FINAL REPORT

Volume 1 of 13 (Text, Figures and Table s 1

- 47 )

STUDY TITLE

A N ORAL (GAVAGE) DEVELOPMENTAL NEUROTOXICITY STUDY OF DECABROMODIPHENYL OXIDE IN RATS

STUDY NUMBER

WIL

- 635002
DATA REQUIREMENT

OPPTS Guideline 870.6300 OECD Guideline 426

STUDY DIRECTOR
Melissa J. Beck, PhD

STUDY INITIATION DATE

3 December 2007

STUDY COMPLETION DATE

20 March 2009

PERFORMING LABORATORY
WIL Research Laboratories, LLC 1407 George Road Ashland, OH 44805

- 8946

SPONSOR

Bromine Science and Environmental Forum

3 7 Square de Meeûs 1000 Brussels, BelgiumWIL

- 635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum



### C OMPLIANCE 

### S TATEMENT

This study, designated WIL

- 635002 , was conducted in compliance with the United States Environmental Protection Agency (EPA) Good Laboratory Practice Standards (40 CFR Part 792 ), 18 September 1989; the Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice [C(97) 186/Final],
26 November 1997; the standard operating procedures of WIL Research Laboratories, LLC and the protoc ol as approved by the S pons or.
The pro tocol was designed to be in accordance with the United States EPA Health Effects
Test Guidelines OPPTS 870.6300 De velopmental Neurotoxicity Study , August 1998, the
United States EPA Data Call

- in Notice, OPPTS, 10 September 1999 and the OECD
Guid eline for Testing of Chemicals, Guideline 4 26 Developmental Neurotoxicity Study
, adopted 16 October 2007.

______________________________________ _____________

Melissa J. Beck, PhD Date Assistant Director, Neurosciences Study DirectorWIL

- 635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum



### T ABLE O F 

### C ONTE NTS

VOLUME 1 Page
Compliance Statement

................................ ................................ ............... 2 Table of Contents

................................ ................................ ....................... 3 Index of Figures

................................ ................................ .......................... 8 Index of Text Tables

................................ ................................ ................ 12
Index of Tables

................................ ................................ ......................... 13 Index of Appendices

................................ ................................ ................. 17
1

. Summary

................................ ................................ ................................ ... 23
1.1

. Objective

................................ ................................ ................................ .... 23
1.2

. Study Design

................................ ................................ .............................. 23
1.3

. Result s

................................ ................................ ................................ ........ 25
1.4

. Conclusions

................................ ................................ ................................ 27
2

. Introduction

................................ ................................ .............................. 28
2.1

. Rationale for Study Design

................................ ................................ ........ 28
2.2

. General Study Information

................................ ................................ ........ 28
2.3

. Key Study Dates

................................ ................................ ........................ 29 3

. Study Design Schematic (Key Phases)

................................ ................... 31
4

. Experimental Procedures

- Materials and Methods

............................. 33
4.1

. Test Substance and Vehicle

................................ ................................ ....... 33
4.1.1

. Test Substance Identification

................................ ................................ ..... 33
4.1.2

. Vehicle Identification

................................ ................................ ................. 33
4.1.3

. Preparation of Vehicle and Dosing Formulations

................................ ...... 34
4.1.4

. Sampling and Analysis

................................ ................................ .............. 35
4.2

. Nicotine Challenge

................................ ................................ ..................... 35
4.3

. Test System

................................ ................................ ................................ 36
4.4

. Organization of Test Groups, Dosage Levels and Treatment Regimen

................................ ................................ ................................ ..... 37
4.5

. Justification of Dosage Levels and Vehicle Selection

............................... 37WIL

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VOLUME 1 (continued) Page

4.6

. F 0 Maternal Generation

................................ ................................ .............. 39
4.6.1

. Animal Receipt and Acclimation

................................ ............................... 39
4.6.2

. Animal Housing

................................ ................................ ......................... 39
4.6.3

. Diet, Drinking Water and Maintenance

................................ ..................... 40
4.6.4

. Environmental Conditions

................................ ................................ ......... 40
4.6.5

. Assignment of Animals to Trea tment Groups and Breeding Procedures

................................ ................................ ................................ .. 41
5

. Parameters Evaluated

................................ ................................ ............. 43 5.1

. F 0 Observations

................................ ................................ .......................... 43
5.1.1

. Clinical Observations and Survival (Both Phases)

................................ .... 43
5.1.2

. Detailed Clinical Observations (Developmental Neurotoxicity Phase)

................................ ................................ ................................ ......... 43
5.1.3

. Body Weights (Both Phases)

................................ ................................ ..... 44
5.1.4

. Food Consumption (Developmental Neurotoxicity Phase)

....................... 44
5.1.5

. Parturition (Both Phases)

................................ ................................ ........... 45
5.1.6

. Macroscopic Examinations (Developmental Neurotoxicity Phase)

.......... 45
5.2

. F 1 Litter Parameters

................................ ................................ ................... 46
5.2.1

. Litter Viability and Deaths (Both Phases)

................................ ................. 46
5.2.2

. Litter Reduction (Developmental Neurotoxicity Phase)

............................ 46
5.2.3

. Clinical Observations (Both Phases)

................................ ......................... 46
5.2.4

. Body Weights (Both Phases)

................................ ................................ ..... 47
5.2.5

. Sex Determination (Both Phases)

................................ .............................. 47
5.2.6

. Calculation of Litter Parameters (Both Phases)

................................ ......... 48
5.2.7

. Weaning and Selec tion (Developmental Neurotoxicity Phase)

................. 48
5.2.8

. Developmental Landmarks, Sensory Function and Neurobehavioral Testing (Developmental Neurotoxicity Phase; Subsets A and B)

................................ ................................ ........................ 51
5.2.8.1

. Balanopr eputial Separation (Subset A)

................................ ...................... 51
5.2.8.2

. Vaginal Patency (Subset s A and B)

................................ ........................... 51
5.2.8.3

. Detailed Clinical Observations (Subset A)

................................ ................ 51WIL

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VOLUME 1 (continued) Page

5. 2.8.4

. Locomotor Activity (Subset A)

................................ ................................ . 52
5.2.8.5

. Acoustic Startle Response (Subset A)

................................ ....................... 53
5.2.8.6

. Biel Maze Swimming Trials (Subsets A and B)

................................ ........ 54 5.2.9

. Macroscopic and Microscopic Examinations (Developmental Neurotoxicity Phase)

................................ ................................ .................. 55 5.2.9.1

. Offspring Not Selected for Behavioral Evaluations

................................ .. 55 5.2.9.2

. Offspring Euthanized Following Completion of the PND 2 2 Learning and Memory Assessments (Subset B) or PND 180 Locomotor Activity Assessment (Subset A) and Not Selected for Neuropathology/Morphometry/Brain Weight Measurements

................... 55 5.2.9.3

. Neuropathology (Subsets C and D)

................................ ........................... 56 5.2.9.3.1

. Macroscopic Examination

................................ ................................ ........ 56 5.2.9.3.2

. Microscopic Examination

................................ ................................ ......... 56 5.2.9.3.3

. Morphometric Analysis

................................ ................................ ............ 58
5.3

. Bioanalytical Analyse s (Bioanalytical Phase)

................................ ........... 59
5.3.1

. Blood Collection

................................ ................................ ........................ 59
5.3.2

. Milk Collection

................................ ................................ .......................... 59
5.3.3

. Sample Preparation and Analysis

................................ .............................. 59
5.3.4

. Eut hanasia

................................ ................................ ................................ .. 60
5.4

. Statistical Methods

................................ ................................ ..................... 60
5.4.1

. Locomotor Activity

................................ ................................ .................... 61
5.4.1.1

. Locomotor Activity Across Sessions PND 13, 17 and 21

......................... 61
5.4.1.2

. Locomotor Activity within Sessions PND 13, 17 and 21

.......................... 62
5.4.1.3

. Locomotor Activity within Sessions PND 61, 120 and 180

...................... 62
5.4.2

. Acoustic Startle Response

................................ ................................ .......... 63
5.4.3

. Biel Water Maze

................................ ................................ ........................ 63
5.4.4

. Pup Body Weights

................................ ................................ ..................... 64
5.4.5

. Maternal Body Weights and Food Consumption

................................ ....... 64
5.5

. Data Retention

................................ ................................ ........................... 65 6

. Results and Discussion

................................ ................................ ............. 66WIL

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VOLUME 1 (continued) Page

6.1

. Analytical Chemistry

................................ ................................ ................. 66 6.2

. Bioanalytical Results (Bioanalytical Phase)

................................ .............. 67 6.3

. F 0 Maternal Gener ation (Developmental Neurotoxicity Phase)

................ 69 6.3.1

. Clinical Observations and Survival

................................ ........................... 69 6.3.2

. Pregnancy Status

................................ ................................ ........................ 70
6.3.3

. Detailed Clinical Observations

................................ ................................ .. 70
6.3.4

. Body Weights

................................ ................................ ............................. 71
6.3.4.1

. Gestation

................................ ................................ ................................ .... 71
6.3.4.2

. Lactation

................................ ................................ ................................ .... 71
6.3.5

. Food Consumption

................................ ................................ ..................... 71
6.3.5.1

. Ges tation

................................ ................................ ................................ .... 71
6.3.5.2

. Lactation

................................ ................................ ................................ .... 71
6.3.6

. Gestation Length and Parturition

................................ ............................... 72
6.3.7

. F 0 Macroscopic Examinations

................................ ................................ ... 72
6.3.7.1

. Unschedule d Deaths

................................ ................................ ................... 72
6.3.7.2

. Females that Failed to Deliver

................................ ................................ ... 73
6.3.7.3

. Females

- Lactation Day 21

................................ ................................ ....... 73
6.4

. F 1 Litter Data (Developmental Neurotoxicity Phase)

................................ 74
6.4.1

. PND 0 Litter Data and Postnatal Survival

................................ ................. 74
6.4.2

. General Physical Condition

................................ ................................ ....... 74
6.4.3

. Offspring Body Weights

................................ ................................ ............ 75
6.4.4

. Necropsies of Pups Found Dead

................................ ................................ 75
6.4.5

. Necropsies of PND 21 Pups Not Selected for Neuropathological Evaluations or Pups Euthanized due to Death of Dam

.............................. 76 6.5

. F 1 Generation

................................ ................................ ............................. 76 6.5.1

. Clinical Observations and Survival

................................ ........................... 76 6.5.2

. Body Weights

................................ ................................ ............................. 77 6.5.3

. Developmental Landmarks, Sensory Function and Behavioral Testing

................................ ................................ ................................ ........ 78
6.5.3.1

. Balanoprepu tial Separation

................................ ................................ ........ 78WIL

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VOLUME 1 (continued) Page

6.5.3.2

. Vaginal Patency

................................ ................................ ......................... 78
6.5.3.3

. Detailed Clinical Observations

................................ ................................ .. 79 6.5.3.4

. Locomotor Activity

................................ ................................ .................... 80
6.5.3.5

. Acoustic Startle Test

................................ ................................ .................. 83
6.5.3.6

. Biel Maze Swimming Trials

................................ ................................ ...... 84
6.5.4

. Macroscopic Examinations (Unscheduled Deaths and Animals Not Selected for Neuropathology)

................................ ................................ .... 85
6.5.4.1

. Unscheduled Deaths

................................ ................................ ................... 85
6.5.4.2

. Animals Euthanized Following PND 22 Learning and Memory Assessments (Subset B)

................................ ................................ ............. 85
6.5.4.3

. Animals Euthanized Following PND 180 Locomotor Activity Assessment (Subset A)

................................ ................................ .............. 86
6.5.5

. Neuropathology

................................ ................................ .......................... 87
6.5.5.1

. PND 21 (Subset C)

................................ ................................ ..................... 87
6.5.5.1.1

. Macroscopic Examinations

................................ ................................ ....... 87
6.5.5.1.2

. Brain Weights/Brain Measurements

................................ ......................... 87
6.5.5.1.3

. Qualitative Histopathology and Brain Morphometry

............................... 87
6.5.5.2

. PND 72 (Subset D)

................................ ................................ .................... 88
6.5.5.2.1

. Macros copic Examinations

................................ ................................ ....... 88
6.5.5.2.2

. Brain Weights/Brain Measurements

................................ ......................... 88
6.5.5.2.3

. Qualitative Histopathology and Brain Morphometry

............................... 89
6.6

. Discussion

................................ ................................ ................................ .. 92
7

. Conclusions

................................ ................................ ............................... 96 8

. Key Study Personnel and Report Submission

................................ ....... 97 9

. Quality Assurance Unit Statement

................................ ......................... 99 9.1

. Phases Inspected

................................ ................................ ........................ 99 9.2

. Approval

................................ ................................ ................................ .. 105 10

. References

................................ ................................ ............................... 106
11

. Deviations from the Protocol

................................ ................................ 109WIL

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### I NDEX O F 

### F IGURES

VOLUME 1 (continued) Page

1. Summary of Body Wei ghts during Gestation [G] (F 0 Females)

.............................. 113

2. Summary of Body Weights during Lactation [G] (F 0 Females)

.............................. 114

3. Summary o f Offspring Weights [G] (F 1 - PND 1

- 21 )

................................ ............. 115

4. Summary o f Body Weights [G] (F 1 - PND 28

- 181 )

................................ ................ 116

5. Summary o f Forelimb Grip Strengt h [G] (F 1 Males)

................................ ............... 117

6. Summa

ry o f Hindlimb Grip Strength [G] (F 1 Males)

................................ .............. 118

7. Summary o f Forelimb Grip Strength [G] (F 1 Females)

................................ ........... 119

8. Summary o f Hindlimb Grip Strength [G] (F 1 Females)

................................ .......... 120

9

. Summary of Motor Activity Total Counts b y Session (F 1 - PND 13, 17 And 21)

................................ ................................ ......................... 121

10

. Summary o f Motor Activity Total Counts (F 1 - PND 13)

................................ ....... 122

11

. Summary o f Motor Activity Total Counts (F 1 - PND 17)

................................ ....... 123

12

. Summary o f Motor Activity Total Counts (F 1 - PND 21)

................................ ....... 124

13

. Summary o f Motor Activity Ambulatory Coun ts b y Session (F 1 - PND 13, 17 And 21)

................................ ................................ ......................... 125

14

. Summa

ry o f Motor Activity Ambulatory Counts (F 1 - PND 13)

............................ 126

15

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 17)

............................ 127

16

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 21)

............................ 128

17

. Summar y o f Motor Activity Total Counts (F 1 - PND 61

- Hour 1)

........................ 129

18

. S ummary o f Motor Activity Total Counts (F 1 - PND 120

- Hour 1)

...................... 130

19

. Summary o f Motor Activity Total Counts (F 1 - PND 180

- Hour 1)

...................... 131WIL

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VOLUME 1 (continued) Page

20

. Summary o f Motor Ac tivity Ambulatory Counts (F 1 - PND 61

- Hour 1)

............. 132

21

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 120

- Hour 1)

........... 133

22

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 180

- Hour 1)

........... 134

23

. Summary o f Motor Activity Total Counts (F 1 - PND 61

- Hour 2

- Animals Dosed With Saline)

................................ ................................ .................... 135

24

. Summary o f Motor Activity Total Counts (F 1 - PND 120

- Hour 2

- Animals Dosed With Saline)

................................ ................................ ................................ ... 136

25

. Summary o f Motor Activity Total Counts (F 1 - PND 180

- Hour 2

- Animals Dosed With Saline)

................................ ................................ ................................ ... 137

26

. S ummary o f Motor Activity Ambulatory Counts (F 1 - PND 61

- Hour 2

- Animals Dosed With Saline)

................................ ................................ .................... 138

27

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 120

- Hour 2

- Animals Dosed With Saline)

................................ ................................ .................... 139

28

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 180

- Hour 2

- Animals Dosed With Saline)

................................ ................................ .................... 140

29

. Summary o f Motor Activity Total Counts (F 1 - PND 61

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ................ 141

30

. Summary o f Motor Activity Total Counts (F 1 - PND 120

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ............................... 142

31

. Summary o f Motor Activity Total Counts (F 1 - PND 180

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ............................... 143

32

. S ummary o f Motor Activity Ambulatory Counts (F 1 - PND 61

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ................ 144

33

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 120

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ................ 145

34

. Summary o f Motor Activity Ambulatory Counts (F 1 - PND 180

- Hour 2

- Animals Dosed With Nicotine)

................................ ................................ ................ 146

35

. Summary o f Startle Response Data [MAX] (F 1 - PND 20)

................................ ..... 147WIL

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VOLUME 1 (continued) Page

36

. Summary o f Startle Response Data [Tmax] (F 1 - PND 20)

................................ ..... 148

37

. Summary o f Startle Response Data [AVE] (F 1 - PND 20)

................................ ...... 149

38

. Summary o f Startle Response Data [MAX] (F 1 - PND 60)

................................ ..... 150

39

. Summary o f Startle Response Data [Tmax] (F 1 - PND 60)

................................ ..... 151

40

. S ummary o f Startle Response Data [AVE] (F 1 - PND 60)

................................ ...... 152

41

. Summary o f Biel Maze Data (F 1 - Mean Time to Escape

- PND 22

- Path A

- Combined Sexes)

................................ ................................ ....................... 153

42

. Summary o f Biel Maze Data (F 1 - Mean No. o f Errors

- PND 22

- Path A

- Combined Sexes)

................................ ................................ ....................... 154

43

. Summary o f Biel Maze Data (F 1 - Mean Time to Escape

- PND 22

- Path B

- Males)

................................ ................................ ................................ ......... 155

44

. Summary o f Biel Maze Data (F 1 - Mean No. o f Errors

- PND 22

- Path B

- Males)

................................ ................................ ................................ ......... 156

45

. Summary o f Biel Maze Data (F 1 - Mean Time to Escape

- PND 22

- Path B

- Female s)

................................ ................................ ................................ ..... 157

46

. Summary o f Biel Maze Data (F 1 - Mean No. o f Errors

- PND 22

- Path B

- Females)

................................ ................................ ................................ ..... 158

47

. Summary o f Biel Maze Data (F 1 - Mean Time t o Escape

- PND 62

- Combined Sexes)

................................ ................................ ................................ ...... 159

48

. Summary o f Biel Maze Data (F 1 - Mean No. o f Errors

- PND 62

- Comb ined Sexes)

................................ ................................ ................................ ........................ 160

49

. Summary o f Brain Morphometry [Level 1] (F 1 Males

- PND 21)

.......................... 161

50

. Summary o f Brain Morphometry [Level 3] (F 1 Males

- PND 21)

.......................... 162

51

. Summary o f Brain Morphometry [Level 5] (F 1 Males

- PND 21)

.......................... 163

52

. Summary o f Brain Morphometry [L evel 1] (F 1 Females

- PND 21)

....................... 164

53

. Summary o f Brain Morphometry [Level 3] (F 1 Females

- PND 21)

....................... 165WIL

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VOLUME 1 (continued) Page

54

. Summary o f Brain Morphometry [Level 5] (F 1 Females

- PND 21)

....................... 166

55

. Summary o f Brain Morphometry [Level 1] (F 1 Males

- PND 72)

.......................... 167

56

. Summary o f Brain Morphometry [Level 3] (F 1 Males

- PND 72)

.......................... 168

57

. Summary o f Brain Morphometry [Level 5] (F 1 Males

- PND 72)

.......................... 169

58

. Summary o f Brain Morphometry [Level 1] (F 1 Females

- PND 72)

....................... 170

59

. Summary o f Brain Morphometry [L evel 3] (F 1 Females

- PND 72)

....................... 171

60

. Summary o f Brain Morphometry [Level 5] (F 1 Females

- PND 72)

....................... 172WIL

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### I NDEX OF 

### T EXT 

### T ABLES

VOLUME 1 (continued) Page

1. Results of Homogeneity and Stability Analyses

................................ ........................ 66

2. Results of Concentration Analyses

................................ ................................ ............ 66

3. Co ncentrations of the Test Substance in Maternal and Pup Plasma

.......................... 67

4. Concentrations of the Test Substance in Maternal Milk Samples

............................. 68

5

. Summary of Level 1 Brain Morphometry

- Males (PND 72)

................................ ... 91WIL

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### I NDEX O F 

### T ABLES

VOLUME 1 (continued) Page



### S UMMARY 

### D ATA 

### - F 0 

### G ENERATION 1. Summary o f Maternal Surv ival a nd Pregnancy Status (F 0 )

................................ ..... 174

2. Summary o f Clinical F indings: Total Occurrence/No. o f Animals (F 0 - Daily Examinations)

................................ ................................ ......................... 175

3. Summary o f Post

- Dose Findings: Total Occurrence/No. o f Animals

..................... 177

4. Summar y o f Detailed Clinical Observations (F 0 - Gestation)

................................ . 178

5. Summary of Detailed Clinical Observations (F 0 - Lactation)

................................ .. 188

6. Summary of Body Weights during Gestation [G] (F 0 )

................................ ............ 198

7. Summary of Body Weight Changes d uring Gestation [G] (F 0 )

............................... 200

8. Summa

ry of Body Weights during Lactation [G] (F 0 )

................................ ............ 203

9. Summary of Body Weight Changes d uring Lactation [G] (F 0 )

............................... 205

10. Summary of Food Consumption during Gestation [G/Animal/Day] (F 0 )

............... 208

11. Summary of Food Consumption during Gestation [G/Kg/Day] (F 0 )

...................... 21 0

12. Summary of Food Consumption during Lactation [G/Animal/Day] (F 0 )

............... 212

13. Summary of Food Consumption during Lactation [G/Kg/Day] (F 0 )

...................... 214

14. Summary of Gestation Lengths [Days] (F 0 )

................................ ............................. 216

15. Summary of Macroscopic Findings (F 0 - Unscheduled Death s)

............................. 217

16. Summary of Macroscopic Findings (F 0 - Failed t o Deliver)

................................ ... 218

17. Summary of Macroscopic Findings (F 0 - Lactation Day 21)

................................ ... 219

18. Summary of Implantation Sites (F 0 )

................................ ................................ ......... 220WIL

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VOLUME 1 (continued) Page



### S UMMARY 

### D ATA 

### - F 1 

### L ITTERS 19. Summary of PN D 0 Litter Data (F 1 )

................................ ................................ ........ 221

20. Summary of Postnatal Survival [% Per Litter] (F 1 )

................................ ................. 222

21. Summary of Pup Clinical Observations (F 1 )

................................ ............................ 224

22. Summary of Offspring Weights [G] (F 1 )

................................ ................................ . 225

23. Summary of Offspring Weight Changes [G] (F 1 )

................................ .................... 228

24. Summary of Pup Necropsy Findings (F 1 - Found Dead Pups)

................................ 231

25. Summary of Pup Necropsy Fi ndings (F 1 - Pups Not Selected for Neuropathology or Euthanized Due t o Death of Dam)

................................ ........... 232



### S UMMARY 

### D ATA 

### - F 1 

### G ENERATION 26. Summary o f Clinical F indings: Total Occurrence/No. o f An imals (F 1 Males

- Subsets A, B and D)

................................ ................................ .............. 233

27. Summary o f Clinical F indings: Total Occurrence/No. o f Animals (F 1 Females

- Subsets A, B and D)

................................ ................................ ........... 235

28. Summary of Body Weights [G] (F 1 )

................................ ................................ ........ 237

29. Summary of Body Weight Changes [G] (F 1 )

................................ ........................... 243

30. Summary of Animal Developmental Parameters

- Balanopreputial Separation (F 1 )

................................ ................................ ................................ .......... 249

31. Summary of Animal Developmental Parameters

- Vaginal Patency (F 1 )

............... 250

32. Summary of Detailed Clinical Observations (F 1 - Subset A)

................................ ................................ ................................ .......... 251

33. Summary of Motor Ac tivity Counts (F 1 - PND 13, 17 a nd 21

- Subset A)

............. 307

34. Summary of Motor Activity Counts (F 1 - PND 61, 120 a nd 180

- Hour 1

- Subset A)

................................ ................................ ................................ .................. 321

35. Summary of Motor Activity Counts (F 1 - PND 61, 120 and 180

- Hour 2

- Animals Dosed w ith Saline

- Subset A)

................................ ................................ ... 333WIL

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VOLUME 1 (con tinued) Page

36. Summary of Motor Activity Counts (F 1 - PND 61, 120 and 180

- Hour 2

- Animals Dose d w ith Nicotine

- Subset A)

................................ ............................... 345

37. Summary of Startle Response Data (F 1 - Subset A)

................................ ................ 357

38

. Summary of Biel Maze Straight Channel Swim Trials (F 1 - PND 22

- Subset B)

................................ ................................ .......................... 369

39

. Summary of Biel Maze Swim Trials (F 1 - PND 22

- Subset B)

.............................. 370

40

. Summary of Biel Maze Straight Channel Swim Trials (F 1 - PND 62

- Subset A)

................................ ................................ .......................... 386

41

. Summary of Biel Maze Swim Trials (F 1 - PND 62

- Subset A)

.............................. 387

42. Summary of M acroscopic Findings (F 1 Males

- Unscheduled Deaths)

.................. 395

43. Summary of Macroscopic Findings (F 1 Females

- Unscheduled Death)

................ 396

44. Summary of Macroscopic Findings (F 1 Males

- Scheduled Euthanasia

- Subset B)

................................ ................................ ................................ ................... 397

45. Summary of Macroscopic Findings (F 1 Females

- Scheduled Euthanasia

- Subset B)

................................ ................................ ................................ ................... 398

46. Summary of Macroscopic Findings (F 1 Males

- Scheduled Euthanasia

- Subset A)

................................ ................................ ................................ .................. 399

47. Summary of Macroscopic Findings (F 1 Females

- Scheduled Euthanasia

- Subset A)

................................ ................................ ................................ .................. 401

VOLUME 2



### S UMMARY 

### D ATA 

### - F 1 

### N EUR OPATHOLOGY 48. Summary of Macroscopic Findings (F 1 - Animals Selected f or Neuropathology

- PND 21

- Subset C)

................................ ................................ 405

49. Summary of Brain Weights and Brain Measu rements (F 1 - Animals Selected f or Neuropathology

- PND 21

- Subset C)

................................ ............................... 407

50. Summary of Micro scopic Findings (F 1 - Animals Selected f or Neuropathology

- PND 21

- Subset C)

................................ ............................... 411WIL

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VOLUME 2 (continued) Page

51. Summary of Brain Morphometry [CM ] (F 1 - Animals Selected f or Neuropathology

- PND 21

- Subset C)

................................ ............................... 415

52. Summary of Macroscopic Findings (F 1 - Animals Selected f or Neuropathology

- PND 72

- Subset D)

................................ ............................... 419

53. Summary of Brain Weights and Brain Measu rements (F 1 - Animals Selected f or Neuropathology

- PND 72

- Subset D)

................................ ............................... 421

54. Summary of Microscopic Findings (F 1 - Animals Selected f or Neu ropathology

- PND 72

- Subset D)

................................ ............................... 425

55. Summary of Brain Morphometry [CM ] (F 1 - Animals Selected f or Neuro pathology

- PND 72

- Subset D)

................................ ............................... 437WIL

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### I NDEX O F 

### A PPENDICES

VOLUME 2 (continued) Page

A. Individual Animal Data

................................ ................................ ............................ 441



### I NDIVIDUAL 

### D ATA 

### - F 0 

### G ENERATI ON A1. Individual Clinical Observat ions (F 0 - Daily Examinations)

............................. 442

VOLUME 3

A2. Individual Clinical Observations (F 0 - Post

- Dosing Observations)

................... 753

A3. Individual Detailed Clinical Observations (F 0 - Gestation)

............................... 974

A4. Individual Detailed Clinica l Observations (F 0 - Lactation)

............................. 1013

A5. Individual Body Weights during Gestation [G] (F 0 )

................................ ....... 1049

A6. Individual Body Weight Changes during Gestation [G] (F 0 )

.......................... 1059

A7. Individual Body Weights during Lactation [G] (F 0 )

................................ ........ 1069

A8. Individual Body Weight Chan ges during Lactation [G] (F 0 )

.......................... 1079

A9. Individual Food Consumption during Gestation [G/Animal/Day] (F 0 )

.......... 1089

A10. Individual Food Consumption during Gestation [G/Kg/Day] (F 0 )

.................. 1099

A11. Individual Food Consumption during Lactation [G/Animal/Day] (F 0 )

........... 110 9

A12. Individual Food Consumption during Lactation [G/Kg/Day] (F 0 )

.................. 1119

VOLUME 4

A13. Individual Gestation Lengths [Days] (F 0 )

................................ ........................ 1130

A14. Individual Macroscopic Findings (F 0 - Unscheduled Deaths)

......................... 1131

A15. Individual Macroscopic Findings (F 0 - Failed t o Deliver)

............................... 1137

A16. Individual Macroscopic Findings (F 0 - Lactation Day 21)

.............................. 1141

A17. Individual Implantation Sites (F 0 )

................................ ................................ .... 1306WIL

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VOLUME 4 (continued) Page



### I NDIVIDUAL 

### D ATA 

### - F 1 

### L ITTERS A18. Individual Litter Viability (F 1 )

................................ ................................ ......... 1316

A19. Individual Pup Via bility (F 1 )

................................ ................................ ............ 1337

A20. Individual Postnatal Survival [% Per Litter] (F 1 )

................................ ............. 1357

A21. Individual Pup Clinical Observations (F 1 )

................................ ....................... 1367

VOLUME 5

A21. Individual Pup Clinical Observations (F 1 ) [continued]

................................ ... 1469

VOLUME 6

A22. Individual Offspring Weights [G] (F 1 )

................................ ............................. 1855

A23. Individual Offspring Weight Changes [G] (F 1 )

................................ ............... 1983

A24. Individual Pup Necropsy Findings (F 1 - Found Dead Pups)

........................... 2108

A25. Individual Pup Necropsy Findings (F 1 - Pups Not Select ed f or Neuropathology or Euthanized Due t o Death of Dam)

................................ ... 2115

VOLUME 7



### I NDIVIDUAL 

### D ATA 

### - F 1 

### G ENERATION A26. Individual Clinical Observations (F 1 Males

- Subsets A, B and D)

................ 2146

A27. Individual Clinical Observations (F 1 Females

- Subsets A, B and D)

............. 2267

A28. Individual Body Weights [G] (F 1 Males)

................................ ......................... 2394

A29. Ind ividual Body Weights [G] (F 1 Females)

................................ ..................... 2409

A30. Individual Body Weight Changes [G] (F 1 Males)

................................ ........... 2424

A31. Individual Body Weight Changes [G] (F 1 Females)

................................ ........ 2439WIL

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VOLUME 7 (continued) Page

A32. Individual Animal Developmental Parameters

- Balanopreputial Separation (F 1 )

................................ ................................ ................................ .. 2454

A33. Individual Animal Developmental Parameters

- Vaginal Patency (F 1 )

........... 2471

VOLUME 8

A34. Individual Detailed Clinical Observations (F 1 - Subset A)

................................ ................................ ................................ .. 2492

VOLUME 9

A35. Individual Motor Ac tivity Counts (F 1 - PND 13, 17 a nd 21

- S ubset A)

........ 2853

A36. Individual Motor Act ivity Counts (F 1 - PND 61, 120 a nd 180

- Hour 1

- Subset A)

................................ ................................ ................................ .......... 2883

A37. Individual Motor Activity Counts (F 1 - PND 61, 120 and 180

- Hour 2

- Animals Dosed w ith Saline

- Subset A)

................................ .......................... 2913

A38. Individual Motor Activity Counts (F 1 - PND 61, 120 and 180

- Hour 2

- Animals Dosed w ith Nicotine

- Subset A)

................................ ....................... 2943

A39. Individual Startle Response Data (F 1 - Subset A)

................................ ............ 2973

A40

. Individual Biel Maze Straight Channel Swim Trials (F 1 - PND 22

- Subset B)

................................ ................................ .................. 2993

A41

. Individual Bie l Maze Swim Trials (F 1 - PND 22

- Subset B)

......................... 3003

A42

. Individual Biel Maze Straight Channel Swim Trials (F 1 - PND 62

- Subset A)

................................ ................................ ................. 3023

A43

. Individual Biel Maze Swim Trials (F 1 - PND 62

- Subset A)

......................... 3033

A44. Individual Macroscopic Findings (F 1 Males

- U nscheduled Deaths)

.............. 3073

A45. Individual Macroscopic Findings (F 1 Females

- Unscheduled Death)

............ 3075

A46. Individual Macroscopic Findings (F 1 Males

- Scheduled Euthanasia

- Subset B)

................................ ................................ ................................ .......... 3076WIL

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VOLUME 9 (continued) Page

A47. Individual Macroscopic Findings ( F 1 Females

- Scheduled Euthanasia

- Subset B)

................................ ................................ ................................ .......... 3151

VOLUME 10

A48. Individual Macroscopic Findings (F 1 Males

- Sc heduled Euthanasia

- Subset A)

................................ ................................ ................................ .......... 3227

A49. Individual Macroscopic Findings (F 1 Females

- Scheduled Euthanasia

- Subset A)

................................ ................................ ................................ .......... 3326


### I NDIVIDUAL 

### D ATA 

### - F 1 

### N EUROPATHOLOGY A50. Individual Macroscopic and Microscopic F indings (F1

- Animals Selected f

or Neuro pathology

- PND 21

- Subset C)

................ 3425

A51. Individual Final Body and Brain Weights and Brain Measu rements (F1

- Animals Selected f

or Neuropathology

- PND 21

- Subset C)

................ 3575

A52. Individual Brain Weights Relative to Final Body Weights [G/100 G] (F1

- Animals Se lected f

or Neuropathology

- PND 21

- Subset C)

................ 3585

A53. Individual Brain Morphomet ry [Cm] (F1

- Animals Selected f or Neuropathology

- PND 2 1

- Subset C)

................................ ............................ 3595

VOLUME 11

A54. Individual Macroscopic and Microscopic F indings (F1

- Animals Selected f

or Neuropathology

- PND 72

- Subset D)

................ 3612

A55. Individual Final Body and Brain Weights and Brain Measurements (F1

- Animals Selected f

or Neuro pathology

- PND 72

- Subset D)

................ 3761

A56. Individual Brain Weights Relative to Final Body Weights [G/100 G] (F1

- Animals Selected f

or Neuropathology

- PND 72

- Subset D)

................ 3771

A57. Individual Brain Morphomet ry [Cm] (F1

- Animals Selected f or Neuro patholo gy

- PND 72

- Subset D)

................................ ............................ 3781

B. Certificate o f Analysis (Sponsor

- Provi d ed Data)

................................ .................. 3809

C

. Analyses o f Dosing Formulations (WIL Research Laboratories, LLC)

................ 3816

D. Nesti ng Material Analysi s

................................ ................................ ...................... 3874WIL

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VOLUME 11 (continued) Page

E. Food and Water Analyses

................................ ................................ ....................... 3881

F

. Animal Room Environmental Conditions

................................ .............................. 3952

G

. Bioanalyt ical Phase Animal Data

................................ ................................ ........... 3966

1. Individual Clinical Observat ions (F 0 - Daily Examinations)

........................... 3967

2. Individual Body Weights during Gestation [G] (F 0 )

................................ ....... 3984

3. Individual Body Weight Changes during Gestation [G] (F 0 )

.......................... 3986

4. Individual Body Weights during Lactation [G] (F 0 )

................................ ........ 3988

5. Individual Body Weight Changes during Lactation [G] (F 0 )

.......................... 3990

6. Individual Gestation Lengths [Days] (F 0 )

................................ ........................ 3992

7. Individual Litter Viability (F 1 )

................................ ................................ ......... 3993

8. Individual Pup Viability (F 1 )

................................ ................................ ............ 3995

9. Individual Postnatal Survival [% Per Litter] (F 1 )

................................ ............. 3997

10. Individual Pup Clinical Observations (F 1 )

................................ ....................... 3999

11. Individual Offspring Weights [G] (F 1 )

................................ ............................. 4025

12. Individual Offspring Weight Changes [G] (F 1 )

................................ ............... 4038

13. Individual P up Necropsy Findings (F 1 - Found Dead Pups)

........................... 4051

H. Scoring Criteria for the Functional Observational Battery

................................ .... 4052

VOLUME 12

I. Pathology Report (WIL Research Laboratories, LLC)

................................ .......... 4058

J. Bioanalytical Method Development (WIL Research Laboratories, LLC)

............ 4072

K

. Plasma Bioanalytical Report ( WIL Research Laboratories, LLC )

........................ 4230

L

. Milk Bioanalytical Report (WIL Research Laboratories, LLC)

............................ 4265WIL

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VOLUME 12 (continued) Page

M

. Statistical Analysis Report (BioSTAT Consultants, Inc.)

................................ ...... 4299

VOLUME 13

N

. WI L Reproductive Historical Control Data Version 2.5 [Crl:CD(SD) Rats]

........ 4431

O

. WIL Developmental Neurotoxicity Method Development and Positive Control Studies

................................ ................................ ................................ ....... 4440

P

. WIL Functional Observational Battery Historical Control Data for Developmental Neurotoxic ity Studies Version 1.1 [Crl:CD(SD) Rats]

................ 4653
Q

. WIL Motor Activity Historical Control Data Version 1.1 [Crl:CD(SD) Rats]

..... 4678

R. Nicotine Challenge Figures

................................ ................................ .................... 4687

S

. WIL Startle Response Historical Control Data Version 1.1 [Crl:CD(SD) Rats]

................................ ................................ ................................ .. 4693

T

. WI L Biel Maze Historical Control Data Version 1.2 [Crl:CD(SD) Rats]

............. 4702

U

. WIL Brain Morphometry Historical Control Data Version 1.3m1 [Crl:CD(SD) Rats]

................................ ................................ ................................ .. 4730

V

. GLP Certificate

................................ ................................ ................................ ....... 4734

W

. Study Protocol

................................ ................................ ................................ ........ 4736WIL

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### 1. S UMMARY 

### 1.1. O BJECTIVE This oral gavage developmental neurotoxicity study in rats with decabromodiphenyl oxide was designed with the following objectives:
1) to determine the potential of the test substance to induce functional and/or morphological insult to the nervous system in the offspring of dams that w ere administered during pregnancy and lactation via oral gavage dosage levels of 1, 10, 100

or 1000 mg/kg/day ;
2) to determine the concentration of the test substance in maternal and neonatal plasma and maternal milk samples on lactation/postnatal day 4 at dosage levels of 1 and
10 mg/kg/day

. The concentration of the test substance in m aternal and neonatal plasma
and maternal milk resulting from oral (gavage) exposure of dams to the test substance in
the same vehicle was already determined in a previous ex posure assessment and dose

range

- finding developmental neurotoxicity study (Beck, 2009 ; WIL

- 635001) at dos ag e levels of 100, 300 and 1000 mg/kg/day.



### 1.2. S TUDY 

### D ESIGN
Four groups of bred female Sprague Dawley Crl:CD(SD) rats (35/group; Groups 2

- 5)

wer e administ ered daily by oral gavage the test substance ( decabromodiphenyl oxide ) in
the vehicle corn oil from gestation day 6 through lactation day 21. Dosage levels were 1, 10, 100 and 1000 mg/kg/day. A concurrent control group of 35 rats (Group 1) received only the vehicle (corn oil) on a comparable regimen. The route of administration, the vehicle and the dosage levels selected for this study were based on the results of an exposure assessment and dose range

- finding developmental neurotoxicity study in the

same strain of rats (Beck, 2009 ; WIL

- 635001). F 0 females were approximately 13 weeks of age at the beginning of test substance administration

. All animals were observed twice daily for appearance and behavio r. Clinical observations were recorded daily (begin ning on gestation day 0 ) and approximatelyWIL

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1 hour following dose administration during the treatment period. B ody weights and food consumption were recorded at appropriate intervals during gestation and lactation

. In addition, detailed clinical observati ons ( DCO ) were conducted out of the home cage
for all dams in each group on gestation days 10 and 15 and on lactation days 10 and 21

. All F 0 females were allowed to deliver and rear their offspring to lactation day 21, at
which time the dams were euthaniz ed and necropsied. F 0 females that failed to deliver were necropsied on post

- mating day 25.

Clinical observations
, body weights and gender identities were recorded for the F 1 pups at appropriate intervals. On postnatal day (PND) 4, litters were culled (r educed) to 8 pups/litter (4 pups/sex, when possible) . Following litter size reduction
, 1 male and /or
1 female pup per litter were randomly assigned to one of the following evaluation subsets (A

- D) until the requisite sample size was met . Subset A of 30 p ups/sex/group was assigned

to DCO ( PND 4, 11, 21, 35, 45 and 60) , acoustic startle response (PND 20 and 60 ), locomotor activity (PND 13, 17, 21 and 61 ) and learning and memory (PND 62 ). A second subset (Subset B) of 20 pups/sex/group was selected for lear ning and memory (PND 22 )

. A
third subset (Subset C) of 15 pups/sex/group was selected for brain weight evaluations (PND 21), and from within this subset, 10 pups/sex from the control and 1000 mg/kg/day groups were designated for neuropathology and morphom etry evaluations (PND 21). Furthermore, 10 pups/sex/group assigned to Subset A and 5 pups/sex/group assigned to
Subset B (representing as many litters as possible) were further assigned to Subset D for brain weight evaluations ( PND 72 ). Within Subset D, 10 pups/sex from the control and 1000 mg/kg/day groups were designated for neuropathology and morphometry evaluations (PND 72) . In addition, 10 males each from the 1, 10 and 100 mg/kg/day groups that were assigned to Subset D were later selected for PND 7 2 mo rphometry evaluation

. The remaining 20 pups/sex/group in Subset A that were not selected for PND 72 evaluation were assigned to PND 120 and 180 locomotor activity. A nicotine challenge was conducted during the PND 61, 120 and 180 locomotor activity as sessments. A nimals were
administered nicotine (20 [PND 61] or 10

- 11 [PND 120 and 180] pups/sex/group) or salineWIL

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(9

- 10 pups/sex/group [PND 61, 120 and 180] ) following the initial 60

- minute test session, and then subjected to a second 60

- minute test session

. Indicators of physical development (balanopreputial separation and vaginal patency) were
evaluated for F 1 animals assigned to Subsets A and B . All F 1 animals not selected for behavioral evaluations were euthanized and necropsied on PND 21 . F 1 animals s elected for learning and memory assessment on PND 22 , and not selected for PND 72 evaluation, and
F 1 animals selected for locomotor activity assessment on PND 120 and 180 were necropsied following completion of these respective assessments

.
An additional b ioanalytical phase, composed of 2 groups of bred female rats (8 rats/group), was conducted to determine the concentration of the test substance in maternal and neonatal plasma and maternal milk . These females were administered the

test substance daily fro m gestation day 6 through lactation day 4 at the 2 lowest dosage
levels ( 1 and 10 mg/kg/day )

. Clinical observations and body weights for the F 0 females were recorded at appropriate in tervals. Clinical observations, body weights and gender
identities were recorded for the F 1 pups at appropriate intervals. Blood samples were
collected from 4 F 0 females /group and their F 1 litters at 8 hours after dose administration

on lactation day/PND 4

. The remaining 4 F 0 females/group had milk collected 8 hours
followi ng dose administration on lactation day 4. All F 0 dams and F 1 pups were euthanized following sample collection and discarded. One F 0 female in the bioanalytical phase that failed to deliver was euthanized on post

- mating day 25 and discarded without
exami nation

. 

### 1.3. R ESULTS Mean concentrations of the test substance in maternal and pup plasma on lactation day/PND 4 at 10 mg/kg/day were 1700 and 2140 ng/mL, respectively . These
concentrations were similar to those noted i n a previous preliminary range

- finding st udy (Beck, 2009 ; WIL

- 635001), in which maternal and pup plasma concentrations were 1334
to 1812 ng/mL and 1998 to 2535 ng/mL, respectively, at the 3 dosage levels used ( 100, 300 and 1000 mg/kg/day ) . Based on these results, no difference s were apparent whe nWIL

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comparing plasma concentrations ( internal exposure s ) between the external dosage levels
of 10 to 1000 mg/kg/day. M ean concentrations of the test substance in maternal and pup plasma at 1 mg/kg/day (510 and 929 ng/mL, respectively) were at least 3

- and 2

- fold
lower , respectively, than those noted at 10 mg/kg/day . Mean concentrations of the test substance in maternal milk samples at 1 and 10 mg/kg/day on lactation day 4 were 5 09 and 1250 ng/mL, respectively. Th ese values were similar to those noted in th e previous preliminary range

- finding study (Beck, 2009 ; WIL

- 635001) at dosage levels of 100 to
1000 mg/kg/day (574 to 628 ng/mL).

There were no test substance

- related mortalities in the F 0 maternal animals during the study. There were no test substance

- re lated clinical findings noted d uring the daily
examinations or at approximately 1 hour following dose administration. Maternal
d etailed clinical observation parameters, as well as maternal body weights and fo o d consumption during gestation and lactation
, were unaffected by test substance
administration.

There were no maternal test substance

- related differences noted between groups when comparing the mean length of gestation, the process of parturition and the internal macroscopic findings. The mean number s of former implantation sites and
unaccounted

- for sites, as well as the mean number of pups born, live litter size and the percentage of males at birth were similar across all groups.

There were no test substance

- related effects on F 1 postnatal survival, clinical observations, body weights, body weight gains or necropsy findings at any dosage level. In addition, startle responsiveness, swimming ability, learning and memory and attainment of sexual developmental landmarks (balanopreputial separation and va ginal patency) were unaffected by maternal test substance administration. There were no test
substance

- related effects on loco motor activity, which was evaluated on PND 13, 17, 21, 61, 120 and 180.WIL

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There were no test substance

- related effects on brain wei ghts, lengths or widths on
PND 21 and 72. There were no histopathologic al alterations related to maternal test substance administration in PND 21 and PND 72 brains or PND 72 central or peripheral nervous system tissues. There were no differences in morph ometric measurements that
were considered related to test substance administration.



### 1.4. C ONCLUSIONS There was no evidence of maternal toxicity at any dosage level of decabromodiphenyl oxide evaluated in this study . Additionally, there were no effects on offsp ring survival and growth, or on any of the neurobehavioral parameters evaluated in this study. Normal patterns of habituation were observed at all relevant ages tested for both locomotor
activit y and auditory startle response . Therefore, under the condit ions of this study, no evidence of developmental neurotoxicity was observed at any dosage level evaluated

. Thus, the no

- observed

- adverse

- effect level (NOAEL) for both F 0 systemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodipheny l oxide was considered to
be 1000 mg/kg/day, the highest dosage level administered in this study. Based on t he mean plasma concentration of the test substance in F 1 pups at 8 hours following maternal dose administration on lactation day 4 , the internal ex posure at dosage levels between 10
and 1000 mg/kg/day was determined in this study and a previous study (Beck, 2009 ; WIL

- 635001) to be 1998 ng/mL to 2535 ng/mL.WIL

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### 2. I NTRODUCTION 

### 2.1. R ATIONALE FOR 

### S TUDY 

### D ESIGN The study design combined the required elements of both the U.S. EPA OPPTS 870.63 00 (1998) and the OECD 426 (200 7 ) guidelines for conducting a developmental neurotoxicity study , and also included an extended 6

- month post

- treatment period (beginning on postnatal day 21) and an increased offspring neuropathology sample size,
as contained in the U.S. EPA Data Call

- in Notice dated 10 September 1999

. The design specific ally covered the OECD element that proposes conducting all behavioral
evaluations in the same 30 F 1 offspring/sex/group
, except the post

- weaning lea rning and

m emory assessment

. Specific U.S. EPA design elements included maternal and offspring detailed clinical observation ( DCO ) assessments, and the specified ages at which testing for all automated behavioral measures were scheduled. In addition, loc omotor activity at 4 and 6 months of age were conducted and based on a publication (Viberg et al., 2003)
that reported changes in locomotor activity at 2, 4 and 6 months of age in mice following a single oral ( gavage ) dose of decabromodiphenyl oxide (purit y not stated) on PND 3

. 

### 2.2. G ENERAL 

### S TUDY 

### I NFORMATION This report presents the data from “ A n Oral (Gavage) Developmental Neurotoxicity Study of Decabromodiphenyl Oxide in Rats

. ” Due to software spacing constraints, t he study title is presented as “ Oral Dev. N eurotoxicity Study of Decabromodiphenyl Oxide
in Rats ” on the report tables. For clarification of the terminology used to differentiate the generations in the report text, the offspring of the F 0 generation were defined as the F 1 litters; pups from the F 1 litters were selected to constitute the F 1 generation.WIL

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The following computer protocols were used for data collection during the study:
Computer Protocol Type of Data Collected
WIL

- 635002

................................ ..... F 0 maternal and F 1 litter data (including F 0 detailed clinical observations) WIL

- 635002A

................................ .. F 1 male post

- weaning body weight, clinical obse rvation, developmental landmark and necropsy (Subset B and Subset A not selected for PND 72 evaluation) data WIL

- 635002D

................................ .. F 1 female post

- weaning body weight, clinical obse rvation, deve lopmental landmark and necropsy (Subset B and Subset A not selected for PND 72 evaluation) data WIL

- 635002X

................................ .. F 1 Subset A FOB, acoustic startle response, locomotor activity (PND 13, 17 and 21) and learning and memory (PND 62) data WIL

- 635002W

................................ . F 1 Subset B (PN D 22) learning and memory data WIL

- 635002N

................................ .. F 1 Subset A locomotor activity data (PND 61, 120 and 180) WIL

- 635002U

................................ .. F 1 Subset C ( PND 21) necropsy and brain weight/measurement/morphometry data WIL

- 635002V

................................ .. F 1 Subset D ( PND 72 ) necropsy and brain weight/measure ment/morphometry data WIL

- 635002T

................................ ... Bioanalytical phase animal data



### 2.3. K EY 

### S TUDY 

### D ATES 1 Date(s) Event(s)
4 December 2007

................................ .... Experimental starting date (animal receipt) 17

- 21 December 2007

............................ F 0 mating 24 December 2007

................................ .. Experimental start date (initiation of F 0 test
s ubstance administration) 24 December 2007

- 4 February 2008

.... F 0 dose administration (developmental neurotoxicity phase) 29 December 2007

- 18 January 2008

.... F 0 dose administration (bioanalytical phase) 12 January

- 12 July 2 008

....................... F 1 developmental indices and be havioral testing 29 January

- 4 February 2008

................. F 0 lactation day 21/F 1 PND 21 necropsies (including F 1 Subset C necropsies)

1 D ate ranges represent the first and last dates of evaluation.WIL

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### K EY 

### S TUDY 

### D ATES 

### ( CONTINUED 

### ) Date(s) Event(s)
9

- 15 February 2008

................................ F 1 necropsies ( Subset B

- following completion of PND 22 learning and memory assessments ) 20

- 26 March 2008

................................ .. F 1 PND 72 (Subset D) necropsies 18 July 2008

................................ ............ Last F 1 necrops y (following PND 180 motor activity assessment) 20 November 2008

................................ . Experimental termination/ completion date (last morphological examination )WIL

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### 3. S TUDY 

### D ESIGN 

### S CHEMAT IC 

### (K EY 

### P HASES 

### ) Acclimation period

Developmental neurotoxicity phase Bioanalytical phase

F 0 clinical observations, body weights and food consumption recorded at appropriate intervals F 0 clinical observations and body weights rec orded at appropriate

intervals
35 mated females per group ( Groups 1

- 5) treated once daily from gestation day 6 through lactation day 21 8 mated females per group ( Groups 2 and 3) treated once daily from gestation day 6 through lactation day 4

F 0 FOB performed on gestation days 10 and 15 and lactation days 10 and 21 All F 0 females allowed to deliver

Female that failed to deliver euthanized and discarded Gross necropsies performed on F 0 animals that were found dead durin g gestation F 1 offspring observed daily; detailed physical examinations and body weights recorded at appropriate intervals All F 0 females allowed to deliver
Gross necropsies performed on F 0 female s that were found d ead following parturition or failed to deliver Gross necropsies performed on F 1 pups that were found dead prior to weaning F 1 pups potentially exposed to test substance in utero and through nursing during lactation

F 1 offspring observed daily; detailed physical examinations and body weights recorded at appropriate intervals Blood samples for plasma analysis collected from 4 F 0 females/group and their litters at 8 hours following dosing on lactation day 4 ; F 0 f emales euthanized and

carcasses discarded following blood collection Milk samples

collected fr om 4 F 0 females/ group at 8 hours following dosing on lactation day 4 ; F 0 females and F 1 pups euthanized and discarded following sample collection F 1 pup s exposed to test substance in utero and through nursing during lactation

F 0 females necropsied on lactation day 21
Gross necropsies performed on F 1 animals that were found dead following weaningWIL

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### S TUDY 

### D ESIG N 

### S CHEMATIC 

### (K EY 

### P HASES 

### ) ( CONTINUED 

### )

Subset C 15 pups/sex/group selected for in situ perfusion; brain weights and brain measurements on PND 21 50 p ups/sex/group selected for post

- weaning developmental landmarks and/or neuropathological eval uation Nonselected pups necropsied on PND 21

Neuropathological and morphometric evaluations conducted for 1 0 pups/sex in control and high

- dose groups

Subset A 30 rats/sex/group selected for startle response evalu ations on PND 20 and 60; loco motor activity evaluations on PND 13, 17, 21 and 61 (including nicotine challenge) ; FOB evaluations on PND 4, 11, 21, 35, 45 and 60; learning and memory evaluation beginning on PND 62 Subset B 20 rats/sex/group selected for learning and memory evaluations beginning on PND 22

Gross necropsy performed on 15 F 1 Subset B rats/ sex/ group 20 rats/sex/group selected for locomotor activit y evaluations on PND 120 and 180; nicotine challenge f ollowed initial 60

- minute test session 10 rats/sex/group from Subset A and 5 rats/sex/group from Subset B designated to Subset D for in situ perfusion; brain weights and measurements on PND 72
Neuropathological and morphometric evaluations co nducted for 10 females in the control and 1000 mg/kg/day groups and 10 males in the control, 1, 10, 100 and 1000 mg/kg/day groups Gross necropsies on PND 185

- 191WIL

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### 4. E XPER IMENTAL 

### P ROCEDURES 

### - M ATERIALS A ND 

### M ETHODS 

### 4.1. T EST 

### S UBSTANCE A N D 

### V EHICLE 

### 4.1.1. T EST 

### S UBSTANCE 

### I DENTIFICATION The test substance
, decabromodiphenyl oxide , was received on behalf of the S ponsor from Wildlife International, LTD.
, Easton, MD , on 8 March 2007
, as follows:

Identification Quantity Received Physical

Description
Decabromodiphenyl oxide Lot no. 7569 Composite * Exp. date: 15 May 2008 CAS no. 1163

- 19

- 5 [WIL log no. 7373A ] 1 bag Gross weight: 5092.5 g Off

- white powder

* Decabromodiphenyl oxide 7569 composite consisted of equal parts of decabromodiphenyl oxide product from the 3 manufacturers (Albemarle Corporation
, ICL

- IP America, Inc., and Chemtura Corporation).

Documentation regarding the purity and stability of the test substance is on file with the
S ponsor and WIL Research Laboratories, LLC. A Certificat e of Analysis for the test
substance was provided by the S ponsor and is presented in Appendix B . The purity of
the test substance was 97.51 %. The test substance was stored at room temperature, protected from light , and was considered stable under these c onditions. A reserve sample
of the test substance (approximately 1 g) was collected on 21 March 2007 , and stored in the Archives of WIL Research Laboratories, LLC.



### 4.1.2. V EHICLE 

### I DENTIFICATION The vehicle used in preparation of the test substance formulations a nd for administration to the control group was corn oil (lot no. 126K0117 , exp. date s : 2 November 2008,
26 December 2008, 7 January 2009
, and 15 January 2009
, received from Sigma Aldrich, Inc., St. Louis, MO ).WIL

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### 4.1.3. P REPARATION OF 

### V EHICLE AND 

### D OSING 

### F ORMULATIONS
For t he vehicle, a sufficient amount of corn oil was dispensed into a glass container . The
vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations. A liquots from the weekly dispensed vehicle were prepared for daily dispensation to the control group. The vehicle was mixed throughout sampling and dose administration procedures.
Dosing formulations were prepared at the test substance concentrations that are indicate d in the following table:

Group Number Test Substance Dosage Level (mg/kg/day) Test Substance

Concentration (mg/mL) 2 Decabromodiphenyl oxide 1 0.2 3 Decabromodiphenyl oxide 10 2 4 Decabromodiphenyl oxide 100 20 5 Decabromodiphenyl oxide 1000 200

The test substance formulations were weight/volume (test substance /vehicle) mixtures. The appropriate amount of the test substance for each formulation was weighed into a
calibrated glass container. Approximately 70% of the total volume of vehicle was added to each container and the formulation was mixed until uniform using a magnetic stirrer. The formulations were then homogenized using a Silverson L4RT or L4RT

- A electronic homogenizer for approximately 5 minutes. Vehicle was added to each container to br ing
the formulations to the calibration mark.

The test substance formulations were prepared approximately weekly as single
formulations for each dosage level, then divided into aliquots for daily dispensation and
stored refrigerated with protection from li ght until use . The test substance formulations
were stirred at room temperature using a magnetic stirrer for a minimum of 2 hours prior

to use.WIL

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The test substance formulations were visually inspected by the study director on
21 December 2007 , and were fou nd to be visibly homogeneous and acceptable for dose
administration

. 

### 4.1.4. S AMPLING A ND 

### A NALYSIS Prior to the initiation of dose administration
, non

- dosing test substance formulations were prepared on 10 December 2007 at concentrations spanning the range of conc entrations to be used on study (0.2 and 200 mg/mL). D uplicate samples ( 1 mL each) for homogeneity
determination were collected from the top, middle and bottom strata of the se non

- dosing formulations. In addition, an aliquot of each non

- dosing formulation , approximately equivalent in volume to that needed for 1 day of dose administration, was collected and stored refrigerated for 10 days. Fo llowing 10 days of refrigerated storage, the aliquots
were remixed for at least 30 minutes and then duplicate sample s (1 mL each) for stability
and res uspens ion homogeneity were collected on 20 December 2007 from the top and bottom strata of these aliquots. Duplicate s amples ( 1 mL each) for concentration analysis were collected from the middle stratum of each dosing fo rmulation (including the control group) prepared for the first (21 December 2007), middle (4 January 2008) and last (25 January 2008) weeks of dose administration

. All analyses were conducted by the Analytical Chemistry Department, WIL Research
Laboratorie s, LLC , using a validated gas chr omatography method with electron capture detection (Coffee, 2008 ; WIL

- 635004 ) . Details about the methodology and results of these analyses are presented in Appendix C , and the re sults are summarized in
Section 6.1

. 

### 4.2. N ICOTIN E 

### C HALLENGE All F 1 males and females selected for PND 61, 120 and 180 locomotor activity evaluations were given an intraperitoneal injection of nicotine or 0.9% saline following
the initial 60

- minute test session. The nicotine and 0.9% saline formulations were prepared using the following components.WIL

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(

- )

- Nicotine hydrogen tartrate salt (lot no. 057K1521 , CAS no 65

- 31

- 6, received from Sigma Aldrich, Inc., St. Louis, MO ) 0.9% Sodium Chloride for Injection, USP (lot no s

. C719583, C729301, C73265 1, C736066 and C740993 , exp. date s : 1 March 2009, 1 July 2009, 1 August 2 009, 1 September 2009 and 1 November 2009, respectively
, manufactured by Baxter Healthcare Corporation, Deerfield, IL ) For preparation of the 0.9% saline formulation, a sufficient amount of 0.9% so dium chloride for injection was transferred into a sterile glass septum vial on each day of dosing. All containers and utensils were sterilized prior to use and all procedures were
conducted with in a laminar flow hood.
For preparation of the nicotine form ulation, a sufficient amount of nicotine was weighed into a tared, weighing vessel. Approximately 70% of the total volume of 0.9% sodium chloride for injection was added to a calibrated glass container, and the nicotine was quantitatively transferred to t he glass container. The formulation was mixed until uniform using a magnetic stirrer. A sufficient amount of 0.9% sodium chloride for injection was then added to the container to bring the formulation to the calibration mark,
and the formulation was stir red again until uniform using a magnetic stirrer. Within a laminar flow hood, the formulation was sterile

- filtered through a 0.22

- μm PVDF syringe filter and capped with a septum. The nicotine formulation was prepared daily, as needed
, and maintained at r oom temperature

. 

### 4.3. T EST 

### S YSTEM Sexually mature, virgin female Sprague Dawley Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh
, NC , were used as the test system on this study.

The animal model selected, the Sprague Dawley Crl:CD(SD) rat, is reco gnized as appropriate for developmental neurotoxicity studies. WIL Research Laboratories, LLC
has reproductive and neurotoxicity historical control data for the Sprague Dawley Crl:CD(SD) rat. This animal model has been proven to be susceptible to the eff ects of developmental neurotoxicants

.WIL

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### 4.4. O RGANIZATION O F 

### T EST 

### G R OUPS 

### , D OSAGE 

### L EVELS A ND 

### T REATMENT 

### R EGIMEN The test and vehicle control substance formulations were administered orally by gavage
to F 0 females , via an appropriately sized flexible, Teflon ®

- shafte d, stainless steel
b all

- tipped dosing cannula (Natume, Japan), once daily from gestation day 6 to lactation day 4 (bioanalytical phase ; total of 20

- 21 doses ) or 21 (developmental neurotoxicity phase ; tot al of 37

- 39 doses ) . A dosage volume of 5 mL/kg was u sed. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day. The F 0 maternal animals were assigned to study groups as follows:

Develo pmental Neurotoxicity Phase (WIL

- 635002 )
Group Number Test Substance Dosage Level (mg/kg/day) Dosage

Volume (mL/kg) Number of

Females 1 Vehicle 0 5 35 2 Decabromodiphenyl oxide 1 5 35 3 Decabromodiphenyl oxide 10 5 35 4 Decabromodiphenyl oxide 100 5 35 5 Decabromodiphenyl oxide 1000 5 35

Bioanalytical Phase (WIL

- 635002T )
Group Number Test Substance Dosage Level (mg/kg/day) Dosage

Volume (mL/kg) Number of

Females 2 Decabromodiphenyl oxide 1 5 8 3 Decabromodiphenyl oxide 10 5 8



### 4.5. J USTIFICA TION OF 

### D OSAGE 

### L EVEL S AND 

### V EHICLE 

### S ELECTION Dosage levels were selected based o n the results of a previous preliminary range

- finding study in rats (Beck, 2009 ; WIL

- 635001). In that study, pregnant female rats were
administered the test substance using cor n oil as the delivery vehicle from gestation day 6 through lactation day 21 at dosage levels of 100, 300 or 1 000 mg/kg/day. An additional
2 groups of f

emale s received either 0 or 1000 mg/kg/day of the test substance in soyaWIL

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phospholipon:Lutrol (SPL) to co mpare systemic exposure when using corn oil and SPL as the delivery vehicles. Furthermore, 2 groups of pups received a single oral (gavage)
dose of the test substance at 20 mg/kg on PND 4 , using either corn oil or SPL as the delivery vehicle , in order to make a systemic exposure comparison between pups that were directly exposed to the test substance on PND 4 and those pups that were indirectly exposed to the test substance via maternal milk

. All groups that received the test substance in corn oil had clin ical findings of light

- colored feces throughout the dosing regimen. However, there were no test
substance

- related changes in mean maternal body weights, body weigh t gains and food consumption during gestation or lactation at any dosage level. In addition , there were no effects on the process of parturition or on survival and growth of the offspring at any dosage level. Plasma level determinations in dams and fetuses on gestation day 20 and in dams and their pups on lactation day 4 revealed a similar inte rnal exposure when dams received 100, 300 or 1000 mg/k g/day. Thus, the plasma uptake of decabromodiphenyl oxide was basically equivalent for these 3 dosage levels. Furthermore, up to a 20

- fold greater internal exposure was observed when the dams were adm inistered the test
substance in cor n oil rather than SPL. Also, slightly higher plasma concentrations of the

test substance were achieved in PND 4 pups exposed to the test substance via maternal milk compared to those pups that received the test substance directly by oral gavage on
PND 4 , whether the delivery vehicle was corn oil or SPL . Therefore, maternal
administration of the test substance in corn oil was expected to produce the greatest level of exposure to the pups. Because the 3 dosage levels that were used in the previous preliminary range

- finding study exhibited a similar internal dose profile
, 4 dosage levels of 1, 10, 100 and 1000 mg/kg/d ay were selected for use in this study with the intent of establishing an internal dose

- response curve betwe en the treatment groups

. The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans

. The number of animals selected for this studyWIL

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was the minimum required to meet the objectives

. The F 1 animals were exposed to the

test substance in utero, as well as via maternal milk while nursing.



### 4.6. F 0 

### M ATERNAL 

### G ENERATION 

### 4.6.1. A NIMAL 

### R ECEIPT A ND 

### A CCLIMATION Two hundred forty

- one sexually mat ure female Sprague Dawley Crl:CD(SD) rats were
received from Charles R iver Laboratories, Inc., Raleigh
, NC on 4 December 2007 . The
animals were 70 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt and weighed the f ollowing day . Each rat was uniquely identified by a Monel meta l ear tag displaying the animal number and housed for
14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for general changes in appearance and behavior. Body weights were recorded prior to the initiation o f mating.
Sexually mature, untreated male Sprague Dawley Crl:CD(SD) rats from the WIL stock animal colony were used to induce pregnancies. These resident males were received from Charles River Laboratories, Inc., Raleigh, NC on 25 September 2007, 13 Novem ber
2007 or 20 November 2007.



### 4.6.2. A NIMAL 

### H OUSING Upon arrival and until pairing, all animals were individually housed in clean, stainless
steel wire

- mesh cages suspended above cage

- board. The cage

- board was changed at least 3 times per week. The animals were paired for mating in the home cage of the resident male. Following positive evidence of mating (see Section 4.6 .5.) , the females were individually housed in plastic maternity cages with nesting material, ground corncob bedding (Bed

- O'Cobs ® ; The Andersons
, Cob Products Division, Maumee, O H ). The nesting material is periodically analyzed by the manufacturer for contaminants. Analyses of the nesting material are provided by the manufacturer and are presented in Appendix D . No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at
WIL Research Laboratories, LLC. The females were housed in these cages through theWIL

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scheduled day of necropsy. Following weaning on PND 21 (developmental neurotoxicity phase only) , pups were housed by litter in plastic maternity cages with nesting material through PND 27 . On PND 28, surviving offspring were housed individually in suspended wire

- mesh cages until euthanasia. Females that did not deliver were necropsied on post

- mating day 25. Animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996)

. The animal facilities at WIL Research Laboratories, LLC ar e accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).



### 4.6.3. D IET 

### , D RINKING 

### W ATER A ND 

### M AINTENANCE The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent
LabD iet ® 5002, is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, LLC. Feed lots used during the study are documented in the study records. Reserve samples (100 g each) were
collected from e ach lot of feed used during the study. Feeders were changed and sanitized once per week. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are mainta ined at WIL Research Laboratories, LLC, and are presented in Appendix E

. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis

- purified (on

- site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.



### 4.6.4. E NVIRONMENTAL 

### C ONDITIONS All rats were housed throughout the acclimation period and during the study in an
enviro nmentally controlled room. The room environmental controls were set to maintain daily averages of 71 F 5 F (22 C 3 C) for temperature and 50% 20% for relative humidity. Room temperature and relative humidity were controlled and monitored using
the Metasys ® DDC Electronic Environmental control system. These data were recordedWIL

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daily and are summarized in Appendix F . Actual mean daily temperature ranged from
69.8 F to 72.2 F ( 21.0 C to 22.3 C) and mean daily relative humidity ranged from 34.0 %

to 54. 2 % during the study. Light timers were calibrated t o provide a 12

- hour light (0600 hours to 1800 hours)/12

- hour dark photoperiod. The light portion of the photoperiod was extended for 53 minutes on 15 March 2008 to complete the PND 22 learning and memory assessment. Air handling units were set to provide a minimum of
1 0 fresh air changes per hour.



### 4.6.5. A SSIGNMENT O F 

### A NIMAL S TO 

### T REATMENT 

### G ROUPS A ND 

### B REEDING 

### P ROCEDURES At the conclusion of the acclimation period, all available females were weighed and
examined in detail for physical abnormalities. At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight (minimum of 220 g) requirements at the time of cohabitation was placed in a suspended wire

- mesh

cage with a resident male from the same strain and source for mating. Resident males were untreated, sexually mature rats that were utilized exclusively for mating. These rats were maintained under similar laboratory conditions as the females. A breeding rec ord containing the male and female identification numbers and the date of cohabitation was prepared. The selected females were approximately 12 weeks old when paired for mating.
Positive evidence of mating was confirmed by the presence of a vaginal copula tory plug or the presence of sperm in a vaginal lavage and verified by a second technician. Each mating pair was examined dail y. The day on which evidence of mating was identified was termed gestation day 0, and the animals were separated.
The experiment al design for the developmental neurotoxicity phase, WIL

- 635002,
consisted of 4 test substance

- treated groups and 1 control group, composed of 35 rats per group. The bioanalytical phase, WIL

- 635002T, consisted of 2 test substance

- treated groups composed o f 8 rats per group. Following completion of the mating period, the bred females were assigned to groups using a WIL Toxicology Data Management SystemWIL

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(WTDMSTM) computer program which randomized the animals based on stratification of the gestation day 0 bod y weights in a block design

. Body weight values ranged from
224 g to 299 g on gestation day 0.WIL

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### 5. P ARAMETERS 

### E VALUATED 

### 5.1. F 0 

### O BSERVATIONS 

### 5.1.1. C LINICAL 

### O BSERVATIONS A ND 

### S URVIVAL 

### (B OTH 

### P HASES 

### ) All rats were observed twice daily, once in the morning and once in the af ternoon, for moribundity and mortality. Individual clinical observations were recorded daily (prior to

test substance administration during the treatment period) for each F 0 female from gestation day 0 until necrops y. Females in the developmental neuroto xicity phase were also observed daily for signs of toxicity approximately 1 hour following dose
administration throughout the treatment period. The absence or presence of findings w as recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor or other difficulties).



### 5.1.2. D ETAILED 

### C LINICAL 

### O BSERVATIONS 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) All dams in each group were observed outside of the home cage on gestation days 10 and
15 and on lactation days 10 and 21. The following parameters were evaluated (refer to Appendix H for a detailed description of the scoring criteria used for each observation) :
Ease of removal from cage Lac rimation/chromodacryorrhea Piloerection Palpebral closure Red/crusty deposits Eye prominence Mobility Convulsions/tremors Grooming Bizarre/stereotypic behavior General body posture Pupillary response Salivation Fur appearance Respiratory rate/character Muc ous membrane/eye color Muscle tone Gait Arousal Urination/defecation Backing

The FOB utilized at WIL Research Laboratories, LLC is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al.
,WIL

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1991; a nd O’Donoghue, 1989)

. Testing was performed by the same technicians,
when ever possible, without knowledge of the animal’s group assignment.



### 5.1.3. B ODY 

### W EIGHTS 

### (B OTH 

### P HASES 

### ) Individual maternal body weights were measured on gestation days 0, 6, 9, 12, 15, 18 and
20 and on lactation days 1, 4, 7, 10, 14, 17 and 21 for animals in the developmental neurotoxicity phase and on gestation days 0, 6, 9, 12, 15, 18 and 20 and on lactation days 1 and 4 for animals in the bioanalytical phase . Group mean body weights were

c alculated for each of these days. Group mean body weight changes were calculated for each corresponding interval of gestation and lactation, and also for gestation days 6

- 20 and for lactation days 1

- 21 (developmental neurotoxicity phase) . Body weights we re also recorded whenever animals were removed from their cages for behavioral assessment. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculat ed for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.



### 5.1.4. F OOD 

### C ONSUMPTION 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### )
Indi vidual maternal food consumption was recorded on gestation days 0, 6, 9, 12, 15, 18
and 20 and on lactation days 1, 4, 7, 10, 14, 17 and 21 . Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals , except f or the intervals of gestation days 6

- 20 and lactation days 1

- 21

. When food consumption could not be determined for an animal during a given interval (due to a n unscheduled death, weighing error, food spillage, obvious erroneous value, etc.), group mean val ues were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.WIL

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### 5.1.5. P ARTURITION 

### (B OTH 

### P HASES 

### )
All femal es were a llowed to deliver naturally and rear their young to PND 4 or weaning (PND 21). During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. Beginning on the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillbirths and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed

. 

### 5.1.6. M ACROSCOPIC 

### E XAMINATIONS 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) A gross necropsy was performed on F 0 females that died d uring the course of the study. The necropsy consisted of examination of the external surface, all orifices and the cranial,
thoracic
, abdominal and p elvic cavities
, including viscera. The number and location of implantation sites and corpora lutea were recorded. Recognizable fetuses were examined externally , euthanized by an intrathoracic injection of sodium pentobarbital (if necessary) and preserved in 10% neutral

- buffered formalin

. Offspring of the dam that was found dead during lactation were euthanized by an intraperitoneal injection of sodium pentobarbital, examined externally and sexed; the stomach was examined for the presence of milk and the carcass was discarded.
All surviving F 0 females with viable pups on lactation day 21 and those that did not deliver (post

- mating day 25) were euthanized by carbon dioxide inhalation. A gross necropsy was performed for each of these females; the thoracic, abdominal and pelvic cavities were opened and the contents were examined. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded

. The number of
unaccounted

- for implantation sites was calculated by subtracting the number of pups born from the number of implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.WIL

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Tissues were preserved in 10% neutral

- buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The
carcass of each female was then discarded.
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).



### 5.2. F 1 

### L ITTER 

### P ARAMETERS 

### 5.2.1. L ITTER 

### V IABILITY A ND 

### D EATHS 

### (B OTH 

### P HASES 

### ) Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. I ntact offspring dying or sent to necropsy due to the death of the dam during PND 0 to 4 were examined externally and sexed; t he stomachs were e xamined for the presence of milk . A detailed gross necropsy was performed on any pup dying after PND 4; tissues were saved in
10% neutral

- buffered formalin for possible histopathological examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.



### 5.2.2. L ITTER 

### R EDUCTION 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) To reduce variability a mong the litters, 8 pups/litter, and when possible 4 /sex, were randomly selected on PND 4. C ulled pups were weighed, euthanized by intraperitoneal injection of sodium pentobarbital on PND 4 and discar ded. If a litter did not meet the sex ratio criteria ( at least 3 pups /sex), the litter was not used for neurobehavioral or neuropathological evaluation

. 

### 5.2.3. C LINICAL 

### O BSERVATIONS 

### (B OTH 

### P HASES 

### ) Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 11 , 14, 17
and 21 , and thereafter at weekly intervals until necropsy. Each pup in the bioanalyticalWIL

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phase received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.



### 5.2.4. B ODY 

### W EIGHTS 

### (B OTH 

### P HASES 

### ) Pups were individually weighed on PND 1, 4, 7, 11 , 14, 17 and 21 , and thereafter at weekly inte rvals until necropsy . Pups were also weighed whenever they were removed from their cages for behavioral testing. Mean pup weights we re presented by s ex for each litter and by group, except for the following instances . Offspring weights that were recorded when the animals were removed from their cages for behavioral testing are maintained in the study records and are not presented in t his report. Pup s in the bioanalytical phase were individually weighed on PND 1 and 4. W hen body weights could not be recorded for a given interval (due to unscheduled death, weighing error, etc.) group values were calculated for that interval using the a vailable d ata. The time periods when body weight values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.



### 5.2.5. S EX 

### D ETERMINATION 

### (B OTH 

### P HASES 

### ) Pups in the developmental neurotoxicity phase were indivi dually sexed on PND 0 (when possible) , 4, 11 and 21. Pups in the bioanalytical phase were individually sexed on
PND 0 (when possible) and 4.WIL

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### 5.2.6. C ALCULATION O F 

### L ITTER 

### P ARAMETERS 

### (B OTH 

### P HASES 

### ) Litter parameters were defined as follows:

Mean Live Litt er Size = Total no. of viable pups PND 0 No. of litters with viable pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 = (% Per Litter) Sum of (Viable pups per litter on PND 0 or PND 4/No. of pups born per litter) No. of litters/group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable pups per litter at end of interval N/Viable pups per litter at start of interval N) No. of litters/group x 100

Where N = PND 0

- 1, 1

- 4 (Pre

- Selection), 4 (Post

- Selecti on)

- 7,

7

- 14, 14

- 21 or 4 (Post

- Selection)

- 21


### 5.2.7. W EANING A ND 

### S ELECTION 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) Each dam and litter remained together until weaning on lactation day 21. Following
standardization of litters ( culling ) on PND 4 , one male and one fem ale pup from each litter were randomly assigned to one of the following evaluation subsets (A

- C). Selected pups retained the dam number and tattoo mark (e.g., 99999

- 01). Each selected pup was uniquely identified by a metal ear tag displaying the identifi cation number following weaning.
Subset A of 30 pups/sex/group was assigned to DCO (PND 4, 11, 21, 35, 45 and 60), acoustic startle response (PND 20 and 60), locomotor activity (PND 13, 17, 21 and 61) and learning and memory (PND 62). A second subset (Su bset B) of 20 pups/sex/group was selected for learning and memory (PND 22). A third subset (Subset C) of
15 pups/sex/group was selected for brain weight evaluations (PND 21), and from within this subset, 10 pup s/sex from the control and 1000 mg/kg/day gro ups were designated for neuropathology and morphometry evaluations (PND 21). Furthermore, 10 pups/sex/group assigned to Subset A and 5 pups/sex/group assigned to Subset B (representing as many litters as possible) were further assigned to Subset D for bra in weight evaluations ( PND 72 ). Within Subset D, 10 pups/sex from the control and 1000 mg/kg/day groups wer e selected for neuropathology and morphometry evaluations (PND 72)

. In addition, 10 males eachWIL

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from the 1, 10 and 100 mg/kg/day groups that were as signed to Subset D were later selected for PND 72 morphometry evaluation

. The remaining 20 pups/sex/group in Subset A that were not selected for PND 72 evaluation were assigned to PND 120 and 180 locomotor activity. A nicotine challenge was conducted
duri ng the PND 61, 120 and 180 locomotor activity assessments . A nimals were either
administered nicotine (20 [PND 61] or 10

- 11 [PND 120 and 180] pups/sex/group) or saline (9

- 10 pups/sex/group [PND 61, 120, and 180] ) following the initial 60

- minute test sessio n, and then subjected to a second 60

- minute test session. Indicators of physical development (balanopreputial separation and vaginal patency) were evaluated for F 1 animals assigned to
Subsets A and B

. All F 1 animals not selected for behavioral evaluation s were euthanized and necropsied on PND 21 . F 1 animals selected for learning and memory assessment on
PND 22, and not selected for PND 72 evaluation, and F 1 animals selected for locomotor activity assessment on PND 120 and 180 were necropsied following co mpletion of these respective assessments

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The following table summarizes the allocation of offspring to subsets for behavioral tests, brain weights
, neuropathology and morphometry evaluation s

. No. Selected (Subset) Age Evaluation 1 5 /sex/group (C ) a PND 21 Brain weights, N europathology
, and Morphometry 30 /sex/group (A)* PND 4, 11, 21, 35, 45 and 60 DCO

30 /sex/group (A)* PND 20 and 60 Auditory startle 30 /sex/group (A)* PND 13, 17, 21 , 61 , 120 and 180 Locomotor a ctivity b

20/sex/group (B) PND 22 Early Learning and Memory** 30 /sex/group (A)* PND 62 Late Learning and Memory** 1 5 /sex/group (D)*** ; a PND 72 Brain weights, Neuropathology
, and Morphometry

*

- The same pup subset was used for DCO , auditory startle, locomotor a ctivity, and PND 6 2 learning and m emory assessment. Except as directed for learning and memory, the same pup s were evaluated at multiple time points.
**

- Different pups were evaluated on PND 22 and 62.
***

- Ten animals/sex/group assigned to this subset were randomly selected from animals previously allocated to Subset A ( behavioral testing ) and 5 animals/sex/group assigned to this subset were randomly selected from animals previously allocated to Subset B ( learning and memory on PND 22 )

. a

- Fifteen pups/sex/grou p were examined for macroscopic neuropathology and had brain weights recorded. From these animals, microscopic neuropathology and morphometry evaluations were initially performed on 10 pups/sex/group in the control and high

- dose groups. Morphometry evalu ations were subsequently also performed on 10 males from the remaining test substance

- treated groups on PND 72

. b

- On PND 61, 120 and 180, a nicotine challenge followed the initial 60

- minute test session. On PND 61, the animals design ated for PND 72 neuropathology and 10 other animals/sex/group (total of 20 animals/sex/group) were administered nicotine and the remaining 10 animals/sex/group were administered 0.9% saline during the nicotine challenge. On PND 120 and 180, 20 animals/sex/group were

ass e ssed for locomotor activity; 10

- 11 animals/sex/group were admini stered nicotine and 9

- 10 animals/sex/group were administered 0.9% saline during the nicotine challenge. Animals that were administered nicotine on PND 61 were also administered nicotine on P ND 120 and 180.WIL

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### 5.2.8. D EVELOPMENT AL 

### L ANDMARKS 

### , S ENSORY 

### F UNCTION A ND 

### N EUROBEHAVIORAL 

### T ESTING 

### ( D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ; S UBSETS 

### A A ND 

### B) The various investigations described herein were used to asses s the maturation and
behavior of the selected F 1 pups. These procedures were concluded when the oldest pups

were 180 days of age. Cumulative litter values include pups that tested positive for the event but were found dead prior to the end of the testing period.



### 5.2.8.1. B ALANOPREPUTIAL 

### S EPARATION 

### (S UBSET 

### A)
Each ma le pup was observed for balanopreputial separation beginning on PND 35 (Korenbrot et al

. , 1977). The day on which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separ ation was present. Individual body weights were recorded on the day of attainment of this landmark.



### 5.2.8.2. V AGINAL 

### P ATENCY 

### (S UBSET S 

### A AND 

### B) Each female pup was observed for vaginal perforation beginning on PND 25 (Adams et
al

. , 1985). The day on which the vagi nal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Individual body weights were recorded on the day of attainment of this
landmark.



### 5.2.8.3. D ETAILED 

### C LINICAL 

### O BSERVATI ONS 

### (S UBSET 

### A) Thirty pups/sex/group were observed outside of the home cage on PND 4, 11, 21, 35, 45
and 60 by the same technicians, whenever possible , without knowledge o f the animal’s group assignment . The same animals were examined at each interval
, ex cept when a n animal was found dead between PND 4 and 21 . In that case, the animal was replaced with another pup of the same age and sex, if available (littermates were used preferentially when available) . Parameters evaluated were identical to those eval uated
for the F 0 dams (see Section 5.1.2. ), except for p iloerection, fur appearance, palpebral closure, eye color, eye prominence, pupillary response, gait, backing and grooming

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These parameters were not recorded for pups on PND 4 and/or 11 due to the ea rly stages of development (palpebral separation had not occurred, incomplete fur growth or inability to complete sufficient movement to determine gait, backing or grooming). However , skin
color was recorded for pups on each day of evaluation. Furthermore , post

- weaning assessments (PND 21, 35, 45 and 60) included forelimb and hindlimb grip strength

measurement.



### 5.2.8.4. L OCOMOTOR 

### A CTIVITY 

### (S UBSET 

### A) Locomotor activity testing was performed in a ro om equipped with a white

- noise generation system set to operate at 70 ± 10 decibels (db). Locomotor activity was
assessed for 30 rats/sex/ group (1 rat/sex/litter from 30 litters/group) on PND 13, 17, 21 and 61 and 20 rats/sex/group (1 rat/sex/litter from 20 litters/group) on PND 120 and 180

. The same animals were tested a t each interval. Locomotor activity was measured automatically using the Kinder Scientific Motor Monitor System (Kinder Scientific, LLC, Poway, C A ). This personal computer

- controlled system utilizes a series of infrared photobeams surrounding a clear, pl astic rectangular cage to quantify an animal’s locomotor activity. Four

- sided black plastic enclosures were used to surround the clear plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by technician s or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. Each animal was tested separately. Data were collected in 5

- minute epochs (print intervals) and the
test session duration was 60 minutes. These data were compiled as six 10

- minute
subintervals for tabulation.
On PND 61, 120 and 180, rats were temporarily removed from the test chamber (for approximately 1 minute) following the initial 60

- minute test session and subjected to a nicotine challenge

. On PND 61, those animals designated for PND 72 neuropathology ( 10 animals/sex/group) and 10 other animals/sex/group (total of 20 animals/sex/group)
were administere d 0.5 mg/kg nicotine . The r emaining animals ( 10 animals/sex/group )

we re administered 0.9% saline. On PND 120 and 180, only 20 animals/sex/group wereWIL

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assessed for locomotor activity , and therefore 10

- 11 animals/sex/group were administered 0.5 mg/kg nicotine and 9

- 10 animals/sex/group were administered 0.9% saline. Animals scheduled for locomotor activity evaluations on PND 61, 120 and 180 received the same compound (nicotine or 0.9% saline) on each day of testing. All doses were administered as a single intraperitoneal injection at a dosage volume of 1 mL /kg. Following
ad ministration of nicotine or 0.9% saline, animals were immediately returned to the test
chamber for a second 60

- minute locomotor activity evaluation

. Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).



### 5.2.8.5. A COUSTIC 

### S TARTLE 

### R ESPONSE 

### (S UBSET 

### A) An acoustic startle response test wa s performed on 30 rats/sex/group (1 rat/sex/litter from 30 litters/group) on PND 20 and 60 using the Kinder Scientific Startle Response System (Kinder Scientific, LLC, Poway, CA ). The same animals were tested at each interval.
Each isolation chamber was composed of high

- quality laminate and medium

- density fiberboard, and measured 10.9 x 14 x 19.5 inches. Each cabinet was equipped with an internal light, fan, viewing lens and a white

- noise generation system. The animal was placed in a rectangular enclosu re of appropriate size, which was then placed into the isolation cabinet. Each enclosure was equipped with a motion sensor.
Acoustic startle response testing was performed in a room equipped with a white

- noise generation system set to operate at 70 10 d ecibels (db). Each test session consisted of a
5

- minute acclimation period in the test cabinet with a 65 5

- db broadband background
white noise. The startle stimulus for each trial was a 115 5

- db mixed

- frequency noise burst stimulus, approximately 20 milliseconds (ms) in duration. Responses were recorded during the first 100 ms following the onset of the startle stimulus for each trial.
Each test session consisted of 50 trials , with an 8

- second intertrial interval. Startle response data were then an alyzed in 5 blocks of 10 trials each. Startle responseWIL

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measurements obtained were maximum response amplitude (MAX), average response amplitude (AVE) and latency to MAX (T MAX ).



### 5.2.8.6. B IEL 

### M AZE 

### S WIMMING 

### T RIALS 

### (S UBSETS 

### A A ND 

### B)
S wimming ability, as well as learni ng and memory were assessed for 20 and
30 rats/sex/group beginning on PND 22 and 62 , respectively (assigned to Subsets B and A, respectively)
, using a water

- filled 8

- unit T

- maze (Biel, 1940). Animals were placed in the maze and were required to traverse t he maze and escape by locating a submerged platform. For the learning and memory phases, the time required to traverse the maze and the numbers of errors for all trials were recorded. An error was defined as any
instance when an animal deviated from the correct channel with all 4 feet. T esting for
Subset B animals was initiated when each animal was 2 2 days of age ; testing for Subset A animals was initiated when each animal was 62 days of age . Animals tested on PND 22 were not used for the second testing interval on PND 62

. Each testing interval consisted of 3 phases that were conducted over 7 consecutive days. Phase 1 was an evaluation of swimming ability and motivation to escape from the maze

. This was performed on day 1 of the Biel maze procedure. F or this evaluation, animals were placed in a straight channel opposite the escape platform, and the time required for each animal to escape was recorded. Each animal underwent 4 trials to evaluate swimming ability and motivation. During each trial, anima ls were given 2 minutes to complete the task successfully

. Upon completing the tria l , the animal was immediately placed at the starting position for another trial until all 4 trials were complete.
Phase 2 of the Biel maze procedure evaluated sequential le arning. This evaluation was conducted on days 2

- 6 of the Biel maze procedure. Animals were allowed 2 trials per day for 2 days to solve the maze that was designated Path A. Animals were then allowed 2 trials per day for 3 consecutive days to solve the P ath B maze (reverse of P ath A).WIL

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Phase 3 of the Biel maze procedure probed the animal for its memory when challenged to solve the Path A maze once again . This evaluation was conducted on day 7 of the Biel maze procedure. Each animal was allowed 2 trials t o solve the Path A maze.
For each trial in Phases 2 and 3, animals were allowed 3 minutes to solve the maze. If an animal did not escape the maze within the allotted 3 minutes, the animal was placed on the escape platform for up to 20 seconds, then remove d from the maze. The minimum intertrial interval for these phases was 1 hour.
Biel maze data were evaluated as the mean time to escape over all trials for each of the
3 phases (i.e., swimming ability and motivation, sequential learning and memory) of the Biel maze procedure. Also, the numbers of errors committed during Phases 2 and 3 were
evaluated.



### 5.2.9. M ACROSCOPIC A ND 

### M ICROSCOPIC 

### E XAMINATIONS 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) 5.2.9.1. O FFSPRING 

### N OT 

### S ELECTED F OR 

### B EHAVIORAL 

### E VALUATIONS
A ll F 1 animals that were not s elected for behavioral evaluations were euthanized by carbon dioxide inhalation and subjected to gross pathology examinations on PND 21

. The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pel vi c cavities, including viscera . Tissues were retained only as deemed necessary by the gross findings.



### 5.2.9.2. O FFSPRING 

### E UTHANIZED 

### F OLLOWING 

### C OMPLETION OF THE



### PND 22 L EARNING A ND 

### M EMORY 

### A SSESSMENTS 

### (S UBSET 

### B) O R 

### PND 180 L OCOMOTOR 

### A CTIVITY 

### A SSESSMENT 

### (S UBSET 

### A) AND 

### N OT 

### S ELECTED FOR 

### N EUROPATHOLOGY 

### /M ORPHOMETRY 

### / B RAIN 

### W EIGHT 

### M EASUREMENTS Offspring scheduled for euthanasia after completion of the learning and memory
evaluations initiated on PND 22 (Subset B) and following completion of the locomotor activity assessment o n PND 180 (Subset A) , but not allocated to Subset D for neuropathology, morphometry and brain weight measurements, were euthanized byWIL

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carbon dioxide inhalation and subjected to a gross pathology examination . A final clinical observation and body weight we re also recorded for animals in Subset A prior to
euthanasia . The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pel vic cavities, including viscera . Organ and tissue samples were preserved in 10% neutral

- buffered formalin as deemed necessary by the gross findings. The carcass of each animal was then discarded.



### 5.2.9.3. N EUROPATHOLOGY 

### (S UBSETS 

### C A ND 

### D ) 5.2.9.3.1. M ACROSCOPIC 

### E XAMINATION On PND 21, 15 rats/sex/group assigned to Subset C were macroscopicall y examine d for neuropathology. On PND 72 , 10 animal s/sex/group from Subset A and
5 animals/sex/group from Subset B , representing as many litters as possible, were randomly selected from those pups dedicated to behavioral evaluation (locomotor activity, acoustic st artle response an d/or learning and memory tests). The selected
animals from Subsets A and B were reassigned to Subset D and macroscopically examined for neuropathology . All animals were anesthetized by an intraperitoneal injection of sodium pentobarbital and perfused in situ with fixative (4% paraformaldehyde/1.4% glutaraldehyde) as described in the WIL Standard Operating
P rocedures (SOPs) . The whole brains were removed (including olfactory bulbs), weighed and the dimensions (length [ excluding the olfact ory bulbs ] and width) were recorded for all Subset C and D animals . Any abnormal coloration or lesions of the
external brain and the spinal cord were recorded. Brains were collected and retained for microscopic examination from all animals n ecropsied on PND 21 and 72 . In addition, the central and peripheral nervous system tissues from the PND 72 pups (Subset D) were dissected and preserved for microscopic examination.



### 5.2.9.3.2. M ICROSCOPIC 

### E XAMINATION Brains from pups selected for microscopic evaluation on PND 21 and 72 were prepared for a qualitative histopathological examination by embedding them in paraffin and then sectioning and staining with hematoxylin and eosin. Sections from all major brainWIL

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regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain [tectum, tegmentum, cerebral peduncles, central gray matter], cerebellum, pons and medulla oblongata [a full coronal section was prepared]) were examined from the first 10 rats/sex in the control and high

- do se groups that met the
criteria for measurement . The criteria for measurement were based on the quali ty of the perfusion, processing and sectioning, and the degree of homology with tissue sections
from other selected animals

. In addition to the qualitati ve histopathological evaluation, a simple morphometric an alysis, as described in Section 5.2.9.3.3. , was performed

. Specific me asurements taken were defined in the WIL SOPs , but included at least
2 me asurements from the neocortical, hippocampal and cerebe llar areas.
Nervous system tissues from the offspring perfused in situ on PND 72 ( Subset D ) were processed for neuropathological evaluation. The following tissues were microscopically
examined using 10 rats/sex in the control and high

- dose groups:
Spinal cord

- at cervical swellings C 3 - C 7 and at lumbar swellings T 13 - L 4 Trigeminal ganglion/nerves a Lumbar dorsal root ganglion at T 13 - L 4 * Lumbar dorsal root fibers at T 13 - L 4 * Lumbar ventral root fibers at T 13 - L 4 * Cervical dorsal root ganglion at C 3 - C 7 * Cervical dorsal root fibers at C 3 - C 7 * Cervical ventral root fibers at C 3 - C 7 * Sciatic nerves (2) + Sural nerves (2) + Tibial nerves (2) + Peroneal nerves (2) + Optic nerves a Eyes a Skeletal muscle (gastrocnemius)

a = Both processed and evaluated micro scopically

+ = 1 processed for microscopic evaluation (2) = Indicates that 2 sections (1 transverse and 1 longitudinal) of the tissue were evaluated from the right hind leg. The tissues from the left hind leg were collected and preserved for possible fut ure evaluation.
* = 4

- 6 tissues were collected at necropsy; 2 tissues were evaluated microscopically

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The central nervous system tissues were embedded in paraffin, and the peripheral nervous system tissues were embedded in plastic (glycol methacrylate)

. Tissues were prepared for histopathological evaluation by sectioning and staining with hematoxylin and eosin. Neuropathologic examinations of tissues from PND 21 and PND 72 were
conducted by Ann Radovsky, DVM, PhD, DACVP, DABT , WIL Research Laboratories, LLC (Appendix I ).



### 5.2.9.3.3. M ORPHOMETRIC 

### A NALYSIS A q uantit ative examination of the brain was conducted on t he first 10 rats/sex in the control and 1000 mg/kg/day groups on PND 21 and PND 72 (as described in
Section 5.2.9.3.2) . As a result of differences noted betw een males in the control and 1000 mg/kg/day groups on PND 72, a q uantit ative examination of the brain was later
conducted on 10 males in the 1, 10 and 100 mg/kg/day groups that were necropsied on
PND 72. These examinations were conducted using the Pax

- ItTM (MIS, Inc., Franklin Park, IL ) image

- capturing computer system and software (version 4. 0), and included the following.

The s pecific levels that were analyzed, designated as Levels 1, 3 and 5, were defined as
follows: Level 1 was a coronal section of rost ral cerebrum, including caudoputamen,
Level 3 was a coronal section of mid

- cerebrum (cerebral cortex, hippocampal formation, thalamus, etc

. ) and Level 5 was a mid

- saggital section of cerebellum and pons. Levels 1, 3 and 5 corresponded to Plates 11, 33 and 79, respectively, in Paxinos and Watson (1998). Measurements of Levels 1 and 3 were bilateral and were averaged for each
animal. Measurements of Level 5 were not paired since the other half of these tissues were sectioned transversely to enable visu aliz ation of cerebellar nuclei.
Measurements were made on homologous sections to ensure that the dimensions of the regions were comparable. In some cases, there was deformation or irregularity of the desired region as a result of perfusion, processing or sect ioning of tissues

. As a result,WIL

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morphometric analysis could not be made for some regions in a few animals . These

cases are presented as “NA ” ( Not Applicable ) on the individual data tables.



### 5.3. B IOANALYTICAL 

### A NALYSES 

### ( B IOANALYTICAL 

### P HASE 

### ) 5.3.1. B LOOD 

### C OLLECTION
Mat ernal blood samples (approximately 0.5 mL each) were collected from the lateral tail
vein of 4 F 0 females/group on lactation day 4 at 8 hours post

- dose administration. The F 1 pups from these same females were euthanized by decapitation on PND 4 , and blood samples (approximately 0.5 mL each) were collected at the same time maternal blood
was collected . The b lood samples from the F 1 pups were pooled by litter. All blood samples were collected into chilled and uniquely labeled tubes containing lithium hepar in as the anticoagulant. Sam ples were maintained on wet ice and protected fro m light until centrifugation.



### 5.3.2. M ILK 

### C OLLECTION On lactation day 4, milk samples were collected from 4 F 0 females/group (different
females than those used for blood collection) at 8 hours post

- dose administration.
F emales were separated from their litters approximately 5.5 hours prior to milk
collection . F ood jars were maintained with the dams during separation

. A 0.05

- mL injection of oxytocin was subcutaneously administered to e ach dam prior to milk
collection. Milk samples (approximately 0.4 mL each) were c ollect ed using gentle

massage into chilled and uniquely labeled tubes , which were then stored in an ice

- water bath until frozen ( at approximately

- 20°C )

. 

### 5.3.3. S AMPLE 

### P REPARATION A ND 

### A NALYSIS Plasma was isolat ed in a refrigerated centrifuge and transferred to uniquely labeled polypropylene tubes. Plasma and milk samples were stored frozen at approximately

- 20°C until analyzed

. Samples were analyzed by the Analytical Chemistry Depa rtment at WIL Research Laboratories, LLC for the presence and concentration of the test substance using a validated GC/MS method presented inWIL

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Appendix J (O’Lear, 2009 ; WIL

- 635003 ). The results of the bioanalyses are presented in Appendices K and L for the plasma and milk analyses, respectively.



### 5.3.4. E UTHANASIA Except for 1 female in the 10 mg/kg/day group that failed to deliver, a ll surviving F 0 females in the bioanalytical phase were euthanized by carbon dioxide inhalation following sample collection. The bio analytical phase female that failed to deliver was euthanized by carbon dioxide inhalation and discarded without examination and sample collection on post

- mating day 25. All remaining F 1 pups (not used for blood collection) were euthanized by an intraperi toneal injection of sodium pentobarbital. All carcasses
were discarded.



### 5.4. S TATISTICAL 

### M ETHODS All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two

- tailed tests (except as noted otherwise) f or minimum

s ignificance levels of 1% and 5%. This was done by comparing each test
substance

- treated group to the control group. Each mean was presented with the standard deviation (S.D.) , standard error (S.E. ; except for DCO parameters ) and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions that were inherent to the types of software used, the means and standard deviations on the summary and individual tables may differ sligh tly. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Statistical analyses were conducted based on techniques re commended by Holson et al. (2008 ). Comparative statistics were not performed on the data from the bioanalytical phase.
Mean gestation lengths, former implantation sites, unaccounted

- for sites, numbers of pups born, live litter sizes, day of attainment of balanopreputial sepa ration or vaginal patency, body weight on the day of attainment, continuous detailed clinical observations and Biel maze straight channel swim data (Phase 1)
, brain weights and dimensions of F 1WIL

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pups and brain morphometric data were analyzed by Dunnett's te st (Dunnett, 1964) to compare the test substance

- treated groups to the control group. Mean litter proportions (percent per litter) of pup viability and males per litter were subjected to the Kruskal

- Wallis nonparametric ANOVA test to determine intergroup differences (Kruskal and Wallis, 1952). If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance

- treated groups to the control group.
Detailed clinical observation data for each test substance

- treated group that yielded scalar and descriptive data were compared to the control group by the Fisher’s Exact Test (Steel and Torrie, 1980).
Statistical analyses of maternal and offspring body weights and body weight gains,
maternal food consum ption and locomotor activity, acoustic startle response and Biel
maze data (Phases 2 and 3) were performed by BioSTAT Consultants, Inc. , Portage, MI , using the SASTM System (SAS Institute, Inc. 2002

- 2003)

. The results of these analyses are presented in Appendix M

. 

### 5.4.1. L OCOMOTOR 

### A CTIVITY 

### 5.4.1.1. L OCOMOTOR 

### A CTIVITY 

### A CROSS 

### S ESSIONS 

### PND 13, 17 AND 

### 21 Cumulative total and ambulatory counts were analyzed with a repeat ed measures analysis of variance (RANOVA). Factors in the model included PUP and LITTER (random effects), fixed effects for treatment group (TRT), SEX and SESSION, and the following interaction terms: TRT*SEX, TRT*SESSION and SEX*SESSION. If TRT*SEX was significant at the 0.05 level
, the analysis was conducted, by sex, with a RANOVA including factors in the model for PUP (random effect), fixed effects for TRT and
SESSION and the interaction terms TRT*SESSION. The covariance structure for the
RANOVA model (s) was selected by comparing Akaike’s Information Criterion (AIC).
The Kenward

- Rogers adjustment for denominator degrees of freedom was applied. In the final RANOVA model, Dunnett’s test was conducted at the 0.05 significance level to compare each test substance

- treated group with the control. If TRT*SESSION wasWIL

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significant, Dunnett’s test was conducted for each of the sessions. If TRT*SESSION was not significant, Dunnett’s test was conducted across the pooled sessions only.



### 5.4.1.2. L OCOMOTOR 

### A CTIVITY WITHIN 

### S ESSIONS 

### PND 13, 17 AND 

### 21 Total and ambulatory counts from six 10

- minute time intervals were analyzed, by session, with a RANOVA. Factors in the model included PUP and LITTER (random effects), fixed effects for TRT, SEX and time interval (TIME)
, and the f ollowing interaction terms: TRT*SEX, TRT*TIME and SEX*TIME. If TRT*SEX was significant
at the 0.05 level
, the analysis was conducted, by sex, with a RANOVA including factors
in th e model for PUP (random effect), fixed effects for TRT and TIME and t he
inte raction terms TRT*TIME. The covariance structure for the RANOVA models(s) was selected by comparing AIC. The Kenward

- Rogers adjustment for denominator degrees
of freedom was applied. In the final RANOVA model, Dunnett’s test was conducted, at
the 0.05 s ignificance level, to compare each test substance

- treated group with the control.
If TRT*TIME was significant, Dunnett’s test was conducted for each of the time
intervals. If TRT*TIME was not significant, Dunnett’s test was conducted across t he pooled ti me intervals only.



### 5.4.1.3. L OCOM OTOR 

### A CTIVITY WITHIN 

### S ESSIONS 

### PND 61, 120 AND 

### 180 For each session, analyses were conducted separately for the initial 60

- minute test session
and the second 60

- minute test session (nicotine challenge). Total and ambulatory counts
f rom six 10

- minute time intervals were analyzed with a RANOVA. Factors in the model
include d PUP (random effect), fixed effects for TRT, SEX and TIME, and the following interaction terms: TRT*SEX, TRT*TIME and SEX*TIME. If TRT*SEX was significant
at the 0 .05 level
, the analysis was conducted, by sex, with a RANOVA including factors
in th e model for PUP (random effect), fixed effects for TRT and TIME and t he
interaction terms TRT*TIME. The covariance structure for the RANOVA model(s) was selected by compar ing AIC. The Kenward

- Rogers adjustment for denominator degrees
of freedom was applied. In the final RANOVA model, Dunnett’s test was conducted, at the 0.05 significance level, to compare each test substance

- treated group with the control.WIL

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If TRT*TIME wa s significant, Dunnett’s test was conducted for each of the time
intervals. If TRT*TIME was not significant, Dunnett’s test was conducted across the pooled time intervals only.



### 5.4.2. A COUSTIC 

### S TARTLE 

### R ESPONSE Each analysis endpoint was analyzed with a RANOVA. Factors in the model include d random effects for PUP and LITTER (PND 20 only), fixed effects for TRT, SEX and trial block (TRIAL), and the following interaction terms: TRT*SEX, TRT*TRIAL and
SEX*TRIAL. If TRT*SEX was significant at the 0.05 level
, the ana lysis was conducted, by sex, with a RANOVA including factors in th e model for PUP (random effect), fixed
effects for TRT and TRIAL and the interaction terms TRT*TRIAL. The covariance structure for the RANOVA model(s) was selected by comparing AIC. The
Ke nward

- Rogers adjustment for denominator degrees of freedom was applied. In the final RANOVA model, Dunnett’s test was conducted, at the 0.05 significance level, to compare each test substance

- treated group with the control. If TRT*TRIAL was significant, Dunnett’s test was conducted for each of the trial blocks. If TRT*TRIAL

was not significant, Dunnett’s test was conducted across the pooled trial blocks only.



### 5.4.3. B IEL 

### W ATER 

### M AZE
L atency and the number of errors for Learning Paths A and B and Memory Path A we re statistically analyzed individually.
Analyses were conducted with a RANOVA. Factors in the model include d random
effects for PUP and LITTER ( PND 22 only ), fixed effects for TRT, SEX and TRIAL, and the following interaction terms: TRT*SEX, TRT*TRIAL and SEX*TRIAL. I f
TRT*SEX was significant at the 0.05 level, the analysis was conducted, by sex, with a RANOVA including factors in th e model for PUP (random effect), fixed effect s for TRT
and TRIAL and the interaction terms TRT*TRIAL. The covariance struct ure for the RANOVA model(s) was selected by comparing AIC. The Kenward

- Rogers adjustment for denominator degrees of freedom was applied. In the final RANOVA model,
Dunnett’s test was conducted, at the 0.05 significance level, to compare each testWIL

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substan ce

- treated group with the control. If TRT*TRIAL was significant, Dunnett’s test

was conducted for each of the trial blocks. If TRT*TRIAL was not significant, Dunnett’s

test was conducted across the pooled trial blocks only.



### 5.4.4. P UP 

### B ODY 

### W EIGHTS Pup body weig hts for the pre

- cull (PND 1 and 4), post

- cull (PND 4, 7, 11, 14, 17 and 21)
, and post

- wean ing ( weekly from PND 28 through 181 ) phases were analyzed separately for each phase with a RANOVA. Factors in the model include d PUP and LITTER (random effects), fix ed effects for TRT, SEX and study day (TIME), and the following interaction terms: TRT*SEX, TRT*TIME and SEX*TIME. If TRT*SEX was significant
at the 0.05 level
, the analysis was conducted, by sex, with a RANOVA including factors in the model for PUP (rand om eff ect), fixed effects for TRT and TIME and the
interaction terms TRT*TIME. The covariance structure for the RANOVA model(s) was selected by comparing AIC. The Kenward

- Rogers adjustment for denominator degrees
of freedom was applied. In the final RAN OVA model, TRT*TIME was evaluated and, if not significant, Dunnett’s test was used to compare each test substance

- treated group with the control across the pooled study days. If TRT*TIME was significant, Dunnett’s test

was conducted, within the framework of the RANOVA, for each of the study days.
Dunnett’s test was conducted a t the 0.05 significance level.
The following pup body weight gains were analyzed with the RANOVA model and methodology described above for body weights: pre

- weaning postnatal days (1

- 4, 4

- 7,
7

- 11, 11

- 14, 14

- 17 and 17

- 21) and post

- weaning postnatal days (weekly from days 28 through 181).



### 5.4.5. M ATERNAL 

### B OD Y 

### W EIGHTS AND 

### F OOD 

### C ONSUMPTION Body weight s and food consumption for gestation d ays 6 to 20 and lacta tion d ays 1 to 21

were analyzed separ ately with a RANOVA. Factors in the model include d ANIMAL (random effect), fixed effec ts for TRT and study day (TIME) and the TRT*TIME
interaction term. The covariance structure was selected by comparing AIC. The
Kenward

- Rogers adjustment for denominato r degrees of freedom was applied. In theWIL

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final RANOVA model, Dunnett’s test was conducted, at the 0.05 significance level, to compare each test substance

- treated group with the control. If TRT*TIME was significant, Dunnett’s test was conducted for each o f the gestation or lactation days. If
TRT*TIME was not significant, Dunnett’s test was conducted across the pooled gestation or lactation days only.
The following maternal body weight gains were analyzed with the RANOVA model and methodology described abo ve for body weights: gestation da y s (0

- 6, 6

- 9, 9

- 12, 12

- 15,
15

- 18 and 18

- 20) and lactation day s (1

- 4, 4

- 7, 7

- 10, 10

- 14, 14

- 17 and 17

- 21).
Total maternal weight gain during the gestation treatment period (gestation days 6

- 20) and lactation (days 1

- 21) were analyzed, individually, with an ANOVA, including a factor in the model for treatment. Pairwise comparisons were made for each individual treated group with the control group through Dunnett’s test at the 0.05 significance level.



### 5.5. D ATA 

### R ETENTION The S ponsor has title to all documentation records, raw data, specimens or other work product generated during the performance of the study. All work product generated by WIL Research Laboratories, LLC, including raw paper data and specimens, are retained
in the Arc hives at WIL Research Laboratories, LLC, as specified in the study protocol. Appropriate supporting documentation for statistical analyses conducted and reported by BioSTAT Consultants, Inc., is maintained in the Archives at W IL Research Laboratories,
LLC.
The reserve sample of the test substance , pertinent electronic storage media and the original final report are retained in the Archives at WIL Research Laboratories, LLC in compliance with regulatory requirements.WIL

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### 6. R ESULTS AND 

### D ISCUSSION Data tables from t he WIL Historical Control database have been included for comparison purposes. The appendix location of these tables is specified in the appropriate section for each endpoint

. 

### 6.1. A NALYTICAL 

### C HEMISTRY Analytical Chemistry Report: Appendix C

Results of the an alyses of dosing formulations are summarized below.
Text Table 1. Results of Homogeneity and Stability Analyses Group 2 (0.2 mg/mL) Group 5

(200 mg/mL) Homogeneity Assessment of the 10 December 2007 Non

- Dosing Formulations Mean Concentration (mg/mL) 0. 193 200 RSD (%) 5.9 5.5 Mean % of Target 96.3 99.8

10

- Day Refrigerated Stability/ Resuspension Homogeneity Assessment of the 10 December 2007 Non

- Dosing Formulations Mean Concentration (mg/mL) 0.180 186 RSD (%) 2.7 6.1 Mean % of Target 90.2 93.2 Mean % of Time Zero 93.6 93.4

Text Table 2 . Results of Concentration Analyses Mean Concentration, mg/mL (% of Target)

Date of Preparation Group 2 (0.2 mg/mL) Group 3

(2 mg/mL) Group 4

(20 mg/mL) Group 5

(200 mg/mL)

21 December 2007 0.197 (98.6) 1.94 (97. 0) 19.8 (99.0) 202 (101)
4 January 2008 0.172 (86.2) 1.77 (88.5) 18.6 (93.0) 187 (93.3)
25 January 2008 0.171 (85.5) 1.74 (86.8) 18.2 (91.1) 187 (93.5)WIL

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Based on these results, the analyzed test substance dosing formulations (preparations)
were within t he acceptable ranges specified in the WIL Research Laboratories, LLC’s
SOPs for suspensions, which are 85% to 115% of the target concentration and an RSD of 10% or less for resuspension homogeneity. Furthermore, test substance formulations

were determined to be stable for 10 days under refrigerated storage

. Therefore, t hese
results confirm that the protocol

- specified dosages of test substance were administered to
the animals.



### 6.2. B IOANALYTICAL 

### R ESULTS 

### (B IOANALYTICAL 

### P HASE 

### ) Individual Animal Data: Appendix G
Plasma Bio an a lytical Report: Appendix K
Milk Bioan a lytical Report: Appendix L
One female in the 10 mg/kg/day group failed to deliver and was euthanized and discarded on post

- mating day 25 . T herefore, milk samples were only collected from 3 females in
th e 10 mg/kg/day group on lactation day 4

. All other females in the bioanalytical phase were gravid and survived to the scheduled euthana sia.
The measured concentrations of the test substance in maternal and pup plasma on lactation day/PND 4 following mater nal administration of the test substance from gestation day 6 to lactation day 4 are presented below. All samples were collected approximately 8 hours post

- dose administration

.
Text Table 3

. Concentrations of the Test Substance in Maternal and Pup Plasma

Dosage Level (mg/kg/day) Day of Sampling Mean Concentration (ng/mL) Standard

Deviation Number of

Samples Analyzed (n) 1 Lactation Day 4 510 89 4 10 Lactation Day 4 1700 540 4 1 PND 4 929 124 4 10 PND 4 2140 257 4WIL

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Text Table 4

. Concentrations of t he Test Substance in Maternal Milk Samples

Dosage Level (mg/kg/day) Day of Sampling Mean Concentration (ng/mL) Standard

Deviation Number of

Samples Analyzed (n) 1 Lactation Day 4 509 164 4 a 10 Lactation Day 4 1250 195 3 b

a = Two of the 4 samples analyz ed were below the lower limit of quantitation (250 ng/mL), and thus were not included in the calculation of the mean. b = Only 3 milk samples were collected because 1 female was nongravid.

In a previous preliminary range

- finding study (Beck, 2009 ; WIL

- 635 001), maternal and

pup plasma concentrations of the test substance (1334 to 1812 ng/mL and 1998 to
2535 ng/mL, respectively) were determined to be similar for dosage levels of 100, 300 or 1000 mg/kg /day . In this study, mean maternal and pup plasma concent ration s of the test
substance were 1700 and 2140 ng/mL, respectively, at approximately 8 hours following
the maternal dose administration of 10 mg/kg/day on lactation day 4 /PND 4. These plasma concentrations were found to be within a similar range of the concentrations noted in the previous preliminary range

- finding study at dosage levels of 100, 300 and
1000 mg/kg/day (Beck, 2009; WIL

- 635001) . Therefore, it was concluded that there is no
difference in plasma concentrations ( internal exposure ) with the ex ternal dosage levels of 10 to 1000 mg/kg/day

. M ean concentration s of the test substance in maternal and pup plasma at approximately 8 hours following maternal dose administration at 1 mg/kg/day ( 510 and 929 ng/mL , respectively ) were at least 3

- and 2

- fold lower , respectively, than
the mean conce ntrations noted at 10 mg/kg/day

. Maternal milk concentrations of the test substance on lactation day 4 ranged from 574 to 628 ng/mL for external dosage levels of 100, 300 and 1000 mg/kg/day in a previous preliminary range

- finding study (Beck, 2009 ; WIL

- 635001). M aternal milk
concentratio ns at 1 and 10 mg/kg/day in this study were 509 and 1250 ng/mL, respectively
, at approximately 8 hours following maternal dose administration on lactation day 4 . H owever, there were mi lk samples from 2 dams in the 1 mg/kg/dayWIL

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group with concentrations that were below the lower limit of quantit ation (250 ng/mL). These milk samples were not included in the calculation of the mean.



### 6.3. F 0 

### M ATERNAL 

### G ENERATION 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P H ASE 

### ) 6.3.1. C LINICAL 

### O BSERVATIONS A ND 

### S URVIVAL Summary Data: Tables 1, 2, 3
Individual Data: Appendix A

- Tables A1, A2

There were no test substance

- related mortalities among the F 0 maternal animals. Six

non

- test substance

- related deaths occurred during the st udy. One , 1 and 4 maternal females in the 10, 100 and 1000 mg/kg/day groups, respectively, were found dead during the study. Female no. 90542 in the 10 mg/kg/day group was found dead on gestation day 15. Female no. 90557 in the 100 mg/kg/day group was f ound dead on gestation day 17 . F emale nos. 90551, 90508 and 90502 in the 1000 mg/kg/day group were f ound dead on gestation day 18 and female no. 90503 in this same group was found dead on lactation day 1. N o remarkable clinical findings were n oted for an y of these females.

Out of the 6 deaths noted above , 3 of the deaths (female nos. 90557, 90551 and 90508)
occurred shortly after dosing on the same date, and 1 other death (female no. 90502) occurred prior to dose administration on the following date

. E vi dence of possible
int ubation error (foamy contents in the esophagus, red discoloration of the lungs and/or lungs not fully collapsed) was noted at necropsy for all of these females ; therefore, these
4 deaths were not cons idered test substance

- related. The re were no macroscopic findings observed for the other 2 females (nos. 90542 and 90503) that were found dead, and thus
these deaths were also not considered to be test substance

- related.

All other F 0 animals in the control, 1, 10, 100 and 1000 mg/kg/day gr oups survived to the scheduled necropsy on lactation day 21 or post

- mating day 25 . No test substance

- related clinical findings were noted at the daily examinations or approximately 1 hour following
dose administration . Findings that were noted in the tes t substance

- treated groups ,WIL

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including hair loss on various body surfaces, mucoid feces and red or clear material around the eye s , nose and mouth , occurred infrequently and/or at similar frequencies in the control group , and therefore were considered to be unrelated to test substance
administration

. Furthermore, these particular clinical findings are very common in this
strain of rat.



### 6.3.2. P REGNANCY 

### S TATUS Summary Data: Table 1 Reproductive Historical Control Data: Appendix N
The pregnancy rates in the control
, 1, 10, 100 and 1000 mg/kg/day groups were 97.1%, 97.1%, 97.1%, 100.0% and 97.1% , respectively. One female each in the control, 1, 10 and 1000 mg/kg/day groups failed to deliver and was determined to be nongravid at

necropsy.


### 6.3.3. D ETAILED 

### C LINICAL 

### O BSERVATIO NS Summary Data: Tables 4, 5
Individual Data: Appendix A

- Tables A3, A4 Positive Control Data: Appendix O (Study n os. 99199, 99263, 99310)
No remarkable differences were apparent between the control and test substance

- treated groups when the detailed c linical observation data were evaluated for gestation days 10
and 15 and lactation days 10 and 21. The mean grooming count in the 10 mg/kg/day group F 0 females was significantly (p<0.05) higher than that in the c ontrol group on gestation day 15 . H owever, because the magnitude of difference was small (0.1 counts)
and because the mean grooming counts at 100 and 1000 mg/kg/day were similar to the control group value , the higher count in the 10 mg/kg/day group was not considered to be
test substance

- related. Thus, no test substance

- related findings were observed for F 0 females regarding the detailed clinical observations.WIL

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### 6.3.4. B ODY 

### W EIGHTS 

### 6.3.4.1. G ESTATION Summary Data: Tables 6, 7 ; Figure 1
Individual Data: Appendix A

- Tables A5, A6

Mean F 0 maternal body weights and body weight gains were unaffected by test substance
administration during gestation. Differences between the control, 1, 10, 100 and
1000 mg/kg/day groups were slight and not statistically significant.



### 6.3.4.2. L ACTATION Summary Data: Tables 8, 9 ; Figure 2
Indiv idual Data: Appendix A

- Tables A7, A8

Mean F 0 maternal body weights and body weight gains were unaffected by test substance
administration during lactation. Differences between the control, 1, 10, 100 and
1000 mg/kg/day groups were slight and not statis tically significant.



### 6.3.5. F OOD 

### C ONSUMPTION 

### 6.3.5.1. G ESTATION Summary Data: Tables 10, 11
Individual Data: Appendix A

- Tables A9, A10

Mean F 0 maternal food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test substance administration during ges tation. Differences between the control, 1, 10, 100 and 1000 mg/kg/day groups were slight and not statistically significant.



### 6.3.5.2. L ACTATION Summary Data: Tables 12, 13
Individual Data: Appendix A

- Tables A11, A12

Mean F 0 maternal food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test substance administration during lactation . Differences between theWIL

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control, 1, 10, 100 and 1000 mg/kg/day groups were slight and not statistically significant.



### 6.3.6. G ESTATION 

### L ENGTH A ND 

### P ARTURITION Summary Data: Table 14
Individual Data: Appendix A

- Table A13 Reproductive Historical Control Data: Appendix N

No test substance

- related effects were noted on mean gestation lengths or the process of parturition at any dosage level . Mean F 0 gestation lengths in the test substance

- treated groups were similar to the control group value. Differences were slight and not statistically significant. The mean gestation lengths in the 1, 10, 100 and 1000 mg/kg/day

groups were 21.9, 21.9, 22.0 and 21.7 days, respecti vely, compared to a mean gestation length of 21.9 days for both the concurrent control group and the WIL historical control data. No signs of dystocia were noted at any dosage level

. 

### 6.3.7. F 0 

### M ACROSCOPIC 

### E XAMINATIONS 

### 6.3.7.1. U NSCHEDULED 

### D EATHS Summary Data: Table 15
I ndividual Data: Appendix A

- Table A14
One, 1 and 4 females in the 10, 100 and 1000 mg/kg/day groups, respectively, were found dead during the study. Female no. 90542 in the 10 mg/kg/day group was found dead on gestation day 15. This female had no macro scopic findings at necropsy and was determined to have 17 normally developing implantations in utero. Female no. 90557 in the 100 mg/kg/day group was found dead on gestation day 17, and female nos. 90551, 90508 and 90502 in the 1000 mg/kg/day group were f ound dead on gestation day 18. Macroscopic findings for these 4 females were indicative of possible intubation error, consisting of foamy contents in the esophagus, dark red discoloration of the lungs and/or lungs not fully collapsed

. Female no. 90503 in the 1000 mg/kg/day group was found dead on lactation day 1 after delivering 10 live pups the previous day . During theWIL

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macroscopic examination, this female was determined to have 4 d ead fetuses retained in

utero.
When the deaths that likely resulted from intubation error were excluded, only single deaths were noted in the 10 and 1000 mg/kg/day groups . N o macroscopic findings were
observed for either of these females that revealed the cause of death. T herefore, these
deaths at 10 and 1000 mg/kg/day were n ot considered test substa nce

- related.



### 6.3.7.2. F EMALES THAT 

### F AILED T O 

### D ELIVER Summary Data: Table 16
Individual Data: Appendix A

- Table A15

One f emale each in the control, 1 , 10 and 1000 mg/kg/day groups ( nos. 90648, 90529,
90546 and 90526 , respectively) failed to deliver and was determined to be nongravid at necropsy. Female nos. 90546 and 90526 in the 10 and 1000 mg/kg/day groups, respectively, had clear fluid in both uterine horns; female no. 90526 also had a cystic ovary. No other internal findings were not ed at necropsy for females that failed to
deliver.



### 6.3.7.3. F EMALES 

### - L ACTATION 

### D AY 

### 21 Summary Data: Table s 17 , 18
Individual Data: Appendix A

- Table s A16 , A17 Reproductive Historical Control Data: Appendix N

N o test substance

- related internal findings were obs erved at any dosage level during the scheduled necropsy on lactation day 21

. The only recorded internal macroscopic findings
observed in th e test substance

- treated groups were dark red areas in the thymus for female no. 90692 in the 1 mg/kg/day group and an absent digit for female no. 90515 in the 10 mg/kg/day group. Because both of these findings occurred in a single animal and
in a manner that was not dose

- related , they were not attribued to test substance
administration

. In addition, dark red areas in the thymus were also noted for 1 female (no. 90612) in the control group. The only other internal findings observed were clearWIL

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fluid and dark red contents in the uterus for female no. 90699 and a firm liver for female
no. 90571 in the control group. The re were no test substance

- related effects on the number of former implantation sites and the number of unaccounted

- for sites

. D ifferences between the control and test substance

- treated groups were slight and not statistically significant.



### 6.4. F 1 

### L ITTER 

### D ATA 

### ( D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ) 6.4.1. PND 0 L ITTER 

### D ATA A ND 

### P OSTNATAL 

### S URVIVAL Summary Data: Tables 19, 20
Individual Data: Appendix A

- Tables A18, A19, A20 Reproductive Historical Control Data: Appendix N
The mean number of pups born, live litter size, p ercentage of males per litter at birth and postnatal survival between birth and PND 0 (relative to number born), PND 0

- 1, 1

- 4 (pre

- selection), 4 (post

- selection)

- 7, 7

- 14, 14

- 21, as well as from birth to PND 4 (pre

- selection) and PND 4 (post

- selection) to P ND 21 were unaffected by F 0 maternal test substance administration at all dosage levels . Differences from the control group were slight, not statistically significant
, and/or did not occur in a dose

- related manner.



### 6.4.2. G ENERAL 

### P HYSICAL 

### C ONDITION Summary Data: Table 21
Individual Data: Appendix A

- Table A21

The numbers of F 1 pups found dead and/or missing, as well as t he general physical
condition of all F 1 pups in this study were unaffected by F 0 maternal test substance
administration. The numbers of p ups that were found dead were 15, 4, 17, 25 and 28 in the control, 1, 10, 100 and 1000 mg/kg/day groups, respectively. One, 2, 1 and 7 pups in
the control, 1, 10 and 1000 mg/kg/day g roups , respectively, were missing and presumed
to have been cannibalized.WIL

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### 6.4.3. O F FSPRING 

### B ODY 

### W EIGHTS Summary Data: Tables 22, 23 ; Figure 3 Individual Data: Appendix A

- Tables A22, A23 Reproductive Historical Control Data: Appendix N

Mean F 1 pup body weights and body weight changes in the 1, 10, 100 and
1000 mg/kg/day group litters were unaffected by test substance administration to the F 0 maternal a nimals throughout the postnatal period (prior to and following culling) . Mean pup body weight gains in the 100 mg/kg/day group were significantly (p<0.05 or p<0.01) higher than the cont rol group values prior to culling ( PND 1

- 4 ) and during PND 4

- 7 and
PND 17

- 21. In addition, mean pup body weights were significantly (p<0.05 or p<0.01) higher in the 100 mg/kg/day group on PND 4 (prior to culling) and in the 10 and
100 mg/kg/day groups on PND 21. However, because slightly higher pup weights are not considered to be toxicologically meaningful and mean pup body weights and body weight gains in the 1000 mg/kg/day group were similar to the control group, the higher pup body weights and /or body weight gains in the 10 and 100 mg/kg/day group s were not attributed

to F 0 maternal test substance administration.



### 6.4.4. N ECROPSIES OF 

### P UPS 

### F OUND 

### D EAD Summary Data: Table 24
Indiv idual Data: Appendix A

- Table A24
The numbers of pups (litters) found dead from PND 0 through PND 21 were 15 ( 10 ), 4 ( 4 ),
17 ( 9 ) , 25(9) and 28(16) in the control, 1, 10, 100 and 1000 mg/kg/day groups, respectively. N o internal findings that could be attributed to F 0 maternal test substance
administration were noted at the necropsies of the aforementioned pups that were found
dead.WIL

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### 6.4.5. N ECROPSIES OF 

### PND 21 P UPS 

### N OT 

### S ELECTED FOR 

### N EUROPATHOLOGICAL 

### E VALUATIONS OR 

### P UPS 

### E UTHANIZED DUE TO 

### D EATH OF 

### D AM Summary Data: Table 25 Individual Data: Appendix A

- Table A25

N o internal findings were found t hat could be attributed to F 0 maternal test substance
administration during the necropsy of pups euthanized on PND 21 or earlier (due to death
of the dam)

. Findings that occurred only in single animals or without relationship to

mater nal test substance ad ministra tion were d ilated renal pelves for 3, 1, 2 and 4 pups in the 1, 10, 100 and 1000 m g/kg/day groups, respectively, dark red discoloration of the right ma n dibular salivary gland for pup no. 90522

- 01 in the 1 mg/kg/day group
, and dark
red areas on the thymus and a small left testis for pup nos. 90509

- 02 and 90553

- 12, respectively, in the 1000 mg/kg/day group

. 

### 6.5. F 1 

### G ENERATION 

### 6.5.1. C LINICAL 

### O BSERVATIONS A ND 

### S URVIVAL Summary Data: Tables 26, 27
Individual Data: Appendix A

- Tables A26, A27
Following weaning of the F 1 pups, 1 male each in the control and 1000 mg/kg/day groups and 1 female in the 1 mg/kg/day group were found dead

. Male no. 90485

- 01 in the
1000 mg/kg/day group (Subset A / Subset D) was found dead while in the motor activity chamber during the second 60

- minute test session on PND 61 following administration of
nicotine

. Male no. 90585

- 05 in the control group (Subset A) and female no. 90601

- 10 in
the 1 mg/kg/day group (Subset A) were found dead on PND 87 and 124, respectively.
There were no clinical findings noted for these animals at the weekly examinations and no internal findings were observed at necropsy. The single deaths in the 1 and
1000 mg/kg/day groups were not considered test substance

- related because mortality was
also noted in the control group and no deaths were noted for F 1 animals in the 10 and
100 mg/kg/day groups

. All other F 1 animals survived to the scheduled necropsies.WIL

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There were no test substance

- related clinical findings at the weekly examinations of the
F 1 animals after PND 21

. Clinical findings noted in all groups , including the control

group, included scabbing and hair loss on various body surfaces and red material around the eyes and nose . T hese findings are commonly observed for laboratory rats of this
strain

. A soft mass in the lateral facial area was noted for female no. 9052 4

- 11 in the
100 mg/kg/day group on the day of scheduled euthanasia
, and a hard mass in the left inguinal area was noted for female no. 90656

- 06 in the 1000 mg/kg/day group beginning 6 weeks prior to the scheduled euthanasia. Because these masses were only noted in single animals
, they were not considered test substance

- related.



### 6.5.2. B ODY 

### W EIGHTS Summary Data: Tables 28, 29 ; Figure 4 Individual Data: Appendix A

- Tables A28, A29, A30, A31

Mean weekly bod y weights and body weight changes in the F 1 males and females following weaning were unaffected by F 0 maternal test substance administration

. All mean weekly body weights and body weight gains in the 1 and 10 mg/kg/day groups were similar to the control g roup values. The only significant (p<0.05) difference in F 1 mean weekly body weight or body weight gain in the 100 mg/kg/day group was a transient reduction in mean body weight gain from PND 42

- 49. In the 1000 mg/kg/day

group, m ean weekly body weights we re slightly lower than the control gro up at the beginning of the post

- weaning period (PND 28 ) and remained lower (decrease ranging from 3.8% to 5.4%) through PND 63. T he differences in mean weekly body weight s were significant (p<0.05 or p<0.01) from PND 42

- 63 as a result of a significantly (p<0.01) lower mean body weight gain in the 1000 mg/kg/day group from PND 28

- 35 and a slightly (not statistically significant) lower mean body weight gain in this group
from PND 35

- 42 . M ean weekly body weight gains in this group were similar to the control group throughout the remainder of the study. Because blood plasma concentrations displayed no internal exposure difference s between external dosage levels
of 10 to 1000 mg/kg/day , and because weekly mea n body weights at 10 andWIL

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100 mg/kg/day were generally similar to or slightly higher than the control group, the slight transient differences in mean body weights an d body weight gains noted in the
1000 mg/kg/day group were not attributed to maternal test substance admin istration.



### 6.5.3. D EVELOPMENT AL 

### L ANDMARKS 

### , S ENSORY 

### F UNCTION A ND 

### B EHAVIORAL 

### T ESTING 

### 6.5.3.1. B ALANOPREPUTIAL 

### S EPARATION Summary Data: Table 30
Individual Data: Appendix A

- Table A32 Reproductive Historical Control Data: Appendix N
Mean ages of attainment of balanoprepu tial separation and mean body weights at the age
of attainment were unaf

fected by F 0 maternal test substance administration . The mean ages of attainment of balanopreputial separation were 43.2, 43.6, 43.7 and 44.2 days in the 1, 10, 100 and 1000 mg/kg/day groups, respectively, compared to 43.0 days in the concurrent control group and 44.9 days in the WIL historical control data. Mean body weights recorded at the age of attainment were 239.9 g , 249.1 g, 241.7 g and 238.5 g in the same respective groups com pared to 241.3 g in the concurrent control group and 228.3 g in the WIL historical control data. Any differences noted were slight and not statistically significant from the concurrent control group.



### 6.5.3.2. V AGINAL 

### P ATENCY Summary Data: Table 31
Individual Data : Appendix A

- Table A33 Reproductive Historical Control Da ta: Appendix N
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by F 0 maternal test substance administration . The mean ages
of attainment of vaginal patency were 32.4, 31.9, 32. 1 and 32.3 days in the 1, 10, 100 and 1000 mg/kg/day groups, respectively, compared to 31.7 days in the concurrent control group and 33.4 days in the WIL historical control data. Mean body weights recorded atWIL

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the ag e of attainment were 114.5 g, 116.3 g, 118.1 g and 113.2 g in the same respective

groups compared to 115.2 g in the concurrent control group and 111.6 g in the WIL
historical control data. Any differences noted were slight and not statistically significan t from the concurrent control group

. 

### 6.5.3.3. D ETAILED 

### C LINICAL 

### O BSERVATIONS Summary Data: Table 32 ; Figures 5, 6, 7, 8 Individual Data: Appendix A

- Table A34
Po sitive Control Data: Appendix O (Study nos. 99199, 99263, 99310)
Hist orical Control Data: Appendix P

No consistent dose

- related trends were noted when detailed clinical observation data
were evaluated at PND 4, 11, 21, 35, 45 and 60 . Findings were considered transient in
the test substance

- treated groups, as they were noted infrequently, similarly in t he control group and/or in a manner that was not dose

- related. Mean urination counts were significantly (p<0.05) lower on PND 11 for the 1 mg/kg/day group females, on PND 21
for all female test substance

- treated group s and on PND 35 for the 10 mg/kg/day g roup females

. B ecause all values were within the range of the WIL historical control data the
lower urination counts wer e not considered test substance

- related. Mean hindlimb grip strength in the 1 mg/kg/day group females (161.7 g ) was significantly (p<0 .05) greater than that in the control group (126.1 g ) on PND 35

. I n the absence of an effect on
hindlimb grip strength at higher dosage levels and at any other time point for this same dosage level
, and because the value was within the range of the WIL hi storical control data (83.5 g to 161.8 g ), the greater hindlimb grip strength in the 1 mg/kg/day group
females was not considered test substance

- related. One female in the 1000 mg/kg/day group had markedly co arse tremors (4.5 mm) on PND 35. Because t remo rs were not observed for any other animal (90 females and 180 total animals [sexes combined]) on PND 35 at any dosage level and were not observed for this animal or any other animal at the other ages of evaluation (PND 4, 11, 21, 45 and 60)
, this single oc currence of tremors
in the 1 female on PND 35 was not considered test substance

- related.WIL

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### 6.5.3.4. L OCOMOTOR 

### A CTIVITY Summary Data: Tables 33, 34, 35, 36; Figures 9 through 34 Individual Data: Appendix A

- Tables A35, A36, A37, A38
Statistic al Analysis Summary: A ppendix M Positive Control Data: Appendi x O (Study nos. 99435, 99441 )
Hist orical Control Data: Appendix Q Nicotine Challenge Figures: Appendix R
Locomotor activity was ass essed with a personel computer

- controlled system that utilizes
a series of infrare d pho to beams to quantify ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams) and total locomotor activity (a
combin ation of fine locomotor skills [ i.e., grooming; int erruption of a single photobeam] and ambulatory locomot or activity). The pattern of habituation (activity or response to a stimulus over time) within each test session and across test sessions was also assessed. Locomotor activity patterns (total activity as well as ambulatory activity counts) in F 1 litters were unaffected by F 0 maternal test substance administration at all dosage levels
when evaluated for 60 minutes on PND 13, 17, 21, 61, 120 and 180 . Values obtained from the 6 subintervals evaluated (0

- 10 minutes, 11

- 20 minutes, 21

- 30 minutes
, 31

- 40 minute s, 41

- 50 minutes and 51

- 60 minutes) and the overall 60

- minute test session
values were comparable to the concurrent control values and /or the WIL historical
control data. Although the mean cumulative total counts on PND 180 in the
1000 mg/kg/day group wer e only slightly lower than that of the concurrent control group
, the difference was sign i ficant (p<0.05). H owever, because mean total counts of both the 1000 mg/kg/day group and the concurrent control group were similar and not statistically significantly different during the first 10 minutes of the test session
, the slightly lower
mean cumulative total counts for F 1 litters in the 1000 mg/kg/day group on PND 180

were not attributed to maternal test substance administration , but rather to slightly greater
habituation

. In addition, evaluation of plasma concentrations of the test substance indicated that there is no difference regarding internal exposure for external dosage levels
of 10 to 1000 mg/kg/day. The mean cumulative total counts for both the 10 andWIL

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100 mg/kg/day groups were determined to be similar statistically to the concurrent control group valu e on PND 180, which further sup ports that slightly greater habituation

was the cause of the lower mean cumulative total counts for F 1 litters in the
1000 mg/kg/day group on PND 180. Other slight d ifferences from the control group

were also noted but were either not statistically significant when analyzed using repeated measures, within the WIL historical control data ranges and/or did not occur in a
dose

- r elated manner. No remarkable shifts in the pattern of habituation occurred in any
of the test substance

- treated groups

. Animals in all groups exhibited the normal habituation response for adul t rats, which is characterized by high levels of activity
duri ng the first 10

- 20 minutes of the hour and then decreasing over successive 10

- minute
subintervals to low levels of act iv i ty at the end of the hour.

A nicotine challenge was conducted on PND 61, 120 and 180 and involved the animals being subjected to a seco nd 60

- minute test session after having received an administered
dose of either nicotine or saline that immediately followed the initial (first) 60

- minute test
session

. The second 60

- minute test session , as the first, was divided into 6 subintervals ( 66

- 75 minutes, 76

- 85 minutes, 86

- 95 minutes , 96

- 105 minutes, 106

- 115 minutes and
116

- 125 minutes)

. When the control group means of animals that received nicotine were compared to the control group means of animals given saline, the expected increase in
activit y as a result of nicotine administration was not observed. Rather, the control group
animals that received nicotine and the control group animals that received saline
exhibited similar counts of total and a mbulatory activity during the first subinterval ( 66

- 75 minute s) of the second 60

- minute test session . This activity did decreas e with
successive subintervals for both nicotine

- administered and saline

- administered control
animals
, and therefore demonstrated the expected habituation response
, but also

sug gested that the dose of nicotine was ineffective in control animals

. The increased activity during the 66

- 75 minute subi nterval , when compared to th e 51

- 60 minute
subinterv al for the same group of animal s , appeared to be the result of handling the
animals between the first and second 60

- minute test sessions

. Because the control group
animals that were administered nicotine prior to the second 60

- minute test session did notWIL

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necessarily respond as expected , it is difficult to fully evaluate the impact of th e challenge portion of this test session. However, animals from each of the treated groups generally did respond in the expected fashion to the nicotine challenge . G roup mean data
for nicotine

- administered animals did not decrease as rapidly over the sec ond 60

- minute
test session as was observed for the saline

- administered animals, and therefore the
nicotine

- administered animals had a greater mean cumulative activity for the entire
60

- minutes when compared to the saline

- administered animals. Moreover, de spite a somewhat slower, although ex pected, habituation in nicotine

- administered animals compared to saline

- administered animals, i t is clear that maternal test substance administration had no effect on the pattern of habituation

.
Results of the second 60

- minute test session revealed that animal s dosed with saline in
the 1000 mg/kg/day group had significantly (p<0.05) lower mean cumulative total counts
on PND 61, 120 and 180 , and significantly (p<0.05) lower mean cumulative ambulatory
counts on PND 180 , whe n compared to the control group values

. These lower mean cumulative and ambulatory counts were attributed to the consistently lower total and ambulatory counts across all subintervals for animal s dosed with saline in the
1000 mg/kg/day group on PND 61, 12 0 and 180 compared to the control group.
However , saline is no t known to cause an effect (when administered to rats by intraperitoneal injection ) that could account for this reduction in activity in adult rats, particularly after these same rats demonstra ted normal activity levels during the first
60

- minute test session (prior to saline administration) . Furthermore, because there were

no changes in activity noted across groups during the second hour of testing for a nimals
dosed with nicotine , it is not bi ologically plausible that saline administration prior to the second hour of testing would manifest an effect. Finally, no difference s in locomotor activity patterns for animals dosed with saline in the 10 and 100 mg/kg/day groups were observed , and intern al exposure to the test substance has been shown to be similar
between dosage levels of 10 to 1000 mg/kg/day (Beck, 2009 ; WIL

- 635001). Based on
the afore

- mentioned reasons
, the de creased activity in animals dose d with saline in the
1000 mg/kg/day group wa s not attributed to maternal test substance administration.WIL

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### 6.5.3.5. A COUSTIC 

### S TARTLE 

### T EST Summary Data: Table 37 ; Figures 35 through 40
Individual Data: Appendix A

- Table A39 Statistical Analysis Summary: Appendix M Positive Control Data: Appendix O (Study no s. 99418, 99440 )
Historical Control Data: Appendix S
The acoustic startle response habituation paradigm was conducted as a longitudinal
assessment with selected F 1 animals evaluated on PND 20 and again at sexual maturity (PND 60 ). Administr ation of the t est substance at 1, 10, 100 and 1000 mg/kg/day to the
F 0 m aternal animals had no significant effect on auditory startle responsiveness. At
PND 20 and 60 , the MAX , T MAX , and AVE values for all trials combined were similar for
the F 1 litters in the control and test substance

- treated groups

. On PND 60, the mean
overall T MAX value in the 1 mg/kg/day group was s ignificant ly (p<0.05 ) lower than the control group mean using repeated measures analysis of variance. However, because the
mean overall T MAX values in the 10, 100 and 1000 mg/kg/day groups were similar to the control group values on PND 60, the difference noted in the 1 mg/kg/day group was not
considered test substance

- related. No other statistically significant differences from the control group were noted . No effects were noted in the pattern of the habituation response over the entire 50

- block test session. Normal habituation to the auditory startle stimulus was observed across all groups; i.e., the greatest response was observed during
the first t rial block, and the response gradually decreased with each successive block of
trials.WIL

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### 6.5.3.6. B IEL 

### M AZE 

### S WIMMING 

### T RIALS Summary Data: Table s 38, 39, 40, 41; Figures 41 throug h 48
I ndividual Data: Appendix A

- Tables A40, A41, A42, A43 Statistical Analysis Su mmary: Appendix M Positive Control Data: Appendix O (Study nos. 99438, 99199) Historical Control Data: Appendix T
Swimming abili ty on day 1 of the Biel maze assessment (PND 22 or 62 ) was similar between the control, 1, 10, 100 and 1000 mg/kg/day gro up s , with no statistically significant differences noted. There were no biologically meaningful trends for the times to criterion (mean time to locate the submerged platform) and the mean numbers of errors
committed during the learning and memory trials betwe en the F 1 litters in the control and
test substance

- treated groups beginning on PND 22 (Subset B) and 62 (Subset A)

. On PND 22, a significant (p<0.05) treatment

- by

- trial interaction was noted for the mean time to escape and the mean number of errors commit ted in Path A (trials 1

- 4) . When Dunnett’s test was conducted for each of the trial s in Path A on PND 22, there were no statistically significant differences between the control and test substance

- treated groups in the mean time to escape. However, a sig nificantly (p<0.05) lower mean number of

errors were committed in the 100 and 1000 mg/ kg/day groups for trials 1 and 3
, respectively, for Path A. In the absence of a consistent trend across trials, the apparent improvement in performance in the 100 and 10 00 mg/ kg/day groups for trials 1 and 3
, respectively, was not considered test substance

- related. S ignificant (p<0.05) treatment

by

- sex interaction s were noted when the mean time to escape and the mean number of
errors committed for Path B (trials 5

- 10) we re evaluated on PND 22 . T herefore, the repeated measures analysis of variance was conducted separately by sex. When analyzed by sex, there were no statistically significant differences in the PND 22 mean time to escape and mean number of errors committed between the control and test
substance

- treated groups for Path B

.WIL

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O n PND 62, a significant (p<0.05 ) treatment

- by

- trial interaction was noted for the mean time to escape from Path B (trials 5

- 10 ). However, when each of th e trials for Path B was evaluated using Dunnett’s test, there were no statistically significant differences in the mean time to escape between the control and test substance

- treated groups.
There were no statistically significant differences noted between the control and test
substance

- tre ated groups in the mean time to escape and the mean number of errors committed during the PND 22 and 62 memory probe (trials 11 and 12).



### 6.5.4. M ACROSCOPIC 

### E XAMINATIONS 

### ( U NSCHEDULED 

### D EATHS AND 

### A NIMALS 

### N OT 

### S ELECTED FOR 

### N EUROPATHOLOGY 

### ) 6.5.4.1. U NSCHEDULED 

### D EATHS Summary Da ta: Tables 42, 43
Individual Data: Appendix A

- Tables A44, A45

Male no. 90585

- 05 in the control group (Subset A) , female no. 90601

- 10 in the
1 mg/kg/day group (Subset A) and male no. 90485

- 01 in the 1000 mg/kg/day group (Subset D) were found dead on PND 87, 124 and 61, respectively. There were no internal findings noted for any of these animals at necropsy. In the absence of a dose response and because mortality also occurred in the control group, the single deaths at
1 and 1000 mg/kg/day were not cons idered test substance

- related.



### 6.5.4.2. A NIMALS 

### E UTHANIZED 

### F OLLOWING 

### PND 22 L EARNING AND 

### M EMORY 

### A SSESSMENTS 

### (S UBSET 

### B) Summary Data: Tables 44, 45 Individual Data: Appendix A

- Tables A46, A47

F 1 pups in Subset B were euthanized following completion of the PND 22 learning and memory assessments

. Hydrocephaly was note d for 1 male in the 1000 mg/kg/day group
, and dilated r enal pelv es were noted for 1, 2 and 1 males in the 10, 100 and
1000 mg/kg/day groups, respectively. In addition, a cysti c kidney was noted for 1 male each in the control, 100 and 1000 mg/kg/day groups and 1 fema le each in the control andWIL

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1000 mg/kg/day groups. Because hydrocephaly was only noted in a single pup of the
274 pups evaluated in the 1000 mg/kg/day group and the 1108 pups evaluated in a ll 4 test
substance

- treated groups in this study (including nonselected pups [PND 21], unscheduled deaths and those pups examined on PND 21 [Subset C], PND 72 [Subset D] or PND 180 [Subset A]) , this finding was not considered test substance

- related. As fo r the other internal findings noted, these occurred in single animals
, at similar incidence s in the control group and/or in a manner that was not dose

- related
, and therefore were also
not attributed to F 0 maternal test substance administration

. No other i nternal findings
were noted.



### 6.5.4.3. A NIMALS 

### E UTHANIZED 

### F OLLOWING 

### PND 180 L OCOMOTOR 

### A CTIVITY 

### A SSESSMENT 

### (S UBSET 

### A) Summary Data: Tables 46, 47
Individual Data: Appendix A

- Tables A48, A49

F 1 pups in Subset A were euthanized following completion of the PND 180 l ocomotor activity assessment. Internal findings noted in a single male and/ or female in the test
substance

- treated groups consisted of small, soft and/or absent testes and epididymides
, cystic kidneys, enlarged or misshapen liver, white areas on the liver
, enlarged lymph nodes, swollen ears or liver , clear contents in the uterus, dark red contents in the urinary bladder, dark red discoloration of the thymus, masses in the mammary gland or adipose
tissue and dilated renal pelves . In addition, 2 females in the 10 mg/kg/day group had an enlarged heart. Because t hese findings occurred in single animals, at similar incidences in the control group and/or in a manner that was not dose

- related , they were not attributed

to F 0 maternal test substance administration

.WIL

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### 6.5.5. N EUROPATHOLOGY 

### 6.5.5.1. PND 21 (S UBSET 

### C) 6.5.5.1.1. M ACROSCOPIC 

### E XAMINATIONS Summary Data: Table 48
Individual Data: Appendix A

- Table A50 Pathology Report: Appendix I

T he necropsies of F 1 animals selected for brain weights on PND 21 revealed no gross observations t hat were considered associated with administration of the test substance. The only finding observed was dark red material covering the entire brain for female

no. 90533

- 15 and male no. 90621

- 02 in the 1 and 1000 mg/kg/day groups, respectively.



### 6.5.5.1.2. B RAIN 

### W EIGH TS 

### /B RAIN 

### M EASUREMENTS Summary Data: Table 49
Indiv idual Data: Appendix A

- Table A51 , A52 Pathology Report: Appendix I

No test substance

- related effects on mean brain weights, lengths and widths were noted in males or females at any dosage level on PND 21. A significantly (p<0.05) lower brain weight relative to final body weight was noted for males in the 100 mg/kg/day group

. H owever, because there were no effects on brain weights relative to final body weights in females at this dosage level, or in ma les and females at 10 and 1000 mg/kg/day, the
difference was not considered test substance

- related.



### 6.5.5.1.3. Q UALITATIVE 

### H ISTOPATHOLOGY A ND 

### B RAIN 

### M ORPHOMETRY Summary Data: Table s 50, 51 ; Figures 49 through 54 Individual Data: Appendix A

- Tables A50, A53 Patholog y Report: Appendix I Historical Control Data: Appendix U
There were no qualitative histopathologic changes in the brains of males or females at any dosage level on PND 21.WIL

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T he thickness of the pons was slightly smaller (p< 0.05) for males in the 1000 mg/k g/day

group when compared to males in the control group

. T he distance (height) between layers of pyramidal neurons in the hippocampus was slightly shorter (p< 0.05) for females
in the 1000 mg/kg/day group
, when compared to females in the control group . Th ese statistically significant reduced measurements fell within the WIL h istorical control data

ranges , and thus the differences were considered spurious.



### 6.5.5.2. PND 72 (S UBSET 

### D) 6.5.5.2.1. M ACROSCOPIC 

### E XAMINATIONS Summary Data: Table 52
Indiv idu al Data: Appendix A

- Tabl e A54 Pathology Report: Appendix I

T he necropsies of F 1 animals sele cted for brain weights on PND 72 revealed no gross observations that were considered associated with administration of the test substance.
Da rk red material covering the entire brain was observed for 2, 2 and 1 males in the 10,
100 and 1000 mg/kg/day groups, respectively, and for 1 female each in the 1 and
1000 mg/kg/day groups. Additionally, absence of the left paraflocculus was noted for
1 male in the 100 mg/kg/day group. All macrosco pic findings were noted in single animals, at a similar incidence in the control group and/or in a manner that was not
dose

- related . T herefore, these findings were not attributed to F 0 maternal test substance
administration

. 

### 6.5.5.2.2. B RAIN 

### W EIGHTS 

### /B RAIN 

### M EASUREMEN TS Summary Data: Table 53
Indiv idu al Data: Appendix A

- Table A55, A56 Pathology Report: Appendix I

No statistically significant differences or test substance

- related effects on mean brain weights, lengths and widths were noted in males or fem ales at an y dosage level on
PND 72 when compared to the control group

.WIL

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### 6.5.5.2.3. Q UALITATIVE 

### H ISTOPATHOLOGY A ND 

### B RAIN 

### M ORPHOMETRY Summary Data: Tables 54, 55 ; Figures 5 5 through 60 Individual Data: Appendix A

- Tables A54, A57 Pathology Report: Appendix I
Historical Contro l Data: Appendix U
Histopathological evaluations were performed in males and females on PND 72 in the control and 1000 mg/kg/day groups only. The observed h istopathologic changes in the brains, spinal cord, spinal ganglia and nerve roots, trigeminal gang lion and nerve, eyes or optic nerves, skeletal muscle or peripheral nerves (sciatic n. , sural n., tibial n., peroneal n.) were all consi dered to be incidental findings or related to some aspect of experimental manipulation (i.e., including perfusion rate/p ressure and/or processing technique ). Because the prevalence, severity and/ or histologic al character of these
incidental tissue alterations were similar between control and 1000 mg/kg/day group animals, no qualitative test substance

- related differences we re found

.

T he initial morphometry evaluation on PND 72 included Level 1 measurements of the height of the hemisphere and the vertical thickness of the cortex in F 1 animals from the
control and 1000 mg/kg/day group s. The mean values for these measurements in males of the 1000 mg/kg/day group were significantly (p<0.05 or p<0.01) reduced from those
of the control group males. However, these mean values for the 1000 mg/kg/day group
males were within the range of values in the WIL h istorical control data. It was further
determined that the mean height of the hemisphere for the study control group males was greater than the maximum mean value in the WIL h istorical control data

. T here were no statistically significant difference s between any of the morphometric measurements in
females of the 1000 mg/kg/day group when compared to the study control group fema les
on PND 72.
Because of the statistically significant differences between the control and
1000 mg/kg/day group males, the Sponsor requested that the morphom etry evaluation be performed with F 1 males from the 1, 10 and 100 mg/kg/day groups on PND 72 toWIL

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determine how the results might compare with that of the study c ontrol and
1000 mg/kg/day group males . Upon evaluating the PND 72 mean values for all morphomet ric measurements from males in all groups, the following was noted:

1. m ean values for the Level 1 height of the hemisphere measurement in males of
the 1, 10 and 100 mg/kg/day group s were similar to each other and to the mean
value of the 1000 mg/kg/day group males . The mean values of all treatment

groups were significantly (p<0.01) lower than t he study control group value;

2. mean values for the Level 1 vertical thickness of t he cortex in males of the 1, 10
and 100 mg/kg/day group s were similar to each other an d were not statistically significant ly different when compared to the study control group ;

3. individual and mean control group values for the height of the hemisphere measurement were almost exclusively above the maximum mean value in the
WIL historical cont rol data. Similarly, 4 of the 10 individual study control group
values for the vertical thickness of the cortex were above the maximum mean value in the WIL historical control data. In addition, there were no statistically significant differences between the 10 and 100 mg/kg/day group males with regard to the vertical thickness of the cortex when compared to the study control

group.
After thoroughly evaluating and comparing individual animal data and group means of body weights, brain weights, brain to fi nal body weight ratios, blood plasma and milk concentrations, sensory function and neurobehavioral testing, macroscopic and microscopic examinations and morphometric analysis collected from this study, the results demonstrate that decabromodiphenyl oxide d oes not cause symptoms of developmental neurotoxicity (e.g. a lack of habituation). However, anatomically, morphometric analysis of male brains indicated that the hemispheric measurements in all treatment groups and the thickness of the cortex in the 1000 mg/kg/day group were statistically significantly different (decreased) when compared to the study control groupWIL

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on PND 72 . Yet, no statistically significant difference s were reported in any treatment group, including females, in regards to functional act ivity or behavioral endpoints, as well as brain weights, lengths and widths. Furthermore, no corresponding differences in microscopic findings were observed in brains of control and 1000 mg/kg/day group
animals. Text Table 5 (shown below) provides a summ ary of Level 1 brain morphometry measurements of males in the study control and test substance

- treated groups and compares the measured mean values with the WIL historical control means and ranges

. The comparison not only shows the aforementioned statisti cally significant differences of
the test substance

- treated groups, but also that the measured mean values of the test
substance

- treated groups are within the range of values of the WIL historical control database. In addition, the comparison reveals that the measured mean values of the study control group actually exceed above the ranges of the WIL historical control database. Furthermore, there was no quantitative difference in the height of the hemisphere between the 1 mg/kg/day group and the other tes t substance

- treated groups, suggesting a lack of dose response. Thus, none of the statistically significant differences of the test
substance

- treated males were considered adverse or associated with F 0 maternal test
substance administration.

Text Table 5

. Summary of Level 1 Brain Morphometry

- Males (PND 72) Dosage Level (mg/kg/day)
0 1 10 100 1000 WIL HC mean (range) Height of Hemisphere (cm) 0.7179 0.6645** 0.6611** 0.6653** 0.6621** 0.6479 (0.5902

- 0.6820 ) Vertical Thickness of Cortex (cm) 0.1813 0.1737 0.1733 0.1733 0.1678* 0.1686 (0.1539

- 0.1839) HC = Historical Control * = p<0.05 ** = p<0.01WIL

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### 6.6. D ISCUSSION This oral gavage developmental neurotoxicity study in rats with decabromodiphenyl oxide was designed with the following objectives:

1) to det ermine the potential of the test substance to induce functional and/or morphological insult to the nervous system in the offspring of dams that were administered during pregnancy and lactation via oral gavage dosage levels of 1, 10, 100 or 1000 mg/kg/day;
2) to determine the concentration of the test substance in maternal and neonatal plasma and maternal milk samples on lactation/postnatal day 4 at dosage levels of 1 and
10 mg/kg/day. The concentration of the test substance in maternal and neonatal plasma and maternal milk resulting from oral (gavage) exposure of dams to the test substance in the same vehicle was already determined in a previous exposure assessment and dose

range

- finding developmental neurotoxicity study (Beck, 2009; WIL

- 635001) at dosage
l evels of 100, 300 and 1000 mg/kg/day.

There were no test substance

- related mortalities in the F 0 maternal animals during the study. There were no test substance

- related clinical findings noted during the daily examinations or approximately 1 hour followin g dose administration. Detailed clinical observation parameters, as well as maternal body weights and food consumption during gestation and lactation
, were unaffected by test substance administration.

There were no maternal test substance

- related differen ces noted between groups when comparing the mean length of gestation, the process of parturition and the internal macroscopic findings. The mean numbers of former implantation sites and
unaccounted

- for sites, as well as the mean number of pups born, live litter size and the percentage of males at birth were similar across groups.
There were no effects on offspring survival or growth, and no remarkable clinical findings were noted for F 1 animals at any dosage level. In addition, attainment of sexualWIL

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Bromine Science and Environmental Forum

develo pmental landmarks (balanopreputial separation and vaginal patency) were unaffected by maternal test substance administration.

Administr ation of the test substance to the F 0 m aternal animals had no significant effect on swimming ability or learning and memo ry. Normal patterns of learning were observed across all groups, as the mean time to escape and number of errors committed were initially relatively high in each path (A and B) and decreased throughout repeated testing. A uditory startle responsiveness wa s also unaffected by maternal test substance
administration , and n ormal habituation to the auditory startle stimulus was observed across all groups ( i.e., the greatest response was observed during the first trial block, and the response gradually decreased with each successive block of trials )

.
There were no test s ubstance

- related effects on loco motor activity when evaluated during

a 6 0

- mi nute test session on PND 13, 17 and 21. The F 1 pups in the current study were
also evaluated for loco motor activity on PND 61, 120 and 180 because changes in
loco motor activity have been previously reported at 2, 4 and 6 months of age in mice and at 2 and 4 months of age in rats, following a single oral gavage dose of decabromodiphenyl oxide to F 1 pups on PND 3 (Viberg et al , 2003 ; Viberg et al., 2007 ). Although this study did not employ direct dosing of pups during the postnatal period, it was determined from a previously performed study (Beck, 2009; WIL

- 635001) that plasma concentrations ( internal exposure ) of the test s ubstance are higher in pups that

r eceiv e the test substance through maternal administration (indirect dosing) compared to

pups directly dosed on PND 4. Following maternal administration of the test substance
in this study, no effects on locomotor activity were noted in F 1 animals during a
60

- minute test session on PND 61, 120 and 180. Furthermore, normal patterns of habituation were observed at all relevant ages (PND 13, 17, 21, 61, 120 and 180) in
which loco motor activity, as well as auditory startle res ponse ( as discussed previously)
,

were evaluated

.
There were no test substance

- related effects on brain weights, lengths or widths on PND 21 and 72. There were no histopathologic al alterations related to test substanceWIL

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Bromine Science and Environmental Forum

administration in PND 21 or PND 72 br ains
, or in PND 72 central or peri pheral nervous system tissues.
There were no differences in morphometric measurements that were considered related to test substance administration. However, on PND 72 the mean values of the height of the hemisphere in ma les from all test substance

- treated groups and the mean value of the thickness of the cortex in males from the 1000 mg/kg/day group were statistically significantly reduced from those of the study control group males. However, these
measured mean values o f the test substance

- treated groups were within the range of
values in the WIL h istorical control data. It was further determined that both the mean height of the hemisphere and the mean thickness of the cortex for the study control group
males were great er than the maximum mean value s in the WIL historical control data

. Furthermore, there were no qualitative changes in brains from males in the
1000 mg/kg/day group compared to the study control group. In addition, t here were no statistically significant differences between any of the morphometric measurements in
females of the 1000 mg/kg/day group when compared to the study control group females
on PND 72. Thus, none of the statistically significant di fferences in the test
substance

- treated males were co nsidered adverse or associated with F 0 maternal test
substance administration.
Mean concentrations of the test substance in maternal and pup plasma on lactation day/PND 4 at 10 mg/kg/day were 1700 and 2140 ng/mL, respectively , at approximately
8 hours foll owing maternal dose administration . In a previous preliminary range

- finding study, similar maternal and pup plasma concentrations of the test substance were found
for d osage levels of 100, 300 and 1000 mg/kg/day (1334 to 1812 ng/mL and 1998 to 2535 ng/mL, respectively). Because the mean plasma concentrations at 10 mg/kg/day were similar to the plasma concentrations previously noted at 100, 300 and
1000 mg/kg/day, it was determined that there is actually no difference in internal

exposure between dosage le vels of 10 to 1000 mg/kg/day. The mean concentrations of the test substance in maternal and pup plasma at 1 mg/kg/day were 510 and 929 ng/mL,WIL

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Bromine Science and Environmental Forum
respectively. Mean concentrations of the test substance in maternal milk samples at 1 and 10 mg/kg/day on lactat ion day 4 were 509 and 1250 ng/mL, respectively; these
values w ere similar to those noted in the previous preliminary range

- finding study at dosage levels of 100 to 1000 mg/kg/day (574 to 628 ng/mL).WIL

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### 7. C ONCLUSIONS There was no evidence of maternal toxicity a t any dosage level of decabromodiphenyl oxide evaluated in this study. Additionally, there were no effects on offspring survival and growth, or on any of the neurobehavioral parameters evaluated in this study. Normal patterns of habituation were observed at all relevant ages tested for both locomotor
activit y and auditory startle response . Therefore, under the conditions of this study, no evidence of developmental neurotoxicity was observed at any dosage level evaluated

. Thus, the no

- observed

- adverse

- ef fect level (NOAEL) for both F 0 systemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodiphenyl oxide was considered to
be 1000 mg/kg/day, the highest dosage level administered in this study. Based on the mean plasma concentration of the test substance in F 1 pups at 8 hours following maternal dose administration on lactation day 4, the internal exposure at dosage levels between 10
and 1000 mg/kg/day was determined in this study and a previous study (Beck, 2009;
WIL

- 635001) to be 1998 ng/mL to 2535 ng/mL.WIL

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Bromine Science and Environmental Forum



### 8. K EY 

### S TUDY 

### P ERSONNEL A ND 

### R EPORT 

### S UBMISSION Report Submitted By :

_____________________________________ _____________

Melissa J. Beck, PhD Date Assistant Director, Neurosciences Study Director
Report Prepared By :

_____________

_________________________ _____________ Jaime L. Bard, MS Date Senior Study Analyst
Report Reviewed By :

______________________________________ _____________ Mark D. Nemec, BS, DABT Date President and Chief Operating Officer

____________________

__________________ _____________ Donald G. Stump, PhD, DABT Date Director, Developmental and Reproductive Toxicology

______________________________________ _____________

Evelyn Tanchevski, BS Date Group Supervisor, Study Analysis and Reports

__ ____________________________________ _____________ Julie S. Varsho, BS, RAC Date Project Specialist, NeurosciencesWIL

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### K EY 

### S TUDY 

### P ERSONNEL 

### A ND 

### R EPORT 

### S UBMISSION 

### ( CONTINUED 

### ) Study Personnel : Sally A. Keets, AS Senior Operations Manager, Vivarium Carol A. Kopp, BS, LAT Manager, Gross Pathology and Developmental Toxicology Laboratory Theresa M. Rafeld Group Manager, Formulations Laboratory Michael A. Safron, AS, HT (ASCP) CM Manager, Histology Daniel W. Sved, PhD Director, Metabolism and Analytica l Chemistry Bennett J. Varsho, MPH , DABT Operations Manager, Developmental and Reproductive Toxicology and the Formulations Laboratory Robert A. Wally, BS, RAC Manager, Reporting and Regulatory Technical Services Brandi L. Zehner, AS Group Supervisor , Sample Processing LaboratoryWIL

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Bromine Science and Environmental Forum



### 9. Q UALITY 

### A SSURANCE 

### U NIT 

### S TATEMENT 

### 9.1. P HASES 

### I NSPECTED

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings

Reported to Management Auditor(s)

03

- Dec

- 2007 Protocol 03

- Dec

- 20 07 17

- Jan

- 2008 K.Mentzer

21

- Dec

- 2007 Vehicle and Test Article

Preparation 21

- Dec

- 2007 17

- Jan

- 2008 M.Salyers

21

- Dec

- 2007 Protocol Amendment I

Review 21

- Dec

- 2007 17

- Jan

- 2008 J.Tooman

16

- Jan

- 2008 Protocol Amendment II

Review 16

- Jan

- 2008 2 8

- Feb

- 2008 K.Mentzer

18

- Jan

- 2008 Animal Care and Equipment 18

- Jan

- 2008 28

- Feb

- 2008 J.Faugl / L.Stepp

22

- Jan

- 2008 Protocol Amendment III

Review 22

- Jan

- 2008 28

- Feb

- 2008 K.Mentzer

31

- Jan

- 2008 Perfusion 01

- Feb

- 2008 26

- Mar

- 2008 A.Lowell

04

- Feb

- 2008 Protocol Amendment IV

Review 04

- Feb

- 2008 26

- Mar

- 2008 K.Mentzer

14

- Feb

- 2008 Pup Functional

Observational Battery 14

- Feb

- 2008 26

- Mar

- 2008 M.Salyers / J.Tooman

03

- Mar

- 2008 Protocol Amendment V

Review 03

- Mar

- 2008 24

- Apr

- 2008 K.Mentzer

12

- Mar

- 2008 Biel Maze 12

- Mar

- 2008 24

- Apr

- 2008 T.DeVan Booth / P.Rusnak

14

- Mar

- 2008 Protocol Amendment VI

Review 14

- Mar

- 2008 24

- Apr

- 2008 K.Mentzer

20

- Mar

- 2008 Perfusion 20

- Mar

- 2008 24

- Apr

- 2008 T.DeVan Booth / P.Rusnak

24

- Mar

- 2008 Body Weights 24

- Mar

- 2008 24

- Apr

- 2008 P.Rusnak

24

- Mar

- 2008 Animal Care and Equipment 24

- Mar

- 2008 24

- Apr

- 2008 P.Rusnak

02

- May

- 2008, 05

- May

- 2008 Study Records (B

- 1, B

- 2,

B

- 3, B

- 4, B

- 5) 05

- May

- 2008 23

- Jun

- 2008 E.Crookshank

08

- May

- 2008 Test Article Dispense 08

- May

- 2008 23

- Jun

- 2008 M.StaufferWIL

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Bromine Science and Environmental Forum

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings

Reported to Management Auditor(s)

09

- May

- 2008 Test Article Administration 09

- May

- 2008 23

- Jun

- 2008 A.Deppe

09

- Jun

- 2008 Trimming of Tissues 09

- Jun

- 2008 23

- Jul

- 2008 P.Rusnak

24

- Jun

- 2008 Body Weights 2 4

- Jun

- 2008 23

- Jul

- 2008 P.Rusnak

02

- Jul

- 2008 Protocol Amendment VII

Review 02

- Jul

- 2008 22

- Aug

- 2008 K.Mentzer

07

- Jul

- 2008 Test Article Dispense 07

- Jul

- 2008 22

- Aug

- 2008 M.Stauffer

09

- Jul

- 2008 Tes t Article Administration

(Nicoti ne Challen ge) 09

- Jul

- 2008 22

- Aug

- 2008 S.Power

17

- Jul

- 2008 Necropsy 17

- Jul

- 2008 22

- Aug

- 2008 P.Rusnak

24

- Jul

- 2008 Protocol Amendment VIII

Review 24

- Jul

- 2008 22

- Aug

- 2008 K.Mentzer

31

- Jul

- 2008, 01

- Aug

- 2008, 03

- Dec

- 2008 Study Records (I

- 1) 03

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

31

- Jul

- 2008, 25

- Nov

- 2008 Study Records (I

- 13) 25

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

31

- Jul

- 2008, 29

- Nov

- 2008 Study Records (I

- 14) 29

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

01

- Aug

- 2008, 29

- Nov

- 2008 Study Records (I

- 23) 29

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

01

- Aug

- 2008, 29

- Nov

- 2008 Study Records (I

- 24) 29

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

04

- Aug

- 2008, 05

- Aug

- 2008, 06

- Aug

- 2008, 15

- Nov

- 2008 Study Reco rds (I

- 8) 15

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

12

- Aug

- 2008, 13

- Aug

- 2008, 14

- Aug

- 2008, 15

- Aug

- 2008, 14

- Nov

- 2008 Study Records (I

- 4) 14

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.ToomanWIL

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Bromine Science and Environmental Forum

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings

Reported to Management Auditor(s)

18

- Aug

- 2008, 19

- Aug

- 20 08, 29

- Nov

- 2008 Study Records (I

- 17) 29

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

21

- Aug

- 2008, 22

- Aug

- 2008, 15

- Nov

- 2008 Study Records (I

- 2) 15

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

22

- Aug

- 2008, 16

- Nov

- 2008, 20

- Nov

- 200 8 Study Records (I

- 9) 20

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

22

- Aug

- 2008, 20

- Nov

- 2008 Study Records (I

- 6) 20

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

25

- Aug

- 2008, 20

- Nov

- 2008 Study Records (I

- 10) 20

- Nov

- 2008 22

- Dec

- 200 8 P.Herrick / J.Tooman

25

- Aug

- 2008, 20

- Nov

- 2008 Study Records (I

- 20) 20

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

25

- Aug

- 2008, 20

- Nov

- 2008 Study Records (I

- 15) 20

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

25

- Aug

- 2008, 20

- No v

- 2008 Study Records (I

- 16) 20

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

27

- Aug

- 2008, 28

- Aug

- 2008, 29

- Aug

- 2008, 01

- Dec

- 2008 Study Records (I

- 18) 01

- Dec

- 2008 26

- Jan

- 2009 P.Herrick / J.Tooman

29

- Aug

- 2008, 02

- Sep

- 2008, 03

- Se p

- 2008, 04

- Sep

- 2008, 01

- Dec

- 2008 Study Records (I

- 19) 01

- Dec

- 2008 26

- Jan

- 2009 P.Herrick / J.Tooman

29

- Aug

- 2008, 02

- Sep

- 2008, 03

- Sep

- 2008 Study Records (A

- 1, A

- 2,

A

- 3) 03

- Sep

- 2008 24

- Oct

- 2008 N.DanielsWIL

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Bromine Science and Environmental Forum

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings

Reported to Management Auditor(s)

08

- S ep

- 2008, 09

- Sep

- 2008, 25

- Nov

- 2008 Study Records (I

- 21) 25

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

09

- Sep

- 2008, 10

- Sep

- 2008, 25

- Nov

- 2008 Study Records (I

- 5) 25

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

10

- Sep

- 2008, 25

- No v

- 2008 Study Records (I

- 25) 25

- Nov

- 2008 22

- Dec

- 2008 P.Herrick / J.Tooman

16

- Oct

- 2008 Protocol Amendment IX 16

- Oct

- 2008 24

- Nov

- 2008 K.Mentzer

14

- Nov

- 2008, 06

- Dec

- 2008 Study Records (Rx

- 1) 06

- Dec

- 2008 26

- Jan

- 2009 L.Fisher / J.Tooman

14

- Nov

- 2008, 17

- Nov

- 2008, 25

- Nov

- 2008, 01

- Dec

- 2008, 02

- Dec

- 2008, 05

- Dec

- 2008, 08

- Dec

- 2008, 09

- Dec

- 2008, 10

- Dec

- 2008, 12

- Dec

- 2008, 13

- Dec

- 2008, 15

- Dec

- 2008, 16

- Dec

- 2008 Draft Report (without

Figures and Analytical, Pathology, Pl asma and Milk, and Statistical Analysis Reports) 16

- Dec

- 2008 26

- Jan

- 2009 A.Lowell / J.Tooman

17

- Nov

- 2008 Draft Report (Analytical

Chemistry Appendix) 17

- Nov

- 2008 22

- Dec

- 2008 N.Daniels

17

- Nov

- 200 8, 18

- Nov

- 2008, 19

- Nov

- 2008 Study Records (I

- 3) 19

- Nov

- 2008 22

- Dec

- 2008 J.Tooman

17

- Nov

- 2008, 18

- Nov

- 2008, 19

- Nov

- 2008 Study Records (I

- 7) 19

- Nov

- 2008 22

- Dec

- 2008 J.Tooman

18

- Nov

- 2008, 19

- Nov

- 2008, 05

- Dec

- 2008 Study Records (N

- 1) 05

- Dec

- 2008 26

- Jan

- 2009 J.ToomanWIL

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Bromine Science and Environmental Forum

Date(s) of Inspection(s) Phase Inspected Date(s) Findings Reported to Study Director Date(s) Findings

Reported to Management Auditor(s)

18

- Nov

- 2008, 19

- Nov

- 2008 Study Records (I

- 11) 19

- Nov

- 2008 22

- Dec

- 2008 J.Tooman

19

- Nov

- 2008 Study Records (N

- 3) 19

- Nov

- 2008 22

- Dec

- 2008 J.Tooman

01

- Dec

- 2008, 09

- Dec

- 2008, 10

- Dec

- 2008, 16

- Dec

- 2008 Draft Report (Figures only) 16

- Dec

- 2008 26

- Jan

- 2009 J.Tooman / A.Lowell

01

- Dec

- 2008 Study Records (I

- 22) 01

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

02

- Dec

- 2008 Study Records (I

- 12) 02

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

02

- Dec

- 2008, 03

- Dec

- 2008 D raft Report (Bioanalytical

Appendix

- Plasma Samples) 03

- Dec

- 2008 26

- Jan

- 2009 E.Crookshank

03

- Dec

- 2008 Draft Report (Bioanalytical

Appendix

- Milk Samples) 03

- Dec

- 2008 26

- Jan

- 2009 E.Crookshank

03

- Dec

- 2008, 04

- Dec

- 2008 Study Records (N

- 2) 0 4

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

04

- Dec

- 2008, 05

- Dec

- 2008 Study Records (N

- 4) 05

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

04

- Dec

- 2008, 05

- Dec

- 2008 Study Records (N

- 5) 05

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

04

- Dec

- 2008, 05

- Dec

- 2008 Study Records (N

- 6) 05

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

05

- Dec

- 2008 Study Records (N

- 7) 05

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

06

- Dec

- 2008 Study Records (TK

- 1) 06

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

08

- Dec

- 2008 Study Records (H

- 1,H

- 2) 08

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

08

- Dec

- 2008 Study Records (P

- 1) 08

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

10

- Dec

- 2008 Draft Report (Statistical

Analysis Report) 10

- Dec

- 2008 26

- Jan

- 2009 J.Tooman

13

- Dec

- 2008, 15

- Dec

- 2008 Draft Report (Pathology

Report) 15

- Dec

- 2008 26

- Jan

- 2 009 J.Tooman

05

- Feb

- 2009 Protocol Amendment X

Review 05

- Feb

- 2009 18

- Mar

- 2009 K.MentzerWIL

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Bromine Science and Environmental Forum
This study was inspected in accordance with the U.S. EPA Good Laboratory Practice Standards (40 CFR Part 792), the OECD Principl es of Good Laboratory Practice , the standard operating procedures of WIL Rese arch Laboratories, LLC
, and the S ponsor's protocol and protocol amendments with the following exceptions. The data located in Appendi x B ( Certificate of Analysis ) were the responsibility of the S ponsor. Qual ity Assurance findings, derived from the inspections during the conduct of the study and from the inspections of the raw data and draft report, are documented and have been reported to the study director. A status report is submitted to management monthly

. This report accurately reflects the data generated during the study. The methods and procedures used in the study were those specified in the protocol, its amendments
, and the standard operating procedures of WIL Research Laboratories, LLC.

The raw data , the retention sample
, and the final report will be stored in the Archives at WIL Research Laboratories, LLC or ano ther location specified by the S ponsor.WIL

- 635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum



### 9.2. A PPROVAL

This study was inspected according to the criteria discussed in Section 9 .1.
Report Aud ited By :

______________________________________ _____________ Elizabeth B. Crookshank, BS Date Compliance Specialist

______________________________________ _____________

Nancy J. Daniels Date Compliance Specialist

_____________________________ _________ _____________ Angela H. Lowell, BS Date Compliance Specialist

______________________________________ _____________

Joel S. Tooman, BS Date Senior Compliance Specialist

Report Released By :

______________________________________ ______ _______ Heather L. Johnson , BS, RQAP

- GLP Date Manager, Quality AssuranceWIL

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Bromine Science and Environmental Forum



### 10. R EFERENCES Adams, J.; Buelke

- Sam, J.; Kimmel, C.A.; Nelson, C.J.; Reiter, L.W.; Sobotka, T.J.; Tilson, H.A.; Nelson, B.K. Collaborative behavioral teratology study: protocol des ign and testing procedure. Neurobehavioral Toxicology and Teratology 1985
, 7 , 579

- 586.
Beck, M.J. An oral (gavage) exposure assessment and oral dose range

- finding developmental neurotoxicity study of decabromodiphenyl oxide in rats (Study No. WIL

- 63500 1). WIL Research Laboratories, LLC, Ashland, OH, 2009

. Biel, W.C. Early age differences in maze performance in the albino rat. Journal of Genetic Psychology 1940
, 56 , 439

- 453.
Coffee, S.T. Development and validation of a GC

- ECD assay for the determin ation of decabromodiphenyl oxide (DeBDPO) concentration in corn oil (Study No. WIL

- 635004 ). WIL Research Laboratories, LLC, Ashland, OH, 2008

. Dunn, O.J. Multiple comparisons using rank sums. Technometrics 1964
, 6(3)
, 241

- 252.
Dunnett, C.W. New table s for multiple comparisons with a control. Biometrics 1964
, 20 , 482

- 491. Gad, S.C. A neuromuscular screen for use in industrial toxicology. Journal of Toxicology and Environmental Health 1982
, 9 , 691

- 704. Haggerty, G.C. Development of tier I neurobeh avioral testing capabilities for incorporation into pivotal rodent safety assessment studies . Journal of the American College of Toxicology

. 1989
, 8(1) , 53

- 69. Holson, R.R.; Freshwater, L.; Maurissen, J.P.J.; Moser, V.C.; Phang, W. Statistical issues a nd techniques appropriate for developmental neurotoxicity testing: A report from the ILSI Risk Science Institute Expert Panel on neurodevelopment al endpoints. Neurotoxicology and Teratology 2008 , doi:10.1016/j.ntt.2007.06.001.
Irwin, S.C. Comprehensive observational assessment: Ia. A systematic quantitative procedure for assessing the behavioral physiological state of the mouse. Psychopharmacologia 1968
, 13 , 222

- 256.
Korenbrot, C.C.; Huhtaniemi, I.T.; Weiner, R.I. Preputial separation as an externa l sign of pubertal development in the male rat. Biology of Reproduction 1977
, 17 , 298

- 303.WIL

- 635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum
Kruskal, W.H.; Wallis, W.A. Use of ranks in one

- criterion variance analysis. Journal of the American Statistical Association 1952
, 47 , 583

- 621.
Moser, V.C.; M cCormick, J.P.; Creason, J.P.; MacPhail, R.C. Comparison of chlordimeform and carbaryl using a functional observational battery . Fundamental and Applied Toxicology 1988
, 11 , 189

- 206.
Moser, V.C.; McDaniel, K.L.; Phillips, P.M. Rat strain and stock comp arisons using a functional observational battery: Baseline values and effects of Amitraz. Toxicology and Applied Pharmacology 1991
, 108 , 267

- 283.

National Research Council. Guide for the Care and Use of Laboratory Animals
, Institute of Laboratory Anima l Resources, Commission on Life Sciences; National Academy Press: Washington, DC, 1996

. O’Donoghue, J.L. Screening for neurotoxicity using a neurologically based examination and neuropathology. Journal of the American College of Toxicology 1989
, 8(1) , 97

- 115. O’Lear, J.R. Development and validation of GC

- ECD assays for the determination of decabromodiphenyl oxide (DeBDPO) concentration in rat plasma and rat milk (Study No. WIL

- 635003). WIL Research Laboratories, LLC, Ashland, OH, 2009

. Paxinos, G; Watson, C. The rat brain in stereotaxic coordinates, 4th Edition; Academic Press: New York, 1998

. Salewski, E. Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. [Staining method for a macroscopic test for implanta tion sites in the uterus of the rat]. Naunyn

- Schmiedebergs Archiv für Experimentelle Pathologie und Pharmakologie 1964
, 247 , 367.

SAS ® Proprietary Software, Version 9.1; SAS Institute, Inc.: Cary, NC, 2002

- 200 3

. Steel, R.G.D.; Torrie, J.H. Princip les and Procedures of Statistics, A Biometrical Approach , 2nd ed.; McGraw

- Hill Book Company: New York, NY, 1980 ; pp 504

- 506.
Viberg, H.; Fredriksson, A.; Jakobsson, E.; Orn, U.; Eriksson, P. Neurobehavioral derangements in adult mice receiving decabrom inated d iphenyl ether (PBDE 209) during a defined period of neonatal brain development. Toxicological Science s 2003
, 76 , 112

- 120.WIL

- 635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum
Viberg, H.; Fredriksson, A.; Eriksson, P. Changes in spontaneous behaviour and altered response to nicotine in the adult rat, after neonatal exposure to the brominated flame retardant, decabrominated diphenyl ether (PBDE 209). Neurotoxicology 2007
, 28(1) , 136

- 42.WIL

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### 11. D EVIATI ONS FROM T HE 

### P ROTOCOL This study was conducted in accordance with the protocol and protocol amend ments,

e xcept for the following:

Protocol Section 8.5.3 states that the test substance would be administered at approximately the same time each day. However, on 4 February 2008 ( lactation day 21), animals were dosed approximately 4.5 to 5 hours later than the ti me that the animals were dosed on the first day of dose administration (24 December 2007) as a result of the timing of the detailed clinical observations for these animals

. Protocol Section 8.7.1 states that animals assigned to the main study (developmenta l neurotoxicity phase) would be observed approximately 1 hour following dose administration. I n the 10 mg/kg/day group, the 1

- hour post

- dosing observation was conducted 1 hour and 16 minutes following dose administration for female no. 90653 on lactation day 15 and for female nos. 90622, 90670 and 90701 on lactation day 14.
Protocol Section 8.9.4 states that blood samples would be collected from the lateral tail vein of each maternal female and via decapitation from the culled offspring in the litters of t hese females on lactation day/PND 4. The culled offspring of female

no. 90714 in the 10 mg/kg/day group were euthanized by an intraperitoneal injection of sodium pentobarbital and discarded. Therefore, the remaining offspring (not culled) of female no. 9 0714 were sent to necropsy, euthanized by decapitation and had blood collected at 8 hours following dose administration of the dam on PND 4.
Protocol Section 8. 10.4.1 states that 30 male and 30 female offspring from each treatment group would be assigned t o Subset A for detailed clinical observations on PND 4, 11, 21, 35, 45 and 60. However, 31 pups/sex were initially selected from the 1 mg/kg/day group and received a detailed clinical observation o n PND 4. As a result, 1 male ( no. 90710

- 05 ) and 1 female ( no. 90710

- 14 ) in the 1 mg/kg/day group were removed from further detailed clinical observations ( on PND 11, 21, 35, 45 or 60 ) . These animals were necropsied on PND 21 with the other pups that were not selected for behavioral evaluations.
Protocol Section 8.10.4.2 states that on PND 120 and 180, 20 animals/sex/group would be assessed for locomotor activity and during the nicotine challenge phase, 10 animals/sex/group would be dosed with nicotine and the remaining 10 animals/sex/group would be dosed with 0. 9% saline. However, in the 10 mg/kg/day group, 9 males were dosed with nicotine and 11 males were dosed with 0.9% saline on PND 120 and 180.WIL

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Protocol Section 8.10.4.2 states that locomotor act ivity would be monitored using 30 rats/sex/group on PND 13, 1 7, 21 and 61. On PND 17, female nos. 90679

- 11 and 90570

- 11 in the control group, no. 90523

- 09 in the 10 mg/kg/day group,

nos. 90590

- 11 and 90565

- 10 in the 100 mg/kg/day group and male no. 90634

- 06 in the 1000 mg/kg/day group were loaded into test chambers for assessment of locomotor activity ; however, th e animals were unloaded and placed in their home cage after approximately 5 minutes because the test session had not start ed . On PND 21, female nos. 90533

- 14 and 90641

- 13 in the 1 mg/kg/da y group, no. 9062 0

- 09 in the 10 mg/kg/day group and male no. 90661

- 02 in the 100 mg/kg/day group were loaded into test chambers for assessment of locomotor activity ; h owever, the animals were unloaded and placed in their home cage after approximately 2 minutes because the test session had not started. On PND 17 and 21, the above

- mentioned animals were placed in the home cage for approximately 15 minutes after unloading, were reloaded and a new session was initiated for each animal.
Protocol Section 8.10.4.4 states that the minimum intertrial interval would be 1 hour. During the PND 62 learning and memory assessment, the time into the maze was not recorded for male nos. 90516

- 01 and 90539

- 06 in the control group and female

no. 90534

- 15 in the 10 mg/kg/day group for trial 6. Therefore, it cannot be determined if the animals rested for at least 1 hour prior to the next trial. However, the animals tested before and after these males had at least a 1 hour rest period between trials.
Protocol Section 8.10.4.4 states that the ti me to escape and number of errors would be recorded during the learning and memory assessment for animals in Subset B and Subset A on PND 22 and 62, respectively. During the PND 22 learning and memory assessment, the time to escape was not recorded for fe male no. 90657

- 14 in the 100 mg/kg/day group for trial 6. During the PND 62 learning and memory assessments, t he time to escape for was not recorded for female no. 90570

- 11 in the control group for trial 10, female no. 90594

- 12 in the 1 mg/kg/day group fo r trial 4, male no. 90670

- 04 in the 10 mg/kg/day group for trial 6 and female nos. 90576

- 15, 90579

- 12 and 90488

- 04 in the 1000 mg/kg/day group for trials 4, 8 and 10, respectively. In addition, the number of errors was not recorded for female

no. 90618

- 15 in the 10 mg/kg/day group for trial 11.
Protocol Section 9.1.5 states that maternal animals that are found dead would be subjected to a gross necropsy, the number and location of implantation sites would be recorded, recognizable fetuses would be examined externally and preserved in 10% neutral

- buffered formalin. However, there is no documentation regarding the description of the uterine contents and examination and retention of the fetuses for female no. 90557 in the 100 mg/kg/day group and female nos. 9 0508 and 90551 in the 1000 mg/kg/day group.WIL

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Protocol Section 9.1.5 states that recognizable fetuses of females that were found dead would be examined externally, euthanized by an intrathoracic injection of sodium pentobarbital and preserved in 10 % neutra l

- buffered formalin. Female

no. 90503 in the 1000 mg/kg/day group was found dead on lactation day 1 with 4 dead fetuses in utero. In addition to the external exam, t he fetuses of this dam were also sexed and crown

- rump lengths were measured.
Protocol Sec tion 9.2.1.1 states that offspring found dead or euthanized in extremis from birth to PND 4 would be examined externally and sexed , and the stomach would be examined for the presence of milk . Female no. 90629

- 15 in the 10 mg/kg/day group was found dead on 14 January 2008 (PND 4), but was not examined until 15 January 2008

. In addition, pup no. 90682

- 01 in the control group was found dead on PND 1 and pup nos. 90662

- 07 and 90662

- 17 in the 1000 mg/kg/day group were found dead on PND 2; however, the stomachs were not examined for the presence of milk. Furthermore, the pups in the following table were found dead on 13 January 2008 and were exa mined for the presence of milk; however, these pups may have also received cranial, thoracic and urinary/reproductive examinations

.

Dam no. Group Pup no. Day of Death (PND) 90658 0 mg/kg/day 1 0 90682 1 mg/kg/day 1 1 90540 1 mg/kg/day 4 4 90673 10 mg/kg/day 13 1 90675 100 mg/kg/day 1, 2, 3, 4, 5, 6, 7, 8, 0 90636 100 mg/kg/day 2 0 90711 1000 mg/kg/day 1, 2 0

Pro tocol Section 9.2.2 states that offspring not previously selected for neuropathology or behavioral evaluations would be euthanized by carbon dioxide inhalation and subjected to a gross necropsy on PND 21

. Male no. 90554

- 04 in the control group was not selected for neuropathology or behavioral evaluations, but was not euthanized and examined until PND 27.

These deviations did not negatively impact the qu ality or integrity of the data or the
outcome of the s tudy.