

500 >500 Preliminary study: Mortality: none Clinical signs: Lethargy (1/1M, 2.5h p.a., NAD on Day 2) Necropsy: NAD Main study: Mortality: none Clinical signs: NAD Body weight: NAD Necropsy: NAD

[20 xx-1180]

4.2.3.1-2

Abbreviations: M = male, F = female, NAD = No abnormality detected, Dose expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Rat (Crl:WI(G1x/BRL/Han)

BR)

SC (Phosphate

buffered saline)

5000

1M/1F preliminary

study

5M/5F main study

5000 >5000 Preliminary study: Mortality: none Clinical signs: Piloerection (1M, 2.5h p.a., recovered 4h p.a.) Body weight: Weight loss (Day1 to Day 4) Necropsy: NAD Main study: Mortality: none Clinical signs: NAD Body weight: Weight loss (4/5 M, Day1-Day8, max.10g) or fluctuations in body weight (1M/1F Day1-Day8) recovering thereafter Necropsy: NAD

[20 xx-1179]

4.2.3.1-3

Abbreviations: M = male, F = female, NAD = No abnormality detected, p.a. = post administration, Dose expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Rat (Crl:WI(G1x/BRL/Han)

BR)

SC (Phosphate

buffered saline, pH 7.4)

0, 50, 125, 500

10M/10F

+ 10M/10F TK satellites

500 >500 Mortality: none Clinical signs: NAD Body weight: NAD Food consumption: NAD Hematology: NAD Coagulation: NAD Serum chemistry: ≥125 µg/kg: Glucose ↑ (M, max. +55%, 24h p.a.), 500 µg/kg: Glucose ↑ (F, +36%, 24h p.a.), cholesterol ↓ (M, -32%, 24h p.a.), NAD 14 day p.a. Organ weights: 50, 125 µg/kg: spleen↓ (M, -25%, Day 2 necropsy) 500 µg/kg: spleen ↓ (F, -24%, Day 2 necropsy), considered to be related to bleeding prior to necropsy, NAD Day 14 necropsy Macroscopy: NAD Microscopy: NAD TK: not evaluated due to the lack of an analytical method at the time of study conduct

[20 xx-1186]

4.2.3.1-4

Abbreviations: M = male, F = female, NAD = No abnormality detected, p.a. = post administration, Doses expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Rat (Crl:WI(G1x/BRL/Han)

BR)

IV (Phosphate

buffered saline)

5000

1M/1F preliminary

study

5M/5F main study

5000 >5000 Preliminary study: Mortality: none Clinical signs: Piloerection (all rats, from 4h p.a.), stained snouts and decreased activity (all rats, Day2), signs recovered on Day 3 Necropsy: NAD Main study: Mortality: none Clinical signs: Piloerection (all rats, from 4h p.a., recovered Day 2), anogential soiling (all rats, from 4h p.a. , recovered Day 4), stained snouts (5M/3F, Day 2, recovered Day 3), chromodacryorrhea (1M, Day2, recovered Day 3) Body weight: Weight loss (5M/4F, Day1-Day4, max. –33g M, -9g F) recovering thereafter Necropsy: NAD

[20 xx-1178]

4.2.3.1-5

Abbreviations: M = male, F = female, NAD = No abnormality detected, p.a. = post administration, Dose expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Rat (Crl:WI(G1x/BRL/Han)

BR)

IV (Phosphate

buffered saline)

0, 1.0, 2.5, 10

10M/10F

+ 10M/10F TK satellites

(dose groups only)

10 >10 Mortality: none Clinical signs: NAD Body weight: NAD Food consumption: NAD Hematology: NAD Coagulation: NAD (insufficent data as consequence of clotted samples) Serum chemistry: 10 µg/kg: ALP ↑ (2F, +85/114%, 24h p.a.), NAD on Day 12 Organ weights: 10 µg/kg: liver ↑ (M:+8%, F:+12%, Day15/16) Macroscopy: NAD Microscopy: 10 µg/kg: liver: glycogen vacuolation (Day 15/16 necropsy) TK: not evaluated due to the lack of an analytical method at the time of study conduct

[20 xx-1184]

4.2.3.1-6

Abbreviations: M = male, F = female, NAD = No abnormality detected, p.a. = post administration, Doses expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Dog (Beagle, Harlan)

SC (Phosphate

buffered saline, pH 7.4)

0, 10, 40, 200

4/4 200 >200 Mortality: none Clinical signs: NAD Body weight: NAD Food consumption: NAD Hematology: NAD Coagulation: NAD Serum chemistry: NAD Organ weights: NAD Macroscopy: NAD Microscopy: Pancreas: 200 µg/kg F: vacuolation of pancreatic islet cells

[20 xx-1187]

4.2.3.1-7

Abbreviations: M = male, F = female, NAD = No abnormality detected, Doses expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.5 – Single-Dose Toxicity (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Doses (µg/kg)

Gender and No.

per Group

Observed Maximum Nonlethal

Dose (µg/kg)

Approximate Lethal Dose

(µg/kg)

Noteworthy Findings Study Number

Location

Dog (Beagle, Harlan)

IV – ca. 30 minutes infusion

(Phosphate buffered saline, pH

7.4)

0, 5, 20, 100

4/4 100 >100 Mortality: none Clinical signs: NAD Body weight: NAD Food consumption: reduced, ≥5 µg/kg: F, Day 1, 2, max. –75%, 20 µg/kg: M, Day 1, max. –23%, 100 µg/kg: M, Day 2, max. –30%, afterwards NAD in M+F Hematology: NAD Coagulation: NAD Serum chemistry: NAD Organ weights: NAD Macroscopy: NAD Microscopy: NAD

[20 xx-1185]

4.2.3.1-8

Abbreviations: M = male, F = female, NAD = No abnormality detected, Doses expressed as peptide content

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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6 REPEAT-DOSE TOXICITY – NONPIVOTAL STUDIES TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Rat / Hsd: SD

SC (0.9% sodium

chloride/aqueous solution)

5-day 70, 1000, 2000

5/5 + 12/12 TK rats*

Not applicable (exploratory study with

limited histopathology)

Mortality: none Clinical signs: none Body weight: 70 µg/kg reduced weight gain Day 1 to Day 3 (M: -87%, F: -93% compared to control) ≥1000 µg/kg weight loss Day 1 to Day 3 (M: up to –17.0 g, F up to –6.0g) Food consumption: ≥70 µg/kg reduced food consumption, maximum Day 1 to Day 3: -44% to -78% in M, -34% to –59% in F Hematology: ≥70 µg/kg: reticulocytes↓ (% of red blood cells and absolute: M: max. –59%) ≥1000 µg/kg: basophils↓ (F: max. –45%) 2000 µg/kg: reticulocytes↓ (% of red blood cells and absolute: F: –25%), hematocrit↑ (M: +4.5%), neutrophils↓ (M: -33%) Coagulation: none

[20 xx-1276]

4.2.3.2-1

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Rat / Hsd: SD

SC (0.9% sodium

chloride/aqueous solution)

5-day 70, 1000, 2000

5/5 + 12/12 TK rats*

Not applicable (exploratory study with

limited histopathology)

Serum chemistry: ≥70 µg/kg: total bilirubin↓ (M: max. –21%), total cholesterol↓ (M: max. –21%) ≥1000 µg/kg: triglycerides↓ (M: max. –40%), urea↑ (M: max. +23%), ALT↑ (F: max. +19%) 2000 µg/kg: potassium↓ (M: -10%) Urinalysis: none (Volume and semiquantitative analysis performed only) Organ weights absolute: ≥70 µg/kg: adrenal gland↑ (M: max. +18%), spleen↓ (M: max. –35%), heart↓ (M: max. –21%), thymus↓ (M: max. –41%) ≥1000 µg/kg: liver↓ (M: max. –18%), heart↓ (F: max. -16%), thymus↓ (F: max. –30%) 2000 µg/kg: adrenal gland↑ (F:+21%), spleen↓ (F:-21%) Macroscopy: none Microscopy ≥70 µg/kg spleen: reduction or loss of extramedullary hematopoiesis, liver: minimal to moderate inflammation in Glisson’s trias

[20 xx-1276]

continued

4.2.3.2-1

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female, * For study 20xx-1276 a specific TK assay was not yet available, therefore actual exposure could not be determined and results are not presented.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Rat / Hsd:SD

and Crl:SD

SC (0.9% sodium

chloride/aqueous solution)

5-day 0, 2000 5M per strain Not applicable (exploratory study with

limited histopathology)

Mortality: none Clinical signs: Hypoactivity (Day 1 to Day 3) Body weight: Weight loss in Hsd:SD and Crl:SD: Day 1 to Day 3: -20.6g/-15.8g Food consumption: Reduced in Hsd:SD and Crl:SD (Day 1 to Day 3: -69%/-63%, Day 3 to Day 5: -14%/-15%) Hematology: Hsd:SD/Crl: SD: reticulocytes↓ (% of red blood cells and absolute: -41%/-60%) Hsd:SD: neutrophils↓ (-44%) Coagulation: Hsd:SD: PT time↑ (+22%) Serum chemistry: Hsd:SD: ALT↑ (+23%), total cholesterol↓ (-22%), triglycerides (-28%)

[20 x-1986] 4.2.3.2-2

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Rat / Hsd:SD

and Crl:SD

SC (0.9% sodium

chloride/aqueous solution)

5-day 0, 2000 5M per strain Not applicable (exploratory study with

limited histopathology)

Urinalysis: Hsd:SD and Crl:SD: Na↓ (partially below detection limit) Organ weights absolute: Hsd:SD and Crl:SD: Liver↓ (-11%/-11%), adrenal gland↑: (+22%/+30%), thymus↓ (-33%/-27%) Hsd:SD: prostate gland ↓(-20%), Crl:SD: Testis↓ (-10%) Macroscopy: none Microscopy: Hsd:SD and Crl:SD: spleen: loss or reduction of extramedullary hematopoiesis Hsd:SD only: liver: minimal inflammation in portal triad

[20 x-1986] continued

4.2.3.2-2

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Rat / Crl:WI(WU)

BR

SC (0.9% sodium

chloride/aqueous solution)

5-day 0, 2000 5M not applicable (exploratory study with

limited histopathology)

Mortality: none Clinical signs: Hypoactivity (Day 1 to Day 4) Body weight: Weight loss (Day to Day 3: -1.8g), later on reduced gain (Day1 to Day 5: -66% of control) Food consumption: Reduced (Day 1 to Day 3: -52%, Day 3 to Day 5: -30%) Hematology: Reticulocytes↓ (% of red blood cells and absolute): (-38/-42%), MCV and MCH↓ (both -7%), neutrophils (%) ↓ (-45%), lymphocytes (%) ↑ (+10%) Serum chemistry: Total cholesterol↓ (-14%), triglycerides (-29%) ↓ Urinalysis: none Organ weights absolute: Spleen↓ (-34%), thymus↓ (-47%) Macroscopy: none Microscopy: Spleen: reduction of extramedullary hematopoiesis (5/5 rats)

[20 x-1987] 4.2.3.2-3

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.6 – Repeat-Dose Toxicity – Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Dog / Marshall Beagle

SC (0.9% sodium

chloride/aqueous solution)

3-day (for each of the 19 dose

levels tested)

(together: 57 days, without

any wash-out period)

0 (controls), 2.5 to 500

(escalating)

2/2 not applicable (no microscopic

investigation)

Mortality: none Clinical signs: noted occassionally as liquid feces (at maximum on 4 of 57 days) and emesis (at maximum on 5 of 57 days) Body weight: males: no body weight loss; females: body weight loss (on Day 58) of 1.1kg (-13%) or 0.9kg (-12% relative to Day1) Food consumption: males: no effect; females: in 1 of two moderately reduced (-26% from Day 13 at 40 µg/kg BID until study end)

[20 x-0233] 4.2.3.2-16

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = Female,

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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7 REPEAT-DOSE TOXICITY TS 2.6.7.7A – Repeat-Dose Toxicity (Rat, 2 weeks)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period Species/Strain: Rat/Hsd:Sprague Dawley SD Duration of Dosing: 2 weeks Test Article: AVE0010 Initial Age: 6-7 weeks Duration of Post-Dose: 2 weeks Study No.: [20xx-1925]

Separate reports for toxicokinetics: [F20 xxKIN0120] (+ amendment 1 [F20 xxKIN0160]), bioanalysis [F20 xxKIN0241] and antibody analysis reports: [F20KIN0002] (+amendment 1 [F20 xxKIN0161])

Date of First Dose: xx xxxxxxxx 20xx Method of Administration: subcutaneous BID (separated by 8h)

Location : 4.2.3.2-4, 4.2.3.2-5, 4.2.3.2-6, 4.2.3.2-7, 4.2.3.2-8, 4.2.3.2-9

Vehicle/Formulation: 0.9% sodium chloride/ aqueous solution GLP Compliance: yes Special Features: none No Observed Adverse Effect Level: 200 µg/kg BID Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200 Number of Animals on Study (Main Study + Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Toxicokinetics: (values after 2nd administration) AUC0-24 (ng.h/mL ) Day 1 Day 14

- -

- -

1.49 1.22

0.38 0.76

31.78 24.09

24.50 11.52

89.30 154.48

298.95 326.61

Cmax (ng/mL) Day 1 Day 14

- -

- -

0.79 0.45

0.21 0.25

11.41 8.31

10.24 6.27

19.23 66.93

100.43 98.29

Anti-drug antibody formation % of ADA positive rats 16 h after last treatment on Day 14 0 0 5 0 0 6 68 25 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = are under the curve, Cmax = maximum concentration, ADA=anti-drug antibody

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A – Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Noteworthy Findings: Body weight gains were decreased at ≥2 µg/kg BID in the treatment and recovery period Food consumption decreased at ≥2 µg/kg BID in the treatment period, but was normal in the recovery period Water consumption was decreased ≥2 µg/kg BID on Day1/2 in both genders followed by an increase on Day 7/8 in females, later on no significant changes were observed Died or Killed Moribund 0 0 0 0 0 0 0 0 Absolute Body Weight (g and %)a Day 1 Day 4 Day 8 Day 11 Day 14 Day 21 Day 27(M) / 26(F)

174.1 196.6 224.8 242.3 258.1 292.4 313.2

149.7 158.2 170.0 177.4 185.3 202.8 213.4

-3.2 -8.2 -8.6 -9.0 -8.6

+0.53 -3.0 -3.2 -3.6 -5.0

-2.8 -8.9 -10 -9.2 -9.6

+3.5 -0.13 +0.18 +0.28 +0.70

-3.3 -11 -12 -10 -10 -9

-10

-1.5 -5.9 -2.3 -2.1 -2.2 -5.1 -6.7

Average Body Weight Gain (g and %)a Days 1 to 4 Days 1 to 8 Days 1 to 11 Days 1 to 14 Days 1 to 21 Days 1 to 27 (M) / 26(F)

22.5 50.7 68.1 83.9

116.4 137.2

8.5 20.3 27.7 35.6 53.2 63.8

-47* -27* -23* -20*

-65* -31 -26 -28

-56* -35* -26* -24*

-65* -25 -17 -11

-73* -40* -27* -25* -19* -19*

-85* -8.9 -5.8 -5.1 -15* -18*

Abbreviations: M = male, F = female a For controls, group means are shown. For treated groups, percent differences from controls are shown. *Significant trend (p<0.05), absolute body weights were not evaluated statistically Note: potential slight differences comparing absolute body weights and body weight gain in corresponding time frame were caused by rounding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A – Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Average Daily Food Consumption (g/day/rat and %)a Days 1 to 4 Days 4 to 8 Days 8 to 11 Days 11 to 14 Days 14 to 21 Days 21 to 27(M) / 26(F)

21.19 21.67 22.46 22.50 22.64 24.28

15.90 15.75 16.19 16.51 16.85 18.28

-19* -15* -12* -12*

-15* -14* -7.7 -8.6

-32* -17* -13* -16*

-23* -6.3* -5.6 -3.3

-38* -17* -12* -15* -2.4 -7.1

-38* -12* -8.3* -8.5* -4.2 -6.1

Average Daily Water Consumption (g/day/rat and %)a Days 1 to 2 Days 7 to 8 Days 14 to 15 Days 21 to 22 Days 27 to 28 (M) / 26 to 27 (F)

28.0

35.35 18.7 28.5 11.2

21.46 19.46 14.6 23.0 8.16

-11 -14 -27

-12

+28* -4.8

-23* -2.8 -6.4

-12

+43* +29

-48* -9.3 +51 -1.8 -46

-36* +52* -0.68 -20 -7.4

Abbreviations: M = male, F = female a For controls, group means are shown. For treated groups, percent differences from controls are shown. *Significant trend (p<0.05), absolute body weights were not evaluated statistically Note: potential slight differences comparing absolute body weights and body weight gain in corresponding time frame were caused by rounding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A – Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Clinical Observations b Hypoactivity 0 0 4 0 10 6 15 12 Ophthalmoscopy (Wk ) Not included in the study Abbreviations: M = male, F = female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A - Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Hematology (Week 2) MCV (fL) 60.30 56.50 60.80 57.40 61.00 58.30* 59.90 58.20* MCH (pg) 19.30 18.80 19.60 18.90 19.30 19.30* 19.30 19.30* Eosinophils (x10*9/L) 0.165 0.226 0.165 0.198 0.195* 0.233 0.462* 0.307 Eosinophils (%) 1.2 2.2 1.2 2.1 1.5* 1.8 3.3* 2.8 Basophils (x10*9/L) 0.242 0.110 0.165 0.067 0.146 0.074 0.124* 0.062 Basophils (%) 1.8 1.0 1.2 0.8 1.2 0.8 1.1 0.5* LU Cells (x10*9/L) 0.157 0.119 0.140 0.114 0.100* 0.109 0.122* 0.106 Coagulation (Week 2) Platelets (x10*9/L) 1118.5 1016.0 927.7* 921.4 835.8* 917.5 935.1* 981.0 PT (s) 11.53 11.84 11.73 12.17 12.16* 12.07 11.91* 12.25* Coagulation (Recovery) PT (s) 11.48 12.20 11.28 11.60* Serum Chemistry (Week 2): ALT (U/L) 19.2 18.3 23.8* 20.7* 26.5* 22.0* 28.0* 22.9* Alkaline Phosphatase (U/L) 251.5 157.6 260.4 183.3 302.1* 180.9 322.9* 183.6 Total Cholesterol (mmol/L) 3.480 2.800 3.330 3.050 2.990* 2.990 3.040* 2.900 Triglycerides (mmol/L) 0.387 0.422 0.466* 0.424 0.532* 0.526 0.531* 0.459 Urea nitrogen (mmol/L) 6.970 7.210 7.350 7.860 6.580 6.830 6.260* 7.630 Creatinine (umol/L) 71.9 67.5 65.3* 61.1* 62.1* 55.5* 61.4* 55.1* Sodium (mmol/L) 144.6 144.6 145.0 145.6 146.3* 145.8 145.5* 145.6 Calcium (mmol/L) 2.596 2.641 2.549 2.656 2.651* 2.693 2.677* 2.670 Abbreviations: M = male, F = female * Significant trend (p<=0.05) through indicated dose level.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A - Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Serum Chemistry (Recovery): Total Protein (g/L) 67.4 64.0 64.2* 64.6 Urinalysis (Week 2): Total Sodium (mmol) 0.4786 0.3498 0.1992* 0.2078* 0.0531* 0.1550* 0.1196 0.1396* Total Potassium (mmol) 0.6820 0.5334 0.5901 0.5369 0.3895* 0.3210* 0.4645* 0.4485* pH Value 7.25 7.40 7.20 7.30 6.85* 7.15 6.85* 7.00* Abbreviations: M = male, F = female * Significant trend (p<=0.05) through indicated dose level.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A - Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Organ Weights a Week 2 Adrenal Gland (g) 0.04641 0.05780 +9 +3.1 +25.9## +3.4 +21.3# +6.8 Adrenal Gland (%) 0.02098 0.03592 +18.5 +6.8 +39.6** +2.8 +35.9** +8.8 Brain (%) 0.80916 1.05 +8.2** +2.9 +8.7** -2.9 +8** ±0 Brain (%) Recovery 0.66236 0.89270 +7.3 +5.5* Heart (g) 0.92615 0.69125 -9.8* -2.0 -11.0* -2.4 -16.9** -8.5* Heart (%) Recovery 0.39875 0.38503 -4.1 +12.1 Kidney (g) 1.73 1.19 -11.6** +5.2 -8.7* +5.0 -11** +4.7 Kidney (g) Recovery 2.04 1.32 -12.8* -5.3 Liver (g) 7.47 5.18 -11..2** -4.1 -11.2** +5.0 -14.3** +0.6 Liver (g) Recovery 9.66 5.99 -12.2# -4.0 Spleen (g) 0.58852 0.46645 -14.9## -16.7# -19.2## -13.9 -20.4## -17# Spleen (%) 0.26648 0.28789 -8.1 -13.2 -10.7# -13.9 -11## -14.9# Testis (%) 1.41 +7.1* +10.6** +10.6** Testis (g) Recovery 3.57 -10.6# Thymus (g) 0.47044 0.31411 -3.9 -16.6 -28.6** -8.2 -25.2** -12.7 Thymus (%) 0.21248 0.19590 +4.4 +14.1 -21** -9.3 -16.2* -11.2 Thyroid gland (g) Recovery 0.02962 0.02518 -25.9* -9.1 Abbreviations: M = male, F = female, LM = light microscopy, Grade 1=minimal, Grade 2=mild, Grade 3=moderate a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by Dunnett’s test at 5% ( * ) or 1% ( ** ) or by Dunn’s test at 5% ( # ) or 1% ( ## ) . b Number of animals with findings. c In recovery animals no difference between treated and control rats.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7A - Repeat-Dose Toxicity (Rat, 2 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 2-week subcutaneous toxicity study in rats with a 2-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 2 20 200

Number of Animals on Study (Main Study+Recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 Gross pathology No treatment-related findings Histopathology b (LM) Number of rats examined (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Injection Site c Infiltrate: mixed inflammatory cell Grade 0 1 2 3

1 4 5 0

1 1 8 0

3 3 4 0

1 1 8 0

2 3 5 0

2 7 1 0

2 0 2 6

1 0 5 4

Additional Information: none Conclusions: NOAEL=200 µg/kg BID Abbreviations: M = male, F = female, LM = light microscopy, Grade 1=minimal, Grade 2=mild, Grade 3=moderate a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by Dunnett’s test at 5% ( * ) or 1% ( ** ) or by Dunn’s test at 5% ( # ) or 1% ( ## ) . b Number of animals with findings. c In recovery animals no difference between treated and control rats.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B – Repeat-Dose Toxicity (Rat, 13 weeks)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period Species/Strain: Rat/ Crl:CD(SD)IGSBR Duration of Dosing: 13 weeks Test Article: AVE0010 (week x to xx: batch xxxxxxxxxxxxx,

from week xx: batch xxxxxxxxxxxxxx) Initial Age: ca. 6 weeks Duration of Post-Dose: 4 weeks Study No.: [20 x-0063] + Amendment 1 [20 x-0063A1] Date of First Dose: xx xxxxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location : 4.2.3.2-14, 4.2.3.2-14a

Vehicle/Formulation : Saline (0.9% NaCl) / aqueous solution GLP Compliance: yes Special Features: none No Observed Adverse Effect Level: 1656.4 µg/kg BID

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4 Number of Animals on Study (Main Study) M: 15 F: 15 M: 10 F: 10 M: 10 F: 10 M: 15 F: 15 M: 10 F: 10 M: 15 F: 15 Toxicokinetics: First assay not including AVE0010 bound to antibodies AUC0-24 (ng.h/mL ) Day 1 - - 3.12 4.53 21.31 19.63 143.19 132.61 293.61 345.91 321.70 535.59 Cmax (ng/mL) Day 1 - - 1.84 2.58 7.75 8.99 65.85 43.20 120.35 113.20 146.00 132.85 Total AVE0010 assay AUC0-24 (ng.h/mL ) Day 1 Day 29 Day 92

- - - - - - 121 3223 17642

113 3363 20637

173 6195 39235

218 6613 28916

255 1822 14348

429 3464 15144

Cmax (ng/mL) Day 1 Day 29 Day 92

- - - - - - 42

835 1837

38 391 1149

67 723 5374

85 864 2461

156 250 1215

118 776 2643

Anti-drug antibody formation % of ADA positive rat Week 14 0 0 70 90 100 90 90 100 80 80 90 100 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = are under the curve, Cmax = maximum concentration, ADA=anti-drug antibodies Total AVE0010 TK assay was only performed with the three higher dose levels

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B – Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5

F: 10+5 M: 10 F: 10 M:

10+5 F:

10+5 Noteworthy Findings: Decreased body weight gain in males at ≥4.2 µg/kg BID, increased body weight gain in recovery period, no significantly decreased body weight (gain) in females Decreased food consumption in males at ≥4.2 µg/kg BID, increased food consumption in recovery period, no relevant effect in females Increased water consumption in both genders at ≥4.2 µg/kg BID Died or Killed Moribund 0 0 0 0 0 0 0 0 0 0 0 0 Absolute Body Weight (g and %)a Week 1 Week 4 Week 8 Week 13 Week 17

232.8 347.5 443.3 496.3 535.3

174.3 223.1 259.7 279.4 285.1

-13 -16 -15 -12

-1.5 -2.9

-0.46 -0.18

-2.3 -7.4 -7.7 -6.3

-0.11 -0.72 -3.04 -5.65

-11 -12 -13 -13 -8.2

+0.23 +3.1 +1.5 -0.93

-11 -11 -9.4 -8.2

+4.7 +6.7 +4.0

+0.75

-11 -9.4 -12 -9.2 +1.7

+0.06 +6.1 +3.3 +1.6 +4.0

Average Body Weight Gain (g and %)a Week 1 to 4 Week 4 to 8 Week 8 to 13 Week 13 to 17 (Recovery rats only)

159.4 95.8 52.9 25.4

73.5 36.6 19.7 7.6

-24***

-15 +14

-9.4 +14 +3.6

-22*** -9.0 +5.9

-9.3 -17 -40

-22***

-18 -12

+98**

+6.1 -8.2 -33 -21

-20*** -2.4 +2.3

+9.9 -13 -42

-18*** -21* +14 +56

+16** -14 -21 +72

Average Daily Food Consumption (g/rat/week and %)a Week 1 Week 4 Week 8 Week 13 Week 17

176.4 177.4 191.2 187.8 150.0

123.1 123.5 124.3 128.7 107.7

-19 -14 -13 -6.0

+0.65 +1.8 +22 +2.2

-8.8 -16 -7.3 -3.4

-5.3 -15 -4.5 -8.9

-23 -12 -8.6 -8.8 +15

-0.65 -0.65 +8.8 +1.1 +8.4

-22 -12 -4.9 -9.6

+4.4 +0.4 +8.0 -5.9

-25 -7.9 -11 -7.5 +15

-2.8 +3.9 +12 +3.2 +14

Abbreviations: M = male, F = female a At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical difference is based on actual data (not percent differences). Statistical evaluation was only performed for average daily body weight gains, * p<0.05, ** p<0.01, ***p<0.001

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B – Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5

F: 10+5 M: 10 F: 10 M:

10+5 F:

10+5 Average Daily Water Consumption (g/rat/week and %)a Week 1 Week 4 Week 8 Week 13 Week 17

163.7 199.3 201.2 202.1 182.5

125.9 156.7 161.4 173.3 159.7

+5.6 +5.8 +11 +8.8

+38 +37 +28 -2.7

+19 +23 +11 +9.6

+36 +31 +21 +8.5

+11 +8.8 +8.5 +3.8 +10

+43 +28 +25 +11 +23

+12 +8.8 +4.0 +9.7

+41 +25 +26 +1.0

+0.92 +17 +25 +23 +18

+51 +49 +45 +15 +41

Abbreviations: M = male, F = female a At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical difference is based on actual data (not percent differences). Statistical evaluation was only performed for average daily body weight gains, * p<0.05, ** p<0.01, ***p<0.001

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B – Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5

F: 10+5 M: 10 F: 10 M: 10 F: 10 M:

10+5 F:

10+5 M: 10 F: 10 M: 10+5

F: 10+5

Clinical Observations Lethargy 0 0 10 10 10 10 15 15 10 10 15 15 Salivation 0 0 10 10 P 10 15 15 10 10 15 15 Paddling (rapid wiping of the face with forepaws) 0 0 10 10 10 10 15 15 10 10 15 15 Mouth rubbing (on cage grid bottoms) 0 0 10 10 10 10 15 15 10 10 15 15 Ophthalmoscopy (Week 4 and 12 ) No treatment related changes Abbreviations: M = male, F = female, P: sign present but not reported by individual animal so that no incidence can be given

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 M: 10 F: 10 M: 10+5 F: 10+5

Hematology Hemoglobin (g/dL) week 4 15.9 16.0 16.4 16.8 16.9*** 16.8* 16.7*** 16.2 16.7*** 17.0** 16.4 16.7 Red Blood Cells (mil/cmm) week 4 7.83 7.90 8.05 8.34* 8.25 8.32* 8.20 8.18 8.26* 8.25 8.04 8.32* Red Blood Cells (mil/cmm) week 13

9.00 8.30 9.17 8.62 9.27 8.64 9.16 8.25 8.96 8.91* 9.19 8.79

PCV (%) week 4 46.3 44.8 47.4 47.9** 48.4* 46.7 48.3* 46.1 48.8** 47.6** 47.1 46.9* RETA (%) week 4 3.3 2.4 3.5 2.8 2.7 1.7*** 2.7 2.0* 2.9 1.9* 3.1 2.1 RETA (%) week 13 2.8 3.1 2.6 2.9 2.2 2.3* 2.2 2.8 2.8 2.1** 2.2 2.4* MCV (fL) week 13 54.4 56.5 54.4 56.6 53.8 55.4 54.1 54.9 54.7 56.0 54.7 54.9# * MCHC (g/dL) week 13 32.9 32.5 33.3 32.8 33.5 33.1 33.6* 33.0 33.2 32.5 33.3 33.1 Reticulocytes (mil/cmm) week 4 0.25 0.19 0.28 0.23* 0.22 0.14** 0.22 0.16 0.24 0.16 0.25 0.17 Reticulocytes (mil/cmm) week 13 0.25 0.25 0.24 0.25 0.20 0.20* 0.20 0.23 0.25 0.19* 0.20 0.21 RDW (%) week 4 11.3 10.5 11.5 10.5 11.5 10.6 11.6 10.6 11.8 10.5 11.8# * 10.8* RDW (%) week 13 13.6 12.2 13.2 11.7 13.0 11.8 12.4* 12.3 13.4 11.4* 13.0 11.8 White Blood Cells (1000/cmm) week 4

12.3 8.6 10.0 7.7 7.6** 5.0*** 9.7 7.4 8.8* 5.6** 9.2 6.0**

White Blood Cells (1000/cmm) week 13

12.7 7.4 10.2 8.1 10.6 6.1 9.2* 8.5 10.4 6.2 7.7*** 6.5

Abbreviations: M = male, F = female Statistical evaluation of terminal sacrificed groups was performed by ANOVA, dose response and Dunnett’s test, except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was done. Statistical evaluation of recovery groups was performed by two-way ANOVA, dose response and Dunnett’s test, except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was performed. *p<0.05, ** p<0.01, ***p<0.001 #Significant dose response test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 M: 10 F: 10 M: 10+5 F: 10+5

Hematology (continued) Neutrophils (1000/cmm) week 4 1.4 0.9 2.5 1.6* 1.9 0.7 1.4 0.8 1.5 1.0 2.2 0.9 Neutrophils (%) week 4 12 11 23* 20* 25** 15 15 12 18 17 24** 15 Neutrophils (%) week 13 15 12 22 17 19 12 20 13 24 18 23# 14 Lymphocytes (1000/cmm) week 4 10.5 7.4 7.1* 5.7 5.3*** 4.0*** 7.9 6.2 6.9* 4.4*** 6.6** 4.8** Lymphocytes (1000/cmm) week 13 10.1 6.2 7.3* 6.3 7.8 5.1 6.6** 7.0 7.5* 4.8 5.6*** 5.2 Lymphocytes (%) week 4 85 86 73* 75** 69*** 79 80 84 78 79 71** 80 Hematology Recovery No treatment-related findings Coagulation Week 4 No treatment-related findings Coagulation Week 13 APTT (s) 21.1 16.4 19.2 16.0 22.2 17.2 20.2 16.4 19.7 16.1 19.9 14.5* Coagulation Recovery No treatment-related findings Abbreviations: M = male, F = female Statistical evaluation of terminal sacrificed groups was performed by ANOVA, dose response and Dunnett’s test, except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was done. Statistical evaluation of recovery groups was performed by two-way ANOVA, dose response and Dunnett’s test, except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was performed. *p<0.05, ** p<0.01, ***p<0.001 #Significant dose response test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 M: 10 F: 10 M: 10+5 F: 10+5

Serum Chemistry AST (IU/L) week 4 95 86 95 93 99 98* 102 93 103 94 98 96 AST (IU/L) week 13 87 89 96 90 91 92 89 92 90 91 86 105# * ALT (IU/L) week 4 47 39 52 47 55 46 65** 36 59* 40 49 42 Alkaline Phosphatase (IU/L) week 4

547 373 651 435 592 379 658 414 651 419 579 503**

Alkaline Phosphatase (IU/L) week 13

238 178 295 177 270 153 304 173 261 174 274 222# *

Alkaline Phosphatase (IU/L) Recovery

248 115 227 136 198# * 105

Sodium (mmol/L) Week 4 146 145 143 145 145 145 145 146 147 145 146#* 145 Potassium (mmol/L) Week 4 4.7 4.2 4.5 4.0 3.8*** 3.9 4.4 4.4 4.0*** 4.1 4.2* 3.8 Potassium (mmol/L) Week 13 4.9 4.7 4.5 4.6 4.3** 5.1 4.4** 4.6 4.6 4.5 4.3*** 4.4 Calcium (mmol/L) Week 4 2.74 2.72 2.72 2.73 2.69 2.68 2.79 2.76 2.74 2.79 2.74 2.77# * Calcium (mmol/L) Week 13 2.82 2.73 2.74* 2.69 2.75* 2.68 2.80 2.66 2.80 2.70 2.77 2.77 Phosphate (mmol/L) Week 4 2.9 2.5 2.8 2.1* 2.4*** 2.3 2.9 2.5 2.6** 2.3 2.6 2.4 Glucose (mmol/L) Week 4 5.5 6.0 5.5 6.8 6.7* 6.5 5.6 5.5 6.2 6.4 5.9 6.2 Bilirubin (umol/L) Week 13 2.0 3.1 2.1 2.5 2.0 2.9 2.4 3.1 2.3 2.9 1.7 1.7** Cholesterol (mmol/L) Week 4 1.9 1.9 2.1 2.0 1.9 1.7 1.9 1.9 2.0 2.1 2.0 2.1# * Cholesterol (mmol/L) Week 13 1.9 1.9 1.9 2.0 1.7 1.9 1.8 2.0 1.8 2.2 1.7 2.1# * Albumin/Globulin Recovery 1.5 1.7 1.6 1.9 1.5 2.1* Abbreviations: See next page

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study (Main Study+recovery) M: 10+5 F: 10+5 M: 10 F: 10 M: 10 F: 10 M: 10+5 F: 10+5 M: 10 F: 10 M: 10+5 F: 10+5

Urinalysis: Specific Gravity Week 4 1.030 1.039 1.038 1.060** 1.045 1.036 1.048* 1.031 1.052** 1.044 1.039 1.040 Specific Gravity Week Recovery 1.055 1.041 1.026** 1.034 1.025** 1.033 Volume (mL) Week 4 5.4 3.1 2.2*** 2.4 2.1*** 2.3 2.3*** 3.7 2.5*** 3.0 3.7* 3.3 Volume (mL) Week 12 7.0 3.3 4.9 3.2 4.4* 2.8 4.4* 3.7 4.7* 3.2 5.2 2.9 Volume (mL) Recovery 2.8 3.9 8.0** 4.5 11.6*** 8.1* Sodium (mmol/L) Week 4 89 107 45** 27*** 58 44** 51* 60** 45** 41** 59 42** Sodium (mmol/L) Week 12 86 116 77 63* 90 69 64 97 65 55** 66# * 88 Total Sodium (mmol) Week 4 0.48 0.31 0.10*** 0.06*** 0.13** 0.11** 0.12*** 0.23 0.11** 0.12** 0.21** 0.14* Total Sodium (mmol) Week 12 0.57 0.40 0.35* 0.19** 0.42 0.25 0.29*** 0.38 0.30** 0.18** 0.34** 0.26 Total Sodium (mmol) Recovery 0.36 0.35 0.76* 0.46 1.11*** 0.65# * Potassium (mmol/L) Week 4 220 201 243 306* 269 167 329* 168 331* 237 262 230 Potassium (mmol/L) Recovery 309 186 128*** 170 124*** 165 Total Potassium (mmol) Week 4 1.15 0.61 0.52*** 0.70 0.54*** 0.38* 0.69*** 0.58 0.85* 0.61 0.89 0.67 Total Potassium (mmol) Week 12 1.30 0.61 0.97** 0.46 0.91*** 0.56 0.66*** 0.49 0.94** 0.46 0.97** 0.50 Total Potassium (mmol) Recovery 0.86 0.68 1.02 0.62 1.43*** 1.08** Abbreviations: M = male, F = female Statistical evaluation of terminal sacrificed groups was performed by ANOVA, dose response and Dunnett’s test., except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was done. Statistical evaluation of recovery groups was performed by two-way ANOVA, dose response and Dunnett’s test., except Bilirubin for which Kruskal-Wallis, Terpstra-Jonckheere and Wilcoxon test was performed. *p<0.05, ** p<0.01, ***p<0.001 #Significant dose response test

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study M: 15 F: 15 M: 10 F: 10 M: 10 F: 10 M: 15 F: 15 M: 10 F: 10 M: 15 F: 15 Organ Weights a Week 13 Adrenal Gland (%) 0.013 0.023 +15.4 +13.0 ±0 +4.4 +7.7 +8.7 +15.4 +4.4 +30.8* +13 Kidney (g) 2.604 1.420 -14.8** +13** -9.5 -0.6 -11.8* +12.5** -9.9* +10* -15.5*** +17.4*** Kidney (%) 0.566 0.542 -4.4 +13.5** -4.2 +4.6 +1.0 +13.5** -3.2 +9.4* -1.1 +17.2*** Kidney (g) Recovery 2.683 1.493 -5-5 +13.3 -2.9 +23.4# * Spleen (g) 0.851 0.522 -7.9 +12.3 -12 -6.9 -22.4** +10.9 -13.4 +1.9 -21.7** +13.8 Spleen (%) 0.185 0.198 +3.8 +13.1 -7.0 -1.5 -10.8 +12.1 -6.5 +1.0 -8.1# * +14.1 Liver (g) 11.381 6.466 -11 +13.2 -12 -8.8 -17.3** +5.0 -9.3 +5.8 -19** +9.8 Liver (%) 2.465 2.468 +0.4 +13.3** -6.4 -4.3 -5.2 +5.8 -3.0 +4.7 -5.0 +9.5 Heart (g) 1.501 0.894 -10.2 +9.6 -7.5 -4.0 -12.1* -0.4 -15.8** -0.2 -19.4*** +1.0 Heart (%) 0.328 0.341 +0.3 +10.3 -2.7 +1.2 -0.3 +0.3 -10.6 -0.6 -6.4# * +0.6 Brain (%) 0.460 0.734 +11.1* +1.2 +5.2 +6.7 +12.2* +3.0 +6.3 -0.3 +16.1** +2.7 Brain (g) Recovery 2.184 1.907 +0.8 +6.7* -0.2 +5.4* Brain (%) Recovery 0.403 0.667 +10.4* *5.8 -1.7 +0.6 Pituitary(g) 0.014 0.013 -7.1 0 -7.1 0 -14.3 +7.7 -7.1 +15.4 -21.4# * +23.1# * Pituitary(%) 0.003 0.005 ±0 ±0 ±0 ±0 ±0 ±0 ±0 ±0 ±0 +20.0# ** Testis (%) 0.794 +9.9 +4.0 +9.8 +5.3 +12.5* Prostate/Seminal Vesicle (g) 2.919 -12.6 -23.4** -27** -14.9 -17.5 Prostate/Seminal Vesicle (%) 0.638 -2.5 -20.1* -17.2 -9.9 -4.7 Abbreviations: M = male, F = female, LM = light microscopy; a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by ANOVA, dose response and Dunnett’s test. * p<0.05, ** p<0.01, ***p<0.001 #Significant dose response test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7B - Repeat-Dose Toxicity (Rat, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous administration toxicity study in rats with a 4-week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 4.1 16.6 165.6 828.2 1656.4

Number of Animals on Study M: 15 F: 15 M: 10 F: 10 M: 10 F: 10 M: 15 F: 15 M: 10 F: 10 M: 15 F: 15 Gross Pathology No treatment-related findings Histopathology (LM) No treatment-related findings Additional Information: none Conclusions: NOAEL = 1656.4 µg/kg BID Abbreviations: M = male, F = female, LM = light microscopy a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by ANOVA, dose response and Dunnett’s test. * p<0.05, ** p<0.01, ***p<0.001 #Significant dose response test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C – Repeat-Dose Toxicity (Rat, 6 months)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period Species/Strain: Rat/Hsd Sprague Dawley Duration of Dosing: 6 months (182/183 days) Test Article: AVE0010 (batch xxxxxxxxxxxxxx) Initial Age: 6 to 7 weeks Duration of Post-Dose: 4 weeks Study No.: [20 x-0085] Date of First Dose: xx xxxxxxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location : 4.2.3.2-15

Vehicle/Formulation : 0.9% sodium chloride/ aqueous solution GLP Compliance : yes Special Features: none No Observed Adverse Effect Level: 2000 µg/kg BID Dose (µg/kg BID) 0 (Vehicle Control) 5 100 2000 Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Toxicokinetics: AUC0-24 (ng.h/mL ) Day 1 Day 7 Day 183

- - 8.77 10.5 15.7

7.74 11.7 12.8

143 146 2735

110 132 3914

711 2815 52597

697 2298 35563

Cmax (ng/mL) Day 1 Day 7 Day 183

- - 4.70 5.22 6.42

4.39 6.08 6.81

57.1 78.7 334

57.2 68.1 427

250 537 3303

386 622 3301

Anti-drug antibody formation % of ADA positive rats Day 7 Day 28 Day 154 Day 210 (M) / 208 (F)

5 5 0 0

5 5 5 0

7 7 47 -

0 0 20 -

0 100 100

-

0 100 100

-

13 100 100 100

0 100 89

100 Additional Information: Values are rounded to 3 significant figures or less. Noteworthy Findings: Mean body weight gain between Day 1 and Day 182 reduced in all groups Decreased food consumption at all dose levels Abbreviations: M = male, F = female, AUC = area under the curve, Cmax = maximum concentration

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C – Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Died or Killed Moribund (main study / TK animals) b 0 / 1 0 / 0 0 / 2 0 / 0 0 / 1 0 / 1 0 / 1 1 / 2 Absolute Body Weight (g) and (%)a Day 1 Day 8 Day 29 Day 92 Day 182 Day end of recovery (M/F: Days 210 / 208)

166.9 211.1 292.1 380.4 444.9 447.2

151.5 171.5 217.3 252.5 282.9 286.4

0

-7* -8* -6 -9*

+2 +3 +1 -1 -5*

0

-5* -6* -4 -7*

+3 +3 -1 -3* -9*

-1

-10* -7* -7

-11* +4

+1 -1 -4* -8*

-11* -2

Average Daily Body Weight Gain (g/animal/day and %)a Day 1 to 8 Day 1 to 29 Day 1 to 92 Day 1 to 182 Day 1 to end of recovery (M/F: Days 210 / 208)

44.2

125.3 213.6 278.5 279.2

20.0 65.8

101.0 132.7 131.2

-33* -19* -9

-14*

+6 0 -6

-15*

-23* -15* -7

-11*

+1

-11* -13* -23*

-45* -15* -11 -18* +10

-14 -15* -22* -27*

0 Average Daily Food Consumption (g/animal/day and %)a Day 1 to 8 Day 22 to 29 Day 85 to 92 Day 176 to 182 Day 183 to 190 Day 190 197 Day 197 to 204 Day 204 to end of recovery (M/F: Days 210 / 208)

20.01 20.74 19.51 20.95 20.83 19.48 20.54 20.01

15.92 17.13 15.73 16.61 16.29 17.55 15.75 15.93

-17* -9 -2 -3

-9* -6 -3 -3

-17* -7* -2 -2

-15* -8* -5 0

-29* -5 -3 -5

+25 +29 +12 +10

-21* -3 -5 -7* +18 +7

+13 -4

Abbreviations: M = male, F = female, TK = Toxicokinetics. Note: Day and time intervals are representative ones of continuous monitoring a At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical difference is based on actual data (not percent differences). b All single intermittent deaths considered to be incidental, based on isolated occurrence

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C – Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period

Dose (µg/kg BID) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Clinical Observations [no. of animals (no. of observations)] Squeaky noises during application - - 2 (2) 1 (1) 14 (78) 12 (100) 20 (179) 18 (227) Ophthalmoscopy (Week M/F: 13/12 and end of dosing) No treatment related findings Abbreviations: M = male, F = female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C - Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Hematology Red Blood Cells (10E12/L) Week 17 8.562 7.833 8.086* 7.785 8.205* 7.572 8.178* 7.829 Neutrophils (10E9/L) Week 17 1.483 1.350 1.608 1.221 1.416 1.383 2.771* 1.097 Neutrophils (%) Week 27 28.13 17.36 18.59* 18.18 20.47* 17.62 19.63* 14.39 Lymphocytes (%) Week 17 80.95 78.85 79.25 80.21 81.96 77.52 73.07* 81.49 Lymphocytes (%) Week 27 65.31 73.95 73.86* 72.43 72.01* 73.47 74.40* 77.73* Lymphocytes (10E9/L) Week 27 7.587 6.700 7.264 5.680 7.713 6.814 8.714* 7.500 Lymphocytes (10E9/L) Recovery 5.140 5.240 7.520* 5.700 Monocytes (10E9/L) Week 17 0.268 0.278 0.332* 0.294 0.342* 0.291 0.352* 0.225 Eosinophils (10E9/L) Week 17 0.254 0.342 0.342 0.351 0.343 0.351 0.301 0.275* LU Cells (%) Week 27 0.57 0.69 0.52 0.70 0.46* 0.60 0.46* 0.71 Coagulation PT (s) Week 27 13.66 14.67 13.82 14.52 13.78 14.73 14.21* 14.74 Serum Chemistry AST (U/L) Week 14 41.3 46.5 41.7 44.1 41.3 38.7* 40.1 42.1* ALP (U/L) Week 14 146.1 122.9 223.8 140.5* 205.5 156.0* 159.9 144.5* Total Bilirubin (umol/L) Week 14 9.29 10.21 7.07* 9.59 7.49* 8.63 7.01* 9.15 Total Bilirubin (umol/L) Week 27 10.43 9.57 10.57 10.82 9.29* 9.50 8.97* 9.90 Total Cholesterol (mmol/L) Week 14 2.573 2.607 2.213 2.940* 2.347 3.273* 2.433 3.047* Triglycerides (mmol/L) Week 14 0.423 0.279 0.609 0.408 0.525 0.309 0.296* 0.311 Triglycerides (mmol/L) Week 27 0.565 0.404 0.575 0.464 0.499 0.420 0.330* 0.357 Abbreviations: M = male, F = female * Significantly different (p<=0.05) from control.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C - Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Serum Chemistry (continued) Glucose (mmol/L) Week 14 5.073 4.960 4.593 4.693 4.720 5.267 4.760 5.527* Glucose (mmol/L) Week 27 6.280 5.667 6.640 5.467 6.247 5.113* 5.507 4.860* Urea Nitrogen (mmol/L) Week 14 5.320 6.087 6.407* 6.500 6.653* 6.473 6.007* 7.367* Urea Nitrogen (mmol/L) Week 27 6.607 7.300 8.367 8.567* 7.700 8.453* 7.500 8.360* Sodium (mmol/L) Week 14 145.53 141.73 144.53 142.87* 144.07 143.40* 145.13 143.73* Sodium (mmol/L) Week 27 146.47 143.60 146.27 144.47 146.40 144.33 147.13 145.13* Calcium (mmol/L) Week 14 2.367 2.395 2.410* 2.460* 2.481* 2.493* 2.477* 2.525* Calcium (mmol/L) Week 27 2.650 2.643 2.685 2.701* 2.712 2.710* 2.665 2.710* Chloride (mmol/L) Week 14 103.9 103.5 102.4 104.1 104.1 105.0* 105.5* 105.1* Phosphate (mmol/L) Week 14 2.383 2.295 2.253 2.219 2.472 2.047* 2.346 2.160* Total Protein (g/L) Week 14 71.5 67.3 69.8 67.4 70.7 71.2* 71.0 73.5* Albumin (g/L) Week 14 35.7 35.5 35.5 35.1 35.1 36.4* 34.3* 37.3* Globulin (g/L) Week 14 35.7 31.8 34.3 32.3 35.5 34.8* 36.7 36.2* Globulin (g/L) Week 27 28.2 26.8 26.8 27.6 28.0 29.3* 28.6 29.5* Albumin/Globulin Week 14 1.003 1.117 1.039 1.090 0.991 1.051* 0.939* 1.033* Albumin/Globulin Week 27 33.7 1.321 35.3 1.271 35.1 1.182* 34.1 1.186* Urinalysis Creatinin (µmol/ColTime) 61.51 37.28 48.42 30.70* 46.61 31.04* 54.88 32.08* Abbreviations: M = male, F = female * Significantly different (p<=0.05) from control.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C - Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period (continued)

Dose (mg/kg/day) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Organ Weights (%) a Adrenal gland (g) 0.04847 0.06177 +37.5## +18.6** +15.8 +9.7 -1.8 +1.6 Adrenal gland (%) 0.01163 0.02303 +48.7## +25.5** +23.4## +21.0** +10.4 +16.1 Brain (g) 1.94 1.82 -3.6 -2.2 -1.5 -3.8* -2.1 -2.7 Brain (%) 0.46558 0.67975 +5.4 +3.5 +4.8 +5.7 +10.3** +11.0** Heart (g) 1.38 0.96563 -2.9 -1.9 -2-2 -5.2 -9.4 -8.2* Heart (%) 0.32993 0.35992 +7.2* -2.4 +4.0 -5.7 +2.1 -8.7* Kidney (g) 2.26 1.54 +4.0 +8.4* 5.3 +6.5 -5.3 +2.6 Kidney (%) 0.53712 0.57254 +14.4** +15.1** +13.0** +17.3** +7.1* +16.5** Liver (g) 10.3 6.33 +1.7 +13.6# -0.1 +8.1 -12.2* -2.4 Liver (%) 2.44 2.35 +12.3## +20.4** +7.4# +18.7** -0.8 +11.1** Spleen (g) 0.74135 0.59395 -18.6** -11.1* -13.6* -12.9* -15.7** -8.0 Spleen (%) 0.17628 0.22081 -11.0** -5.7 -7.5 -3.8 -4.6 +4.5 Pituitary gland (%) 0.00300 0.00648 +20* -2.5 +3.3 +4.8 +17.0 -7.6 Prostate (g) 3.35 -10.2 -15.2# -12.2# Testis (%) 0.86432 +11.7## +11.7## -2.9 Gross Pathology No treatment-related findings Abbreviations: a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by Dunnett’s test at 5% ( * ) or 1% ( ** ) or by Dunn’s test at 5% ( # ) or 1% ( ## ) . b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=mild, Grade 3=moderate c In recovery animals no difference between treated and control rats.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7C - Repeat-Dose Toxicity (Rat, 6 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 6-month subcutaneous toxicity study in rats with a 4 week recovery period (continued)

Dose (mg/kg/day) 0 (Vehicle Control) 5 100 2000

Number of Animals on Study (Main Study + recovery) M: 15+5 F: 15+5 M: 15 F: 15 M: 15 F: 15 M: 15+5 F:15+5 Histopathology (LM) b Number of rats examined 15 15 15 15 15 15 15 15 Injection Site c Reaction: fibroblastic Grade 0 2 3

14 1 0

9 4 2

13 1 1

9 4 2

7 6 2

8 7 0

7 6 2

10 5 0

Testis c Atrophy: seminiferous tubule Stasis : spermatid Mineralization Necrosis: seminiferous tubule Inflammation: chronic

2 1 0 0 0

2 1 0 0 0

1 1 1 0 0

5 3

4* 1 1

Additional Information: none Conclusions: NOAEL = 2000 µg/kg BID Abbreviations: a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by One-Sided Exact Fisher test at 5% ( * ) . b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=mild, Grade 3=moderate c In recovery animals no difference between treated and control rats.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7D – Repeat-Dose Toxicity (Rat, 4 weeks)

Test Article: Lixisenatide Report Title: ZP10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks Species/Strain: Rat Crl:CD (SD) IGS BR Duration of Dosing: 4 weeks Test Article: AVE0010 (batch xxxxxxxxx) Initial Age: 5 weeks Duration of Post-Dose: None Study No.: [20 x-2059]

Separate toxicokinetics GMPK report [F20 xKIN0227] Separate bioanalytical report MDS report [F20 xKIN0249] Separate antibody determination report no. [DIV1156]

Date of First Dose: xx xxxx 20xx Method of Administration: Intravenous (bolus) Location: 4.2.3.2-10, 4.2.3.2-11, 4.2.3.2-12, 4.2.3.2-13 Vehicle/Formulation : Sodium citrate buffer (pH 5.3) / aqueous solution GLP Compliance: Yes Special Features: None, BID dosing with an interval of approximately 8 hours No Observed Adverse Effect Level: 30 µg/kg BID Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30 Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Toxicokinetics: * AUC0-24 (ng.h/mL )* Day 1 Day 28

- - 1.64 2.05

1.37 1.65

6.36 8.07

6.74 7.01

24.9 29.3

20.2 26.3

Cmax (ng/mL)* Day 1 Day 28

- - 3.95 4.34

3.35 4.27

13.3 16.0

16.4 16.0

66.1 74.1

54.9 68.7

Anti-drug antibody formation: no specific anti-AVE0010 antibodies were detected in any group Day 29: Response after addition of soluble AVE0010 to serum / Response without addition of soluble AVE0010

0.94 0.88 0.98 0.93 0.96 0.94 0.92 0.98

Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = are under the curve, Cmax = maximum concentration * Note: The time period between blood sampling and bioanalytic drug level determination was approximately x years due to technical reasons. The long-term stability of AVE0010 in EDTA plasma was proven at –xx°C and –xx °C only for x months. The toxicokinetic parameters for the 4-week IV rat study are therefore given with the restriction, that stability of AVE0010 in EDTA plasma at –xx°C was not investigated for the period of x years. The values are therefore not included in the toxicokinetics overview table.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7D – Repeat-Dose Toxicity (Rat, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks

Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Noteworthy Findings: Decreased weight gain in males in week 1, slightly decreased food consumption in week 1, water consumption increased (week 3) Died or Killed Moribund 0 0 0 0 0 0 0 0 Absolute Body Weight (g and %)a Week 1 Week 2 Week 3 Week 4

294.2 314.2 328.9 334.3

202.1 210.8 220.2 223.4

-3.2 -1.6 -0.5 -1.1

+1.9 ±0 -1.1 -1.8

-5.0 -3.4 -2.8 -3.2

+1.3 -1.1 -1.3 -1.4

-0.6 +1.0 +1.1 ±0

+3.6 +3.3 +2.5 +1.0

Average Body Weight Gain (g per week %)a Week 1 Week 1+2 Week 1 to 3 Week 1 to 4

24.6 44.6 59.3 64.7

11.4 20.1 29.5 32.7

-31 -7.8

-0.17 -3.2

+32 -1.5 -8.8 -13

-34 -9.4 -4.7 -6.8

+46 +1.0 -1.4 -1.8

-20

-0.22 +0.67 -4.9

+14 +6.5 -1.0 -10

Average Daily Food Consumption (g per cage and %)ab Week 1 Week 2 Week 3 Week 4

173 155 154 135

118 115 114 108

-5.2 # +2.6 +1.9 -0.74

-4.2 # -1.7 -3.5 -8.3

-8.7 -3.9 -3.9 -7.4

-5.1 -1.7 -5.3 -11

-6.4 +6.5 +2.6 +0.74

-4.2 # +3.5 ±0 -7.4

Average Daily Water Consumption (g/rat/day and %)a Week 3 (assessed during 7 days in week 3 only)

27.4

24.3

+97

+152

+61

+85

+86

+89

Abbreviations: M = male, F = female a For controls, group means are shown. For treated groups, percent differences from controls are shown. b 5 rats were housed per cage. # Value from one cage only (other cage excluded due to excessively wet residue) Only body weight was evaluated statistically (and showed no significant differences)

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TS 2.6.7.7D – Repeat-Dose Toxicity (Rat, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks

Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Clinical Observations Piloerection 3 1 10 10 10 10 10 10 Abnormal gait 0 0 10 10 10 10 10 10 Hypoactivity 0 0 10 10 10 10 10 10 Irregular/fast respiration 0 0 10 10 10 10 10 10 Hunched posture 0 0 8 10 10 9 10 10 Eyelids partially closed 0 0 2 3 9 5 10 9 Increased vocalization 0 0 0 0 0 1 0 3 Walking on toes 0 0 0 0 0 6 0 0 Ophthalmoscopy (Week 4 ) No noteworthy findings Additional information: Number of animals showing clinical signs decreased with progressing treatment, from Day 27 no animal showed clinical signs Abbreviations: M = male, F = female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7D - Repeat-Dose Toxicity (Rat, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks

Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Hematology (Week 4) Hematocrit (L/L) 0.452 0.424 0.447 0.430 0.455 0.442** 0.450 0.439** Lymphocytes (x10*9/L) 12.70 11.57 8.88** 10.90 10.26** 10.94 9.77** 11.68 Basophils (x10*9/L) 0.06 0.04 0.02* 0.05 0.04* 0.04 0.04* 0.05 Coagulation (Week 4) PT (s) 13.3 13.9 13.9 14.2 14.2 14.4 14.7** 14.5* Serum Chemistry (Week 4): Bilirubin (umol/L) 2 2 2 2 2 2 2* 2 Urea nitrogen (mmol/L) 4.19 5.12 5.84** 5.75 5.51** 5.58 5.21** 6.03 Glucose (mmol/L) 6.68 5.32 6.71 5.20 6.54 4.92 7.64* 5.46 Phosphate (mmol/L) 2.33 1.95 2.38 1.88 2.38 2.01 2.44 2.25** Total Protein (g/L) 65 70 65 70 67 69 67 67** Albumin (g/L) 31 37 31 35** 30 34** 29 33** Alpha 1 Globulin (g/L) 13 12 12 12 13 11 12 11** Alpha 2 Globulin (g/L) 4 5 5** 5 5** 4 5** 4 Beta Globulin (g/L) 14 14 14 15** 15 15** 16 15** Albumin/Globulin Ratio 0.90 1.10 0.90 0.99** 0.83 0.98** 0.82 0.99**

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7D - Repeat-Dose Toxicity (Rat, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks

Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Urinalysis (Week 4): pH 7.1 6.7 7.4 7.1 7.3 7.0 7.7* 6.8 Specific Gravity (g/L) 1033 1034 1027 1019++ 1033 1037 1026 1036 Sodium (mmol/L) 102.1 107.9 39.9** 23.0 53.8** 39.3++ 65.2** 63.3++ Chloride (mmol/L) 108.0 110.8 66.4 42.1++ 86.2 72.7+ 89.0 88.2 Abbreviations: M = male, F = female Statistical evaluation was performed by William’s or Dunnett’s test. * p<0.05, ** p<0.01 William’s test + p<0.05, ++ p<0.01 Dunnett’s test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7D - Repeat-Dose Toxicity (Rat, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by intravenous (Bolus) Administration to CD rats for 4 weeks

Dose (µg/kg BID) 0 (Vehicle Control) 3 10 30

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Organ Weights a Week 4 Adrenal Gland (g) 0.0572 0.0618 +0.4 +13.1 +6.1 +11.8 ±0 +16.5* Spleen (g) 0.6490 0.4991 -15.4* -6.9 -13.3* -5.2 -10.8* -9.5 Lung (g) 1.362 1.197 -2.8 -15.5 +8.9 -9.4 +3.4 -3.3# Gross Pathology No treatment-related findings Histopathology (LM) No treatment-related findings Additional Information: none Conclusions: NOAEL = 30 µg/kg BID Abbreviations: M = male, F = female, LM = light microscopy a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by William’s, Dunnett’s or Shirley’s test. * p<0.05 William’s test + p<0.05, ++ p<0.01 Dunnett’s test # p<0.05 Shirley’s test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7E – Repeat-Dose Toxicity (Dog, 4 weeks)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks Species/Strain: Dog / Harlan Beagle Duration of Dosing: 4 weeks Test Article: AVE0010 (batch xxxxxxxxx)

Initial Age: 18 to 22 weeks Duration of Post-Dose: None Study No.: [20 x-2060]

Separate antibody determination report no. [DIV1157] Date of First Dose: xx xxxx 20xx Method of Administration: Subcutaneous Location : 4.2.3.2-17, 4.2.3.2-18 Vehicle/Formulation: Saline (0.9% NaCl) /aqueous solution / BID dosing with an interval of approximately 8 hours GLP Compliance: Yes Special Features: Non No Observed Adverse Effect Level: 200 µg/kg BID Dose (µg/kg BID) 0 (Vehicle Control) 10 40 200 Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Toxicokinetics: Sampling on Day 1 and 28, sample analysis not performed due to lack of analytical method at the time of study conduct AUC0-24 (ng.h/mL ) - - - - - - - - Cmax (ng/mL) - - - - - - - - Anti-drug antibody formation: no significant amounts of anti-AVE0010 antibodies were detected, but the variation in dog was too high for a definitive assessment on antibody response Day 29: Response after addition of soluble AVE0010 to serum / Response without addition of soluble AVE0010

0.96 0.90 0.79 0.81 0.87 0.62 0.72 0.85

Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = area under the curve, Cmax = maximum concentration

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7E – Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 Noteworthy Findings: Reduced body gain in females at ≥10 µg/kg BID in week 1 and in males and females at 200 µg/kg BID in week 2 Reduced food consumption in all groups most pronounced in the first 2 study weeks being statistically significant at 200 µg/kg BID in males and females between week 1 and 4 Died or Killed Moribund 0 0 0 0 0 0 0 0 Group mean Body Weight Gain (kg) per group Week 1 Week 2 Week 3 Week 4 Week 1 to 4

0.23 0.53 0.43 0.40 1.60

0.37 0.27 0.13 0.40 1.17

0.77 0.37 0.30 0.43 1.87

0.13 0.60 0.20 0.27 1.20

0.27 0.30 0.20 0.23 1.00

0.00 0.30 0.27 0.33 0.90

0.27 0.10 0.27 0.57 1.20

0.07 0.10 0.33 0.33 0.83

Group mean Body Weight Gain (kg per week and %)a Week 1 Week 2 Week 3 Week 4 Week 1 to 4

0.23 0.53 0.43 0.40 1.60

0.37 0.27 0.13 1.40 1.17

+235 -30 -30 +7.5 +18

-65

+122 +54 -33 +2.6

+17 -43 -53 -43 -37

-100 +11

+108 -18 -23

+17 -81 -37 +43 -24

-81 -63

+154 -18 -29

Average Daily Food Consumption (g/day and %)a Week 1 Week 2 Week 3 Week 4 Week 1 to 4

2810 2805 2804 2809

11228

2183 2456 2461 2585 9685

-29 -12 -6.5 -4.0 -13

-26 -22 -8.0 -15 -17

-24 -14 -14 -17 -17

-31 -24 -17 -16 -22

-40 -26 -19 -9.4 -23*

-33 -32 -22 -19 -26*

Abbreviations: M = male, F = female a For controls, group means are shown. For treated groups, percent differences from controls are shown. * p<0.05 (average daily food consumption and body weight gain week 1 to 4 was evaluated statistically)

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TS 2.6.7.7E – Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg/BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Clinical Observations There were no noteworthy clinical signs Ophthalmoscopy (Wk ) No treatment related changes Abbreviations: M = male, F = female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7E - Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg/BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Hematology (Week 4) Hematocrit (L/L) 0.375 0.390 0.359 0.345* 0.361 0.343* 0.358 0.377* Hemoglobin (g/dL) 12.6 13.2 11.9 11.2$$ 11.9 11.4$$ 11.9 12.6 Coagulation (Week 4) No treatment-related findings Serum Chemistry (Week 4): Phosphate (mmol/L) 2.58 2.60 2.46 2.59 2.61 2.49 2.78* 2.70 Chloride (mmol/L) 109 109 108 106* 110 111 110 109 Urinalysis (Week 4): pH 6.3 5.4 7.0 7.5 7.0 5.9 6.1 7.4** Abbreviations: M = male, F = female Statistical evaluation was performed by William’s test and Dunnett’s test. * p<0.05, ** p<0.01 William’s test $$ p<0.01 Dunnett’s test

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7E - Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Organ Weights (%) a Week 4 Heart (g) 68.1 48.8 -12.2* -1.4 -6.6* -12.5 -8.7* -0.6 Liver (g) 320 283 -9.1 -23** +5.0 -23.3** -0.6 -13.4** Gross Pathology No treatment-related changes Histopathology (LM) b Number of dogs examined M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Injection Site 1 Inflammation: subcutaneous Grade 0 1 2 Fibroblast Proliferation: subcutaneous Grade 0 1 2

2 1 0

2 1 0

2 1 0

3 0 0

3 0 0

3 0 0

3 0 0

3 0 0

1 0 2

0 1 2

1 1 1

2 1 0

2 1 0

2 1 0

1 0 2

0 2 1

Injection Site 2 Inflammation: subcutaneous Grade 0 1 2 Fibroblast Proliferation: subcutaneous Grade 0 1 2

2 1 0

2 1 0

3 0 0

3 0 0

2 1 0

2 1 0

3 0 0

3 0 0

2 1 0

1 2 0

2 1 0

2 1 0

2 1 0

2 1 0

2 1 0

2 0 1

Abbreviations: M = male, F = female a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by William’s test. * p<0.05, ** p<0.01 b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7E - Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Histopathology (LM) b Number of dogs examined M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Injection Site 3 Inflammation: subcutaneous Grade 0 1 2 Fibroblast Proliferation: subcutaneous Grade 0 1

3 0 0

2 1

3 0 0

3 0

2 1 0

1 2

1 1 1

2 1

2 1 0

3 0

1 1 1

1 2

1 1 1

3 0

1 2 0

0 3

Injection Site 4 Inflammation: subcutaneous Grade 0 1 2 Fibroblast Proliferation: subcutaneous Grade 0 1 2

1 2 0

1 2 0

2 1 0

1 2 0

2 0 1

2 1 0

2 1 0

2 1 0

2 1 0

1 2 0

2 0 1

2 1 0

2 0 1

2 0 1

1 2 0

1 2 0

Abbreviations: M = male, F = female a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by William’s test. * p<0.05, ** p<0.01 b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight

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TS 2.6.7.7E - Repeat-Dose Toxicity (Dog, 4 weeks) (continued)

Test Article: Lixisenatide Report Title: ZP-10A: Toxicity study by subcutaneous injection administration to beagle dogs for 4 weeks (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Histopathology (LM) b Number of dogs examined M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F:3 Injection Site 5 Inflammation: subcutaneous Grade 0 1 Fibroblast Proliferation: subcutaneous Grade 0 1 2

2 1

2 1 0

2 1

1 2 0

2 1

3 0 0

1 2

2 1 0

1 2

0 2 1

1 2

1 2 0

2 1

2 1 0

2 1

1 2 0

Injection Site 6 Inflammation: subcutaneous Grade 0 1 Fibroblast Proliferation: subcutaneous Grade 0 1

2 1

2 1

1 2

2 1

3 0

3 0

2 1

2 1

0 3

0 3

3 0

3 0

2 1

2 1

2 1

1 2

Additional Information: none Conclusions: NOAEL = 200 µg/kg BID Abbreviations: M = male, F = female a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by William’s test. * p<0.05, ** p<0.01 b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight

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TS 2.6.7.7F – Repeat-Dose Toxicity (Dog, 13 weeks)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period Species/Strain: Dog / Marshall beagle Duration of Dosing: 13 weeks Test Article: AVE0010 (Day x to xx: batch xxxxxxxxxxxxxxx, from

Day xx: batch xxxxxxxxxxxxxx) Initial Age: 9 to 10 months Duration of Post-Dose: 4 weeks Study No.: [20 x-1926] + Amendment 1 [20 x-1926-amend01] Date of First Dose: xx xxxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location : 4.2.3.2-19, 4.2.3.2-19a

Vehicle/Formulation : Saline (0.9% NaCl) / aqueous solution GLP Compliance: yes Special Features: none No Observed Adverse Effect Level: 20 µg/kg BID males, 250 µg/kg BID females Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250* Number of Animals on Study (main study + recovery) M: 3+2 F: 3+2 M: 3 F: 3 M: 3 F: 3 M: 3+2 F: 3+2 Toxicokinetics: First assay not including AVE0010 bound to antibodies AUC0-24 (ng.h/mL ) Day 1 - - 233 203 1230 1289 4112 3528 Cmax (ng/mL) Day 1 - - 24.8 25.1 126.1 160.4 347.4 404.6 Total AVE0010 assay AUC0-24 (ng.h/mL ) Day 1 Day 28 Day 91

- - 215 392

2700

197 322 957

1019 4906

21607

1395 1749 3252

3578 1438 8289

3225 3037

11072 Cmax (ng/mL) Day 1 Day 28 Day 91

- - 22 41

160

24 35 77

122 356

1098

193 137 239

300 134 470

341 222 618

Anti-drug antibody formation % ADA positive dogs Week 4 Week 13 Week 17

0 0 0

0 20 0

100 100

67 100

100 100

100 100

80 60

100

100 80 0

Additional Information: Values are rounded to three significant figures or less. Abbreviations: M = male, F = female, AUC = area under the curve, Cmax = maximum concentration * Mid dose was reduced from 300 µg/kg BID to 100 µg/kg BID from Day 29 and high dose was redcued from 1000 µg/kg BID to 400 µg/kg BID from Day 15 and to 250 µg/kg from day 29 due to substantial body weight effects

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7F – Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250*

Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Noteworthy Findings: Reduced body weight at ≥20 µg/kg BID triggering dose reduction in the mid and high dose group after 4-week treatment Reduced food consumption at ≥20 µg/kg BID in males and less severe in females, mainly in the first 4 weeks of the study Died or Killed Moribund 0 0 0 0 0 0 0 0 Group Mean Body Weight Gain (kg) per group Day 1-15 Day 15-29 Day 29-92 Day 1-92** Day 92-120

0.14 0.07 0.53 0.74 0.51

0.10 0.05 0.10 0.25 -0.05

-0.02 0.04 0.65 0.67

-0.16 -0.01 0.46 0.29

-0.22 0.07 0.55 -0.15

-0.35 0.10 0.61 0.37

-0.50 -0.40 0.87 -0.05 1.22

-0.31 -0.27 0.18 -0.39 1.05

Group Mean Body Weight Gain (kg and %)a Day 1-10 Day 1-29 Day 29-92 Day 1-92** Day 92-120

0.11 0.21 0.53 0.74 0.51

0.12 0.16 0.10 0.25 -0.05

n.a. -90 +23 -9.5

n.a n.a.

+360 +16

n.a

n.a. +3.8 n.a.

n.a

n.a. +510 +48

n.a n.a. +64 n.a.

+137

n.a

n.a. +90 n.a. n.a.

Abbreviations: M= male, F = female a At end of indicated period. For controls, group means are shown. For treated groups, percent differences from controls are shown. No statistics was performed. * Mid dose was reduced from 300 µg/kg BID to 100 µg/kg BID from Day 29 and high dose was reduced from 1000 µg/kg BID to 400 µg/kg BID from Day 15 and to 250 µg/kg from day 29 due to substantial body weight effects ** Differences compared to additive values of Day 1 to 28/29 and Day 29 to 91/92 are caused by rounding n.a.: not applicable, percentage of body weight gain compared to control cannot be calculated due to body weight loss/negative values

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7F – Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250*

Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Average Daily Food Consumption ( g/dog/week and %)a Day 1-7 Day 1-28 Day 29-91 Day 1-91** Day 92-119

2247 2354 2385 2375 2412

1651 1537 1577 1565 1729

-28 -15 -7.6 -9.9

-31

-0.98 +20 +14

-73 -45 -16 -25

-20 +11 +37 +29

-74 -29 +3.0 -6.6

+45.3

-61

-0.20 +38 +27 +61

Additional Information: none Abbreviations: M= male, F = female a At end of indicated period. For controls, group means are shown. For treated groups, percent differences from controls are shown. No statistics was performed. * Mid dose was reduced from 300 µg/kg BID to 100 µg/kg BID from Day 29 and high dose was reduced from 1000 µg/kg BID to 400 µg/kg BID from Day 15 and to 250 µg/kg from day 29 due to substantial body weight effects ** Differences compared to additive values of Day 1 to 28/29 and Day 29 to 91/92 are caused by rounding n.a.: not applicable, percentage of body weight gain compared to control cannot be calculated due to body weight loss/negative values

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7F – Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250*

Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Clinical Observations Appeared thin (from week 4 at 20 µg/kg (1F), and from week 1/2 at ≥100 µg/kg BID, sign persisted at 250 µg/kg BID up to week 16)

0 0 0 1 3 2 3 4

Vomiting (rarely, i.e. max. two times during the study in most affected dogs, only one F high dose dog showed 5x vomiting)

0 0 0 2 0 2 2 3

Salivation (single occasion) 0 0 0 0 0 1 0 0 Liquid or soft feces (single occasion) 1 1 0 1 1 0 0 0 Red discharge on pen floor (single occasion) 0 0 0 0 1 0 0 0 Ophthalmoscopy (Week 4, week 12, week 16) Left and right pupillary light reflex negative Week 4 Week 12 Week 16

0 0 0

0 0 0

0 0 0

0 0 0

1 0 0

0 0 0

0 0 0

0 0 0

Electrocardiography (Week 4, week 13 ) No treatment-related changes Abbreviations: M = male, F = female * Mid dose was reduced from 300 µg/kg BID to 100 µg/kg BID from Day 29 and high dose was redcued from 1000 µg/kg BID to 400 µg/kg BID from Day 15 and to 250 µg/kg from day 29 due to substantial body weight loss

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7F - Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: (continued) AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period

Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250*

Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Hematology (Week 4, week 13, week 17) No treatment-related findings Coagulation (Week 4, week 13, week 17) No treatment-related findings Serum Chemistry (Week 4, week 13, week 17) No treatment-related findings Urinalysis (Week 4, week 13, week 17) No treatment-related findings Additional Information: None Abbreviations: M = male, F = female

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TS 2.6.7.7F - Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued) Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250* Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Organ Weights (%) Week 13, week 17 No treatment-related findings Gross Pathology No treatment-related findings Histopathology (LM) a Number of dogs examined only main group had noteworthy findings, but not recovery dogs M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 Liver Vacuolation: hepatocyte Grade 0 1 2 3

0 1 1 1

0 0 2 1

0 1 1 1

0 0 2 1

1 1 1 0

0 2 1 0

0 2 1 0

1 2 0 0

Testis Dilatation: tubular Grade 0 1 2

3 0 0

3 0 0

1 2 0

0 1 2

Sperm stasis: segmental Grade 0 1

3 0

3 0

1 2

0 3

Abbreviations: M = male, F = female a Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight, Grade 3=moderate, Grade 4=moderately severe

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7F - Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued) Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250* Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Histopathology (LM) a (continued) Number of dogs examined only main group had noteworthy findings, but not recovery dogs M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3

Injection Site: Left anterior flank Inflammation/Fibrosis: subdermal Grade 0 1 2 3 4

0 2 1 0 0

0 3 0 0 0

0 0 3 0 0

0 3 0 0 0

0 0 0 2 1

0 0 1 0 2

0 0 1 0 2

0 0 1 1 1

Injection Site: Left posterior flank Inflammation/Fibrosis: subdermal Grade 0 1 2 3 4

0 1 1 1 0

1 2 0 0 0

0 0 2 1 0

0 1 1 1 0

0 0 0 1 2

0 0 1 0 2

0 0 1 1 1

0 0 1 1 1

Injection Site: Right anterior flank Inflammation/Fibrosis: subdermal Grade 0 1 2 3 4

1 2 0 0 0

1 2 0 0 0

0 0 2 1 0

0 1 0 1 1

0 0 1 2 0

0 0 0 3 0

0 0 1 2 0

0 0 2 1 0

Abbreviations: M = male, F = female a Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight, Grade 3=moderate, Grade 4=moderately severe

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TS 2.6.7.7F - Repeat-Dose Toxicity (Dog, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 13-week subcutaneous administration toxicity study in the dog followed by a 4 week treatment free period (continued) Dose (µg/kg BID) 0 (Vehicle Control) 20 300/100* 1000/400/250* Number of Animals on Study M: 5 F: 5 M: 3 F: 3 M: 3 F: 3 M: 5 F: 5 Histopathology (LM) a (continued) Number of dogs examined only main group had noteworthy findings, but not recovery dogs M: 3 F: 3 M: 3 F: 3 M: 3 F: 3 M: 3 F: 3

Injection Site: Right posterior flank Inflammation/Fibrosis: subdermal Grade 0 1 2 3 4

1 1 1 0 0

1 2 0 0 0

0 0 2 1 0

0 1 1 1 0

0 0 0 1 2

0 0 1 0 2

0 0 0 1 2

0 1 1 0 1

Conclusions: NOAEL = 20 µg/kg BID for males, 250 µg/kg BID for females Abbreviations: M = male, F = female a Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight, Grade 3=moderate, Grade 4=moderately severe

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7G – Repeat-Dose Toxicity (Dog, 12 months)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs Species/Strain: Dog / Marshall Beagle Duration of Dosing: 12 months Test Article: AVE0010 (batch xxxxxxxxxxxxxx from xx xxx to xx

xxxxxxxx 20xx, and batch xxxxxxxxxxxxxxxx from xx xxxxxxxx 20xx to xx xxxx 20xx)

Initial Age: 7 to 8 months Duration of Post-Dose: none Study No.: [20 x-0064] and Amendment 1 [20 x-0064-amend01] Date of First Dose: xx xxxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location : 4.2.3.2-20, 4.2.3.2-20a

Vehicle/Formulation : 0.9% sodium chloride/aqueous solution GLP Compliance : yes Special Features: dose titration (step-wise escalation) in mid and high dose; all dose groups started with 2 µg/kg BID, mid dose reached definitive dose after 31 days, high dose after 85 days No Observed Adverse Effect Level: 2 μg/kg for males / 1000 μg/kg for females Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000 Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5* M: 4 F: 4 M: 4 F: 4 Toxicokinetics: AUC0-24 (ng.h/mL ) Day 1, 31 (mid dose), 85 (high dose) Day 184, 216 (mid dose), 268 (high dose) Day 371, 370 (mid and high dose)

- - 19.3 150

126

18.6 24.7

21.7

2650 33300

25600

3230 31000

33800

120000 247000

161000

339000 43700

33900

Cmax (ng/mL) Day 1, 31 (mid dose), 85 (high dose) Day 184, 216 (mid dose), 268 (high dose) Day 371, 370 (mid and high dose)

- - 2.05 12.0

7.16

1.95 3.01

3.25

217 2060

1300

263 2060

1630

17400 13500

7270

42300 3050

1950

Anti-drug antibody formation % ADA positive dogs Day 184, 216 (mid dose), 268 (high dose) Day 371, 370 (mid and high dose)

25**

100**

50**

25**

75

75

60

75

100

100

100

100

100

100

100

100 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = area under the curve, Cmax = maximum concentration; * One additional female was added after 3 month of definitive dosing (replaced a female necropsied prematurely due to an eating disorder). ** Overall the assay revealed 21% of false positive results, a possible reason for non-specific interaction (50% considered as false positive in controls) was considered to be food or age of animals.

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5** M: 4 F: 4 M: 4 F: 4 Noteworthy Findings: Reduced body weight gain (relative to pretest) in males at ≥200 µg/kg BID and in females at 1000 µg/kg BID (based on lower body weight gain during the first 3 month of study duration) Reduced food consumption in males at ≥200 µg/kg BID and in females at 1000 µg/kg BID Died or Killed Moribund 0 0 0 0 0 0 0 0 Absolute Body Weight (kg and %)a Day 1 Day 28 Day 92 Day 183 Day 372

8.90 9.75

10.75 10.93 12.20

8.05 8.60 9.48 9.55

10.15

-2 -8

-14* -12 -7

-5

-15* -16* -18* -18

-1 -7

-15* -13* -17*

0

-7* -8*

-17* -9

-4

-15* -25* -22* -25*

-5

-15* -27* -25* -27*

Average Body Weight Gain (kg and %)a Days 1 to 28 Days 28 to 92 Days 92 to 183 Days 183 to 372 Days 1 to 372

0.85 1.00 0.18 1.27 3.30

0.55 0.88 0.07 0.60 2.10

-73 -73

+139 +38 -19

weight lossb

-37 weight lossb

-18 -69

-71 -92

+133 -58 -61

weight lossb

-13 weight lossb

+125 -42

weight lossb weight lossb

+122 -51 -84

weight lossb weight lossb

+186 -50

weight loss Average Weekly Food Consumption (g and %)a Days 1 to 377

2.333

1.895

-3

-1

-7

-1

-17

-13

Abbreviations: M = male, F = female, a At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical difference is based on actual data (not percent differences). b The animals of the respective dose groups did not gain weight in the time period mentioned. They experienced a weight loss, which is to be considered as “negative weight gain”. Therefore a calculation of a “percent difference in weight gain compared to controls” was not performed and “weight loss” was entered in the table. * significant trend (p≤0.05), statistics only performed for absolute weight ** One additional female was added after 3 month of definitive dosing (replaced a female necropsied prematurely due to an eating disorder).

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5* M: 4 F: 4 M: 4 F: 4 Clinical Observations: number of affected dogs / number of observations in study No adverse clinical sign was noted during entire study duration. Vocalization 4/43 3/4 4/14 4/11 4/84 4/70 4/168 4/88 Salivation - - - 2/2 1/1 - 1/1 - Regurgitation - - - - - - 1/1 1/1 Increased resistance to dosing - - - - 1/1 - 2/1 - Absence of food intake 3/1 1/1 1/1 1/1 3/3 3/3 2/4 3/4 Liquid feces 1/6 - 4/25 5/16 4/2 2/16 4/7 2/2 Feces discolored (red, black, brown) 1/1 - - 1/5 - - 1/3 2/2 Emesis 2/8 1/1 4/5 3/6 4/7 4/4 4717 4/17 Warmness to touch - - - - - - - 1/4 Reduced skin elasticity - - - 1/4 - - - - Paleness of oral mucous membranes - - - 1/3 - - - - Administration site (swelling) 1/9 - - - - - 1/6 2/8 Administration site scabbed 1/16 - - - - - - 1/2 Administration site red 1/9 - - - - - - 1/4 Administration site discharge (yellow) 1/4 - - - - - - 1/3 Ophthalmoscopy (weeks 12, 26, 40, and 52) No treatment-related changes Electrocardiography (weeks 12, 26, 39, and 52) No treatment-related changes Abbreviations: M = male, F = female, - = sign absent * One additional female was added after 3 month of definitive dosing (replaced a female necropsied prematurely due to an eating disorder).

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5** M: 4 F: 4 M: 4 F: 4 Hematology Hemoglobin (g/L) (Week 12) 156.5 153.8 142.8* 134.4 143.0* 147.0 137.5* 149.8 Coagulation No treatment-related findings Serum Chemistry Globulin (g/L) (Week 39) 23.5 23.5 22.0 24.5 23.5 23.3 26.5 27.0* Urinalysis No treatment-related findings Abbreviations: M = male, F = female *Significantly different (p<=0.05) from control. ** One additional female was added after 3 month of definitive dosing (replaced a female necropsied prematurely due to an eating disorder).

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Organ Weights (%) a Week 53 Brain (%) 0.63735 0.74280 +6.2 +13.6 +21.4* +10.3 +21.1* +23.5 Heart (g) 95.73 76.01 -11.6 -3.2 -4.4 -1.8 -20.7* -11.8 Pituitary gland (g) 0.07750 0.07250 -12.3 -6.9 +13.6 +3.4 -28.6** -13.8 Thymus (g) 5.38 4.40 -42* +3.2 -16 +18.9 -21.8 -53.9* Thyroid Gland (g) 0.82750 0.71000 -16 -14.8 -31.7** -7.4 -15.7 -29.6 Gross Pathology No treatment-related findings

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Histopathology (LM) b Number of dogs examined M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Testis Atrophy: tubule Grade 0 1 3 4 Vacuolation: tubule Grade 0 1 2 3 4 Stasis: spermatid Grade 0 1 2 3 Hypospermatogenesis Grade 0 2 3 4 Fibrosis: tubule Grade 0 2

2 2 0 0

1 2 1 0 0

2 2 0 0

1 1 2 0

4 0

3 1 0 0

4 0 0 0 0

4 0 0 0

0 2 2 0

4 0

1 1 0 2

2 0 1 0 1

2 2 0 0

0 0 2 2

4 0

1 0 3 0

1 0 0 3 0

0 0 1 3

0 0 1 3

3 1

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Histopathology (LM) b (continued) Number of dogs examined M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Epididymis Oligospermia Grade 0 3 4 5 Aspermia Grade 5 Dilatation: initial segment and efferent ducts Grade 0 2 3 Degeneration: epithelium of initial segment Grade 0 2 3

4 0 0 0

0

4 0 0

4 0 0

4 0 0 0

0

4 0 0

4 0 0

0 2 1 1

1

1 1 2

2 0 2

0 0 3 1

1

0 1 3

0 1 3

Injection Site: Left Fibrosis: subcutaneous Grade 0 1 2 3 4 5

4 0 0 0 0 0

1 0 1 1 1 0

4 0 0 0 0 0

4 0 0 1 0 0

0 1 1 1 0 1

1 0 0 1 1 1

0 0 0 0 2 2

1 0 0 1 2 1

Abbreviations: see next page

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.7G - Repeat-Dose Toxicity (Dog, 12 months) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 12-month twice a day (BID) subcutaneous toxicity study in dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 200 1000

Number of Animals on Study (Main Study) M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Histopathology (LM) b (continued) Number of dogs examined M: 4 F: 4 M: 4 F: 5*** M: 4 F: 4 M: 4 F: 4 Injection Site: Right Fibrosis: subcutaneous Grade 0 1 2 3 4 5

3 0 1 0 0 0

1 0 0 3 0 0

3 0 1 0 0 0

5 0 0 0 0 0

1 0 1 1 1 0

0 0 0 1 2 1

0 0 0 1 0 3

0 0 0 1 2 1

Additional Information: None Conclusions: NOAEL = 2 μg/kg for males / 1000 μg/kg for females Abbreviations: a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by Dunnett’s test at 5% ( * ) or 1% ( ** ) . b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=mild, Grade 3=moderate, Grade4=marked, Grade5=severe *** One additional female was added after 3 month of definitive dosing (replaced a female necropsied prematurely due to an eating disorder).

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8 GENOTOXICITY: IN VITRO TS 2.6.7.8A - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxx

Test Article: Lixisenatide Report Title: ZS42-0010: Reverse Mutation in four Histidine-requiring Strains of Salmonella typhimurium and one Tryptophan- requiring Strain of Escherichia coli Test for Induction of: Reverse mutation in bacterial strains No. of Independent Assays: 2 Test Article (Batch): xxxxxxxxx Test system: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA

No. of Duplicate Cultures: 3 (vehicle controls: 5) No. of Cells Analyzed/Culture: NA

Report No.: [20 x-1183]

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9-mix) Vehicle: For Test Article: 100 mM sodium phosphate buffer

For positive controls: DMSO or water

Location: 4.2.3.3.1-1 GLP Compliance: yes Date of Treatment: xx-xxx-20xx (first exp.)

Treatment: Treat and plate xx-xxx-20xx (second exp.) Genotoxic Effects: None (with and without S9 mix)

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP 2uvrA

0b 1.6

10.80 ± 3.63 15.33 ± 4.16

80.00 ± 15.38 81.00 ± 5.00

15.20 ± 5.12 17.67 ± 7.77

18.80 ± 5.07 15.67 ± 3.21

15.00 ± 3.08 17.00 ± 4.36

8 11.33 ± 3.06 84.00 ± 5.00 10.33 ± 4.04 10.67 ± 2.08 19.67 ± 4.73 Without ZS42-0010 40 11.67 ± 1.53 71.67 ± 8.02 17.00 ± 4.00 13.67 ± 5.51 16.33 ± 5.69 S9 mix 200 13.67 ± 3.06 84.67 ± 9.02 13.67 ± 3.51 12.33 ± 1.53 16.67 ± 5.86

1000 16.00 ± 5.00 85.00 ± 17.52 16.00 ± 2.00 9.67 ± 0.58 14.67 ± 2.08 5000 12.33 ± 3.51 69.33 ± 13.05 16.67 ± 8.39 10.33 ± 1.53 23.67 ± 3.79 Positive control a 165.33 ± 45.63 647.33 ± 21.50 2234.67 ± 105.63 1467.00 ± 97.63 41.33 ± 11.37

Abbreviations: DMSO = dimethyl sulfoxide; NA = not applicable; SD = Standard deviation. a = Positive controls (without S9 mix): 2-Nitrofluorene, 25 µg/mL for TA98; 4-Nitroquinoline 1-oxide, 1 µg/mL for TA100; N-methyl-N´-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535 and 7.5 µg/mL for WP2uvrA; ICR191, 1 µg/mL for TA1537;b = vehicle control (100 mM sodium phosphate buffer). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8A - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: ZS42-0010: Reverse Mutation in four Histidine-requiring Strains of Salmonella typhimurium and one Tryptophan- requiring Strain of Escherichia coli

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA 0b 14.20 ± 3.11 72.80 ± 8.41 16.40 ± 2.97 6.00 ± 0.71 16.40 ± 1.52 1.6 18.33 ± 2.52 72.00 ± 4.58 16.67 ± 3.21 9.67 ± 2.08 19.00 ± 7.00

With 8 15.33 ± 4.62 79.67 ± 11.55 17.00 ± 5.29 7.67 ± 3.06 14.67 ± 2.08 S9 mix ZS42-0010 40 11.67 ± 4.51 68.00 ± 11.14 17.33 ± 5.86 7.33 ± 1.53 11.33 ± 2.08

200 16.00 ± 1.73 67.67 ± 6.51 17.33 ± 4.04 10.33 ± 1.53 12.00 ± 5.57 1000 16.00 ± 1.00 83.67 ± 18.58 14.00 ± 6.24 10.33 ± 0.58 11.67 ± 0.58 5000 15.67 ± 1.15 84.00 ± 18.19 13.33 ± 3.21 6.67 ± 2.08 14.33 ± 1.15 Positive control a 173.33 ± 18.77 241.33 ± 40.22 37.33 ± 10.69 30.33 ± 8.33 24.67 ± 7.57

Abbreviations: DMSO = dimethyl sulfoxide; SD = Standard deviation. a = Positive controls (with S9 mix): 2-Aminoanthracene, 5 µg/mL for TA98 and TA1535, 3.5 µg/mL for TA100 and TA1537, 20 µg/mL for WP2uvrA; b = vehicle control (100 mM sodium phosphate buffer). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.8A - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: ZS42-0010: Reverse Mutation in four Histidine-requiring Strains of Salmonella typhimurium and one Tryptophan- requiring Strain of Escherichia coli:

Metabolizing system Test Article Concentration

(µg/mL)

Second mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA 0b 29.20 ± 4.66 75.40 ± 6.19 11.20 ± 3.56 8.00 ± 3.67 12.80 ± 5.12 156.25 25.00 ± 8.19 58.33 ± 5.77 11.00 ± 4.58 6.00 ± 2.00 10.67 ± 4.73 312.5 23.67 ± 3.06 75.33 ± 14.22 15.00 ± 3.00 6.33 ± 1.15 12.00 ± 0.00

Without ZS42-0010 625 23.33 ± 8.74 63.67 ± 4.51 9.67 ± 1.53 7.00 ± 4.36 8.00 ± 1.73 S9 mix 1250 25.00 ± 4.36 70.67 ± 4.73 10.67 ± 3.21 7.33 ± 1.53 10.33 ± 2.52

2500 30.00 ± 11.14 72.33 ± 6.66 13.00 ± 4.36 8.00 ± 1.00 15.00 ± 3.61 5000 27.67 ± 6.03 (T) 56.67 ± 13.61 11.67 ± 6.51 4.67 ± 1.15 7.67 ± 2.52 Positive control a 1394.00 ± 194.28 699.00 ± 77.95 2431.00 ± 50.86 965.00 ± 239.82 974.67 ± 40.28 0b 22.00 ± 6.04 78.40 ± 9.26 10.40 ± 4.72 5.40 ± 1.14 13.00 ± 2.00 156.25 22.00 ± 5.57 80.00 ± 7.55 10.67 ± 2.08 5.00 ± 1.00 14.00 ± 4.58 312.5 19.67 ± 3.79 58.00 ± 10.82 9.67 ± 3.06 6.33 ± 3.51 8.00 ± 2.00

With ZS42-0010 625.0 24.00 ± 5.57 69.33 ± 12.01 6.67 ± 0.58 6.33 ± 1.53 12.33 ± 6.66 S9 mix 1250 19.33 ± 1.53 68.33 ± 13.65 8.33 ± 0.58 10.00 ± 1.00 15.33 ± 4.04

2500 22.67 ± 2.31 68.33 ± 12.50 12.67 ± 1.53 7.67 ± 2.52 10.67 ± 1.53 5000 20.00 ± 5.57 63.33 ± 9.61 9.67 ± 3.06 8.67 ± 6.66 11.67 ± 5.51 Positive control a 610.33 ± 149.53 293.00 ± 25.94 49.33 ± 16.77 11.33 ± 3.79 32.67 ± 8.33

Conclusion: ZS42-0010 was found negative in this bacterial reverse mutation tests in the absence and presence of metabolic activation. Abbreviations: SD = Standard deviation T: Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies. a = Positive controls (without S9 mix): 2-Nitrofluorene, 25 µg/mL for TA98; 4-Nitroquinoline 1-oxide, 1 µg/mL for TA100; N-methyl-N´-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535 and 7.5 µg/mL for WP2uvrA; ICR191, 1 µg/ml for TA1537. Positive controls (with S9 mix): 2-Aminoanthracene, 5 µg/mL for TA98 and TA1535; 3.5 µg/mL for TA100 and TA1537; 20 µg/mL for WP2uvrA; b = vehicle control (100 mM sodium phosphate buffer). Toxic effects and precipitate: For all strains no precipitation was observed. Toxicity (slight thinning of the bacterial background growth) was observed in strain TA98 (withS9 mix) at 5000 µg/mL. No toxicity was observed in the other strains. Comments: No relevant increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test Test for Induction of: Reverse mutation in bacterial strains No. of Independent Assays: 2 Test Article (Batch): xxxxxxxxxxxxxx Test system: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA

No. of Duplicate Cultures: 3 No. of Cells Analyzed/Culture: NA

Report No.: [20 x-1342]

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9 mix)

Location: 4.2.3.3.1-2 GLP Compliance: yes

Vehicle: For Test Article: water

For positive controls: DMSO or water

Date of Treatment : xx-xxx-20xx, (first exp.); xx-xxx-20xx (second exp.)

Treatment: Treat and plate method. The strain WP2uvrA with S9 mix was treated using the preincubation method, because common positive controls gave no appropriate results using the treat and plate method in this part of assay.

Genotoxic Effects: None (with and without S9 mix)

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA Negative control 16.0 ± 1.7 145.3 ± 8.1 11.0 ± 1.7 21.7 ± 3.5 13.3 ± 4.5 0b 16.0 ± 3.0 152.0 ± 11.5 7.7 ± 1.5 27.3 ± 4.6 12.7 ± 4.0 50 15.0 ± 3.0 130.7 ± 22.7 6.7 ± 0.6 25.7 ± 11.6 13.0 ± 1.7

Without AVE0010 160 15.0 ± 1.0 145.0 ± 3.0 6.7 ± 2.1 24.0 ± 2.6 13.3 ± 3.8 S9 mix 500 13.7 ± 1.5 155.7 ± 2.5 9.7 ± 2.9 25.3 ± 2.5 13.7 ± 4.5

1600 17.3 ± 4.6 131.0 ± 8.7 6.0 ± 1.0 20.0 ± 1.0 13.3 ± 6.1 5000 15.3 ± 4.7 133.0 ± 7.0 9.3 ± 3.2 27.7 ± 4.7 13.3 ± 3.5 Positive control a 1121.7 ± 114.7 1484.0 ± 96.8 1694.3 ± 34.1 1543.0 ± 109.3 170.7 ± 12.7

Abbreviations: DMSO = dimethyl sulfoxide; NA = not applicable; SD = Standard deviation; Negative control = bacteria alone. a = Positive controls (-S9 mix): 2-nitrofluorene, 2.5 µg/mL for TA98; 4-Nitroquinoline N-oxide, 1 µg/mL for TA100 and WP2uvrA; N-methyl-N-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535; ICR191, 1 µg/mL for TA1537. b = vehicle control (water). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA Negative control 18.7 ± 3.8 144.7 ± 7.6 9.3 ± 3.5 9.3 ± 2.1 10.3 ± 2.5 0b 15.3 ± 2.1 154.0 ± 7.5 7.7 ± 4.7 7.7 ± 3.1 9.7 ± 2.1 50 15.7 ± 3.8 139.0 ± 6.1 8.7 ± 1.2 10.0 ± 1.0 8.7 ± 1.2

With AVE0010 160 14.3 ± 4.0 154.0 ± 4.6 11.3 ± 3.1 9.7 ± 1.5 9.3 ± 5.8 S9 mix 500 12.0 ± 1.0 147.3 ± 20.4 7.7 ± 3.2 8.7 ± 3.2 13.0 ± 2.6

1600 18.3 ± 2.1 153.7 ± 9.7 9.7 ± 0.6 8.3 ± 3.1 11.3 ± 2.1 5000 16.3 ± 6.7 149.0 ± 19.7 8.0 ± 1.7 6.0 ± 2.0 10.7 ± 1.5 Positive control a 124.3 ± 4.0 424.3 ± 16.9 57.3 ± 9.1 30.7 ± 4.0 8.3 ± 3.2c

Abbreviations: SD = Standard deviation; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 0.5 µg/mL for TA98; 1.5 µg/mL for TA100, TA1535, and TA1537; 30 µg/mL for WP2uvrA; b = vehicle control (water); c = Positive control not valid. Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test

Metabolizing system Test Article Concentration

(µg/plate)

First mutagenicity experiment (preincubation method) Number of His+ Revertant Colonies (Mean ± SD)

WP2uvrA Negative control 8.7 ± 3.5 0b 9.0 ± 0.0 50 6.0 ± 0.0 (T+ P)

With AVE0010 160 12.3 ± 3.2 (T+ P) S9 mix 500 8.3 ± 1.2 (T+ P)

1600 8.0 ± 2.0 (T+ P) 5000 10.7± 2.3 (T+ P) Positive control a 94.0 ± 22.3

Abbreviations: SD = Standard deviation T = Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies; P = Precipitation; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 30 µg/plate for WP2uvrA; b = vehicle control (water). Toxic effects and precipitate: For strain WP2uvrA precipitation and toxicity was observed within a concentration range of 50 to 5000 µg/plate. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test

Metabolizing system Test Article Concentration

(µg/plate)

Repeat of first mutagenicity experiment (preincubation method) Number of His+ Revertant Colonies (Mean ± SD)

WP2uvrA Negative control 8.7 ± 3.1 0b 6.7 ± 1.5 0.16 9.7 ± 2.3

With AVE0010 0.5 11.0 ± 2.6 S9 mix 1.6 7.7 ± 1.5

5 7.3 ± 3.8 16 9.3 ± 2.1 50 10.0 ± 2.0 (T+ P) Positive control a 76.7 ± 10.1

Abbreviations: SD = Standard deviation T = Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies; P = Precipitation; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 30 µg/plate for WP2uvrA; b = vehicle control (water). Toxic effects and precipitate: For strain WP2uvrA precipitation and toxicity was observed at 50 µg/plate. Comments: Strain WP2uvrA was repeated due to high toxicity down to the lowest concentration in the first experiment (preincubation method). No increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test

Metabolizing system Test Article Concentration

(µg/mL)

Second mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA Negative control 14.0 ± 5.2 145.0 ± 14.4 6.3 ± 2.3 27.3 ± 3.2 11.0 ± 1.0 0b 13.0 ± 3.5 157.0 ± 25.2 11.0 ± 6.1 20.7 ± 4.7 12.3 ± 1.5 312.5 12.7 ± 4.5 161.0 ± 17.3 9.3 ± 3.5 22.3 ± 2.5 12.0 ± 7.0

Without AVE0010 625 12.3 ± 1.2 138.0 ± 13.0 8.7 ± 3.8 21.7 ± 3.5 13.0 ± 2.0 S9 mix 1250 16.0 ± 3.6 134.3 ± 7.4 7.3 ± 3.2 20.7 ± 2.1 8.7 ± 6.0

2500 12.7 ± 4.2 145.3 ± 12.2 11.0 ± 1.0 22.7 ± 5.5 9.7 ± 4.0 5000 13.3 ± 1.5 143.7 ± 2.5 7.7 ± 1.5 20.0 ± 5.6 13.0 ± 3.5 Positive control a 696.0 ± 26.2 1478.3 ± 41.6 2229.7 ± 32.7 1748.0 ± 65.0 100.0 ± 3.6 Negative control 12.7 ± 2.1 140.7 ± 5.8 9.3 ± 3.2 11.3 ± 2.5 n.p. 0b 17.3 ± 3.8 157.7 ± 7.8 8.7 ± 2.5 5.7 ± 0.6 n.p. 312.5 19.0 ± 3.0 147.3 ± 6.5 8.0 ± 2.6 7.7 ± 1.5 n.p.

With AVE0010 625 16.0 ± 1.7 142.3 ± 5.7 7.0 ± 2.0 8.7 ± 3.8 n.p. S9 mix 1250 17.0 ± 4.4 142.0 ± 11.3 7.0 ± 3.0 8.3 ± 3.2 n.p.

2500 15.7 ± 2.5 141.7 ± 13.3 5.3 ± 2.1 9.3 ± 2.1 n.p. 5000 16.3 ± 5.9 132.0 ± 6.1 6.0 ± 1.0 10.0 ± 1.7 n.p. Positive control a 97.3 ± 9.1 498.7 ± 62.1 51.0 ± 9.2 26.7 ± 6.7 n.p.

Abbreviations: SD = Standard deviation; n.p.= not performed; Negative control = bacteria alone; a = Positive controls (without S9 mix): 2-nitrofluorene, 2.5 µg/mL for TA98; 4-Nitroquinoline N-oxide, 1 µg/mL for TA100 and WP2uvrA; N-methyl-N-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535; ICR191, 1µg/mL for TA1537. Positive controls (with S9 mix): 2-aminoanthracene, 0.5 µg/mL for TA98; 1.5 µg/mL for TA100, TA1535, and TA1537; b = vehicle control (water). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8B - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation test

Metabolizing system Test Article Concentration

(µg/plate)

Second mutagenicity experiment (preincubation method) Number of His+ Revertant Colonies (Mean ± SD)

WP2uvrA Negative control 7.7 ± 1.5 0b 6.0 ± 2.6 1.56 9.0 ± 3.0

With AVE0010 3.13 7.0 ± 1.7 S9 mix 6.25 11.0 ± 2.0

12.5 7.7 ± 2.1 25 7.3 ± 4.0 50 8.0 ± 0.0 (T+ P) Positive control a 71.7 ± 1.2

Conclusion: AVE0010 was found negative in this bacterial reverse mutation tests in the presence and absence of metabolic activation. Abbreviations: SD = Standard deviation T = Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies; P = Precipitation; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 30 µg/plate for WP2uvrA; b = vehicle control (water). Toxic effects and precipitate: For strain WP2uvrA precipitation and toxicity was observed at 50 µg/plate. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay Test for Induction of: Reverse mutation in bacterial strains No. of Independent Assays: 2 Test Article (Batch): xxxxxxxxxxxxxx Test system: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA

No. of Duplicate Cultures: 3 No. of Cells Analyzed/Culture: NA

Report No.: [20 x-0234]

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9 mix)

Location: 4.2.3.3.1-3 GLP Compliance: yes

Vehicle: For Test Article: water

For positive controls: DMSO or water

Date of Treatment: xx-xxx-20xx(first exp.); xx-xxx-20xx(second exp.)

Treatment: Treat and plate method. Except strain WP2vrA with S9 mix was treated using the preincubation method, because common positive controls gave no appropriate results using the treat and plate method.

Genotoxic Effects: None (with and without S9-mix)

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA Negative control 12.7 ± 2.1 133.7 ± 9.1 10.7 ± 2.1 8.3 ± 2.5 11.7 ± 2.3 0b 11.3 ± 4.0 125.3 ± 7.2 10.0 ± 2.0 8.3 ± 3.2 6.7 ± 3.5 50 14.3 ± 3.5 134.3 ± 5.0 5.0 ± 2.0 7.0 ± 2.6 7.3 ± 3.2

Without AVE0010 160 11.0 ± 2.6 136.7 ± 5.5 9.3 ± 4.2 5.7 ± 5.0 5.7 ± 1.2 S9 mix 500 18.0 ± 2.0 126.7 ± 3.5 7.7 ± 1.5 7.0 ± 1.0 5.3 ± 1.5

1600 14.3 ± 3.8 133.7 ± 12.7 8.3 ± 0.6 6.3 ± 2.5 7.0 ± 2.0 5000 14.7 ± 5.0 123.3 ± 21.1 8.3 ± 2.3 8.3 ± 5.5 7.0 ± 2.6 Positive control a 960.7 ± 42.4 1321.0 ± 108.4 2215.3 ± 59.2 1482.7 ± 51.8 62.3 ± 14.6

Abbreviations: DMSO = dimethyl sulfoxide; NA = not applicable; SD = Standard deviation; Negative control = bacteria alone; a = Positive controls (without S9 mix): 2-nitrofluorene, 2.5 µg/mL for TA98; 4-Nitroquinoline N-oxide, 1 µg/mL for TA100, and WP2uvrA; N-methyl-N-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535; ICR191, 1 µg/mL for TA1537; b = vehicle control (water). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay

Metabolizing system Test Article Concentration

(µg/mL)

First mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2uvrA Negative control 16.0 ± 2.6 127.7 ± 6.0 7.7 ± 1.5 4.7 ± 2.1 n.p. 0b 13.3 ± 2.3 115.3 ± 6.0 10.0 ± 2.6 6.3 ± 0.6 n.p. 50 9.7 ± 2.5 126.0 ± 18.3 9.3 ± 2.3 4.0 ± 1.7 n.p.

With AVE0010 160 11.7 ± 1.2 138.3 ± 18.0 9.0 ± 4.6 7.0 ± 3.0 n.p. S9 mix 500 11.7 ± 3.1 137.7 ± 7.6 5.3 ± 2.3 6.7 ± 1.5 n.p.

1600 11.7 ± 2.3 140.3 ± 21.1 8.3 ± 2.5 7.3 ± 3.5 n.p. 5000 14.3 ± 6.4 138.3 ± 17.8 9.3 ± 2.1 3.7 ± 1.5 n.p. Positive control a 50.0 ± 5.3 347.3 ± 16.2 23.7 ± 0.6 20.0 ± 2.6 n.p.

Abbreviations: SD: Standard deviation; n.p.= not performed; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 0.5 µg/mL for TA98; 1.5 µg/mL for TA100, TA1535, and TA1537; b = vehicle control (water). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay

Metabolizing system Test Article Concentration

(µg/mL)

Repeat of the first mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA1535 Negative control 11.3 ± 6.1 0b 9.3 ± 0.6 50 6.7 ± 4.0

With AVE0010 160 12.3 ± 1.5 S9 mix 500 10.3 ± 0.6

1600 14.0 ± 3.6 5000 10.3 ± 3.5 Positive control a 42.0 ± 3.0

Abbreviations: SD = Standard deviation; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 1.5 µg/mL for TA1535; b = vehicle control (water). Toxic effects and precipitate: No precipitation and no toxicity were observed. Comments: Strain TA1535 was repeated, because the number of revertant colonies of the positive control was only slightly above a two-fold increase compared to the vehicle control. No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay

Metabolizing system Test Article Concentration

(µg/plate)

First mutagenicity experiment (preincubation method) Number of His+ Revertant Colonies (Mean ± SD)

WP2uvrA Negative control 11.7 ± 4.0 0b 11.7 ± 3.5 0.16 9.3 ± 0.6 0.5 7.7 ± 2.5 1.6 11.7 ± 2.1

With AVE0010 5 11.0 ± 4.4 S9 mix 16 12.0 ± 3.6

50 12.3 ± 2.5 160 13.0 ± 5.3 500 12.0 ± 3.0 (T) 1600 11.0 ± 2.6 (T) 5000 10.0 ± 1.0 (T) Positive control a 110.7 ± 7.6

Abbreviations: SD = Standard deviation T = Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 30 µg/plate for WP2uvrA; b = vehicle control (water). Toxic effects and precipitate: For strain WP2uvrA toxicity was observed within a concentration range of 500 to 5000 µg/plate. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay

Metabolizing system Test Article Concentration

(µg/mL)

Second mutagenicity experiment (treat and plate method) Number of His+ Revertant Colonies (Mean ± SD)

TA98 TA100 TA1535 TA1537 WP2 uvrA Negative control 12.7 ± 3.2 152.7 ± 38.2 7.7 ± 2.1 10.3 ± 6.7 16.7 ± 7.5 0b 17.3 ± 10.2 162.7 ± 7.5 8.0 ± 5.6 9.3 ± 0.6 11.3 ± 5.0 312.5 13.3 ± 2.3 166.0 ± 18.0 11.0 ± 4.4 9.3 ± 5.8 13.0 ± 2.6

Without AVE0010 625 13.7 ± 6.4 155.7 ± 22.8 8.3 ± 3.2 9.0 ± 1.7 15.3 ± 3.2 S9 mix 1250 14.3 ± 2.3 166.0 ± 12.8 9.3 ± 3.1 12.0 ± 1.0 9.7 ± 1.5

2500 16.0 ± 7.2 157.7 ± 1.5 6.3 ± 0.6 10.0 ± 3.0 13.7 ± 1.2 5000 13.0 ± 6.1 149.3 ± 10.4 7.3 ± 4.5 12.3 ± 0.6 11.7 ± 2.5 Positive control a 1134.7 ± 18.8 1626.0 ± 16.4 2462.7 ± 89.0 2182.0 ± 61.6 102.3 ± 4.2 Negative control 13.0 ± 4.4 135.0 ± 7.5 9.0 ± 1.0 8.3 ± 1.5 n.p. 0b 14.0 ± 4.4 157.0 ± 4.4 10.0 ± 1.0 5.7 ± 1.5 n.p. 312.5 14.0 ± 4.4 142.7 ± 3.2 9.3 ± 1.5 3.7 ± 2.1 n.p.

With AVE0010 625 15.7 ± 2.5 153.7 ± 8.6 9.0 ± 3.5 6.3 ± 1.2 n.p. S9 mix 1250 14.7 ± 2.1 149.3 ± 17.4 8.3 ± 1.5 6.3 ± 3.1 n.p.

2500 10.7 ± 1.2 164.0 ± 8.2 7.0 ± 3.0 4.7 ± 1.5 n.p. 5000 15.3 ± 2.9 121.7 ± 6.7 6.7 ± 1.5 5.3 ± 2.5 n.p. Positive control a 70.3 ± 12.7 341.0 ± 34.2 36.3 ± 3.2 20.0 ± 1.7 n.p.

Abbreviations: SD = Standard deviation; n.p.= not performed; Negative control = bacteria alone; a = Positive controls (without S9 mix): 2-nitrofluorene, 2.5 µg/mL for TA98; 4-Nitroquinoline N-oxide, 1 µg/mL for TA100 and WP2uvrA; N-methyl-N-nitro-N-nitrosoguanidine, 2.5 µg/mL for TA1535; ICR191, 1 µg/mL for TA1537. Positive controls (with S9 mix): 2-aminoanthracene, 0.5 µg/mL for TA98; 1.5 µg/mL for TA100, TA1535, and TA1537; b = vehicle control (water). Toxic effects and precipitate: For all strains no precipitation and no toxicity was observed. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8C - Genotoxicity: In Vitro – Ames Test, batch xxxxxxxxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Bacterial reverse mutation assay

Metabolizing system Test Article Concentration

(µg/plate)

Second mutagenicity experiment (preincubation method) Number of His+ Revertant Colonies (Mean ± SD)

WP2uvrA Negative control 9.0 ± 1.7 0b 10.7 ± 4.6 7.8 11.0 ± 2.0 15.6 7.0 ± 1.0

With AVE0010 31.25 11.7 ± 2.3 S9 mix 62.5 10.3 ± 2.1

125 11.7 ± 3.5 250 11.3 ± 2.1 500 10.3 ± 4.0 (T) Positive control a 191.7 ± 25.0

Conclusion: AVE0010 was found negative in this bacterial reverse mutation tests in the presence and absence of metabolic activation. Abbreviations: SD = Standard deviation T = Toxicity by an inhibition of bacterial background growth and or by a decrease of the number of revertant colonies; Negative control = bacteria alone; a = Positive controls (with S9 mix): 2-aminoanthracene, 30 µg/plate for WP2uvrA; b = vehicle control (water). Toxic effects and precipitate: For strain WP2uvrA toxicity was observed at 500 µg/plate. Comments: No increase in the number of revertant colonies was observed.

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TS 2.6.7.8D - Genotoxicity: In vitro – Mammalian chromosome aberration test in human lymphocytes batch xxxxxxxxx

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes Test for Induction of: Chromosome aberrations No. of Independent Assays: 2 Test Article (Batch): xxxxxx batch xxxxxxxxx Test system: Human lymphocytes No. of Replicate Cultures: 2

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9 mix)

No. of Cells Analyzed/Concentrations: 200 Report No.: [MAF0100]

Vehicle: For Test Article: water For positive controls: water Location: 4.2.3.3.1-4 Treatment: 3 hours (with S9 mix) and 3 and 20 hours (without S9 mix), 20 hours sampling time

Date of Treatment: xx-xxx-20xx (first exp.) and xx-xxx-20xx (second exp.)

GLP Compliance: yes

Genotoxic Effects: None (with and without S9 mix)

Metabolizing system

Test Article Concentration (µM)

Mitotic index data - First main experiment

3-hour treatment (without S9 mix) Mean Mitotic index (%)

3-hour treatment (withS9 mix) Mean Mitotic index (%)

0.0b 9.8 8.2 0.39 8.7 8.0

Without 0.78 7.7 7.2 and ZP10A 1.56 8.7 7.5 with 3.13 9.0# 8.6

S9 mix 6.25 7.6# 6.4 12.5 7.2# 7.0# 25.0 3.7 a 7.0# 50.0 5.8 a 6.2#

Abbreviations: a =Test article causing clotting, with resulting in debris on the slides, therefore concentration could not be selected for metaphase analysis;. b = vehicle control (water); # = evaluated concentrations. Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed, however at 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). Toxicity (reduction in the mitotic index) was decreased to 38% at 25 µM and 59% at 50 µM (without S9 mix, 3-h treatment) and to 76% at 50 µM (with S9 mix, 3-h treatment). Comments: None.

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TS 2.6.7.8D - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes

Metabolizing system

Test Article Concentration (µM)

Mitotic index data - Second main experiment

20-hour treatment (without S9 mix) Mean Mitotic index (%)

3-hour treatment (with S9 mix) Mean Mitotic index (%)

0.0b 10.3 11.5 0.39 9.5 not tested

Without 0.78 8.9 not tested and ZP10A 1.56 8.9# not tested with 3.13 11.2# 11.2

S9 mix 6.25 8.5# 11.0 12.5 a 10.2# 25.0 a 8.3# 50.0 a 10.8#

Abbreviations: a = Test article causing clotting, with resulting in debris on the slides, therefore concentration could not be selected for metaphase analysis; b = vehicle control (water); # = evaluated concentrations. Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed, however at 12.5, 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). No toxicity (reduction in the mitotic index) was observed. Comments: None.

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TS 2.6.7.8D - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes

Metabolizing system

Test Article Concentration (µM)

First main experiment 3-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

0.0b 1.0 9.8 Without ZP10A 3.13 1.5 9.0 S9 mix 6.25 1.0 7.6

12.5 1.5 7.2 Mitomycin C

(positive control µg/mL) 0.2 24.0 *** c not scored

0.0b 1.0 8.2 With ZP10A 12.5 0.5 7.0

S9 mix 25.0 1.5 7.0 50.0 1.0 6.2 CPA (positive control µg/mL) 6.0 25.0 *** c not scored

Abbreviations: a = % of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water); c = 50 cells per culture were analyzed due to high levels of aberrations seen; CPA = cyclophosphamide . Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001 was performed. Toxic effects and precipitate: No precipitation was observed, however at 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). Toxicity (reduction in the mitotic index) was decreased to 38% at 25 µM and 59% at 50 µM (without S9 mix, 3-h treatment) and to 76% at 50 µM (with S9 mix, 3-h treatment). Comments: No increase in the number of cells with structural aberrations was observed.

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TS 2.6.7.8D - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes

Metabolizing system

Test Article Concentration (µM)

Second main experiment 20-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

0.0b 0.5 10.3 Without ZP10A 1.56 0.0 8.9 S9 mix 3.13 0.5 11.2

6.25 0.5 8.5 Mitomycin C

(positive control µg/mL) 0.1 12.5 *** not scored

Second main experiment 3-hour treatment 0.0b 0.5 11.5

With ZP10A 12.5 1.0 10.2 S9 mix 25.0 1.5 8.3

50.0 1.0 10.8 CPA (positive control µg/mL) 6.0 13.3 *** c not scored

Abbreviations: a = % of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water); c = 50 cells per culture were analyzed due to high levels of aberrations seen; CPA = cyclophosphamide. Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001 was performed. Toxic effects and precipitate: No precipitation was observed, however at 12.5, 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). No toxicity (reduction in the mitotic index) was observed. Comments: No increase in the number of cells with structural aberrations was observed.

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TS 2.6.7.8D - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes

Metabolizing system

Test Article Concentration (µM)

First main experiment 3-hour treatment

Polyploid cells per 1000 cells scored Mean Relative Mitotic Index (%) 0.0b 0.0 100.0 0.39 not scored 89.0 0.78 not scored 79.0

Without ZP10A 1.56 not scored 89.0 S9 mix 3.13 not scored 92.0#

6.25 not scored 78.0# 12.5 1.0 73.0# 25.0 a 38.0 50.0 a 59.0 0.0b 1.0 100.0 0.39 not scored 98.0 0.78 not scored 88.0

With ZP10A 1.56 not scored 91.0 S9 mix 3.13 not scored 105.0

6.25 not scored 78.0 12.5 not scored 85.0# 25.0 not scored 85.0# 50.0 2.0 76.0#

Abbreviations: a = Test article causing clotting, with resulting in debris on the slides, therefore concentration could not be selected for metaphase analysis ; b = vehicle control (water); # = evaluated concentrations;. Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed, however at 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). Toxicity (reduction in the mitotic index) was decreased to 38% at 25 µM and 59% at 50 µM (without S9 mix, 3-h treatment) and to 76% at 50 µM (with S9 mix, 3-h treatment). Comments: None.

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TS 2.6.7.8D - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: ZP10A: In vitro mammalian chromosome aberration test in human lymphocytes

Metabolizing system

Test Article Concentration (µM)

Second main experiment 20-hour treatment

Polyploid cells per 1000 cells scored Mean Relative Mitotic Index (%) 0.0b 2.0 100.0 0.39 not scored 92.0 0.78 not scored 86.0 1.56 not scored 86.0#

Without ZP10A 3.13 not scored 109.0# S9 mix 6.25 1.0 83.0#

12.5 a a 25.0 a a 50.0 a a Second main experiment 3-hour treatment 0.0b 2.0 100.0 3.13 not scored 97.0

With ZP10A 6.25 not scored 96.0 S9 mix 12.5 not scored 89.0#

25.0 not scored 72.0# 50.0 1.0 94.0#

Conclusion: ZP10A did not induce structural chromosomal aberrations in human lymphocytes in the presence and absence of metabolic activation.

Abbreviations: a = Test article causing clotting, with resulting in debris on the slides, therefore concentration could not be selected for metaphase analysis; b = vehicle control (water); # = evaluated concentrations. Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed, however at 12.5, 25 and 50 µM, test article debris was found on the slides, limiting the top concentration for evaluation (without S9 mix). No toxicity (reduction in the mitotic index) was observed. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In vitro – Mammalian chromosome aberration test in human lymphocytes batch xxxxxxxxxxxxxx

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010 Test for Induction of: Chromosome aberrations No. of Independent Assays: 1 Test Article (Batch): xxxxxxxxxxxxxx Test system: Human lymphocytes No. of Replicate Cultures: 2

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9-mix)

No. of Cells Analyzed/Concentrations: 200 Report No.: [20 x-1343]

Vehicle: For Test Article: water For positive controls: nutrient medium Location: 4.2.3.3.1-5 Treatment: 3 hours (with S9 mix) and 3 and 20 hours (without S9 mix), 20 hours sampling time

Date of Treatment: xx-xxx-20xx GLP Compliance: yes

Genotoxic Effects: None

Metabolizing system

Test Article Concentration (µg/mL)

Toxicity – Main experiment

3-hour treatment Mean Mitotic index (%)

20-hour treatment Mean Mitotic index (%)

Negative control n.d. n.d. 0.0b 14.2 12.1 39.1 n.d. n.d. 78.2 n.d. n.d.

Without AVE0010 156.2 n.d. n.d. S9 mix 312.5 11.2 16.2

625.0 11.2 11.4 1250.0 11.7# 13.1# 2500.0 8.5# 11.4# 5000.0 10.4# 9.3#

Abbreviations: n.d. = not determined; # = evaluated concentrations; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. No biological relevant toxicity (clear reduction in the mitotic index) was observed without S9 mix. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Toxicity – Main experiment

3-hour treatment Mean Mitotic index (%)

Negative control n.d. 0.0b 12.0 39.1 14.0 78.2 12.5 156.2 13.4

With AVE0010 312.5 13.1# S9 mix 625.0 13.0#

1250.0 8.3# 2500.0 7.0 5000.0 0.0

Abbreviations: n.d. = not determined; # = evaluated concentrations; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. Strong toxicity (reduction in the mitotic index) was observed at 2500 µg/mL (57.9% decrease) and at 5000 µg/mL (0% survival). Although the mitotic index was only reduced to 57.9% compared to the vehicle control at 2500 µg/mL, the cultures could not be evaluated for cytogenetic damage due to poor metaphase quality in combination with reduced cell number. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 3-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

Negative control 2.5 15.7 0.0b 2.0 14.2

Without AVE0010 1250.0 2.5 11.7 S9 mix 2500.0 1.5 8.5

5000.0 0.0 10.4 EMS (positive control) 660.0 8.5 *** 13.4 Negative control 1.0 13.8 0.0b 1.0 12.0

With AVE0010 312.5 2.0 13.1 S9 mix 625.0 1.5 13.0

1250.0 1.5 8.3 CPA (positive control) 30.0 8.5 *** 7.7

Abbreviations: EMS = ethylmethane sulfonate; CPA = cyclophosphamide; Negative control = cells alone; a = % of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water). Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001. Toxic effects and precipitate: No precipitation was observed. No relevant toxicity (clear reduction in the mitotic index) was observed without S9 mix. Strong toxicity (reduction in the mitotic index) was observed at 2500 µg/mL (57.9% decreases) and at 5000 µg/mL (0% survival). Although the mitotic index was only reduced to 57.9% compared to the vehicle control at 2500 µg/mL, the cultures could not be evaluated for cytogenetic damage due to poor metaphase quality in combination with reduced cell number. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 20-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

Negative control 1.5 16.4 0.0b 1.5 12.1

Without AVE0010 1250.0 2.5 13.1 S9 mix 2500.0 0.0 11.4

5000.0 1.0 9.3 EMS (positive control) 550.0 10.0 *** 8.0

Abbreviations: EMS = ethylmethane sulfonate; Negative control = cells alone; a = number of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water). Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001. Toxic effects and precipitate: No precipitation was observed. No relevant toxicity (clear reduction in the mitotic index) was observed. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 3-hour treatment

Polyploid cells per 500 cells scored Mean Relative Mitotic Index (%) Negative control 0.0 100.0 0.0b 1.0 100.0

Without AVE0010 1250.0 1.0 82.0 S9 mix 2500.0 0.0 59.5

5000.0 1.0 72.9 EMS (positive control) 660.0 2.0 85.4 Negative control 1.0 100.0 0.0b 1.0 100.0

With AVE0010 312.5 0.0 108.8 S9 mix 625.0 0.0 107.9

1250.0 1.0 68.8 CPA (positive control) 30.0 0.0 56.0

Abbreviations: EMS = ethylmethane sulfonate; CPA = cyclophosphamide; Negative control = cells alone; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. No relevant toxicity (clear reduction in the mitotic index) was observed without S9-mix. Strong toxicity (reduction in the mitotic index) was observed at 2500 µg/mL (57.9% decrease) and at 5000 µg/mL (0% survival). Although the mitotic index was only reduced to 57.9% compared to the vehicle control at 2500 µg/mL, the cultures could not be evaluated for cytogenetic damage due to poor metaphase quality in combination with reduced cell number. Comments: None.

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TS 2.6.7.8E - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in vitro in human lymphocytes with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 20-hour treatment

Polyploid cells per 500 cells scored Mean Relative Mitotic Index (%) Negative control 2.0 100.0 0.0b 0.0 100.0

Without AVE0010 1250.0 1.0 107.9 S9 mix 2500.0 0.0 94.2

5000.0 0.0 76.4 EMS (positive control) 550.0 1.0 48.5

Conclusion: AVE0010 did not induce structural chromosomal aberrations in human lymphocytes in the presence and absence of metabolic activation.

Abbreviations: EMS = ethylmethane sulfonate; Negative control = cells alone; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. No toxicity (clear reduction in the mitotic index) was observed. Comments: None.

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes batch xxxxxxxxxxxxxx

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010 Test for Induction of: Chromosome aberrations No. of Independent Assays 1 Test Article (Batch): xxxxxxxxxxxxxx Test system: Human lymphocytes No. of Replicate Cultures: 2

Metabolizing System: Liver homogenate from rats pretreated with Aroclor (S9 mix)

No. of Cells Analyzed/Concentrations: 200 Report No.: [DSE 20 x-0386]

Vehicle: For Test Article: water For positive controls: EMS: nutrient medium, CPA: saline (0.9%NaCl [w/v]) Location: 4.2.3.3.1-6 Treatment: 3 hours (with S9 mix) and 3 and 20 hours (without S9 mix), 20 hours sampling time

Date of Treatment: xx-xxx-20xx GLP Compliance: yes

Genotoxic Effects: None

Metabolizing system

Test Article Concentration (µg/mL)

Toxicity – Main experiment

3-hour treatment Mean Mitotic index (%)

20-hour treatment Mean Mitotic index (%)

Negative control n.d. n.d. 0.0b 13.8 14.2 312.5 14.2 13.7 468.8 n.d. 13.1

Without AVE0010 625.0 12.9 13.1 S9 mix 937.3 n.d. 12.5#

1250.0 10.0# 9.5# 1875.0 n.d. 6.9# 2500.0 7.9# 6.4 5000.0 7.7# n.d.

Abbreviations: EMS = ethylmethane sulfonate; CPA = cyclophosphamide; n.d. = not determined; #= evaluated concentrations; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed after 3 hours exposure at 2500 µg/mL (57.2% decrease), and at 5000 µg/mL (55.8% decrease). After 20 hours exposure toxicity was noted at 1875 µg/mL (48.6% decrease), and at 2500 µg/mL (44.7% decrease). Comments: None

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Toxicity – Main experiment

3-hour treatment Mean Mitotic index (%)

Negative control n.d. 0.0b 12.0 312.5 12.9#

With AVE0010 625.0 9.5# S9 mix 1250.0 6.5#

2500.0 2.1 5000.0 0.0

Abbreviations: n.d. = not determined; # = evaluated concentrations; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed at 1250 µg/mL (54.2% decrease). Comments: None.

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 3-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

Negative control 2.0 13.1 0.0b 1.0 13.8

Without AVE0010 1250.0 2.5 10.0 S9 mix 2500.0 3.5 *** e 7.9

5000.0 0.5 7.7 EMS (positive control) 660.0 12.5 *** 12.2 Negative control 1.0 13.0 0.0b 2.0 12.0

With AVE0010 312.5 1.0 12.9 S9 mix 625.0 1.0 9.5

1250.0 1.0 6.5 CPA (positive control) 22.5 15.0 *** 8.5

Abbreviations: EMS = ethylmethane sulfonate; CPA = cyclophosphamide; Negative control = cells alone; a = % of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water); e = 200 cells per culture were analyzed due to inhomogeneous data. Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed without S9 mix at 2500 µg/mL (57.2% decrease), and at 5000 µg/mL (55.8% decrease). With S9 mix toxicity was noted at 1250 µg/mL (54.2% decrease). Comments: At 2500 µg/mL without S9 mix statistically significant higher frequencies as compared to the corresponding vehicle control was observed. Although 3.5% aberrant cells were statistically significant compared to the low response (1% aberrant cells) in the vehicle control, the response is within the historical control data range. Therefore, the statistical significance has to be regarded as being biological irrelevant.

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 20-hour treatment

Number of cells with structural aberrationsa (Mean %)

Mean Mitotic Index (%)

Negative control 0.0 14.4 0.0b 2.5 14.2

Without AVE0010 937.3 1.0 12.5 S9 mix 1250.0 0.5 9.5

1875.0 1.0 6.9 EMS (positive control) 660.0 37.0 *** c 6.8

Abbreviations: EMS = ethylmethane sulfonate; Negative control = cells alone; a = % of cells with chromosome aberrations excluding gaps per 200 cells scored; b = vehicle control (water); c = 50 cells per culture were analyzed due to high levels of aberrations seen. Statistical significance: For the incidence of aberrant cells, Fisher Exact test *** = significant at p <0.001. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed at 1875 µg/mL (48.6% decrease). Comments: No increase in the number of cells with structural aberrations was observed.

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 3-hour treatment

Polyploid cells per 500 cells scored Mean Relative Mitotic Index (%) Negative control 0.0 100.0 0.0b 1.0 100.0

Without AVE0010 1250.0 0.0 72.5 S9 mix 2500.0 2.0 57.2

5000.0 1.0 55.8 EMS (positive control) 660.0 0.0 93.5 Negative control 1.0 100.0 0.0b 1.0 100.0

With AVE0010 312.5 0.0 107.5 S9 mix 625.0 0.0 79.2

1250.0 0.0 54.2 CPA (positive control) 22.5 0.0 65.4

Abbreviations: EMS = ethylmethane sulfonate; CPA = cyclophosphamide; Negative control = cells alone; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed without S9 mix at 2500 µg/mL (57.2% decrease), and at 5000 µg/mL (55.8% decrease). With S9 mix toxicity was noted at 1250 µg/mL (54.2% decrease). Comments: No increase in the number of cells with structural aberrations was observed.

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TS 2.6.7.8F - Genotoxicity: In Vitro – Mammalian chromosome aberration test in human lymphocytes (continued)

Test Article: Lixisenatide Report Title: Chromosome aberration test in human lymphocytes in vitro with AVE0010

Metabolizing system

Test Article Concentration (µg/mL)

Main experiment 20-hour treatment

Polyploid cells per 500 cells scored Mean Relative Mitotic Index (%) Negative control 0.0 100.0 0.0b 0.0 100.0

Without AVE0010 937.3 1.0 88.0 S9 mix 1250.0 0.0 66.5

1875.0 2.0 48.6 EMS (positive control) 660.0 0.0 47.4

Conclusion: AVE0010 did not induce structural chromosomal aberrations in human lymphocytes in the presence and absence of metabolic activation.

Abbreviations: EMS = ethylmethane sulfonate; Negative control = cells alone; b = vehicle control (water). Statistical significance: Not applicable. Toxic effects and precipitate: No precipitation was observed. Clear toxicity of about or below 50% of control (reduction in the mitotic index) was observed at 1875 µg/mL (48.6% decrease). Comments: None.

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9 GENOTOXICITY: IN VIVO TS 2.6.7.9A - Genotoxicity: In vivo micronucleus test (mouse)

Test Article: Lixisenatide Report Title: ZP10A Mouse micronucleus test Test for Induction of: Chromosome damage Treatment Schedule: Single administration Test Article (Batch): xxxxxx

xxxxxxxxx Species/Strain/Sex: Mouse CD-1/male Age: approx. 35 days

Sampling Time: 24 and 48 hours after treatment (24 hours for positive controls)

Cells Evaluated: Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE)

Method of Administration: Intravenous, 10 mL/kg (oral, 20 mL/kg for positive control)

Report No.: [MUT0212]

No. of Cells Analyzed/Animal: 2000 PCE Vehicle/Formulation: 0.9% NaCl (buffered with sodium citrate pH 5.3)

Location: 4.2.3.3.2-1

Special Features: None Date of Treatment: xx-xxx-20xx GLP Compliance: yes Toxic / Cytotoxic Effects: None Genotoxic Effects: None Evidence of Exposure: Systemic exposure ensured by intravenous administration

Test Article Dose (mg/kg) Number of animals PCE/( PCE +NCE) %

(Meana) MPCE per 2000 PCE

(Mean) 24-hours sampling time

Vehicle 0.0 7 7 7 7 5

39 38 40 35 41

0.4 0.4 0.1 0.3

18.0**

AVE0010 1.25 2.5 5.0

Mitomycin C 12.0 48-hours sampling time

Vehicle 0.0 7 7

39 38

0.6 0.6 AVE0010 5.0

Conclusion: AVE0010 administered by the intravenous route was found negative in the rat bone marrow micronucleus test. Abbreviations: MPCE = Micronucleated polychromatic erythrocytes, PCE = Polychromatic erythrocytes, NCE = Normochromatic erythrocytes, a =occasional apparent errors of +1% may occur due to rounding of values. Statistical significance: ** p<0.01. Method for genotoxicity: one-sided Wilcoxon´s sum of ranks test and Jonckheere´s test for trend. Clinical Observation: No clinical signs were observed during the treatment period.

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10 CARCINOGENICITY TS 2.6.7.10A – Dose Range Finding Toxicity - Nonpivotal Studies

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Mouse SC BID (Saline, 0.9%

sodium chloride)

14 days 20, 200, 1000, 2000

10/10 + 44/44 TK

Not applicable (exploratory study with

limited histopathology)

Mortality: none Clinical signs: 20 µg/kg: Hypoactivity (1M), 200+1000 µg/kg: bluish discolorated injection site (1F) Body weight: ≥200 µg/kg: increased gain Day 1 to 14 (F: 49x to 78x) ≥1000 µg/kg: reduced gain Day 1 to 5 (M:-92%/-94%) Food consumption: ≥20 µg/kg reduced Day 1 to 10 (F: -10% to –23%) ≥200 µg/kg reduced Day 1 to 5 (M: -6% to –23%) 2000 µg/kg reduced Day 5 to 10 (M: -9%)

[20 x-1952] 4.2.3.4.2-1

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = female, NAD = no abnormalities detected * percentages related to organ weight - body weight ratio, isolated significances in lower dose groups only not included

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TS 2.6.7.10A – Dose Range Finding Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Mouse SC BID (Saline, 0.9%

sodium chloride)

14 days 20, 200, 1000, 2000

10/10 + 44/44 TK

Not applicable (exploratory study with

limited histopathology)

Water consumption: ≥20 µg/kg reduced Day 1 to 2 (M+F: -24% to –65%) ≥1000 µg/kg reduced Day 7 to 8(M: -51%) Hematology: ≥20 µg/kg: neutrophils↑ (M: +2.7% to +28%), ≥200 µg/kg: platelets↓ (F: -21% to –25%), 2000 µg/kg: RBC↓ (M: -11%), hemoglobin ↓ (M: -9%), hematocrit ↓ (M: -9%), WBC ↓ (M: -21%), lymphocytes ↓ (M: -30%), monocytes ↓ (M: 31%)

[20 x-1952] continued

4.2.3.4.2-1

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = female, NAD = no abnormalities detected * percentages related to organ weight - body weight ratio, isolated significances in lower dose groups only not included

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TS 2.6.7.10A – Dose Range Finding Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration (Vehicle/

Formulation)

Duration of Dosing

Doses (µg/kg BID)

Gender and No. per Group

NOAEL (µg/kg BID)

Noteworthy Findings Study Number

Location

Mouse SC BID (Saline, 0.9%

sodium chloride)

14 days 20, 200, 1000, 2000

10/10 + 44/44 TK

Not applicable (exploratory study with

limited histopathology)

Serum chemistry: ≥1000 µg/kg BID: bilirubin ↓ (M: -18 to –20%), calcium ↑ (F: +3 to +4%) 2000 µg/kg BID: triglycerides ↓ (M: -38%), chloride↑ (M: +2.5%), albumin/globulin ratio↓(M: -10%), ALT ↓ (F: -25%) Organ weights*: 2000 µg/kg BID: Thymus ↓ (M: -35%) Macroscopy: NAD Microscopy: NAD TK values are included in section 3 Overview of toxicokinetics Data

[20 x-1952] continued

4.2.3.4.2-1

Abbreviations: NOAEL: No-Observed-Adverse-Effect-Level, M = Male, F = female, NAD = no abnormalities detected * percentages related to organ weight - body weight ratio, isolated significances in lower dose groups only not included

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TS 2.6.7.10B – Carcinogenicity Range Finding Toxicity (mouse, 13 weeks)

Test Article: Lixisenatide Report Title: AVE0010: 13-week twice daily dosing subcutaneous range-finding study in mice Species/Strain: Mouse/ Crl:CD-1 (ICR)BR Duration of Dosing: 13 weeks Test Article: AVE0010 (week x to xx: batch xxxxxxxxxxxxx,

from week xx: batch xxxxxxxxxxxxxx) Initial Age: 7 to 8 weeks Duration of Post-Dose: none Study No.: [20 x-0062] Date of First Dose: xx xxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location: 4.2.3.4.2-2

Vehicle/Formulation: Saline (0.9% NaCl) / aqueous solution GLP Compliance: yes Special Features: none No Observed Adverse Effect Level: 1656 µg/kg BID Dose (µg/kg BID) 0 (Vehicle Control) 16.6 165.6 828.2 1656 Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Toxicokinetics: Assay not including AVE0010 bound to antibodies* AUC0-24 (ng.h/mL ) Day 1 - - 26.9 22.4 384.1 259.2 2009.5 1613.6 2848.0 2386.7 Cmax (ng/mL) Day 1 - - 14.2 11.7 214.9 138.9 834.1 456.6 1127.4 931.8 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = area under the curve, Cmax = maximum concentration, ADA=anti-drug antibodies * Valid assessment of TK at later time points not possible due to antibody formation. A subsequent 13-week mouse TK study was performed with a modified TK assay analysing total AVE0010

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 16.6 165.6 828.2 1656

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Noteworthy Findings: Body weight gain was in general increased in both genders at ≥16.6 µg/kg BID. Food consumption was reduced in week 1 at ≥16.6 µg/kg BID in females and ≥165.6 µg/kg BID in males Water consumption was reduced in week 1 at ≥16.6 µg/kg BID in females and ≥165.6 µg/kg BID in males From week 2 food and water consumption was rather increased compared to control Died or Killed Moribund 0 0 0 0 0 0 1# 0 0 1## Absolute Body Weight (g and %)a Week 1 Week 4 Week 8 Week 13

36.4 37.9 39.3 40.2

28.4 29.4 31.0 32.3

-1.4

-0.53 +1.5 +2.7

+1.8 +6.5 +6.8 +7.1

+3.6 +5.8 +6.9 +9.2

+2.5 +6.5 +7.4 +5.3

-4.4 +1.1 +2.3 2.0

+1.1 +11 +9.4 +9.0

+2.5 +7.4 +10 +11

+1.4 +10 +10 +11

Average Body Weight Gain (g and %)ab Day1 to week 4 Week 4 to 8 Week 8 to 13 Day 1 to week 13

1.8 1.3 1.0 4.1

1.5 1.6 1.3 4.3

+33 +69 +40 +46

+133* +13 +15 +56

+44 +46 +80 +54

+127* +25 -46 +42

+100 +54 -80 +39

+180***

-6.3 ±0.0 +60

+28

+100 +20 +49

+187***

+13 +31

+81** Average Daily Food Consumption (g/mouse/week and %)a Week 1 Week 2 Week 1 to 4 Week 5 to 8 Week 9 to 13 Week 1 to 13

37.8 37.5 37.1 36.8 35.5 36.4

41.5 41.4 41.6 40.5 40.2 40.7

+5.8 +12 +10 +7.3 +11 +9.6

-9.2 -6.5 -4.8 +8.9 +6.2 +3.7

-4.8 +12 +10 +17 +19 +16

-5.1 +3.9 +4.1 +9.9 +7.0 +7.1

-19 +2.4 +1.6 +6.8 +6.5 +5.0

-26 -13 -13 -2.2 -6.2 -7.1

-17 +1.9 +2.2 +8.4 +6.8 +5.8

-27 -11 -12 ±0.0 -3.0 -4.9

Abbreviations: See next page

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 16.6 165.6 828.2 1656

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Average Daily Water Consumption (g/mouse/week and %)a Week 1 Week 2 Week 1 to 4 Week 5 to 8 Week 9 to 13 Week 1 to 13

42.7 37.3 38.4 37.6 36.0 37.2

44.9 46.1 44.5 44.2 44.3 44.3

+9.4 +22 +18 +18 +31 +21

-13 +1.1 -5.2 -1.6 -2.5 -2.9

-8.7 +17 +7.6 +15 +23 +16

-11 -4.8

-0.67 +8.8 +5.0 +4.5

-20 +29 +6.8 +2.7 -2.2 +2.2

-24 -15 -13 -7.0 -11 -10

-18 +18 +8.6 +15 +5.8 +9.4

-30 -16 -14 -6.8 -14 -12

Abbreviations: M = male, F = female a At end of dosing period. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical difference is based on actual data (not percent differences). Statistical evaluation was performed for average daily body weight gains, food consumption and water consumption intervals only, * p<0.05, ** p<0.01, ***p<0.001 (two-way ANOVA, dose response and Dunnett’s test) b Potential slight differences between weight gain values and related absolute weight values are caused by rounding # death on Day 88 associated to anesthesia at blood sampling, ## euthanized on Day 11, death considered to be caused by dosing injury

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 16.6 165.6 828.8 1656

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Clinical Observations Opaque/small eye 1 0 1 0 0 0 0 0 0 0 Damaged ear 1 0 0 0 0 0 0 0 0 0 Damaged tail or sores/lesion at tail 1 2 1 2 1 1 0 1 0 1 Thinning fur (dorsal, head or body) 1 3 2 1 5 0 2 1 2 2 Protruding eye 0 4 0 1 0 2 0 2 1 1 Excessive activity 0 1 0 0 0 0 0 0 0 0 Hair loss (head) 0 0 0 1 0 0 0 0 0 0 Sores / lesions (back, urogenital are, shoulders, eyelid or body) 0 0 3 0 2 0 1 1 0 0 Obese 0 0 0 0 0 0 1 0 0 0 Protruding penis 0 0 0 0 0 0 2 0 0 0 Impaired mobility 0 0 0 0 0 0 0 0 0 1 Abbreviations: M = male, F = female

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 16.6 165.6 828.2 1656

Number of Animals on Study M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10

Hematology (Week 13) No treatment-related findings Coagulation (Week 13) No treatment-related findings Serum Chemistry (Week 13) Urea nitrogen (mmol/L) 7.9 7.9 5.7* 7.1 7.0 5.9 9.7 5.4* 7.3 6.5 Creatinin (µmol/L) 31 34 32 34 32 32 32 36 33 37# * Total Protein (g/L) 51 50 54 53 52 51 52 54* 54 53 Albumin (g/L) 28 32 31 35 31 34 31 36* 31* 35 Triglycerides (mmol/L) 1.91 1.73 1.27* 1.63 1.64 1.23 1.12** 1.32 1.23* 1.49 Abbreviations: M = male, F = female Statistical evaluation was performed by ANOVA, dose response and Dunnett’s test. * p<0.05, ** p<0.01, ***p<0.001 # Significant dose response test.

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (mg/kg/day) 0 (Vehicle Control) 16.6 165.6 828.2 1656

Number of Animals on Study (Main Study) M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 M: 10 F: 10 Organ Weights (%) a Week 13 Kidney (g) 0.519 0.358 +13.1 +7.3 +25.2** +7.8 +6.2 -1.7 +15.4 +8.7 Kidney (%) 1.246 1.088 +12.8 -0.2 +16.6* +2.0 +7.1 -7.6 +7.4 -0.4 Liver (g) 1.877 1.559 +5.5 +12.9 +21** +10.3 +9.5 +10.6 +20.4 +21.7* Liver (%) 4.529 4.722 +4.0 +5.0 +12.1* +4.4 +9.8 +4.1 +11.5 +11.5# * Epididymides (g) 0.120 0 +21.7* -1.7 +5 Gross Pathology b Number of mice examined 10 10 10 10 10 10 9 10 10 9 Injection site: Neck Red Red area

0 0

0 0

1 0

0 0

0 0

0 1

1 0

0 0

1 0

0 0

Injection site: Right hip Sore

0

0

1

0

0

0

0

0

0

0

Injection site: Left hip Red Sore Dark area

0 0 0

0 0 0

0 1 0

0 0 1

0 0 0

0 0 0

1 1 0

1 0 0

0 0 0

0 0 0

Abbreviations: M = male, F = female a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by ANOVA, dose response and Dunnett’s test. * p<0.05, ** p<0.01, ***p<0.001 # Significant dose response test. b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight, Grade 3=moderate

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TS 2.6.7.10B - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks) (continued)

Test Article: Lixisenatide Report Title: 13-week twice daily dosing subcutaneous range-finding study in mice

Dose (mg/kg/day) 0 (Vehicle Control) 16.6 165.6 828.2 1656

Histopathology (LM) b Number of mice examined 10 10 10 10 10 10 9 10 10 9 Injection site: 1 Inflammation/Fibrosis, Grade 0 subdermal 1 2

8 2 0

8 2 0

7 3 0

8 2 0

8 1 1

3 6 1

7 2 0

2 8 0

5 5 0

2 6 1

Injection site: 2 Inflammation/Fibrosis, Grade 0 subdermal 1 2

7 3 0

7 3 0

7 3 0

4 6 0

5 5 0

2 7 1

6 3 0

0 7 3

6 4 0

0 5 4

Number of mice examined 10 10 10 10 10 10 9 10 10 9 Injection site: 3 Inflammation/Fibrosis, Grade 0 subdermal 1 2

8 2 0

8 2 0

9 1 0

3 7 0

8 2 0

0

10 0

7 1 1

0 8 2

6 4 0

0 2 7

Number of mice examined 10 10 10 10 10 10 9 10 10 9 Liver Glycogen vacuolation Grade 0 1 2 3

0 3 6 1

0 3 4 3

2 2 5 1

0 5 3 2

0 5 4 1

0 3 2 5

0 4 2 3

0 2 3 5

0 1 7 2

0 1 1 7

Additional Information: none Conclusions: NOAEL=1656 µg/kg BID Abbreviations: M = male, F = female a Both absolute and relative weights differed from controls in the direction indicated. Number indicates percent difference for the absolute and relative organ weight. Statistical evaluation was performed by ANOVA, dose response and Dunnett’s test. * p<0.05, ** p<0.01, ***p<0.001 # Significant dose response test. b Number of animals with findings. LM = light microscopy, Grade 1=minimal, Grade 2=slight, Grade 3=moderate

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TS 2.6.7.10C - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks, toxicokinetics)

Test Article: Lixisenatide Report Title: AVE0010: 3-month subcutaneous toxicokinetic study in mice Species/Strain: Mouse/Crl ICR: CD1 Duration of Dosing: 91 days (antibody animals), 92 days

(toxicokinetic animals) Test Article: AVE0010 (batch xxxxxxxxxxxxxx)

Initial Age: 6 to 7 weeks Duration of Post-Dose: none Study No.: [20 x-0443] Date of First Dose: xx xxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location: 4.2.3.4.2-3

Vehicle/Formulation: Saline (0.9% NaCl) / aqueous solution GLP Compliance: yes Special Features: none No Observed Adverse Effect Level: not determined (toxicokinetics study) Dose (µg/kg BID) 0 (Vehicle Control) 200 1000 2000 Number of Animals on Study (antibody / toxicokinetic mice) M: 20 / 12 F: 20 / 12 M: 20 / 66 F: 20 / 66 M: 20 / 66 F: 20 / 66 M: 20 / 66 F: 20 / 66 Toxicokinetics: Total AVE0010 assay AUC0-24 (ng.h/mL ) Day 1 / 2 Day 92 / 93 - - 192

351 145 337

927 9521

787 6980

1590 20351

1490 13039

Cmax (ng/mL) Day 1 / 2 Day 92 / 93 - - 136

229 121 162

602 972

597 3017

1023 4216

1018 2597

Anti-drug antibody formation % of ADA positive mice Day 92 0 0 40 40 60 90 100 60 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, F = female, AUC = are under the curve, Cmax = maximum concentration, ADA=anti-drug antibodies * Total AVE0010 assay was only performed with the three higher dose levels

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TS 2.6.7.10C - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks, toxicokinetics) (continued)

Test Article: Lixisenatide Report Title: AVE0010: 3-month subcutaneous toxicokinetic study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 200 1000 2000

Number of Animals on Study (antibody and toxicokinetic mice) M: 32 F: 32 M: 86 F: 86 M: 86 F: 86 M: 86 F: 86

Noteworthy Findings: Body weight gain was increased at ≥200 µg/kg BID Food consumption was decreased at ≥200 µg/kg BID up to Day 9, at ≥1000 µg/kg BID up to Day 22 (males) and 15 (females), at 2000 µg/kg BID males still showed a decrease up to Day 36 Died or Killed Moribund 1# 0 1# 0 3# 0 6# 2# Absolute Body Weight (g and %)a Day 9 Day 29 Day 57 Day 92

29.18 32.06 34.15 34.62

24.66 26.89 28.74 29.50

+2.4 +4.6 +7.0 +8.9

+1.5 +4.5 +4.4 +6.7

-0.86 +4.9 +7.6 +9.2

+1.1 +6.2 +6.3 +9.0

+0.75 +5.9 +8.6 +10

+0.85 +5.4 +5.9 +8.7

Average Daily Body Weight Gain (g and %)ab Day 1-9 Day 1 to 15 Day 1 to 22 Day 1 to 92

2.51 3.35 4.23 7.92

0.48 1.28 1.78 5.32

+11 +3.6 +28* +33*

+190 +87*

+104* +47*

-12 -2.4 +34* +39*

+158 +95*

+139* +59*

-11 -3.6 +31* +39*

+121 +84*

+108* +55*

Average Daily Food Consumption (g /d/mouse and %)ac Day 1 to 9 Day 9 to 15 Day 15 to 22 Day 29 to 36 Day 85 to 92

5.33 5.12 5.11 5.68 5.19

4.85 4.64 4.82 5.11 4.79

-9.0* +0.20 +2.0 -0.70 +4.4

-11* -3.9 +3.1 +3.9 +2.3

-17* -4.7* -3.3* -3.0 +2.1

-16* -6.0* +2.1 +5.5 +4.4

-19* -5.7* -3.1* -4.4* +2.1

-19* -6.7* +1.0 +4.3 +3.6

Abbreviations: M = male, F = female a For controls, group means are shown. For treated groups, percent differences from controls are shown. b Average daily body weight gain was statistically significantly increased at ≥ 200 µg/kg BID also between Day 1 to 29, 1 to 36, 1 to 43, 1 to 50, 1 to 57, 1 to 64, 1 to 71, 1 to 78, 1 to 85 (not shown in the table) c Average daily food consumption was not statistically significantly changed in any group between Day 22 to 29, and after Day 36. # Origin of death not determined histopathologically, deaths were assumed to be caused by handling injury during frequent SC administration * Significant trend (p<= 0.05), only average body weight gain and food consumption was evaluated statistically

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TS 2.6.7.10C - Carcinogenicity Range Finding Toxicity (mouse, 13 weeks, toxicokinetics) (continued)

Test Article: Lixisenatide Report Title: (continued) 3-month subcutaneous toxicokinetic study in mice

Dose (µg/kg BID) 0 (Vehicle Control) 200 1000 2000

Number of Animals on Study (Main Study) M: 32 F: 32 M: 86 F: 86 M: 86 F: 86 M: 86 F: 86 Clinical Observations Lesion at administration site 0 0 0 0 0 0 1 0 Partial alopecia 1 1 0 1 0 4 0 1 Unkempt coat 0 0 1 0 0 0 0 1 Cold to touch 0 0 0 0 1* 0 0 0 Corneal opacity 0 0 0 0 1 0 0 0 Eye cloudy 0 0 1 0 0 0 0 0 Eye discharge 0 0 2 0 2 0 0 0 Eyelids partially closed 0 0 1 0 0 0 0 0 Eyelid reddened 0 0 1 0 0 0 0 0 Flaccidity 0 0 0 0 1* 0 0 0 Limited limb use 0 1 0 1 0 0 2 0 Loss of limb use 0 1 1* 0 1 0 1 0 Motor activity absent 0 0 1* 0 0 0 0 0 Motor activity decreased 0 0 0 0 2 0 0 0 Penis prolapsed 0 0 1* 0 0 0 0 0 Piloerection 0 0 3 1 3 2 5 1 Recumbent posture 0 0 1* 0 0 0 0 0 Scab 1 0 0 1 0 2 2 0 Tremors 0 0 0 0 0 0 1 0 Additional information: * sign only seen in intermittent deaths Abbreviations: M = male, F = female

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TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in mice Species/Strain: Mouse / Crl:CD-1 Duration of Dosing: 2 years Master reference/Report No.: [CAR0085] Initial Age: 6 to 7 weeks Method of Administration: subcutaneous BID (split by approximately

8 hours) Location: 4.2.3.4.1-1

Date of First Dose: xx xxxxxxxxx 20xx Vehicle/Formulation: saline (0.9% NaCl) GLP Compliance: Yes Treatment of Controls: 2 control groups with vehicle saline (0.9%

NaCl)

Basis for High-Dose Selection: High dose was selected to provide an exposure exceeding highest human AUC by more than 25-fold Special Features: none Daily Dose (µg/kg BID) 0 (2 control groups) 40 200 1000 Gender M F M F M F M F AUC 0-24h (ng.h/mL) Day 176 < LLOQ < LLOQ 67.3 51.2 1970 706 20000 36700 Antibody positive [%] 0 43% 78% 96% Number of animals at start 120 120 60 60 60 60 60 60 Died/Sacrificed Moribund 27 / 30 33 / 28 31 33 32 36 32 25 Terminal Sacrifice 33 / 30 27 / 32 29 27 28 24 28 35 Survival (%) 55 / 50 45 / 53.3 48.3 45 46.7 40.0 46.7 58.3 Clinical observations [no of animals with sign] Abdomen

distended

2 / 5

7 / 2

7

11

11

24

23

27 Corneal epithelial opacity 3 / 3 2 / 2 5 4 5 2 6 9 Coat unkempt 3 / 4 10 / 4 9 10 6 8 9 6 Cold to touch 1 / 0 6 / 5 6 5 4 9 3 7 Paleness; whole body 4 / 2 9 / 7 6 9 2 12 3 11 Respiration laboured 0 / 0 9 /5 2 8 3 10 1 5 Hair loss; neck dorsal 2 / 0 1 / 2 1 3 1 5 0 3 Scab; neck dorsal 6 / 8 2 / 6 5 6 3 7 4 5 Abbreviations: ND: Not Determined M: male F: female - No noteworthy findings. a At 6 months. 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).

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TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in mice Daily Dose (µg/kg BID) 0 (2 control groups) 40 200 1000 Gender M F M F M F M F Humane euthanasia 1 / 2 8 / 8 2 7 1 12 1 6 Moribund euthanasia 0 / 0 1 / 0 1 0 3 0 1 0

Body Weight (%)a Week 1 29.77 24.37 +1 +1 0 -1 -2 -2 Week 8 33.96 28.11 +5* +5* +7* +7* +7* +9* Week 13 35.58 29.32 +4* +6* +7* +7* +7* +10* Week 26 37.68 30.77 +4* +7* +6* +9* +6* +8* Week 52 38.89 32.37 +3* +5* +7* +6* +7* +8* Week 78 39.83 33.82 +1 +2 +6* +7* +7* +7* Week 100 39.42 34.55 -1 +4 +6* +4 +8* +7* Body Weight Gain (g) 1 to 26 Weeks 7.90 6.37 +15* +31* +27* +48* +35* +47* 1 to 52 Weeks 9.16 7.99 +12* +17* +27* +29* +34* +38* 1 to 100 Weeks 9.96 10.01 -10 +13 +23* +18* +33* +30* Abbreviations: ND: Not Determined M: male F: female - No noteworthy findings. a At 6 months. 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).

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TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in mice Daily Dose (µg/kg BID) 0 (control) 40 200 1000 Gender M F M F M F M F Food Consumption (g/d/mouse) and (%) a At week 1

5.97

4.99

+2

-2

+2

+1

0

-1

At week 8 5.10 4.57 +2 -1 +2 +7* -2 +6*

At week 53 5.09 4.55 -2 +4 0 +5 -5* +1

At week 101 5.34 4.96 -1 +4 +1 +7 +4* +2 Additional Information: a At 6 months. For controls, group means are shown. For treated groups, percent differences from controls are shown. Statistical significance is based on actual data (not on percent differences). Abbreviations: ND: Not Determined M: male F: female

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TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse (continued)

Test Article: Lixisenatide Report Title: Subcutaneous carcinogenicity study in mice (continued) Daily Dose (µg/kg BID) 0 (control 1&2) 40 200 1000 Gender M F M F M F M F Number of Animals with Neoplastic Lesions: Thyroid gland: c-cell focal hyperplasia 1/3 4/2 3 3 8 2 4 11 Thyroid gland: c-cell adenoma 0/0 0/0 1 0 1 0 4* 1 Uterus, endometrium: adenocarcinoma 0/0 0 3** 2 Abbreviations: M: male F: female * Significantly different (p=0.0039) from pooled controls; ** Significantly different (p=0.0224) from pooled controls

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TS 2.6.7.10D – Carcinogenicity – 2-year study in the mouse (continued)

Test Article: Lixisenatide Report Title: Subcutaneous carcinogenicity study in mice (continued) Daily Dose (µg/kg BID) 0 (control 1&2) 40 200 1000 Gender M F M F M F M F Noteworthy Findings: Hematology Not performed Gross Pathology No treatment-related findings Organ weight No treatment-related findings Histopathology- Incidence Non-Neoplastic Lesions:

Parotid gland: basophilic hypertrophy foci 3/0 3/3 57 57 56 58 53 58 Additional Information: none Conclusions: NOAEL= 40 µg/kg BID (males) and 200 µg/kg BID (females) Abbreviations: M: male F: female

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in rats Species/Strain: Rat/Crl: Sprague-Dawley Duration of Dosing: 2 years Master reference/Report No.: [CAR0084] Initial Age: 6 to 7 weeks Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location: 4.2.3.4.1-2

Date of First Dose: xx xxxxxx 20xx Vehicle/Formulation: saline (0.9% NaCl) GLP Compliance: Yes (Non-GLP for ADA titer, neutralization and cross reactivity only)

Treatment of Controls: 2 control groups; saline (0.9% NaCl) Basis for High-Dose Selection: High dose was selected to provide an exposure exceeding highest human AUC by more than 25-fold Special Features: assay for neutralization of ADAs, cross-reactivity on GLP-1 and glucagon and ADA-titer included Daily Dose (µg/kg BID) 0 (pooled controls 1&2) 40 200 1000 Gender M F M F M F M F AUC Plasma levels (ng.h/mL) Day 4/5 LLOQ 0.05 ng/mL Day 86/87 Day 359/360

- - -

- - -

66.7 15500 8290

69.5 8340 6620

242 53100 39400

268 31200 31700

863 20400 45700

823 14100 32800

Anti-Drug-Antibodies (ADAs, %) Day 93 Day 366

- 17

- 94

97 97

100 94

100 91

“ male no. 64: 0.1 ng/mL; male no. 206: 0.14 ng/mL “” male no.67: 0.15 ng/mL; male no. 205: 0.15 ng/mL “”” female nos. 268, 270 and 275: 0.05, 0.08 and 1.13 ng/mL, respectively; plasma concentration Plasma levels at termination (ng/mL) na na na na na na na na Number of animals at start 120 120 60 60 60 60 60 60 Died/Sacrificed Moribund 64 81 32 44 37 36 35 33 Terminal Sacrifice 56 39 28 16 23 24 25 27 Survival (%) 53.3/40% 40/25% 46.7% 26.7% 38.3% 40.0% 41.7% 45.0% Clinical Observations [no. of animals] Salivation, marked - - 3 1 2 5 2 3 Salivation, mild - - 1 18 6 16 8 24 Salivation, moderate 1 - 39 38 46 38 51 52 Abdomen distended, mild - - 1 9 5 6 6 6 Abdomen distended, moderate - 1 4 2 4 2 3 6 Abdomen distended, marked - 1 - - - - - - Incoordinated gait, stilted, hind limbs both 7 17 1 24 4 24 1 27

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in rats Daily Dose (µg/kg BID) 0 (pooled controls 1&2) 40 200 1000 Gender M F M F M F M F Humane euthanasia 25 38 10 12 17 18 15 17 Moribund euthanasia 10 7 4 6 8 3 9 7 Absolute Body Weight [g] & (%)a Week 2 Week 8 Week 13 Week 26 Week 41 Week 43 Week 50 Week 52/ 51 (M / F) Week 65 Week 78 Week 91 Week 104

229.1 449.1 533.8 646.5 711.5 683.2 734.4 702.0 779.8 798.7 800.8 771.2

181.1 256.6 280.5 323.6 353.5 342.1 376.4 364.7 421.8 444.8 464.2 463.9

-5*

-10* -9* -9*

-10* -5* -9* -4*

-10* -11* -12* -11*

+1 -2* -2*

-10* -12* -8*

-14* -10* -15* -16* -15* -11*

-7*

-10* -11* -12* -13* -8*

-12* -7*

-13* -12* -14* -13*

-1 -3* -6*

-13* -16* -13* -18* -15* -21* -21* -23* -18*

-10* -11* -12* -13* -14* -9*

-13* -8*

-14* -16* -15* -17*

0

-3* -4*

-13* -15* -12* -18* -16* -20* -21* -20* -18*

Absolute body weight change [g]; (%)a Day 1 to 92 Day 1 to 183 Day 1 to 351 Day 1 to 540 Day 1 to 722 (study end)

372.7 482.3 531.6 628.0 599.8

125.6 160.6 206.4 286.5 307.1

-13 -13 -12 -14 -15

-5

-15 -17 -24 -17

-16 -17 -16 -15 -17

-12 -21 -25 -31 -27

-16 -16 -16 -19 -21

-10 -21 -26 -32 -27

na # = not applicable; bw gains were negative for controls in week 42 to 43 vs. positive or almost unchanged bw gains for treated groups Abbreviations: ND: Not Determined M: male F: female - No noteworthy findings. a At 6 months. 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).

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous carcinogenicity study in rats Daily Dose (µg/kg BID) 0 (pooled controls 1&2) 40 200 1000 Gender M F M F M F M F Average weekly BW Gain [g/animal/week]; (%)a Week 1 to 41 Week 42 to 43 Week 44 to 50 Week 51 to 52 / 50 to 51 (M / F) Week 1 to 104

13.19 -13.50 3.43 -16.2 5.78

4.75 -6.40 3.27 -5.85 2.94

-13* na # -47* na # -15*

-20* na # -60* na # -15*

-17* na # -52* na # -16*

-26* na # -73* na # -26*

-17* na # -38* na # -21*

-25* na # -72* na # -26*

na # = not applicable; bw gains were negative for controls in week 42 to 43 vs. positive or almost unchanged bw gains for treated groups Abbreviations: ND: Not Determined M: male F: female - No noteworthy findings. a At 6 months. 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).

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat (continued)

Test Article: Lixisenatide Report Title: Subcutaneous carcinogenicity study in rats (continued) Daily Dose (µg/kg BID) 0 (controls 1) 40 200 1000 Gender M F M F M F M F Average Daily Food Consumption [g/animal/day]

& (%)a Week 2 Week 8 Week 13 Week 26 Week 41 Week 43 Week 50 Week 52 Week 66 Week 79 Week 92 Week 105

23.98 28.28 27.66 28.75 30.46 25.96 30.29 25.19 29.61 30.47 29.94 29.21

17.91 18.41 18.18 19.66 18.62 15.21 20.55 17.74 19.62 21.89 20.31 22.83

-19* -10* -7* -9*

-10* +7* -9* +8* -6*

-10* -9* -11

-13* -4 -3*

-12* -2

+22* -10* +3 -3 -4 -2 +4

-22* -11* -10* -10* -10* +4* -10* +7* -12* -11* -13* -2

-18* -1 -6*

-14* -3

+21* -7* +6 +2 -9* -8* +1

-26* -11* -7* -9*

-10* +6* -8* +9* -9*

-12* -9*

-13*

-18* -5* -7*

-13* -1

+19* -10* +4 -1 -9* 0 -8

Abbreviations: ND: Not Determined M: male F: female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat (continued)

Test Article: Lixisenatide Report Title: Subcutaneous carcinogenicity study in rats (continued) Daily Dose (mg/kg/day) 0 (control 1&2) 40 200 1000 Gender M F M F M F M F Number of Animals with Neoplastic Lesions: Thyroid gland: c-cell focal hyperplasia 20/17 21/12 26 28 29 25 30 33 Thyroid gland: c-cell adenoma 12/14 10/7 34* 20* 24* 30* 37* 26* Thyroid gland: c-cell carcinoma 0/0 0/0 0 0 3 1 1 2 Abbreviations: ND: Not Determined M: male F: female * Significantly different (p<0.0001) from pooled controls.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10E – Carcinogenicity – 2-year study in the rat (continued)

Test Article: Lixisenatide Report Title: Subcutaneous carcinogenicity study in rats (continued) Daily Dose (mg/kg/day) 0 (control 1&2) 40 200 1000 Gender M F M F M F M F Noteworthy Findings: Hematology Not performed Gross Pathology No treatment-related findings Histopathology-Non-Neoplastic Lesions No treatment-related findings Additional Information: none Conclusions: NOAEL= No NOAEL for c-cell hyperplasias and adenomas and 40 µg/kg BID for c-cell carcinomas Abbreviations: M: male F: female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10F – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in mice

Test Article: Lixisenatide Report Title: 1-Day QD and BID subcutaneous toxicity study in mice with a 5-day recovery Species/Strain: Crl:CD-1(ICR) Duration of Dosing: Once / Twice Master reference/Report No.: [DIV1333] Initial Age: 6 to 7 weeks Method of Administration: subcutaneous QD or BID (split by

approximately 8 hours) Location: 4.2.3.4.3-8

Date of First Dose: xx xxxxx 20xx Vehicle/Formulation: saline (0.9% NaCl) GLP Compliance: Yes Treatment of Controls: saline (0.9% NaCl) Special Features: Measurement of Calcitonin in plasma, Assessment of proliferation markers (RNA level) in thyroid Daily Dose (µg/kg) 0 2000 QD 2000 BID Gender M F M F M F Noteworthy Findings: - Clinical signs None - Body weight Body weight decrease in both males and females treated at 2000 µg/kg/ QD and 2000µg/kg/ BID (up to 10% at BID) - Food consumption Decrease in both males and females treated at 2000 µg/kg/ QD and BID on Day-interval 1 to 2 (up to 60% BID) - Plasma Calcitonin concentrations Both QD and BID injection resulted in overall increased mean calcitonin values during the test period of 24 hours when

compared to mean control or pre-test values (approximately 2-fold). This calcitonin increase was statistically significant in males from 3 to 24 hours after QD and at 8.17 hours after BID injection and in females at 9 hours after both, QD and BID injection.

- Proliferation markers No treatment-related effects in gene expression profile Additional Information: none Conclusions: Lixisenatide caused an increased Calcitonin release Abbreviations: M: male F: female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10G – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in rats

Test Article: Lixisenatide Report Title: 1-Day QD and BID subcutaneous toxicity study in rats with 5-day recovery Species/Strain: Crl:CD(SD) Duration of Dosing: Once / Twice Master reference/Report No.: [DIV1332] Initial Age: 6 to 7 weeks Method of Administration: subcutaneous QD or BID (split by

approximately 8 hours) Location: 4.2.3.4.3-9

Date of First Dose: xx xxxxx 20xx Vehicle/Formulation: saline (0.9% NaCl) GLP Compliance: Yes Treatment of Controls: saline (0.9% NaCl) Special Features: Measurement of Calcitonin in plasma, Assessment of proliferation markers (RNA level) in thyroid Daily Dose (µg/kg) 0 2000 QD 2000 BID Gender M F M F M F Noteworthy Findings: - Clinical signs None - Body weight Decreased one day after dosing (Day 2) at 2000 μg/kg QD and 2000 μg/kg BID (up to 13%), as compared to the controls.

Mean body weight turned to almost normal values during the recovery period (until Day 6) - Food consumption Decreased on Day-interval 1 to 2 at 2000 μg/kg QD and BID (up to 89%) as compared to controls, with subsequent

overcompensation for males on Day interval 5 to 6 or turn to normal for females between Day-intervals 2 to 5 and 5 to 6. - Plasma Calcitonin concentrations No treatment-related effects - Proliferation markers No treatment-related effects in gene expression profile Additional Information: none Conclusions: No increased Calcitonin levels were observed after QD or BID administration of lixisenatide Abbreviations: M: male F: female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.10H – Carcinogenicity – Mechanistic studies: Calcitonin release and gene expression in knock out mice

Test Article: Lixisenatide Report Title: Exploratory 14-day subcutaneous (BID) toxicity study in GLP1R (-/-) KO and wild-type mice Species/Strain: Mouse, CD1 and GLP-1R (-/-) KO Duration of Dosing: 2 weeks Master reference/Report No.: [TSA1481] Initial Age: 6 to 7 weeks Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location: 4.2.3.4.3-10

Date of First Dose: xx xxxx 20xx Vehicle/Formulation: saline (0.9% NaCl) GLP Compliance: No Treatment of Controls: saline (0.9% NaCl) Special Features: Measurement of Calcitonin in plasma CD1 mice GLP-1R (-/-) knockout mice Daily Dose (µg/kg BID) 0 1000 0 1000 Gender M F M F M F M F Noteworthy Findings: - Clinical signs None - Body weight CD1 mice: slight decrease in mean group body weight (D-1 to D8)

KO mice: slight decrease in mean group body weight (D8 to D15) - Food consumption CD1 mice: decrease in mean absolute food consumption over the treatment period - Plasma Calcitonin concentrations CD1 mice: statistically significant higher mean plasma calcitonin concentration in both sexes at the end of the 14-day dosing

period, when compared to control CD1 mice. KO mice: no calcitonin release, plasma calcitonin concentration remained low and similar to values observed in CD1 and KO mice from control groups

Additional Information: none Conclusions: increase in plasma calcitonin concentration in CD1 mice, but no increase in treated GLP1R (-/-) KO mice Abbreviations: M: male F: female

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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11 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - NONPIVOTAL STUDIES TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rat/Hsd: Sprague Dawley

SC BID (0.9% sodium

chloride)

GD 6 to 17 70, 1000, 2000 6F Maternal data Mortality: none Clinical signs: ≥70 µg/kg: Hypoactivity (sleepiness), bristling coat 2000 µg/kg: reddish vaginal discharge (1F) Body weight: ≥70 µg/kg: body weight constant or decreased (GD6 to 9), later on body weight gain ↓ Food consumption: ≥70 µg/kg: Food consumption ↓ Macroscopic: NAD

[20 x-1923] 4.2.3.5.2-1

Abbreviations: M = male, F = female, GD = gestation day, NAD = no abnormality detected

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rat/Hsd: Sprague Dawley

SC BID (0.9% sodium

chloride)

GD 6 to 17 70, 1000, 2000 6F Caesarean section: 70 µg/kg: postimplantation loss (1F, considered incidental) 1000 µg/kg: Early resorptions↑, dead fetuses↑ 2000 µg/kg: total postimplantation and/or preimplantation loss (5F) Fetal data ≥70 µg/kg: fetal body weights (and placental weights) ↓, crown-rump lengths ↓ External examination: NAD

[20 x-1923] continued

4.2.3.5.2-1

Abbreviations: M = male, F = female, GD = gestation day, NAD = no abnormality detected

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rabbit (Himalayan)/ Chbb:HM(SPF) Kleinrusse

SC BID (0.9% sodium

chloride)

GD 6 to 18 40, 200, 1000 6F Maternal data Mortality: 1000 µg/kg: 2F euthanized on Day 24 after premature delivery Clinical signs: ≥40 µg/kg: Hypoactivity including decreased reactivity and hypotonicity of skeletal muscles, bristling coat, decreased hay consumption, decreased defecation 1000 µg/kg: diarrhea(2F), reddish discolored tray bedding (1F) Body weight: ≥40 µg/kg: Body weight ↓ (Day 6 to 10), later on during treatment no or minimal weight gain Food consumption: ≥40 µg/kg: Food consumption ↓ Macroscopic: 1000 µg/kg (euthanasias Day 24): large gall bladder (2F)

[20 x-1924] 4.2.3.5.2-5

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rabbit (Himalayan)/ Chbb:HM(SPF) Kleinrusse

SC BID (0.9% sodium

chloride)

GD 6 to 18 40, 200, 1000 6F Caesarean section: NAD Fetal data 40 µg/kg: Thoracogastroschisis and related skeletal malformations (1 fetus) 1000 µg/kg: fetal body weights↓, crown-rump length ↓ External examination: NAD

[20 x-1924] continued

4.2.3.5.2-5

Abbreviations: M = male, F = female, GD = gestation day, NAD = no abnormality detected

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rat (Hsd: Sprague

Dawley SD)

SC BID (0.9% sodium

chloride)

GD 6 until LD 3 2.5, 35, 500 6F Maternal data (F0 generation) Mortality: none Clinical signs: ≥2.5 µg/kg: decreased motor activity, piloerection Body weight: ≥2.5 µg/kg: Body weight loss (GD 6 to 7:-6.0 to –20.7g), lower weights during further pregnancy (GD 21: -7.6 to –13.3% of control), weight gain normal during lactation Food consumption: ≥2.5 µg/kg: Food consumption ↓ (GD 6 to 9): max. –38% to –82% on GD 6 to 7 Gestation/Parturition: NAD

[DPP0025] 4.2.3.5.3-1

Abbreviations: M = male, F = female, GD = gestation day, LD = lactation day, PND = post-natal day, NAD = no abnormality detected.

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rat (Hsd: Sprague

Dawley SD)

SC BID (0.9% sodium

chloride)

GD 6 until LD 3 2.5, 35, 500 6F Lactation: ≥2.5 µg/kg: decreased nursing and nesting behaviour associated with decreased activity, less suckling of pups, slightly increased cannibalism Macroscopy: NAD Pup data (F1 generation) Mortality: 500 µg/kg: live birth index/PND 4 viability reduced (-8/-12%) Clinical signs: 500 µg/kg: necrosis of tail tip (2 pups from 2 litters, PND 2-4) Body weight: ≥2.5 µg/kg: birth weight↓ (-10% to –27% of control), weight gain↓ (-6% to –42%, PND 0 to 4)

[DPP0025] continued

4.2.3.5.3-1

Abbreviations: M = male, F = female, GD = gestation day, LD = lactation day, PND = post-natal day, NAD = no abnormality detected.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.11 - Reproductive and Developmental Toxicity - Nonpivotal Studies (continued)

Test Article: Lixisenatide

Species/ Strain

Method of Administration

(Vehicle/ Formulation)

Duration of Dosing

Doses (µg/kg BID)

No. per Group

and Gender (M/F)

Noteworthy Findings Study No. Location

Rat (Hsd: Sprague

Dawley SD)

SC BID (0.9% sodium

chloride)

GD 6 until LD 3 2.5, 35, 500 6F Visceral examination: NAD Skeletal examination: 2.5 µg/kg: 1 pup with multiple malformations of shoulder girdle, long bones of forelimb, wavy ribs 35 µg/kg: 4 pups with wavy/thickened ribs (incidental as attributed to decreased suckling -lower milk content in stomach- not at high dose and within historical range), 500 µg/kg: 3 pups with sternebra anomalies

[DPP0025] continued

4.2.3.5.3-1

Abbreviations: M = male, F = female, GD = gestation day, LD = lactation day, PND = post-natal day, NAD = no abnormality detected.

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12 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - FERTILITY AND EARLY EMBRYONIC, DEVELOPMENT TO IMPLANTATION

TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat Species/Strain: Rat / Crl:CD (SD) IGSBR Duration of Dosing: M: from 4 weeks prior to pairing until study week 8, F: from 2

weeks prior to pairing until Day 6 of gestation Test Article: AVE0010 (batch xxxx xxxxxxxxx)

Initial Age: M: 9 to 11 weeks, F: 11 to 13 weeks

Study No. [20 x-0550] + Amendment 1 [20 x-0550A1]

Date of First Dose: xx xxxxx 20xx (M), xx xxx 20xx (F)

Day of Mating: Day 0 Location: 4.2.3.5.1-1, 4.2.3.5.1-1a

Special Features: none Day of C-Section: Gestation day 13 GLP Compliance: yes Method of Administration: subcutaneous BID (split by approximately 8 hours) No Observed Adverse Effect Level: 414 µg/kg BID

Vehicle/Formulation: 0.9% sodium chloride

Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414 No. of males/females on study 24/24 24/24 24/24 24/24 No. died or sacrificed moribund 0/0 0/0 0/0 0/0

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TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414 MALES: Clinical Observations Subdued behavior 0 24 24 24 Salivation 0 7 12 13 Teeth abnormalities 0 0 0 1 Teeth damage 1 0 0 1 Red leg 0 1 0 0 Colored tears 1 0 0 1 Hair loss 2 0 0 1 Raised hair 1 1 0 0 Skin/hair staining 8 13 15 14 Thinning fur 3 1 3 0 Skin lesion 0 0 2 0 FEMALES: Clinical Observations Subdued behavior 0 24 24 24 Salivation 0 6 3 9 Pale teeth 5 6 2 5 Colored tears 0 1 0 0 Hair loss 3 0 1 0 Skin staining 15 16 19 20 Thinning fur 3 0 6 7

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TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation (continued)

Test Article: Lixisenatide

Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414

MALES: No. of Males on Study 24 24 24 24 Premating Body Weight (g and %) Day 29 a 465.1 -6.5 -7.9 -9.2 Premating Average Daily Body Weight Gain (g and %) a Study Day 1 to 15 Study Day 15 to 29 Study Day 29 to 53

46.8 47.6 43.6

-36*** -33*** -6.4

-38*** -40*** +11

-42*** -48*** -6.0

Premating Average Daily Food Consumption (g/animal/period and %) a Study Days 1 to 7 Study Days 8 to 14 Study Days 15 to 21 Study Days 22 to 28

193.6 192.5 201.4 203.5

-15*** -12*** -16*** -14***

-22*** -15*** -19*** -16***

-30*** -12*** -19*** -18***

Male Sex Organ Weights (%) not assessed Necropsy Observations - NTF NTF NTF Histomorphologic evaluation of male sex organs not evaluated Abbreviations: NTF = No test article-related findings a 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 difference). Statistically significant ANOVA dose response and Dunnett’s test: *P<0.05, **P<0.01, ***P<0.001

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TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation (continued) Test Article: Lixisenatide

Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414

FEMALES: No. Died or Sacrificed Moribund 0 0 0 0 No. Evaluated 24 (all pregnant) 24 (23 pregnant) 24 (22 pregnant) 24 (all pregnant) Premating Body Weight (%) a – Study Day 29 257.2 -1.4 -1.4 ±0 Premating Average Daily Body Weight Gain (g and %)a Study Days 1 to 29

29.1

-5.8

-0.7

+8.2 Gestation Average Daily Body Weight Gain (g and %)a GD 0-3 GD 3-6 GD 6-10 GD 10-13

11.5 12.7 15.7 15.6

+21 ±0 -21 +1.9

-19 -7.1 +11 -4.5

-7.8 -45*

+51** +2.6

Pre-pairing Average Daily Food Consumption (g/animal/period and %)a Study Days 1 to 7 Study Days 8 to 14 Study Days 15 to 21 Study Days 22 to 28

122.0 127.7 127.4 131.0

-0.2 +1.4

-13.1* -12**

-0.9 -0.9 -19** -11**

-0.6 -0.5

-27*** -14***

Gestation Average Daily Food consumption (g/animal/day and %)a

GD 0 to 5 GD 6 to 12

22.2 25.4

-5.4 +1.2

-14*** +3.1

-12** +6.3

Necropsy Observations - NTF NTF NTF Organ weights not assessed - - - - Abbreviations: GD = gestation day, NTF = No test article-related findings a 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 difference). Statistically significant ANOVA dose response and Dunnett’s test: *P<0.05, **P<0.01, ***P<0.001

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 177

TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation (continued)

Test Article: Lixisenatide

Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414

FEMALES: Mating Index a 100 95.8 100 100 Pregnancy Index b 100 100 91.7 100 Fertility Index c 100 95.8 91.7 100 No. times in estrus – predosing 3.2 3.3 3.1 3.1 No. times in estrus – dosing before pairing 3.2 3.0 2.7 2.3 Proportion of M/D days – predosing ND ND ND ND Proportion of M/D days – dosing before pairing ND ND ND ND No. with aberrant M/D days – predosing ND ND ND ND No. with aberrant M/D days – dosing ND ND ND ND Median time to insemination (days) 2.5 3 3 3 No. with viable embryos 24 23 22 24 No. with total resorptions 0 0 0 0 No. with ≥ 1 non-viable embryo per no. pregnant at cesarean section

1 0 0 0

Abbreviations: ND not determined a Mating index = No. inseminated females/ No. females paired with males b Pregnancy index = No. pregnant females / No. inseminated females. c Fertility index = No. pregnant females / No. females paired with males.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 178

TS 2.6.7.12A - Reproductive and Developmental Toxicity - Fertility and Early Embryonic, Development to Implantation (continued)

Test Article: Lixisenatide

Report Title: AVE0010: Subcutaneous study of fertility and early embryonic development in the rat (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2 29 414

LITTERS (mean per litter ± SD): No. Evaluated 24 23 22 24 Corpora lutea 14.7±2.7 14.7±2.5 13.9±1.9 14.8±3.3 Implantations 13.4±1.8 13.3±2.9 13.0±1.9 13.7±1.5 Pre-implantation loss (% ) a 7.2 ±12.0 9.4±14.1 6.4±7.9 5.4±10.8 Total embryos 12.6±2.0 12.7±2.5 12.5±1.9 12.8±1.8 Viable embryos 12.6±2.0 12.7±2.5 12.5±1.9 12.8±1.8 Dead embryos 0.0±0.2 0.0±0.0 0.0±0.0 0.0±0.0 Resorptions 0.8±1.4 0.5±0.8 0.4±0.6 0.9±0.9 Resorptions (% ) 5.9 3.5 3.2 6.4 Post-implantation loss (% ) 6.0±9.2 3.5±5.2 3.2±4.6 6.6±6.3 Additional Information: Conclusions: no adverse effects on fertility or early embryonic development at any dose level tested, NOAEL=414 µg/kg BID Abbreviations: SD = standard deviation a Percentages = averages of individual litter percents.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 179

13 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - EFFECTS ON EMBRYO-FETAL DEVELOPMENT

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats Species/Strain: Rat / Hsd: Sprague Dawley SD Duration of Dosing: GD 6 to GD 17 Test Article: AVE0010 (batch xxxxxxxxx) Initial Age: 9 to 11 weeks at mating Day of Mating: Day 0 Study No.: [20 xx-0551]

Separate reports for toxicokinetics [F20xxKIN0022] (+ amendment 1 [F20xxKIN0022-amend01]) and bioanalytics [F20xxKIN0309]

Date of study in-vivo initiation (mating): xx xxxx 20xx Day of C-Section: GD 20 Location: 4.2.3.5.2-2, 4.2.3.5.2-3, 4.2.3.5.2-3a, 4.2.3.5.2-4

Special Features: none Method of Administration: subcutaneous BID (split by approximately 8 hours) GLP Compliance: yes No Observed Adverse Effect Level: below 2.5 µg/kg BID for maternal and developmental toxicity

Vehicle/Formulation: 0.9% sodium chloride

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Females Toxicokinetics:

AUC(0-24h) ( ng.h/mL) Day 7 of treatment - 3.9 49.4 581.4 Cmax (ng/mL) Day 7 of treatment - 3.5 18.8 150.8 No. Inseminated 32 30 30 30 No. Pregnant 20 21 21 16 No. Died or Killed Moribund 0 0 0 0 No. with Live Fetus(es) 20 21 21 16 No. with Total Litter Loss 0 0 0 0 No. with ≥ 1 Resorption 11 9 15 8 Clinical Signs of Toxicity - Decreased motoractivity

Piloerection Decreased motoractivity

Piloerection Decreased motoractivity

Piloerection Abbreviations: AUC = area under the curve, - No noteworthy findings, GD = gestation day, pregnancy data refers to main group rats

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 180

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal, Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500

Females (cont.) Absolute Body Weights (g and %)a on GD 9 on GD 13 on GD 16 on GD 18 on GD 20

255.9 272.7 290.1 323.9 353.6

-6.0* -8.1* -6.0* -7.9* -6.5*

-7.3* -8.0* -6.7* -9.8* -9.9*

-8.5* -8.4* -6.5* -9.4* -7.1*

Body Weight Gains/Losses (g)

GD 0 to 6 GD 6 to 9# GD 0 to 9 GD 0 to 13 GD 0 to 16 GD 0 to 18 GD 0 to 20

28.6 6.7 35.3 52.2 69.6

103.4 133.1

27.8 -0.5

27.2* 37.3* 59.4* 85.0*

117.4*

26.3 -4.9

21.4* 35.0* 55.1* 76.6*

102.9*

24.4* -9.9

14.5* 30.1* 51.4* 73.6*

108.8* Food Consumption (g/100g body weight/day and %)a

GD 0 to 3 GD 3 to 6 GD 6 to 9 GD 9 to 13 GD 13 to 16 GD 16 to 18 GD 18 to 20

7.88 8.27 7.57 7.66 7.74 7,81 7.39

-2.8 -1.2 -23* -19* -19* -7.7* +11*

-4.6 -5.0* -37* -18* -17* -9.2* +14*

-6.0* -5.4* -45* -19* -17* -9.3* +18*

Abbreviations: F = female, GD = gestation day, a 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). # not statistically evaluated, * significant trend (p≤0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 181

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal, Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500

Females (cont.) Necropsy Observations - Clear fluid in uterus horns (2F)

Clear fluid in uterus horns (1F) -

No. Corpora Lutea 15.9 15.8 16.0 16.1 No. Implantations 14.8 14.7 14.2 15.3 % Pre-implantation Loss# 7.3 6.6 10.9 5.4

Abbreviations: F = female, GD = gestation day, a 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). # not statistically evaluated, * significant trend (p≤0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 182

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters Total Fetuses# 14.0 14.0 12.5 14.7

Live Fetuses 14.0 14.0 12.5 14.6 Dead Fetuses 0.0 0.0 0.0 0.1 Resorptions# 0.8 0.7 1.7 0.6 Viable Fetuses (%) a# 94.4 95.0 87.3 95.7 Post Implantation Loss (%) a# 5.6 5.0 12.7 4.3 Avg. fetal body weight (g) 3.41 3.28 3.35 3.06* Avg. Male fetal body weight (g) 3.46 3.33 3.39 3.18* Avg. Female fetal body weight (g) 3.36 3.23 3.29 2.94* Avg. Fetal Crown-rump length (mm) 36.9 36.2 36.7 35.0*

Sex ratio of fetuses (% male) 48.4 54.9 51.4 49.1

Abbreviations: a % of implantations # not statistically evaluated, * significant trend (p≤0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Fetal Gross External and visceral Anomalies

General Anasarca MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/136

- -

General Hemorrhage MIN Litter Incidence Fetal Incidence

1/20

1/143

- -

- -

- -

General retarded MIN Litter Incidence Fetal Incidence

2/20

2/143

6/21

7/153

4/21

4/136

4/16

4/121 Fetal Skeletal Anomalies Skull Mandible incisors not visible MIN Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Maxilla incisors not visible MIN Litter Incidence Fetal Incidence

- -

2/21

2/153

2/21

2/135

2/16

2/121 Individual skull bones unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

1/20 1/143

4/21 5/153

2/21 2/135

4/16 4/121

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 184

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Shoulder girdle Clavicle incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Clavicle Misshapen MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Scapula bent MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Scapula incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Scapula short Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Spina scapula bent Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 185

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Forelimbs Humerus bent MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Humerus incomplete ossific. OSS Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Humerus short MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Radius bent MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Radius incomplete ossific. OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Radius short MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Ulna bent MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Ulna incomplete ossific. OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Ulna short MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 186

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Forepaws 1st to 5th digit distal phalanx unossified OSS Litter Incidence Fetal Incidence

- -

- -

1/21 1/135

- -

Metacarpal 2 unossified OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Metacarpal 5 unossified OSS Litter Incidence Fetal Incidence

4/20

5/143

7/21

12/153

7/21

9/135

9/16

19/121* Sternebrae Sternebrae unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

15/20 29/143

16/21 53/153

16/21 48/135

15/16 70/121*

Ribs Rib abnormalities MIN Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Rib incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Rib wavy and/or thicked MIN Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significant higher than control, Jackknife-t-test (p<0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 187

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Ribs (continued) Rib 14th rib supernumerary short or full MIN Litter Incidence Fetal Incidence

12/20 22/143

9/21 21/153

7/21 11/135

4/16 5/121

Cervical/thoracic/lumbar/sacral/ caudal vertebrae

Cer. Vertebrae arch. unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

1/20 1/143

1/21 2/153

1/21 1/135

- -

Thoracic vertebrae arch. unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21 1/135

- -

Thoracic vertebrae centrum unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

4/20 4/143

2/21 2/153

3/21 4/135

1/16 1/121

Lumbar vertebrae arch. unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21 1/135

- -

Lumbar vertebrae centra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21 1/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 188

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous Embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Sacral vertebrae arch. unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

2/21 2/153

2/21 2/135

- -

Sacral vertebrae centra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

2/21 2/153

2/21 2/135

- -

Caudal vertebrae centra unossified of less than 4 OSS Litter Incidence Fetal Incidence

12/20 28/143

16/21 52/153

14/21 49/135

15/16 74/121*

Pelvic girdle Ilium bent MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Ilium incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Ischium unossified OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Pubis incomplete ossification OSS Litter Incidence Fetal Incidence

1/20

1/143

1/21

1/153

2/21

2/135

- -

Pubis unossified OSS Litter Incidence Fetal Incidence

- -

- -

2/21

2/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significant higher than control, Jackknife-t-test (p<0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 189

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous Embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Hindlimbs Femur bent MAL Litter Incidence Fetal Incidence

- -

1/21

1/153

1/21

1/135

- -

Femur incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Femur short MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Fibula bent MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Fibula incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Fibula short MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Tibia bent MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Tibia incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Tibia short MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/135

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 190

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous Embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Hindpaws 1st to 5th digit – distal phalanx – unossified OSS Litter Incidence Fetal Incidence

1/20 1/143

3/21 4/153

3/21 3/135

3/16 3/121

Metatarsal 5 unossified Litter Incidence Fetal Incidence

1/20

1/143

2/21

2/153

3/21

3/135

1/16

1/121

Bouin investigation Abdomen fluid-filled MIN Litter Incidence Fetal Incidence

- -

1/21

1/141

- -

- -

General retarded MIN Litter Incidence Fetal Incidence

1/20

1/137

2/21

3/141

3/21

3/128

1/16

2/112 Anophthalmia MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/128

- -

Microphthalmia MAL Litter Incidence Fetal Incidence

- -

1/21

1/141

- -

- -

Diaphragm hernia MAL Litter Incidence Fetal Incidence

- -

- -

1/21

1/128

- -

Liver hemorrhagic MIN Litter Incidence Fetal Incidence

1/20

1/137

2/21

2/141

1/21

1/128

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 191

TS 2.6.7.13A - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rats (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous Embryo-fetal toxicity study in rats (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 35 500 Litters (cont.) live fetuses

Bouin investigation Renal pelvis dilated MIN Litter Incidence Fetal Incidence

1/20

1/137

1/21

1/141

1/21

1/128

1/16

1/112 Ureter dilated MIN Litter Incidence Fetal Incidence

1/20

1/137

- -

- -

1/16

1/112 Litters dead fetuses

Skeletal Several bones incomplete ossification or unossified Litter Incidence Fetal Incidence

- -

1/21 1/1 dead fetus

- -

- -

Multiple malformations: Majority of bones incomplete or unossified Litter Incidence Fetal Incidence

- -

- -

- -

1/16 1/1 dead fetus

Conclusion: NOAEL for maternal and developmental toxicity below 2.5 µg/kg BID Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 192

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits Species/Strain: Rabbit / Chbb: Himalayan (SPF) Kleinrusse Duration of Dosing: GD 6 to GD 18 Test Article: AVE0010 (batch

xxxxxxxxx) Initial Age: 5 to 7 months at mating Day of Mating: Day 0 Study No.: [20 x-0552]

Separate reports for toxicokinetics: [F20xxKIN0024] (+ amendment 1 [F20xxKIN0024-amend01]) and bioanalytics: [F20xxKIN0250]

Date of study in-vivo initiation (mating): xx xxxx 20xx Day of C-Section: GD 29 Location: 4.2.3.5.2-6, 4.2.3.5.2-7, 4.2.3.5.2-7a, 4.2.3.5.2-8

Special Features: none Method of Administration: subcutaneous BID (split by approximately 8 hours) GLP Compliance: yes No Observed Adverse Effect Level: below 2.5 µg/kg BID for fetal and maternal toxicity

Vehicle/Formulation: 0.9% sodium chloride

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Females Toxicokinetics:

AUC(0-24h) ( ng.h/mL) Day 7 of treatment - 27.2 292.5 3899.2 Cmax (ng/mL) Day 7 of treatment - 4.14 48.83 499.70 No. Inseminated 20 20 20 20 No. Pregnant 19 19 18 20 No. Died or Killed Moribund 0 0 0 0 No. with Live Fetus(es) 19 19 16 20 No. with Total Litter Loss 0 0 1 0 No. with ≥ 1 Resorption 5 8 7 10 No. of Abortions (premature delivery) 0 0 1 0

Clinical Signs of Toxicity -

Decreased motor activity Piloerection

Decreased hay consume Reduced feces

Decreased motor activity Piloerection

Decreased hay consume Reduced feces

Decreased motor activity Piloerection

Decreased hay consume Reduced feces

Abbreviations: - = finding absent, AUC = area under the curve, - No noteworthy findings, GD = gestation day

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 193

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryofetal, Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250

Females (cont.) Absolute Body Weights (g and %)a on GD 6 on GD 10 on GD 13 on GD 16 on GD 19

on GD 23

2669 2673 2692 2759 2765 2793

+2.0 -0.6 -2.0 -3.7* -3.4* -0.6

+1.6 -2.9 -3.3* -5.4* -4.6* -1.1

+0.9 -6.7* -6.2* -9.6* -10* -2.4

Body Weight Gains/Losses (g)

GD 0 to 6 GD 6 to 10# GD 0 to 10 GD 0 to 13 GD 0 to 16 GD 0 to 19 GD 0 to 29

46 4

49 68

135 141 316

28 -65 -37* -58* -38* -24* 183

43 -117 -74* -66* -62* -34* 184

46 -199 -153* -121* -153* -160* 208

Abbreviations: F = female, GD = gestation day, a 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). # not statistically evaluated, * significant trend (p≤0.05), number in brackets (mid-dose group): values excluding females with total litter loss

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 194

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryofetal, Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250

Food Consumption (g/100g body weight/day and %)a

GD 0 to 3 GD 3 to 6 GD 6 to 10 GD 10 to 13 GD 13 to 16 GD 16 to 19 GD 19 to 23 GD 23 to 26 GD 26 to 29

3.19 3.17 3.35 3.11 2.67 3.03 3.03 3.00 3.24

+1.6 +3.2 -65* -48* -48* -48* +28* +21* +18*

+7.5 +4.4 -85* -52* -56* -54* +37* +26* +17*

+10* +12 -94* -58* -71* -69* +37* +44* +27*

Necropsy Observations - Missing junction of uterus horn to vagina (1F) - -

No. Corpora Lutea 8.1 8.2 7.9 (8.2) 8.7 No. Implantations 8.0 7.1 6.8 (7.2) 8.2 % Pre-implantation Loss# 1.3 13.8 16.2 (13.0) 6.5

Abbreviations: - = finding absent, F = female, GD = gestation day, a 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). # not statistically evaluated, * significant trend (p≤0.05), number in brackets (mid-dose group): values excluding females with total litter loss

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 195

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters Total Fetuses# 7.6 6.5 6.2 (6.6) 7.0

Live Fetuses 7.6 6.5 6.2 (6.6) 6.8* Dead Fetuses 0 0 0 0.2 Resorptions# 0.4 0.6 0.7 (0.6) 1.2 Viable Fetuses (%) a# 95.6 91.2 87.4 (92.9) 81.2 Post Implantation Loss (%) a# 4.4 8.8 12.6 (7.1) 18.8 Avg. fetal body weight (g) 40.01 41.04 39.28 40.35 Avg. Male fetal body weight (g) 40.20 41.16 40.07 40.71 Avg. Female fetal body weight (g) 39.67 39.91 38.86 40.53 Avg. Fetal Crown-rump length (mm) 94.4 95.6 95.2 95.8

Sex ratio of fetuses (% male) 46.2 53.6 50.6 45.2 Abbreviations: a % of implantations # not statistically evaluated, * significant trend (p≤0.05), number in brackets (mid-dose group): values excluding females with total litter loss

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 196

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters (cont.) live fetuses

Fetal Gross External Anomalies Forepaws/digits Paw hyperflexion MIN Litter Incidence Fetal Incidence

1/19

1/145

8/19* 8/122

1/16

2/103

4/20

5/135 Fetal Visceral Anomalies General Abdomen fluid filled MIN Litter Incidence Fetal Incidence

2/19

2/145

- -

- -

- -

Thoracic fluid-filled MIN Litter Incidence Fetal Incidence

- -

- -

1/16

1/103

- -

Arteries Carotid artery malpositioned branch MIN Litter Incidence Fetal Incidence

7/19 10/145

4/19 12/122

5/16 6/103

7/20 10/135

Innominate artery short MIN Litter Incidence Fetal Incidence

2/19

2/145

1/19

2/122

3/16

3/103

2/20

2/135 Subclavian artery malpositioned branch MIN Litter Incidence Fetal Incidence

1/19 1/145

1/19 1/122

- -

1/20 1/135

Abbreviations: - = finding absent, Abbreviations: MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 197

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters (cont.) live fetuses

Fetal Visceral Anomalies (cont.) Heart Ventricular septum – septum defect MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/20 1/135

Pericardium hydropericardium MIN Litter Incidence Fetal Incidence

- -

- -

- -

1/20

2/135 Lung Lung abnormalities MIN Litter Incidence Fetal Incidence

6/19

9/145

6/19

12/122

5/16

17/103

9/20

18/135 Kidney Kidney malpositioned MIN Litter Incidence Fetal Incidence

- -

- -

1/16

1/103

- -

Abdomen Gallbladder absent MAL Litter Incidence Fetal Incidence

- -

- -

1/16

1/103

2/20

2/135 Gallbladder small MIN Litter Incidence Fetal Incidence

- -

- -

1/16

1/103

2/20

2/135 Stomach abnormalities MIN Litter Incidence Fetal Incidence

1/19

1/145

3/19

4/122

1/16

1/103

1/20

2/135 Abbreviations: - = finding absent, Abbreviations: MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 198

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters (cont.) live fetuses

Fetal Skeletal Anomalies Skull Skull splitting of bone MIN Litter Incidence Fetal Incidence

- -

4/19

5/122*

4/16

5/103*

- -

Skull sutural bone MIN Litter Incidence Fetal Incidence

3/19

5/144

4/19

10/122

4/16

5/103

3/20

4/135 Skull hole small MIN Litter Incidence Fetal Incidence

3/19

3/144

1/19

1/122

1/16

1/103

1/20

1/135 Parietal additional suture MIN Litter Incidence Fetal Incidence

1/19

1/144

5/19

7/122

2/16

2/103

1/20

1/135 Forepaws 5th digit middle phalanx incomplete ossification OSS Litter Incidence Fetal Incidence

1/19 1/144

2/19 3/122

3/16 4/103

- -

Sternebrae Sternebrae abnormalities MAL Litter Incidence Fetal Incidence

8/19

12/144

7/19

14/122

7/16

19/103

13/20

32/135* Sternebrae unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

15/19 57/144

14/19 36/122

10/16 25/103

13/20 39/135

Abbreviations: - = finding absent, Abbreviations: MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 199

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters (cont.) live fetuses

Fetal Skeletal Anomalies (cont.) Ribs Rib at 7th cervical arch short MIN Litter Incidence Fetal Incidence

4/19

7/144

1/19

2/122

1/16

1/103

1/20

1/135 Rib abnormalities MAL Litter Incidence Fetal Incidence

- -

2/19

2/122

- -

- -

Rib 13th rib supernumerary short or full MIN Litter Incidence Fetal Incidence

1/19 1/144

2/19 2/122

- -

7/20 7/135*

Thoracic vertebrae Thoracic vertebrae centrum unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/19 1/122

- -

- -

Caudal vertebrae Caudal vertebrae centra abnormalities MAL Litter Incidence Fetal Incidence

- -

1/19 2/122

- -

- -

Caudal vertebrae centra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

3/19 3/144

3/19 3/122

2/16 2/103

3/20 4/135

Abbreviations: - = finding absent, Abbreviations: MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 200

TS 2.6.7.13B - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (1) (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 2.5 25 250 Litters (cont.) live fetuses

Fetal Skeletal Anomalies (cont.) Caudal vertebrae (cont.) Caudal vertebrae centra ossification of less than 15 OSS Litter Incidence Fetal Incidence

10/19 17/144

11/19 35/122*

12/16 23/103*

15/20 40/135*

Multiple malformations Litter Incidence Fetal Incidence

- -

2/19

2/122

2/16

2/103

1/20 a

1/135 a Litters Dead fetuses

Skeletal Individual skeletal bones unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/1 with premature

delivery 4/6 dead fetuses

1/3 1/3

Sternebrae fused MAL Litter Incidence Fetal Incidence

- -

- -

1/1 with premature delivery

1/6 dead fetuses

- -

Ribs at the left 7th cervical arch short Litter Incidence Fetal Incidence

- -

- -

- -

1/3 1/3

Ribs at the left 7th cervical arch short Litter Incidence Fetal Incidence

- -

- -

- -

1/3 1/3

Conclusions: NOAEL for fetal and maternal toxicity below 2.5 µg/kg BID

Abbreviations: Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test). a Based on a reevaluation conducted after completion of the study, the fetus at the 250 µg/kg BID was not considered as having multiple malformation.

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 201

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous embryo-fetal toxicity study in rabbits Species/Strain: Rabbit / Chbb: HM (SPF) Himalayan Duration of Dosing: GD6 to GD 18 Test Article: AVE0010 (batch

xxxxxxxxx) Initial Age: 4-6 months (at mating) Days of Mating: Day 0 Study No.: [20 x-1086] Date of study in-vivo initiation (mating): xx xxx 20xx Day of C-Section: GD 29 Location: 4.2.3.5.2-9 Special Features: none Method of Administration: subcutaneous BID (split by approximately 8 hours) GLP Compliance: yes No Observed Adverse Effect Level: 0.15 µg/kg BID for maternal, and 2.5µg/kg BID for fetal toxicity

Vehicle/Formulation: 0.9% sodium chloride / aqueous solution

Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Females Toxicokinetics:

AUC(0-24h) ( ng.h/mL) Day 7 of treatment - 0.929 16.0 41.5 Cmax (ng/mL) Day 7 of treatment - 0.263 2.22 8.43 No. Inseminated 20 20 20 20 No. Pregnant 20 18 19 19 No. Died or Killed Moribund 0 0 0 0 No. with Live Fetus(es) 20 18 17 19 No. with Total Litter Loss (resorptions only) 0 0 1 0

No. with ≥ 1 Resorption 5 3 11 6 No. of Abortions (Premature delivery) 0 0 1 0 Clinical Signs of Toxicity - - Decreased motoractivity

Piloerection Decreased motoractivity

Piloerection Abbreviations: - = finding absent, AUC = area under the curve, GD = gestation day, F = females

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 202

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5

Females (cont.) Absolute Body Weights (g and %)a on GD 6 on GD 10 on GD 19 on GD 23

2.453 2.463 2.563 2.590

+0.9 ±0.0 +0.9 +1.4

+5.6* +1.7 -1.8 +2.3

+6.6* +1.9 -2.4 +3.4

Body Weight Gains/Losses (g)

GD 0 to 6 GD 6 to 10# GD 0 to 10 GD 0 to 13 GD 0 to 16 GD 0 to 19 GD 0 to 29

56 10 66 90

153 167 315

49 -12 37* 59

128 159 327

61 -86 -25* -25* -11* -12* 225*

49 -104 -55* -49* -33* -63* 210*

Food Consumption (g/100g body weight/day and %)a

GD 0 to 3 GD 3 to 6 GD 6 to 10 GD 10 to 13 GD 13 to 16 GD 16 to 19 GD 19 to 23 GD 23 to 26 GD 26 to 29

3.38 3.77 3.16 3.22 3.14 3.57 3.47 3.34 3.46

+1.8 +1.1 -17* -5.6* -22* -5.9

+7.8* +4.2 +2.6

+13 -1.1 -55* -40* -51* -53* +19* +19* +11*

+3.6 -3.2 -70* -50* -63* -64* +13* +22* +17*

Abbreviations: - = finding absent, F = female, GD = gestation day, # not statistically evaluated, * significant trend (p≤0.05) a 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).

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 203

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5

Females (cont.) Water consumption (g/100g body weight/day and %)a GD 6 to 8 GD 8 to 10 GD 15 to 17 GD 17 to 19 GD 22 to 24

7.7 7.6 7.7 7.8 7.5

-6.5 -5.3 -3.9 -2.6 +9.3

-26* -9.2* -18* -21* +5.3

-44 -18* -40* -41* +12

No. Corpora Lutea 7.3 7.4 8.0 7.1 No. Implantations 6.7 6.9 7.4 6.5 % Pre-implantation Loss# 6.8 6.5 8.4 8.1

Abbreviations: F = female, GD = gestation day a 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). # not statistically evaluated, * significant trend (p≤0.05)

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 204

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Total Fetuses # 6.4 6.7 6.0 (6.3) 6.1

Live Fetuses 6.2 6.6 5.9 (6.2) 6.1 Dead Fetuses # 0.2 0.1 0.1 (0.1) 0.0 Resorptions # 0.3 0.2 1.5 (1.1) 0.4 Viable Fetuses (%) a# 93.3 95.4 79.5 (84.1) 95.0 Post Implantation Loss (%) a# 6.7 4.6 20.5 (15.9) 5.0 Avg. Fetal Body Weight (g) 40.64 40.45 38.30 40.98 Avg. Male Fetal Body Weight (g) 40.20 40.41 37.46 40.81 Avg. Female fetal body weight (g) 40.41 40.10 38.42 40.49 Avg. Fetal crown-rump length (mm) 96.9 97.6 95.5 97.6 Sex ratio of fetuses (% male) 50.4 50.8 49.6 56.8

a % of implantations # not statistically evaluated In brackets: values excluding females with total litter loss

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 205

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses

Fetal Gross External Anomalies

General General retarded MIN Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Head Cranium acephaly MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Forepaws/digits Paw hyperflexion MIN Litter Incidence Fetal Incidence

1/20

1/123

- -

2/17

4/106

1/19

3/116 Forepaw – digit – brachydactyly MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

1/19

1/116 1st digit – ectodactyly MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Trunk Thorax/abdomen – thoracogastroschisis MAL Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 206

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Visceral Anomalies Neck Trachea absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Arteries Innominate artery absent MIN Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Innominate artery short MIN Litter Incidence Fetal Incidence

4/20

5/123

4/18

5/119

6/17

12/106

2/19

4/116 Carotid artery absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Carotid artery malpositioned branch MIN Litter Incidence Fetal Incidence

7/20 12/123

6/18 9/119

7/17 10/106

4/19 5/116

Subclavian artery malpositioned branch MIN Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

1/19 1/116

Veins Anterior vena cava absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons; NE = not evaluable

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 207

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Visceral Anomalies (cont.) Great vessels Aorta malpositioned MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Aortic arch retroesophageal MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/19

1/116 Aortic arch retrotracheal MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/19

1/116 Ductus arteriosus absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Pulmonary artery branch malpositioned MAL Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Heart Heart microcardia MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Pericardium hypopericardium MIN Litter Incidence Fetal Incidence

- -

1/18

1/119

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 208

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Visceral Anomalies (cont.) Lung Lung small MAL Litter Incidence Fetal Incidence

- -

1/18

1/119

- -

- -

Lung anomalies MIN Litter Incidence Fetal Incidence

8/20

16/123

12/18

23/119

9/17

24/106

10/19

20/116 Diaphragm Diaphragm hernia MAL Litter Incidence Fetal Incidence

- -

1/18

1/119

- -

- -

Kidney Kidney malpositioned MIN Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Abdomen Gallbladder absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

1/19

1/116 Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 209

TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies Skull Skull hole small MIN Litter Incidence Fetal Incidence

- -

- (1/15 #DC)

- (1/102 #DC)

3/11# (1/6 DC)

3/58#* (1/48 DC)

3/19

3/116* Skull splitting of bone MIN Litter Incidence Fetal Incidence

2/20

2/123

- (1/15 #DC)

- (1/102 #DC)

- -

6/19

7/116 Skull sutural bone MIN Litter Incidence Fetal Incidence

1/20

2/123

1/3# (4/15 DC)

3/17#NE (8/102 DC)

1/11# (2/6 DC)

1/58# (5/48 DC)

2/19

2/116 Frontal additional suture MIN Litter Incidence Fetal Incidence

- -

1/3#

1/17#NE

- -

- -

Parietal additional suture MIN Litter Incidence Fetal Incidence

- -

- (1/15 #DC)

- (1/102 #DC)

3/11# (3/6 DC)

5/58# (4/48 DC)

2/19

2/116 Shoulder girdle Spina scapula misshapen MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Forepaws Metacarpal 1 absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Forepaw phalanx anomalies MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

1/19

1/116 Forepaw phalanx unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

- -

1/19 1/116

Forepaw 5th digit middle phalanx incomplete OSS Litter Incidence Fetal Incidence

- -

- -

- (1/6 #DC) - (1/48 #DC)

- -

Sternebrae Sternebrae anomalies MAL Litter Incidence Fetal Incidence

4/20

4/123

- (2/15 #DC)

- (2/102 #DC)

4/11# (4/6 DC)

5/58# (7/48 DC)

3/19

4/116 Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Sternebrae (cont.) Sternebrae unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

17/20 40/123

1/3# (11/15 DC) 1/17#NE (33/102 DC)

10/11# (5/6 DC) 26/58# (11/48 DC)

17/19 49/116

All sternebrae absent or rudimentary MAL Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Ribs Rib anomalies MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/19

1/116 Rib 13th rib supernumerary short or full MIN Litter Incidence Fetal Incidence

2/20 2/123

- -

2/11# 3/58#

3/19 4/116

At 7th cervical arch cervical short MIN Litter Incidence Fetal Incidence

- -

- (1/15 #DC)

- (4/102 #DC)

- (1/6 #DC)

- (1/48 #DC)

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Vertebral column Thoracic vertebrae scoliosis MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/19

1/116 Cervical vertebrae Cervical vertebrae arch. Anomalies MAL Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Thoracic vertebrae Thoracic vertebrae centrum anomalies MAL Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

1/19 1/116

Thoracic vertebrae centrum unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Thoracic vertebrae (cont.) Thoracic vertebrae arch. Anomalies MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/19 1/116

Lumbar vertebrae 5th lumbar centrum unilateral ossification OSS Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

6th lumbar centrum unilateral ossification Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

Sacral vertebrae 1st sacral centrum absent MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Sacral vertebrae (cont.) 2nd sacral centrum unilateral ossification OSS Litter Incidence Fetal Incidence

1/20 1/123

- -

- -

- -

All sacral arches misshapen MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Caudal vertebrae Caudal vertebrae centra anomalies MAL Litter Incidence Fetal Incidence

2/20 2/123

- (1/15 #DC) - (1/102 #DC)

1/11# 1/58#

1/19 1/116

Caudal vertebrae centra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

3/20 3/123

- (1/15 #DC) - (1/102 #DC)

- -

- -

Caudal vertebrae centra ossification of less than 15 OSS Litter Incidence Fetal Incidence

12/20 23/123

1/3# (10/15 DC) 2/17#NE (17/102 DC)

6/11# (5/6 DC) 14/58# (14/48 DC)

13/19 33/116

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters live fetuses (cont.)

Fetal Skeletal Anomalies (cont.) Pelvic girdle Pubis small MAL Litter Incidence Fetal Incidence

1/20

1/123

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Dead fetuses

Fetal Gross External Anomalies General General retarded dead Litter Incidence Fetal Incidence

2/3 with dead fetuses

2/3 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

- -

General markedly stunted, dead Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Skeletal Forepaws/Digits 1st digit ectrodactyly MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

4th digit ectrodactyly MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

5th digit ectrodactyly MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Dead fetuses

Skeletal (cont.) Skull Individual skull bones unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

1/3 with dead fetuses 1/3 dead fetuses

- -

- -

- -

Parietal additional suture MIN Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

- -

Forepaws Metacarpal 1 absent MAL Litter Incidence Fetal Incidence

- -

1/1 with dead fetuses

1/1 dead fetuses

- -

- -

Metacarpal 1 incomplete ossification OSS Litter Incidence Fetal Incidence

1/3 with dead fetuses 1/3 dead fetuses

- -

- -

- -

Metacarpal 2 absent MAL Litter Incidence Fetal Incidence

- -

1/1 with dead fetuses

1/1 dead fetuses

- -

- -

Metacarpal 3 absent MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Abbreviations: see next page

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Dead fetuses

Skeletal (cont.) Forepaws (cont.) Metacarpal 4 absent MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Metacarpal 5 absent MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Forepaw phalanx unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/1 with dead fetuses 1/1 dead fetuses

1/1 with dead fetuses 1/1 dead fetuses

- -

Sternebrae Sternebra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

1/3 with dead fetuses 1/3 dead fetuses

- -

- -

- -

Ribs All ribs multiple abnormalities MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Dead fetuses

Skeletal (cont.) Vertebral column All vertebrae multiple abnormalities MAL Litter Incidence Fetal Incidence

1/3 with dead fetuses 1/3 dead fetuses

- -

- -

- -

Caudal vertebrae Caudal vert. Centra ossification of less than 15 OSS Litter Incidence Fetal Incidence

2/3 with dead fetuses 2/3 dead fetuses

- -

1/1 with dead fetuses 1/1 dead fetuses

- -

Pelvic girdle Pubis incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/1 with dead fetuses

1/1 dead fetuses

- -

- -

Pubis unossified OSS Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

- -

1/1 with dead fetuses

1/1 dead fetuses

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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TS 2.6.7.13C - Reproductive and Developmental Toxicity - Effects on Embryo-fetal Development, Rabbits (2) (continued)

Test Article: Lixisenatide Report Title: Subcutaneous embryo-fetal toxicity study in rabbits (continued) Dose (µg/kg BID) 0 (Vehicle Control) 0.15 1 2.5 Litters Dead fetuses

Skeletal (cont.) Hindlimbs Talus unossified OSS Litter Incidence Fetal Incidence

1/3 with dead fetuses

1/3 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

1/1 with dead fetuses

1/1 dead fetuses

- -

Conclusions: NOAEL for maternal toxicity = 0.15 µg/kg BID; NOAEL for embryo-fetal toxicity = 2.5 µg/kg BID Remarks: # Skeletons of 15/18 litters at 0.15 µg/kg BID and 6/17 litters at 1 µg/kg BID were more or less decomposed due to inadvertently exaggerated clearing with potassium hydroxide. Parts of these skeletons (head, vertebral column including tail, sternebrae, shoulder and pelvic girdle including long bones of the fore and hind limbs) could be examined in all or almost all fetuses of these litters. Findings in these decomposed skeletons are included in brackets and described as DC=decomposed skeletons) Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significantly higher than control (p<0.05, Jackknife-t-test), DC = decomposed skeletons NE = not evaluable

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14 REPRODUCTIVE AND DEVELOPMENTAL TOXICITY - EFFECTS ON PRE- AND POST-NATAL DEVELOPMENT, INCLUDING MATERNAL FUNCTION

TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Species/Strain: Rat / Hsd: Sprague Dawley SD Duration of Dosing: GD6 until LD20 (F0 rats) Test Article: AVE0010 (batch xxx

xxxxxxxxxxx) Initial Age: 9 to 11 weeks Day of Mating: Day 0 Master Reference/Report No.: [DPN0327] Date of First Dose: xx xxxxxxxxx 20xx Method of Administration: subcutaneous BID (split by

approximately 8 hours) Location: 4.2.3.5.3-2

Special Features: none Vehicle/Formulation: 0.9% sodium chloride / aqueous solution GLP Compliance: yes Litters Culled/Not Culled: randomly 4M/4F pups per litter culled whenever possible

Day of Necropsy: F0: LD21-23 (delivering rats), GD25-GD27 (non delivering rats), F1: PND4 or PND21 (non selected pups), selected F: GD 14 (inseminated) or 14 days post cohabitation (non-inseminated), selected M: following GD14 caesarean section of F1 females

No Observed Effect Level: 2 µg/kg BID for pre-postnatal development of F1-rats, no NOEL defined for dams Dose (µg/kg BID) 0 (Control) 2 20 200 F0 Females

No. Pregnant 18 23 20 22 No Inseminated 24 24 24 24 No. Died or Sacrificed Moribund 0 0 0 0 No. Aborted 0 0 0 0 Clinical Observations:

-

Decreased motoractivity including sleepiness and decreased reactivity Piloerection

Decreased motoractivity including sleepiness and decreased reactivity Piloerection

Decreased motoractivity including sleepiness and decreased reactivity Piloerection

Necropsy Observations No compound-related findings Abbreviations: - = finding absent, GD: Gestation day, LD: Lactation day, PND: Post natal day, SD: standard error, TA: test article, - : no noteworthy findings, +: observation present Additional information: AVE0010 concentrations for the low dose formulation (formulation analysis) were below acceptance criteria (90 to 110 % of nominal concentration), based on study director conclusion the administered dose levels were most likely distinctly above 75% of the nominal dose; 75% of nominal dose corresponds to 1.5 µg/kg

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200

F0 Females

Mean body weight gains in gram per day (SD)

GD6-7 2.8 (4.91) -2.5* (4.98) -8.9* (3.43) -15.0* (6.07)

GD7-9 4.2 (1.59) 3.1 (1.74) 4.6 (2.16) 6.0* (2.16)

GD9-12 4.2 (1.79) 3.6 (1.35) 3.7 (1.52) 3.9 (1.33)

GD12-15 5.2 (1.61) 4.1 (2.18) 4.6 (2.46) 4.2 (1.69)

GD15-18 11.9 (4.41) 9.1* (3.53) 9.5* (2.10) 8.0* (2.94)

GD18-21 12.5 (3.82) 9.4 (3.34) 11.0 (4.34) 8.9* (3.97)

GD6-18 6.3 (1.68) 4.5* (1.46) 4.5* (0.95) 3.8* (0.92)

GD0-21 6.6 (1.39) 5.1* (1.32) 5.4* (1.13) 4.6* (1.03)

LD0-4 0.7 (3.14) 1.6 (2.99) 2.3 (2.95) 1.7 (1.91)

LD4-7 2.0 (2.78) 2.0 (3.16) 1.0 (1.63) 3.5 (2.41)

LD7-11 2.0 (3.38) 1.6 (1.85) 1.9 (2.08) 2.5 (1.97)

LD11-14 1.1 (3.17) 0.4 (2.53) 2.2 (2.43) 1.3 (3.50)

LD14-18 -1.3 (2.99) 0.2 (1.71) -0,6 (1.12) -0.5 (2.15)

LD18-21 -0.7 (3.07) -1.8 (2.14) -1.8 (1.71) -1.8 (1.79)

LD0-21 0.6 (0.44) 0.7 (0.45) 0.9* (0.59) 1.1* (0.36) Abbreviations: GD: Gestation day, LD: Lactation day, SD: standard deviation Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 F0 Females

Average daily food consumption in g/day (SD) GD6-7 20.1 (3.00) 14.5* (4.36) 9.2* (3.25) 5.4 * (3.39) GD7-9 20.5 (1.87) 15.7* (2.43) 15.6* (3.31) 13.7* (2.17) GD9-12 21.0 (2.59) 17.0 (1.90) 16.6* (2.10) 16.2* (1.72) GD12-15 20.5 (2.00) 17.4* (1.93) 17.6* (1.71) 17.0* (1.89) GD15-18 23.9 (2.83) 20.7* (2.93) 20.0* (1.91) 19.3* (1.72) GD18-21 24.7 (2.40) 21.8* (2.33) 21.8* (2.71) 21.0* (1.77) GD6-18 21.4 (2.04) 17.6* (1.88) 16.9* (1.72) 15.9* (1.38) GD0-21 20.5 (1.60) 17.9* (1.60) 17.7* (1.55) 17.0* (1.17) LD0-4 28.1 (7.43) 24.8 (8.38) 27.5 (5.93) 24.7 (5.67) LD4-7 40.8 (9.08) 37.6 (10.79) 36.9 (7.56) 36.3 (7.40) LD7-11 48.3 (11.22) 42.7* (10.95) 43.5* (9.42) 42.3* (8.83) LD11-14 54.2 (13.80) 48.6* (12.68) 49.8* (11.07) 47.4* (11.21) LD14-18 56.1 (16.13) 52.8 (14.56) 53.1* (11.65) 51.3* (13.00) LD18-21 67.8 (17.36) 62.8 (19.23) 64.3 (15.35) 62.5 (17.44) LD0-21 48.5 (11.82) 44.2 (11.96) 45.2* (9.79) 43.4* (10.14) Mean Duration of Gestation (days) 22.2 22.6 22.4 22.6 No. with live pups 18 22 17 21 No. with total litter loss 0 1 3 1 Gestation Index (%) 100 95.6 85 95.5 Pregnancy index (%) 75 95.8 83.3 91.7 Implantations (mean ± SD) 11.8 ± 5.08 10.0 ± 4.48 11.7 ± 4.58 9.5 ± 4.51

Abbreviations: GD = Gestation day, LD: Lactation day, SD: standard deviation, Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 F1 Litters No. Litters Evaluated 18 22 17 21 (Preweaning) Mean No. of Implantations 11.8 10.0 11.7 9.5 Live pups PND0 180 171 174 148 Dead pups 4 10 3 12 Viability index at PND4 (pre-culling) 98.0 90.3 99.1 91.5 Viability index at PND21 (weaning) 100.0 100.0 98.8 95.0 Viable pups PND4 (pre-culling) 177 164 172 143 PND4 (culling) 122 124 117 119 PND21 122 124 116 117 Body Weight Change (g) PND4-7 Males and females (combined) 1.97 1.91 1.82 1.81 Males 2.12 1.97 1.85* 1.90* Females 1.91 1.91 1.80 1.75 Body Weight Change (g) PND7-11 Males and females (combined) 2.14 1.91 2.04 1.99 Males 2.33 1.99* 2.05* 2.00* Females 2.04 1.88 2.04 1.98 Abbreviations: GD = Gestation day, PND = Postnatal day Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 F1 Litters (Preweaning)

Body Weight Change (g) PND11-14 Males and females (combined) 2.21 2.05 2.04 1.95

Males 2.33 2.09 2.13 2.05 Females 2.14 2.00 1.95 1.85* Body Weight Change (g) PND14-21 Males and females (combined) 2.89 3.08 2.95 2.97 Males 3.03 3.19 3.05 3.15 Females 2.81 2.96 2.82 2.85 Body Weight Change (g) PND0-21 Males and females (combined) 2.16 2.15 2.11 2.12 Males 2.29 2.22 2.17 2.21 Females 2.09 2.10 2.05 2.05 Live birth index 98.6 88.9 97.2 89.8 Sex ratios (% males) 49.2 59.0 44.8 49.7

Clinical signs** Insufficient suckling (9) Bent tail (1 pup)

Insufficient suckling (13), not suckled (1 pup once) Each 1 pup: Tail shortened, part of tail missing, tail tip necrotic

Increased incidence of insufficient suckling (31)

Increased incidence of insufficient suckling (16) Tail deformed (1), necrotic tail tip (2) shortened tail (1), wavy (9) or bent (1) tail

Abbreviations: PND: Postnatal day Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test), ** pup incidence in brackets

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 F1 Litters (Preweaning)

Visceral examinations % of pup incidence 32 (59/184) 28 (50/181) 33 (59/177) 22 (37/160) Post implantation loss 0.2 0.3 0.1 0.3

Post implantation survival index 11.6 9.7 11.6 9.2 Pups delivered 184 181 177 160 Dead pups 4 10 3 12 Pups found dead PND0 to PND4 3 7 2 5 Eye opening median day 1st observed Males 14.6 14.4 14.7 14.5 Females 14.5 14.5 14.5 14.4 Pupil constriction % responding on test day PND21 Males 63/63 72/72 58/58 59/59 Females 59/59 52/52 58/58 58/58 Pinna separation median Day 1st observed Males 1.8 1.8 1.6 1.6 Females 1.8 1.8 1.6 1.6 Coat growth start median Day 1st observed Males 3.8 4.1 4.6* 4.5* Females 3.8 3.9 4.6* 4.5* Incisor eruption median Day 1st observed Males 9.5 9.6 10.1 9.6 Females 9.4 9.9 10.0 9.6 Abbreviations: PND: Postnatal day Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 Examination of F1 generation Generation Clinical signs after weaning No relevant signs none none none (After weaning) Mortality after weaning 0 0 0 1# Sensitive evaluation and behavioral testing Vaginal opening (median) day achieved 37.2 40.3 34.5 34.3* Body weight at achievement (g) 118.5 117.5 111.9 111.6 Balanopreputial separation (median) day achieved 34.8 34.8 34.8 34.2* Body weight at achievement (g) 143.4 131.8 132.3 126.7 Auditory startle overall responses PND 21 Males 63/63 72/72 58/58 59/59 Females 59/59 52/52 58/58 58/58 Righting reflex PND21 Males 63/63 72/72 58/58 59/59 Females 59/59 52/52 58/58 58/58

Water maze test (group means of success among 5 passages per rat)

Males learning 3.9 3.8 4.0 3.6 Females learning 4.0 3.4 3.5 4.0 Males re-learning 2.8 2.4 2.8 2.7 Females re-learning 2.1 2.8 2.9 2.8 Abbreviations: PND: Postnatal day, g: grams Additional Information: * significant trend (p≤0.05) through indicated dose level (step-down trend test), # accidental death

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 F1 Males Generation Body weight changes (g) (After weaning) PND0-7 4.8 5.1 4.9 4.9 PND7-14 6.8 6.4 7.0 6.8

PND14-21 6.5 6.9 6.3 6.8 PND21-28 5.0 5.5 6.9* 6.3*

PND28-35 8.8 7.4* 7.0* 7.4* PND35-42 5.9 5.3 5.5 4.9* PND42-49 4.3 3.7* 3.8* 3.7*

PND49-56 3.6 2.8* 2.6* 2.1* PND56-63 2.0 2.0 2.4 2.3

PND77-84 1.6 1.9 2.0 1.3

Average daily food consumption (g/day) PND21-28 17.7 17.6 19.8 18.7

PND28-35 24.4 23.1 23.3 23.5 PND35-42 25.6 24.6 25.2 25.0

PND42-49 25.5 24.3 25.0 24.5 PND49-56 30.4 23.5 24.8 26.0 PND56-63 25.3 30.3 24.5 21.6

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 F1 Females Generation Body weight changes (g) (After weaning) PND0-7 4.0 4.0 4.2 3.8 PND7-14 5.1 5.1 5.2 5.3

PND14-21 4.3 4.5 4.0 4.0 PND21-28 2.4 3.3* 3.7* 3.4*

PND28-35 4.0 3.1 3.4 3.1 PND49-56 2.2 1.7 1.4 0.3* GD0-4 4.6 4.7 4.7 5.0

GD4-8 3.2 3.8 3.2 3.7 GD8-11 4.9 5.0 5.4 4.9

GD11-14 4.1 5.2* 5.4* 5.4* Abbreviations: PND: Postnatal day, GD: Gestation day Additional Information: Weaning was performed on Day 21 * significant trend (p≤0.05) through indicated dose level (step-down trend test), # no significant body weight changes from PND77 in males and between PND49/PND69 in females

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 F1 Females Generation Average daily food consumption (g/day) (After weaning) PND21-28 13.9 14.9 15.6 14.9 PND28-35 16.8 16.3 16.9 16.5

PND35-42 17.3 18.3 18.4 17.7 PND42-49 17.5 17.7 17.6 17.8

PND49-56 18.5 17.8 18.6 17.7 PND56-63 18.2 18.3 18.6 18.9 GD0-4 19.3 19.5 19.4 20.1

GD4-8 20.0 20.5 20.4 20.5 GD8-11 20.6 20.9 21.7 21.4

GD11-14 21.3 21.8 22.0 22.1 Abbreviations: PND: Postnatal day, GD: Gestation day Additional Information: Weaning was performed on Day 21 * significant trend (p≤0.05) through indicated dose level (step-down trend test), # no significant body weight changes from PND77 in males and between PND49/PND69 in females

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 F1 Generation F1 Generation Males Reproductive Indices

Mating index % 100 95.8 100 100

Fertility index % 95.5 79.2 87.5 91.3

Pregnancy index % 95.5 82.6 87.5 91.3

F1 Generation females assigned to reproductive assessment

No. evaluated 22 24 24 23

Average times in estrus Cyclicity of females was not impaired in all groups as evidenced by the females’ readiness to mate

No. of days to inseminate 6.7 4.7 6.0 4.3

No. females placed into cohabitation 22 24 24 23

Mating index 100 95.8 100 100

Pregnancy index 95.5 86.4 87.5 91.3

Fertility index 95.5 79.2 87.5 91.3

No. with viable litter 21 18 21 21

No. with total litter loss 0 1 0 0

No. Corpora lutea 17.5 19.0 17.9 18

No. implantations 15.0 16.8 16.6 16.5

Pre-implantation loss 2.6 2.2 1.3 1.6

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters No. evaluated 21 18 21 21

Total embryos per litter 13.9 16.2 15.7 15.9

Live 13.9 16.2 15.7 15.9

Dead 0 0 0 0

Resorptions 1.0 0.7 0.9 0.6

Females with > 1 resorption 12 11 11 8

Post-implantation loss 1.0 0.7 0.9 0.6

Conclusions: NOEL = 2 µg/kg for pre-postnatal effects (F1 generation)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 live fetuses (pups)

Fetal visceral Anomalies

Abdomen Stomach anomalies MIN Litter Incidence Fetal Incidence

- -

- -

1/12 1/55

- -

Stomach malpositioned MAL Litter Incidence Fetal Incidence

- -

- -

1/12 1/55

- -

Urogenital system Bladder distended MIN Litter Incidence Fetal Incidence

1/12 3/55

- -

- -

1/10 1/24

Fetal Skeletal Anomalies Skull Sutural bone MIN Litter Incidence Fetal Incidence

1/12 1/55

1/12 1/40

- -

3/10 3/24

Forepaws Phalanx incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

- -

1/10 1/24

Sternebrae Sternebrae unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

1/12 1/55

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 live fetuses (pups)

Fetal skeletal Anomalies (continued)

Ribs At 7th cervical arch cervical ,full MAL Litter Incidence Fetal Incidence

- -

1/12 1/40

- -

- -

At 7th cervical arch cervical, short MIN Litter Incidence Fetal Incidence

1/12 2/55

- -

- -

- -

Rib anomalies MAL Litter Incidence Fetal Incidence

- -

1/12 1/40

- -

- -

Rib 14th supernumerary short or full MIN Litter Incidence Fetal Incidence

3/12 5/55

4/12 4/40

- -

1/10 1/24

Thoracic vertebrae Thoracic vertebrae anomalies MAL Litter Incidence Fetal Incidence

- -

1/12 1/40

- -

- -

Caudal vertebrae Caudal vertebrae centra ossification of less than 15 OSS Litter Incidence Fetal Incidence

2/12 2/55

- -

1/12 1/55

1/10 1/24

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 live fetuses (pups)

Fetal skeletal Anomalies (continued)

Hindlimbs Talus unossified OSS Litter Incidence Fetal Incidence

- -

- -

1/12 1/55

- -

Hindpaws Phalanx incomplete ossification OSS Litter Incidence Fetal Incidence

1/12 1/55

- -

1/12 1/55

- -

Litters F1 dead fetuses (pups)

Fetal visceral Anomalies

General General incomplete examination due to autolysis Litter Incidence Fetal Incidence

1/2 1/4

2/7 2/10

2/3 2/4

6/8 9/13

Urogenital system Bladder distended MIN Litter Incidence Fetal Incidence

1/2 1/4

- -

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 dead fetuses (pups)

Fetal skeletal Anomalies Shoulder girdle Spina scapula bent MIN Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Scapula bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Scapula short MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/8

1/13 Clavicle bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Forelimbs Humerus bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Ulna bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Radius bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 dead fetuses (pups)

Fetal skeletal Anomalies (continued)

Forepaws Phalanx incomplete ossification OSS Litter Incidence Fetal Incidence

2/2 4/4

5/7

8/10

3/3 3/4

8/8

12/13 Sternebrae Sternebra unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

- -

- -

2/8 2/13

Ribs Ribs anomalies MAL Litter Incidence Fetal Incidence

- -

1/7

1/10

- -

- -

Ribs wavy and/or thickened MIN Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Rib 14th rib supernumerary short or full MIN

Litter Incidence Fetal Incidence

- -

- -

- -

1/8 1/13

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 dead fetuses (pups)

Fetal skeletal Anomalies (continued)

Thoracic vertebrae Thoracic vertebrae cent. unossified or incomplete ossification OSS Litter Incidence Fetal Incidence

- -

1/7 1/10

- -

- -

Thoracic vertebrae anomalies MAL Litter Incidence Fetal Incidence

- -

1/7

1/10

- -

- -

Lumbar vertebrae All lumbar vertebrae absent or rudimentary MAL Litter Incidence Fetal Incidence

- -

1/7 1/10

- -

- -

Sacral vertebrae All sacral vertebrae absent or rudimentary MAL Litter Incidence Fetal Incidence

- -

1/7 1/10

- -

- -

Caudal vertebrae Caudal vertebrae centra ossification of less than 15 OSS Litter Incidence Fetal Incidence

2/2 4/4

7/7 9/10

3/3 4/4

8/8 12/13

Caudal vertebrae anomalies MAL Litter Incidence Fetal Incidence

- -

1/7

1/10

- -

- -

Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding, * significant higher than control, Jackknife-t-test (p<0.05)

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TS 2.6.7.14 - Reproductive and Developmental Toxicity - Effects on Pre- and Post-natal, Development, Including Maternal Function (continued)

Test Article: Lixisenatide Report Title: AVE0010: Subcutaneous pre- and postnatal developmental toxicity study in rats Dose (µg/kg BID) 0 (Control) 2 20 200 Litters F1 dead fetuses (pups)

Fetal skeletal Anomalies (continued)

Pelvic girdle Ischium misshapen MAL Litter Incidence Fetal Incidence

- -

- -

- -

1/8

1/13 Hindlimbs Femur bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Tibia bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Fibula bent MAL Litter Incidence Fetal Incidence

- -

- -

- -

2/8

2/13 Calcaneus unossified OSS Litter Incidence Fetal Incidence

- -

1/7

1/10

1/3 1/4

- -

Talus unossified OSS Litter Incidence Fetal Incidence

2/2 4/4

7/7

10/10

3/3 4/4

8/8

12/13 Hindpaws Phalanx incomplete ossification OSS Litter Incidence Fetal Incidence

2/2 4/4

7/7

10/10

3/3 4/4

8/8

12/13 Abbreviations: - = finding absent, MAL = Malformation, MIN = Minor anomaly, OSS = Ossification finding

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15 STUDIES IN JUVENILE ANIMALS TS 2.6.7.15A – Studies in juvenile animals – Exploratory study in male juvenile dogs

Test Article: Lixisenatide Report Title: AVE0010 - Exploratory 2-week twice a day (BID) subcutaneous pharmacokinetic study in juvenile male dogs Species/Strain: Dog / Marshall Beagle Duration of Dosing: 2 weeks Test Article: AVE0010 (batch xxx)

Initial Age: 18 to 19 weeks Duration of Post-Dose: 2 weeks Study No.: [DIV1328]

Date of First Dose: xx xxxxx 20xx Method of Administration: Subcutaneous Location : 4.2.3.5.4-1 Vehicle/Formulation: Saline (0.9% NaCl) /aqueous solution / BID dosing with an interval of approximately 8 hours GLP Compliance: No Special Features: No No Observed Adverse Effect Level: Not applicable Dose (µg/kg BID) 0 (Vehicle Control) 5 20 200 Number of Animals on Study (Main Study) M: 3 M: 3 M: 3 M: 3 Toxicokinetics: Sampling on Day 1 and 14 AUC0-24 (ng.h/mL ) – Day 14 - 31500 136000 755000 Cmax (ng/mL) – Day 14 - 3820 17000 84400 Anti-drug antibody formation: Day – 4 and Day 14 Detection of antibodies on Day 14, incidence 0/3 0/3 1/3 1/3 Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, AUC = area under the curve, Cmax = maximum concentration

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TS 2.6.7.15A – Studies in juvenile animals – Exploratory study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – Exploratory 2-week twice a day (BID) subcutaneous pharmacokinetic study in juvenile male dogs (continued)

Dose (µg/kg BID) 0 (Vehicle Control) 5 20 200

Number of Animals on Study (Main Study) M: 3 M: 3 M: 3 M: 3 Noteworthy Findings: Wet food offered at 20 and 200 µg BID due to low pellet consumption Died or Killed Moribund 0 0 0 0 Group mean Body Weight Gain (kg) per group Week 1 Week 2

0.37 0.40

0.03 0.14

-0.30 0.07*

-0.93* 0.23*

Group mean Body Weight Gain (kg per week and %)a Week 1 Week 2

0.37 0.40

-92 -65

N/A -82

N/A -42

Food Consumption (g) Pellets Wet Food Total

13555

- 13555

9527

- 9527

3679

11541 15220

2707

11681 14388

Abbreviations: M = male; N/A: not appropriate a For controls, group means are shown. For treated groups, percent differences from controls are shown. * p<0.05

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TS 2.6.7.15A – Studies in juvenile animals – Exploratory study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – Exploratory 2-week twice a day (BID) subcutaneous pharmacokinetic study in juvenile male dogs (continued)

Dose (µg/kg/BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 M: 3 M: 3 M: 3 Clinical Observations

Mucoid / soft feces Liquid feces, mild Liquid feces, moderate Feces discolored red Salivation, mild Salivation, marked Vomiting, mild Vomiting, moderate Vomiting, foamy Redness (oral mucosa, ears, eyes) Vocalization Reduced skin elasticity Absent food intake Paleness of oral mucosa Activity decreased, slight Activity decreased, moderate Weakness Emaciated Increased resistance to dosing Skin: scab Administration site: scratching Trembling Twitching Cold to touch Uncoordinated gait Recumbent posture

3

3

1

1

3 3 1

3 3

2 1

1

3 1 1 2 1 1

3 2 2 2 1 1 1 1 1 2 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1

Abbreviations: M = male

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TS 2.6.7.15A – Studies in juvenile animals – Exploratory study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – Exploratory 2-week twice a day (BID) subcutaneous pharmacokinetic study in juvenile male dogs (continued)

Dose (µg/kg/BID) 0 (Vehicle Control) 10 40 200

Number of Animals on Study (Main Study) M: 3 M: 3 M: 3 M: 3 Hematology (Week 2) No treatment-related findings Coagulation (Week 2) No treatment-related findings Serum Chemistry (Week 2) No treatment-related findings Additional Information: none Abbreviations: M = male

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs

Test Article: Lixisenatide Report Title: AVE0010 - 8 Month twice a day (BID) subcutaneous toxicity study in juvenile male dogs with a 2-month recovery period Species/Strain: Dog / Marshall Beagle Duration of Dosing: 8 months Test Article: AVE0010 (batch xxxx)

Initial Age: 16 to 17 weeks Duration of Post-Dose: 2 months Study No.: [TXC1462]

Date of First Dose: xx xxxxxx 20xx Method of Administration: Subcutaneous Location : 4.2.3.5.4-2 Vehicle/Formulation: Saline (0.9% NaCl) / aqueous solution / BID dosing with an interval of approximately 8 hours GLP Compliance: Yes Special Features: Pair-fed vehicle control No Observed Adverse Effect Level: 5 µg/kg BID

Dose (µg/kg/d) 0 (Vehicle Control)

0 (pair-fed vehicle control)

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Toxicokinetics: Sampling on Day 7, 91 and 245 AUC0-24 (ng.h/mL ) Day 7 - - 29.9 125 1050 522

Day 91 - - 40.5 121 4070 1420 Day 245 - - 49.4 2360 32500 4660

Cmax (ng/mL) Day 7 - - 3.31 14.1 76.8 59.3 Day 91 - - 4.97 13.4 228 115 Day 245 - - 5.48 120 1580 303

ADA-positive dogs Day 85 - 1 1 1 3 4 Day 245 - 1 1 3 4 6

Additional Information: Values are rounded to 3 significant figures or less. Abbreviations: M = male, AUC = area under the curve, Cmax = maximum concentration, ADA=anti-lixisenatide antibodies, a twice daily with doses of 5, 20 and 200 µ/kg, respectively per application; b

application once daily

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (cont.)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control)

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Noteworthy Findings: Wet food offered at 20 and 200 µg BID due to low pellet consumption Died or Killed Moribund 0 0 0 0 0 0 Absolute body weight (kg and %) c Day 1 Day 29

Day 92 Day 183 Day 245 Day 301/303 d

5.43 6.97 10.1 11.1 11.3 11.7

0

-10 -7 -3 -2 0

0 -4 -8 -5 -6 -

-1 -9 -9 -6 -3 -

-1 -9 -8 -3 0 -1

-2 -8 -8 -5 -3 -4

Group mean Body Weight Gain (kg per time period and %) c Day 1 to 29 Day 29 to 92

Day 92 to 183 Day 183 to 245 Day 245 to 301/303 d

1.54 3.13 1.0 0.17 0.43

-45 0

+32 +65 +67

-19 -17 +27 -53

-

-38 -11 +33

+147 -

-38 -5

+48 +194 -37

-31 -7

+19 +165 -46

Average Weekly Food Consumption (%) Week 1 Week 4 Week 12 Week 26 Week 36 Week 44

95.6 99.9

100.0 100.0 100.0 100.0

NA NA NA NA NA NA

77.0 98.1 89.2 99.3

100.0 -

63.7 93.2 91.0 97.9 92.5

-

52.3 91.7 98.4

100.0 100.0 100.0

63.0 94.0 94.9 99.6 99.7

100.0 Abbreviations: M = male; NA not applicable as pair-fed control received the same amount which was fed by the 400 µ/g/day group, this amount was consumed by approximately 100% a twice daily with doses of 5, 20 and 200 µ/kg, respectively per application; b application once daily c For vehicle control, group means are shown. For pair-fed vehicle control and treated groups, percent differences from vehicle control are shown. d day 301 for vehicle and pair-fed vehicle control, Day 303 for 400 and 200 µg/kg/day * significant trend (p≤0.05), statistics only performed for absolute weight

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued) Test Article: Lixisenatide

Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (cont.)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control)

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Clinical Observations: number of affected dogs

Soft feces Liquid feces, mild Liquid feces, moderate Liquid feces, marked Feces discolored red Salivation, mild Vomiting, mild Vomiting, moderate Vomiting, marked Vomiting, red/brown Redness (oral mucosa, ears, eyes, skin, genital area)

Vocalization

2 - - - 1 - 1 - - - 2 5

3 - - - 1 1 1 - - - 3 3

2 - - - 1 - 3 - - - 2 3

1 - 1 - 4 1 2 2 - - 3 4

5 6 1 1 5 - 5 1 - 1 5 6

- 6 1 - 1 - 5 2 1 1 3 5

Abbreviations: M = male

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued) Test Article: Lixisenatide

Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (cont.)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control)

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Clinical Observations (continued): number of affected dogs

Reduced skin elasticity Absent food intake Emaciated Paleness of oral mucosa Activity decreased, mild Activity decreased, moderate Trembling Twitching Warm to touch Uncoordinated gait Skin: scab/ulceration Skin: nodule Ulceration oral mucosa Hematoma Administration site: scratching Administration site: induration/swelling Administration site: scab Heart rate increased Watery discharge, eye Hair loss, hindlimbs Red material in cage

- - - 1 - - - - - - - - - - - 1 2 - - - -

- - - - 1 - - - - - 1 - - - - 2 1 1 - - -

- - - - - - - - - - - - - - - 2 1 - - - -

- - - - - - - - - - - - - - - 3 - - 1 1 -

6 1 4 1 6 2 3 1 2 2 3 - 1 1 2 5 1 - 1 - 1

1 - - - 1 - - - - - 1 1 - - - 4 2 - - - -

Abbreviations: M = male

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (continued)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Ophthalmoscopy (week 13 and 34) No treatment-related findings Electrocardiography (week 12 and 34, end of recovery) No treatment-related findings Testicular volume No treatment-related findings Hematology (Day 85, 245, 300-302) Red cell distribution width (%) Day 85 13.4 13.4 13.9* 14.0* 14.8* 14.1

Day 245 12.8 12.8 12.9 13.2* 13.6* 12.9 Day 300-302 12.3 12.9 - - 13.4 12.4

Reticulocyte count (1012/L) Day 85 0.0527 0.0588 0.0423 0.0583 0.1105* 0.0550 Day 245 0.0338 0.0393 0.0333 0.383 0.0612 0.0240* Day 300-302 0.0275 0.0700 - - 0.0685 0.0175

Platelet count (109/L) Day 85 298.2 302.7 373.8 350.3 456.5* 407.2* Day 245 265.5 276.5 302.3 350.0* 419.2* 341.0* Day 300-302 207.0 231.5 - - 305.5 279

Coagulation (Day 85, 245, 300-302) No treatment-related findings Serum Chemistry (Day 85, 245, 300-302) Triglyceride (mmol/L) Day 85 0.413 0.432 0.500 0.445 0.457 0.438

Day 245 0.257 0.317* 0.390* 0.343* 0.408* 0.368* Phosphate (mmol/L) Day 245 1.260 1.375 1.745 1.573 1.553 1.507* Urinalysis (Day 85, 245, 300-302) No treatment-related findings Additional Information: none Abbreviations: M = male * significantly different (p≤0.05) from vehicle control

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (continued)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Organ weights No treatment-related findings Gross Pathology animals at end of treatment/recovery animals Injection site: Focus/area, red 1/- -/- 1 2 2/- 2/-

Texture, gelatinous -/- -/- - - 2/- 4/- Histopathology (LM) Testis

Tubular dilation Grade 0 4/2 4/2 3 1 1/2 1/2 Grade 1 - - 1 1 - 1/- Grade 2 - - - 1 2/- 2/- Grade 3 - - - 1 1/- -

Tubular vacuolation Grade 0 - - - - -/1 - Grade 1 4/2 4/2 3 1 1/1 2/2 Grade 2 - - 1 2 2/- 2/- Grade 3 - - - 1 1/- -

Sperm stasis Grade 0 3/2 4/1 4 3 3/1 1/1 Grade 1 1/- -/1 - 1 1/1 3/1

Abbreviations: M = male a highest grade for each animal recorded

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.15B – Studies in juvenile animals – 8-Month Toxicity Study in juvenile dogs (continued)

Test Article: Lixisenatide Report Title: AVE0010 – 8 Month twice a day (BID) subcutaneous toxicology study in juvenile male dogs with a 2-month recovery period (continued)

Dose (µg/kg/day) 0 (Vehicle Control)

0 (pair-fed vehicle control

10a 40 a 400 a 200b

Number of Animals on Study (Main Study) M: 4+2 M: 4+2 M: 4 M: 4 M: 4+2 M: 4+2 Histopathology (LM) (continued) Epididymis

Dilation of initial segment Grade 0 4/2 4/2 4 4 2/2 1/2 Grade 1 - - - - 1/- - Grade 2 - - - - 1/- 3/-

Degeneration of initial segment Grade 0 4/2 4/2 4 4 3/2 1/2 Grade 1 - - - - - 2/- Grade 2 - - - - 1/- 1/-

Oligospermia Grade 0 4/2 4/2 4 4 3/2 3/2 Grade 2 - - - - - 1/- Grade 3 - - - -- 1/- -

Injection site (right and left)a Mixed cellular infiltrate Grade 0 2/1 2/1 2 1 -/1 -/1

Grade 1 2/1 2/1 1 - -/1 -/1 Grade 2 - - 1 1 - 1/- Grade 3 - - - 2 4/- 3/-

Fibrosis Grade 0 3/2 2/2 3 1 -/1 1/1 Grade 1 - 1/- - 1 - - Grade 2 - - - - -/1 -/1 Grade 3 1/- 1/- - 1 1/- 1/- Grade 4 - - 1 1 3/- 2/-

Bone mineral density evaluation No treatment-related finding Femur length evaluation No treatment-related finding Additional Information: none Abbreviations: M = male, a highest grade for each animal recorded

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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16 LOCAL TOLERANCE TS 2.6.7.16A - Local Tolerance AVE0010 batch xxxxxxxx

Test Article: Lixisenatide Report Title: AVE0010: Local intravenous, intra-arterial, paravenous, subcutaneous and intramuscular tolerance study in rabbits

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, IV, PV, IA SC, IM, IV, IA: 0.5 mL/site

PV: 0.1 mL/site

4F (SC+IV)

4F (IM+PV)

4F (IA)

Mortality: none Clinical signs: none except short twitching of 2F at injection (1F treated IV+SC, 1F treated IM+PV) Body weight: Reduction Day 1 to 2 (average –5%) Macroscopy and microscopy: SC: moderate tolerability Contrary to controls minimal to mild necrosis and fibroblastic reactions and increased infiltration with mixed inflammatory cells.

IM: poor tolerability (intolerability)

Compared to controls, obviously increased necrosis with mineralization and infliltration of mixed inflammatory cells.

[20 x-0519] 4.2.3.6-1

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intraveneous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.16A - Local Tolerance AVE0010 batch xxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010: Local intravenous, intra-arterial, paravenous, subcutaneous and intramuscular tolerance study in rabbits (cont.)

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, IV, PV, IA SC, IM, IV, IA: 0.5 mL/site

PV: 0.1 mL/site

4F (SC+IV)

4F (IM+PV)

4F (IA)

IV: good tolerability

Only slight difference between controls and AVE0010 treated sides. From the latter 2/4 sides showed mild fibroblastic reaction.

PV: poor tolerability (intolerability)

Contrary to controls, vascular necrosis in 4/4 AVE0010-treated sides with infiltration with mixed inflammatory cells.

IA: limited tolerability

Contrary to controls, moderate to marked insudation of the periarterial connective tissue with infiltration of mixed inflammatory cells

[20 x-0519] continued

4.2.3.6-1

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intraveneous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.16B - Local Tolerance AVE0010 batch xxxxxxxx

Test Article: Lixisenatide Report Title: AVE0010 (BR477/38): Local subcutaneous, intramuscular and paravenous tolerance study in rabbits

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, PV SC, IM: 0.5 mL/site

PV: 0.1 mL/site

4F SC

4F (IM+PV)

Mortality: none Clinical signs: none Body weight: Reduction Day 1 to 2 (average –4%) Macroscopy and microscopy: SC: good Tolerability

control site: NAD; AVE0010 site: hemorrhage (2F) 1/4 AVE0010 treated sides showed a slight necrosis in the panniculus muscle at 24 h. After 120 h regeneration was seen IM: good tolerability control site hemorrhage (1F), visible injection canal (1F); AVE0010 site: hemorrhage (2F), visible injection track (2F) Compared to controls, muscular necrosis was minimally higher.

[20 xx-0771] + Amendment [20 xx-0771-amend01]

4.2.3.6-2 4.2.3.6-2a

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intravenous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.16 B - Local Tolerance AVE0010 batch xxxxxxxx (continued)

Test Article: Lixisenatide Report Title: AVE0010 (BR477/38): Local subcutaneous, intramuscular and paravenous tolerance study in rabbits

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, PV SC, IM: 0.5 mL/site

PV: 0.1 mL/site

4F SC

4F (IM+PV)

PV: poor tolerability control site: visible injection site (2F), AVE0010 site: minimally reddened injection area (3F), hemorrhage (1F) Contrary to controls, vascular necrosis with insudation of perivascular tissue and infiltration of mixed inflammatory cell infiltration after 24 h. Fibroblastic reaction after 120 h.

[20xx-0771] + Amendment [20 xx-0771-

amend01]

continued

4.2.3.6-2 4.2.3.6-2a

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intravenous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.16C - Local Tolerance Placebo of AVE0010

Test Article: Placebo of Lixisenatide Report Title: Placebo of AVE0010: Local intravenous, intra-arterial, paravenous, subcutaneous and intramuscular tolerance study in rabbits

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, IV, PV, IA SC, IM, IV, IA: 0.5 mL/site

PV: 0.1 mL/site

4F (SC+IV)

4F (IM+PV)

4F (IA)

Mortality: none Clinical signs: none except short twitching at IV and IA injection (all F) Body weight: PV, IM: slight loss Day 1 to 2 (1F: -3%) Macroscopy and microscopy: SC: moderate tolerability Compared to controls increased necrosis and infiltration with mixed inflammatory cells and presence of mild fibroblastic reactions. IM: intolerability Compared to controls, obviously increased muscular necrosis and infliltration with mixed inflammatory cells.

[20 xx-0327] 4.2.3.6-3

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intravenous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.16C - Local Tolerance Placebo of AVE0010 (continued)

Test Article: Placebo of Lixisenatide Report Title: Placebo of AVE0010: Local intravenous, intra-arterial, paravenous, subcutaneous and intramuscular tolerance study in rabbits

Species/Strain or In Vitro Assay

Method of Administration

Doses (mg/kg/day)

or Concentration

Gender and No. per Group

Noteworthy Findings Study Number

Location

Rabbit/New Zealand White SC, IM, IV, PV, IA SC, IM, IV, IA: 0.5 mL/site

PV: 0.1 mL/site

4F (SC+IV)

4F (IM+PV)

4F (IA)

IV: good tolerability Difference to controls (necrosis in 1/4) is considered to be iatrogenic (injection procedure). PV: poor tolerability (intolerability) Contrary to controls, necrosis in 4/4 AVE0010-treated sides with perivenous insudation and infliltration of mixed inflammatory cells. IA: limited tolerability Compared to controls, obviously increased insudation of the periarterial connective tissue.

[20 xx-0327] continued

4.2.3.6-3

Abbreviations: F female, SC subcutaneous, IM intramuscular, IV intravenous, PV paravenous, IA intraarterial

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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17 OTHER TOXICITY STUDIES TS 2.6.7.17A - Other Toxicity Studies – Antigenicity – B-cell response

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses (µg/kg)

Gender and No. per Group

Noteworthy Findings Study Number

Location

B-cell response on Day 0, 35 and 63 after start of dosing; plasma analyses by means of ELISA (ELISA plates coated by 1 µg peptide), reference: rabbit reference antiserum obtained by immunisation with peptide very closely related to AVE0010 (only difference extra-xxx at position xx)

Mice / BALB/cJ

Oral

SC

5 times

each administration separated by a

period of 2 weeks

19950

1.995

10F

10F

Oral: none of the mice showed any noteworthy antibody titer SC: 2/10 mice showed anti-AVE0010 antibodies in plasma on Day 63

[DIV1155] 4.2.3.7.1-1

Abbreviations: F female

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TS 2.6.7.17B - Other Toxicity Studies – Antigenicity – T-cell response

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses Gender and No.

per Group Noteworthy Findings Study

Number Location

T-cell proliferation measured in cells gained from draining of poplietal and inguinal lymph nodes primed with AVE0010. [3H]thymidine incorporation determined after incubation (re-stimulation) of lymphocytes with AVE0010, GLP-1 and PBS/tuberculin purified peptide derivative as negative/positive control

Mice / BALB/cJ

Injection in hindpaw pads and base of tail

single In-vivo priming

65 nmol peptide/mouse

(approx. 324 µg/ mouse)

65 nmol peptide/animal + 100 µL FCA

In vitro re-stimulation

0.06 to 136 µM

9F

5F

AVE0010 (and GLP-1) did not stimulate T-cell proliferation in lymphocytes of mice primed with AVE0010 without FCA AVE0010 (and GLP-1) stimulated T-cell proliferation dose-dependently in lymphocytes of mice primed with AVE0010 with FCA and decreased T-cell proliferation at high concentrations of >5.1 µM AVE0010 and >45 µM GLP-1

[DIV1158] and

amendment 1

[DIV1158-amend01]

4.2.3.7.1-2 4.2.3.7.1-2a

Abbreviations: F female, GLP-1 glucagon-like peptide-1, PBS phosphate-buffered saline, FCA Freunds complete adjuvants

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.17C - Other Toxicity Studies – Degradation products – 2-week mouse study

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

General toxicity study to qualify degradation products (batch xxxxxxxxxxxxxx, stored under xxxxxxxxxxxxxxxxx at xxxx for xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxx%)

Mouse/ Crl:CD-1(ICR)

SC BID 14-day 2, 20, 200 10M/10F Mortality: none Clinical signs: no signs related to treatment Body weight: ≥20 µg/kg: Day 0 to 14: increased gain (M/F: +84% and above), Day 14: absolute weight ↑ (M:+8%) Food consumption: NAD Water consumption: NAD Hematology: 200 µg/kg: hematocrit ↓ (F: -4%) Serum chemistry: ≥20 µg/kg: phosphate ↑ (M/F: +15% to +29%), triglycerides ↓ (M: -22% to –33%)

[TSA1242] 4.2.3.7.6-1

Abbreviations: M male, F female, NAD = no abnormalities detected * percentages related to organ weight - body weight ratio, isolated significances in lower dose groups only not included

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.17C - Other Toxicity Studies – Degradation products – 2-week mouse study (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

General toxicity study to qualify degradation products (batch xxxxxxxxxxxxxx, stored under xxxxxxxxxxxxxxxxx at xxxx for xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxx%)

Mouse/ Crl:CD-1(ICR)

SC BID 14-day 2, 20, 200 10M/10F Organ weights*: ≥2 µg/kg: liver↓ (M: -7% to –12%, non dose-related), spleen↓ (M: -13% to –19%, non dose-related), ≥20 µg/kg: heart ↓ (M: -15% and –12%, non dose-related), 200 µg/kg: lung ↓ (M: -6%) Macroscopy: NAD Microscopy: NAD

[TSA1242] continued

4.2.3.7.6-1

Abbreviations: M male, F female, NAD = no abnormalities detected * percentages related to organ weight - body weight ratio, isolated significances in lower dose groups only not included

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.17D - Other Toxicity Studies – Batch qualification – 2-week rat study

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

General toxicity study to qualify batch xxxxxxxxxxxxxx including impurities included in batch (xxxxxxxxxxxxxxxxxxxxx xxx%)

Rat / Hsd:Sprague

Dawley

SC BID 14-day 2, 20, 200 10M/10F Mortality: none Clinical signs: none Body weight: ≥2 µg/kg: decreased gain (M: -24% to –29%), decreased final weight (M: -6% to –9%) Food consumption: ≥2 µg/kg: decreased between Day 1 to 8 (M: -9% to –20%); F: -8% to –16%) ≥20 µg/kg: decreased between Day 8 and 14 (M: -13%) Water consumption: ≥2 µg/kg: increased between Day 7 and 8 (M: +19% to +49%, F: +33% to +70%)

[TSA1331] 4.2.3.7.6-2

Abbreviations: M male, F female, A/G albumin/globulin ratio, * percentages related to organ weight - body weight ratio, NAD = no abnormalities detected, isolated significances in lower dose groups only not included

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

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TS 2.6.7.17D - Other Toxicity Studies – Batch qualification – 2-week rat study (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

Rat / Hsd:Sprague

Dawley

SC BID 14-day 2, 20, 200 10M/10F Hematology: ≥2 µg/kg: reticulocytes ↓(M: -19% to –25%) LU cells ↓ (M: -32% to –39%) ≥20 µg/kg: hemoglobin ↑ (F: +5%), neutrophils ↓ (F: -18%/-19%) Coagulation: ≥2 µg/kg: PT-time ↑ (M: +5 to +12%) ≥20 µg/kg: Fibrinogen ↓ (M: -14/-26%) Serum chemistry: ≥2 µg/kg: ALT↑ (M+F: +11% to +49%), ALP↑ (F: +13% to +42%), bilirubin↑ (F: +18% to +35%), triglycerides↑ (F: +24% to +54%), α1-globulin↓ (M: -11% to –20%) ≥20 µg/kg: Na↑ (M: +1%/+2%), globulin↓ (M: -5%/-6%), A/G↑ (M: +7%/+8%),α1-globulin↓ (F: -9%/-10%) 200 µg/kg: AST↑ (M: +28%), ALP↑ (M: +25%), calcium↑ (F: +2%), phosphate (F: +11%)

[TSA1331] continued

4.2.3.7.6-2

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TS 2.6.7.17D - Other Toxicity Studies – Batch qualification – 2-week rat study (continued)

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

Rat / Hsd:Sprague

Dawley

SC BID 14-day 2, 20, 200 10M/10F Urinalysis: ≥2 µg/kg: sodium↓ (M: -41% to –56%), potassium↓ (M+F: -16% to –48%), specific weight↓ (M: -1%) ≥20 µg/kg: volume↑ (M: +19%/ +28%) 200 µg/kg: pH↓ (M: -4%), sodium↓ (F: -44%) Organ weights*: ≥2 µg/kg: adrenal gland ↑ (M+F: +14% to +48%), spleen↓ (M: -17% to –23%), testis↑ (M: +9% to +11%), thymus↓ (M: -13% to –24%) ≥20 µg/kg: liver↑ (M: +6%/+7%), prostate↓ (M: -18%), spleen↓ (F: -10%/-12%) 200 µg/kg: thymus↓ (F: -19%) Macroscopy: NAD Microscopy: NAD

[TSA1331] continued

4.2.3.7.6-2

Abbreviations: M male, F female, A/G albumin/globulin ratio, * percentages related to organ weight - body weight ratio, NAD = no abnormalities detected, isolated significances in lower dose groups only not included

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TS 2.6.7.17E - Other Toxicity Studies – Batch qualification – 13-week rat study

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses

(µg/kg BID) Gender and No.

per Group Noteworthy Findings Study

Number Location

General toxicity study to qualify batch xxxxxxxxxxxxxxx, stored under xxxxxxxxxxxxxxxxx at xxxx (xxxxxxxxxxxxxxxx xxx%) and containing xxxx% of the impurity xxxxxxxxxx

xxxxxxxxxxxx); batch xxxxxxxxxxxxxxx (xxxxxxxxxxxx) included as reference

Rat / Crl:Cd

Sprague Dawley

SC BID 13-weeks 200 10M/10F Mortality: none Clinical signs: Salivation, scab formation at injection site Body weight: Reduction of mean body weight (M: -8% to -13% beginning on Day 8; F: -8% and -7% starting at Day 22) Food consumption: Absolute food consumption reduced (M: -23 and -24% on Day 8, -7% to -15% up to Day 36 and -3 to -5% until the end of treatment; F: -22 and -29% on Day 8, up to -14% on Day 22, slight increase up to +10% until the end of treatment) Hematology: none Coagulation: none Serum chemistry: none Urinalysis: none Calcitonin profile: none Organ weights: none Macroscopy: NAD Microscopy: minimal SC fibrosis at injection site

[TXC1482] 4.2.3.7.6-3

Abbreviations: M male, F female, SC = subcutaneous, NAD = no abnormalities detected, isolated significances in lower dose groups only not included

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 265

TS 2.6.7.17F - Other Toxicity Studies – Toxicology Safety assessment AVE0010 - drug substance

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses Gender and No.

per Group Noteworthy Findings Study

Number Location

Local lymph node assay

Mouse / Balb/c N

dermal (dorsum of both ears)

3-day (daily) 0, 2.5 mg per mouse ear

(10% solution in 1,2 propanediol)

4F No sensitizing properties [IST0273] 4.2.3.7.7-1

In vitro corrosivity

Corrsitex test In vitro - - - Not performed (compound not compatible with test reagent)

[IST0273] 4.2.3.7.7-1

Cutaneous irritation

Rabbit / New Zealand White

dermal 4 hours under semi-oclusive

bandage

500 mg per rabbit

3M Not irritant to skin [IST0273] 4.2.3.7.7-1

Ocular irritation

HET-Cam Test

In vitro 5 minutes incubation

100 mg - (4 eggs per compound)

Irritant in vitro [IST0273] 4.2.3.7.7-1

Abbreviations: F female, M male

2.6.7 Nonclinical Toxicology Tabulated Summary xx-xxx-20xx Lixisenatide (AVE0010)

Property of the sanofi-aventis group - strictly confidential Page 266

TS 2.6.7.17G - Other Toxicity Studies – Toxicology Safety assessment AVE0010 - xxxxxxxxxxxxx

Test Article: Lixisenatide Species/

Strain Method of

Administration Duration of

Dosing Doses Gender and No.

per Group Noteworthy Findings Study

Number Location

Local lymph node assay

Mouse / Balb/c N

dermal (dorsum of both ears)

3-day (daily) 0, 6.25 mg per mouse ear

(25% solution in 1,2 propanediol)

4F No sensitizing properties [IST0274] 4.2.3.7.7-2

In vitro Corrosivity

Corrositex test

In vitro Max. 4 hours incubation

500 mg - (3 replicates) Non-corrosive in vitro [IST0274] 4.2.3.7.7-2

Cutaneous irritation

Rabbit / New Zealand White

dermal 4 hours under semi-oclusive

bandage

500 mg per rabbit

3M Not irritant to skin [IST0274] 4.2.3.7.7-2

Ocular irritation

HET-Cam Test

In vitro 5 min incubation 100 mg - (4 eggs per compound)

Irritant in vitro [IST0274] 4.2.3.7.7-2

Abbreviations: F= female, M=male