FINAL REPORT

Volume 1 of 13

(Text, Figures and Tables 1-47)

STUDY TITLE

AN 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

37 Square de Meeûs

1000 Brussels, Belgium
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

COMPLIANCE STATEMENT

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 protocol as approved by the Sponsor.

The protocol was designed to be in accordance with the United States EPA Health Effects 

Test Guidelines OPPTS 870.6300 Developmental Neurotoxicity Study, August 1998, the 

United States EPA Data Call-in Notice, OPPTS, 10 September 1999 and the OECD 

Guideline for Testing of Chemicals, Guideline 426 Developmental Neurotoxicity Study, 

adopted 16 October 2007.

______________________________________ _____________

Melissa J. Beck, PhD Date

Assistant Director, Neurosciences

Study Director
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

TABLE OF CONTENTS

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. Results........................................................................................................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...............................37
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

4.6. F0 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 Treatment Groups and Breeding 

Procedures..................................................................................................41

5. Parameters Evaluated .............................................................................43

5.1. F0 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. F1 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 Selection (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. Balanopreputial Separation (Subset A)......................................................51

5.2.8.2. Vaginal Patency (Subsets A and B)...........................................................51

5.2.8.3. Detailed Clinical Observations (Subset A)................................................51
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 22 

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 Analyses (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. Euthanasia ..................................................................................................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.............................................................................66
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

6.1. Analytical Chemistry .................................................................................66

6.2. Bioanalytical Results (Bioanalytical Phase) ..............................................67

6.3. F0 Maternal Generation (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. Gestation ....................................................................................................71

6.3.5.2. Lactation ....................................................................................................71

6.3.6. Gestation Length and Parturition...............................................................72

6.3.7. F0 Macroscopic Examinations ...................................................................72

6.3.7.1. Unscheduled Deaths...................................................................................72

6.3.7.2. Females that Failed to Deliver...................................................................73

6.3.7.3. Females - Lactation Day 21 .......................................................................73

6.4. F1 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. F1 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. Balanopreputial Separation........................................................................78
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. Macroscopic 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 ................................................................109
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

INDEX OF FIGURES

VOLUME 1 (continued) Page

1. Summary of Body Weights during Gestation [G] (F0 Females)..............................113

2. Summary of Body Weights during Lactation [G] (F0 Females)..............................114

3. Summary of Offspring Weights [G] (F1 - PND 1-21) .............................................115

4. Summary of Body Weights [G] (F1 - PND 28-181)................................................116

5. Summary of Forelimb Grip Strength [G] (F1 Males)...............................................117

6. Summary of Hindlimb Grip Strength [G] (F1 Males)..............................................118

7. Summary of Forelimb Grip Strength [G] (F1 Females)...........................................119

8. Summary of Hindlimb Grip Strength [G] (F1 Females)..........................................120

9. Summary of Motor Activity Total Counts by Session 

(F1 - PND 13, 17 And 21).........................................................................................121

10. Summary of Motor Activity Total Counts (F1 - PND 13).......................................122

11. Summary of Motor Activity Total Counts (F1 - PND 17).......................................123

12. Summary of Motor Activity Total Counts (F1 - PND 21).......................................124

13. Summary of Motor Activity Ambulatory Counts by Session 

(F1 - PND 13, 17 And 21).........................................................................................125

14. Summary of Motor Activity Ambulatory Counts (F1 - PND 13)............................126

15. Summary of Motor Activity Ambulatory Counts (F1 - PND 17)............................127

16. Summary of Motor Activity Ambulatory Counts (F1 - PND 21)............................128

17. Summary of Motor Activity Total Counts (F1 - PND 61 - Hour 1) ........................129

18. Summary of Motor Activity Total Counts (F1 - PND 120 - Hour 1) ......................130

19. Summary of Motor Activity Total Counts (F1 - PND 180 - Hour 1) ......................131
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

20. Summary of Motor Activity Ambulatory Counts (F1 - PND 61 - Hour 1) .............132

21. Summary of Motor Activity Ambulatory Counts (F1 - PND 120 - Hour 1) ...........133

22. Summary of Motor Activity Ambulatory Counts (F1 - PND 180 - Hour 1) ...........134

23. Summary of Motor Activity Total Counts (F1 - PND 61 - Hour 2 -

Animals Dosed With Saline)....................................................................................135

24. Summary of Motor Activity Total Counts (F1 - PND 120 - Hour 2 - Animals 

Dosed With Saline)...................................................................................................136

25. Summary of Motor Activity Total Counts (F1 - PND 180 - Hour 2 - Animals 

Dosed With Saline)...................................................................................................137

26. Summary of Motor Activity Ambulatory Counts (F1 - PND 61 - Hour 2 -

Animals Dosed With Saline)....................................................................................138

27. Summary of Motor Activity Ambulatory Counts (F1 - PND 120 - Hour 2 -

Animals Dosed With Saline)....................................................................................139

28. Summary of Motor Activity Ambulatory Counts (F1 - PND 180 - Hour 2 -

Animals Dosed With Saline)....................................................................................140

29. Summary of Motor Activity Total Counts (F1 - PND 61 - Hour 2 -

Animals Dosed With Nicotine)................................................................................141

30. Summary of Motor Activity Total Counts (F1 - PND 120 - Hour 2 - Animals 

Dosed With Nicotine)...............................................................................................142

31. Summary of Motor Activity Total Counts (F1 - PND 180 - Hour 2 - Animals 

Dosed With Nicotine)...............................................................................................143

32. Summary of Motor Activity Ambulatory Counts (F1 - PND 61 - Hour 2 -

Animals Dosed With Nicotine)................................................................................144

33. Summary of Motor Activity Ambulatory Counts (F1 - PND 120 - Hour 2 -

Animals Dosed With Nicotine)................................................................................145

34. Summary of Motor Activity Ambulatory Counts (F1 - PND 180 - Hour 2 -

Animals Dosed With Nicotine)................................................................................146

35. Summary of Startle Response Data [MAX] (F1 - PND 20).....................................147
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

36. Summary of Startle Response Data [Tmax] (F1 - PND 20).....................................148

37. Summary of Startle Response Data [AVE] (F1 - PND 20)......................................149

38. Summary of Startle Response Data [MAX] (F1 - PND 60).....................................150

39. Summary of Startle Response Data [Tmax] (F1 - PND 60).....................................151

40. Summary of Startle Response Data [AVE] (F1 - PND 60)......................................152

41. Summary of Biel Maze Data (F1 - Mean Time to Escape - PND 22 -

Path A - Combined Sexes) .......................................................................................153

42. Summary of Biel Maze Data (F1 - Mean No. of Errors - PND 22 -

Path A - Combined Sexes) .......................................................................................154

43. Summary of Biel Maze Data (F1 - Mean Time to Escape - PND 22 -

Path B - Males).........................................................................................................155

44. Summary of Biel Maze Data (F1 - Mean No. of Errors - PND 22 -

Path B - Males).........................................................................................................156

45. Summary of Biel Maze Data (F1 - Mean Time to Escape - PND 22 -

Path B - Females) .....................................................................................................157

46. Summary of Biel Maze Data (F1 - Mean No. of Errors - PND 22 -

Path B - Females) .....................................................................................................158

47. Summary of Biel Maze Data (F1 - Mean Time to Escape - PND 62 -

Combined Sexes)......................................................................................................159

48. Summary of Biel Maze Data (F1 - Mean No. of Errors - PND 62 - Combined 

Sexes)........................................................................................................................160

49. Summary of Brain Morphometry [Level 1] (F1 Males - PND 21)..........................161

50. Summary of Brain Morphometry [Level 3] (F1 Males - PND 21)..........................162

51. Summary of Brain Morphometry [Level 5] (F1 Males - PND 21)..........................163

52. Summary of Brain Morphometry [Level 1] (F1 Females - PND 21).......................164

53. Summary of Brain Morphometry [Level 3] (F1 Females - PND 21).......................165
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

54. Summary of Brain Morphometry [Level 5] (F1 Females - PND 21).......................166

55. Summary of Brain Morphometry [Level 1] (F1 Males - PND 72)..........................167

56. Summary of Brain Morphometry [Level 3] (F1 Males - PND 72)..........................168

57. Summary of Brain Morphometry [Level 5] (F1 Males - PND 72)..........................169

58. Summary of Brain Morphometry [Level 1] (F1 Females - PND 72).......................170

59. Summary of Brain Morphometry [Level 3] (F1 Females - PND 72).......................171

60. Summary of Brain Morphometry [Level 5] (F1 Females - PND 72).......................172
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

INDEX OF TEXT TABLES

VOLUME 1 (continued) Page

1. Results of Homogeneity and Stability Analyses........................................................66

2. Results of Concentration Analyses ............................................................................66

3. Concentrations 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) ...................................91
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

INDEX OF TABLES

VOLUME 1 (continued) Page

SUMMARY DATA - F0 GENERATION

1. Summary of Maternal Survival and Pregnancy Status (F0).....................................174

2. Summary of Clinical Findings: Total Occurrence/No. of Animals 

(F0 - Daily Examinations).........................................................................................175

3. Summary of Post-Dose Findings: Total Occurrence/No. of Animals.....................177

4. Summary of Detailed Clinical Observations (F0 - Gestation) .................................178

5. Summary of Detailed Clinical Observations (F0 - Lactation)..................................188

6. Summary of Body Weights during Gestation [G] (F0)............................................198

7. Summary of Body Weight Changes during Gestation [G] (F0)...............................200

8. Summary of Body Weights during Lactation [G] (F0) ............................................203

9. Summary of Body Weight Changes during Lactation [G] (F0)...............................205

10. Summary of Food Consumption during Gestation [G/Animal/Day] (F0)...............208

11. Summary of Food Consumption during Gestation [G/Kg/Day] (F0)......................210

12. Summary of Food Consumption during Lactation [G/Animal/Day] (F0)...............212

13. Summary of Food Consumption during Lactation [G/Kg/Day] (F0) ......................214

14. Summary of Gestation Lengths [Days] (F0).............................................................216

15. Summary of Macroscopic Findings (F0 - Unscheduled Deaths).............................217

16. Summary of Macroscopic Findings (F0 - Failed to Deliver) ...................................218

17. Summary of Macroscopic Findings (F0 - Lactation Day 21)...................................219

18. Summary of Implantation Sites (F0).........................................................................220
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

SUMMARY DATA - F1 LITTERS

19. Summary of PND 0 Litter Data (F1)........................................................................221

20. Summary of Postnatal Survival [% Per Litter] (F1).................................................222

21. Summary of Pup Clinical Observations (F1)............................................................224

22. Summary of Offspring Weights [G] (F1).................................................................225

23. Summary of Offspring Weight Changes [G] (F1)....................................................228

24. Summary of Pup Necropsy Findings (F1 - Found Dead Pups)................................231

25. Summary of Pup Necropsy Findings (F1 - Pups Not Selected for 

Neuropathology or Euthanized Due to Death of Dam) ...........................................232

SUMMARY DATA - F1 GENERATION

26. Summary of Clinical Findings: Total Occurrence/No. of Animals 

(F1 Males - Subsets A, B and D)..............................................................................233

27. Summary of Clinical Findings: Total Occurrence/No. of Animals 

(F1 Females - Subsets A, B and D)...........................................................................235

28. Summary of Body Weights [G] (F1)........................................................................237

29. Summary of Body Weight Changes [G] (F1)...........................................................243

30. Summary of Animal Developmental Parameters - Balanopreputial 

Separation (F1)..........................................................................................................249

31. Summary of Animal Developmental Parameters - Vaginal Patency (F1)...............250

32. Summary of Detailed Clinical Observations 

(F1 - Subset A)..........................................................................................................251

33. Summary of Motor Activity Counts (F1 - PND 13, 17 and 21 - Subset A).............307

34. Summary of Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 1 -

Subset A) ..................................................................................................................321

35. Summary of Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 2 -

Animals Dosed with Saline - Subset A)...................................................................333
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 1 (continued) Page

36. Summary of Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 2 -

Animals Dosed with Nicotine - Subset A)...............................................................345

37. Summary of Startle Response Data (F1 - Subset A)................................................357

38. Summary of Biel Maze Straight Channel Swim Trials 

(F1 - PND 22 - Subset B)..........................................................................................369

39. Summary of Biel Maze Swim Trials (F1 - PND 22 - Subset B)..............................370

40. Summary of Biel Maze Straight Channel Swim Trials 

(F1 - PND 62 - Subset A)..........................................................................................386

41. Summary of Biel Maze Swim Trials (F1 - PND 62 - Subset A)..............................387

42. Summary of Macroscopic Findings (F1 Males - Unscheduled Deaths) ..................395

43. Summary of Macroscopic Findings (F1 Females - Unscheduled Death)................396

44. Summary of Macroscopic Findings (F1 Males - Scheduled Euthanasia -

Subset B)...................................................................................................................397

45. Summary of Macroscopic Findings (F1 Females - Scheduled Euthanasia -

Subset B)...................................................................................................................398

46. Summary of Macroscopic Findings (F1 Males - Scheduled Euthanasia -

Subset A) ..................................................................................................................399

47. Summary of Macroscopic Findings (F1 Females - Scheduled Euthanasia -

Subset A) ..................................................................................................................401

VOLUME 2

SUMMARY DATA - F1 NEUROPATHOLOGY

48. Summary of Macroscopic Findings (F1 - Animals Selected 

for Neuropathology - PND 21 -Subset C)................................................................405

49. Summary of Brain Weights and Brain Measurements (F1 - Animals Selected 

for Neuropathology - PND 21 - Subset C)...............................................................407

50. Summary of Microscopic Findings (F1 - Animals Selected 

for Neuropathology - PND 21 - Subset C)...............................................................411
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 2 (continued) Page

51. Summary of Brain Morphometry [CM] (F1 - Animals Selected 

for Neuropathology - PND 21 - Subset C)...............................................................415

52. Summary of Macroscopic Findings (F1 - Animals Selected 

for Neuropathology - PND 72 - Subset D)...............................................................419

53. Summary of Brain Weights and Brain Measurements (F1 - Animals Selected 

for Neuropathology - PND 72 - Subset D)...............................................................421

54. Summary of Microscopic Findings (F1 - Animals Selected 

for Neuropathology - PND 72 - Subset D)...............................................................425

55. Summary of Brain Morphometry [CM] (F1 - Animals Selected 

for Neuropathology - PND 72 - Subset D)...............................................................437
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

INDEX OF APPENDICES

VOLUME 2 (continued) Page

A. Individual Animal Data ............................................................................................441

INDIVIDUAL DATA - F0 GENERATION

A1. Individual Clinical Observations (F0 - Daily Examinations).............................442

VOLUME 3

A2. Individual Clinical Observations (F0 - Post-Dosing Observations)...................753

A3. Individual Detailed Clinical Observations (F0 - Gestation)...............................974

A4. Individual Detailed Clinical Observations (F0 - Lactation).............................1013

A5. Individual Body Weights during Gestation [G] (F0) .......................................1049

A6. Individual Body Weight Changes during Gestation [G] (F0)..........................1059

A7. Individual Body Weights during Lactation [G] (F0)........................................1069

A8. Individual Body Weight Changes during Lactation [G] (F0)..........................1079

A9. Individual Food Consumption during Gestation [G/Animal/Day] (F0) ..........1089

A10. Individual Food Consumption during Gestation [G/Kg/Day] (F0)..................1099

A11. Individual Food Consumption during Lactation [G/Animal/Day] (F0)...........1109

A12. Individual Food Consumption during Lactation [G/Kg/Day] (F0)..................1119

VOLUME 4

A13. Individual Gestation Lengths [Days] (F0)........................................................1130

A14. Individual Macroscopic Findings (F0 - Unscheduled Deaths).........................1131

A15. Individual Macroscopic Findings (F0 - Failed to Deliver)...............................1137

A16. Individual Macroscopic Findings (F0 - Lactation Day 21)..............................1141

A17. Individual Implantation Sites (F0)....................................................................1306
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 4 (continued) Page

INDIVIDUAL DATA - F1 LITTERS

A18. Individual Litter Viability (F1).........................................................................1316

A19. Individual Pup Viability (F1)............................................................................1337

A20. Individual Postnatal Survival [% Per Litter] (F1).............................................1357

A21. Individual Pup Clinical Observations (F1).......................................................1367

VOLUME 5

A21. Individual Pup Clinical Observations (F1) [continued] ...................................1469

VOLUME 6

A22. Individual Offspring Weights [G] (F1).............................................................1855

A23. Individual Offspring Weight Changes [G] (F1)...............................................1983

A24. Individual Pup Necropsy Findings (F1 - Found Dead Pups)...........................2108

A25. Individual Pup Necropsy Findings (F1 - Pups Not Selected for 

Neuropathology or Euthanized Due to Death of Dam)...................................2115

VOLUME 7

INDIVIDUAL DATA - F1 GENERATION

A26. Individual Clinical Observations (F1 Males - Subsets A, B and D)................2146

A27. Individual Clinical Observations (F1 Females - Subsets A, B and D).............2267

A28. Individual Body Weights [G] (F1 Males).........................................................2394

A29. Individual Body Weights [G] (F1 Females).....................................................2409

A30. Individual Body Weight Changes [G] (F1 Males)...........................................2424

A31. Individual Body Weight Changes [G] (F1 Females)........................................2439
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 7 (continued) Page

A32. Individual Animal Developmental Parameters - Balanopreputial 

Separation (F1)..................................................................................................2454

A33. Individual Animal Developmental Parameters - Vaginal Patency (F1)...........2471

VOLUME 8

A34. Individual Detailed Clinical Observations 

(F1 - Subset A)..................................................................................................2492

VOLUME 9

A35. Individual Motor Activity Counts (F1 - PND 13, 17 and 21 - Subset A)........2853

A36. Individual Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 1 -

Subset A)..........................................................................................................2883

A37. Individual Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 2 -

Animals Dosed with Saline - Subset A) ..........................................................2913

A38. Individual Motor Activity Counts (F1 - PND 61, 120 and 180 - Hour 2 -

Animals Dosed with Nicotine - Subset A).......................................................2943

A39. Individual Startle Response Data (F1 - Subset A)............................................2973

A40. Individual Biel Maze Straight Channel Swim Trials 

(F1 - PND 22 - Subset B)..................................................................................2993

A41. Individual Biel Maze Swim Trials (F1 - PND 22 - Subset B) .........................3003

A42. Individual Biel Maze Straight Channel Swim Trials 

(F1 - PND 62 - Subset A) .................................................................................3023

A43. Individual Biel Maze Swim Trials (F1 - PND 62 - Subset A).........................3033

A44. Individual Macroscopic Findings (F1 Males - Unscheduled Deaths)..............3073

A45. Individual Macroscopic Findings (F1 Females - Unscheduled Death)............3075

A46. Individual Macroscopic Findings (F1 Males - Scheduled Euthanasia -

Subset B) ..........................................................................................................3076
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 9 (continued) Page

A47. Individual Macroscopic Findings (F1 Females - Scheduled Euthanasia -

Subset B) ..........................................................................................................3151

VOLUME 10

A48. Individual Macroscopic Findings (F1 Males - Scheduled Euthanasia -

Subset A)..........................................................................................................3227

A49. Individual Macroscopic Findings (F1 Females - Scheduled Euthanasia -

Subset A)..........................................................................................................3326

INDIVIDUAL DATA - F1 NEUROPATHOLOGY

A50. Individual Macroscopic and Microscopic Findings 

(F1 - Animals Selected for Neuropathology - PND 21 - Subset C)................3425

A51. Individual Final Body and Brain Weights and Brain Measurements 

(F1 - Animals Selected for Neuropathology - PND 21 - Subset C)................3575

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

(F1 - Animals Selected for Neuropathology - PND 21 - Subset C)................3585

A53. Individual Brain Morphometry [Cm] (F1 - Animals Selected for 

Neuropathology - PND 21 - Subset C)............................................................3595

VOLUME 11

A54. Individual Macroscopic and Microscopic Findings 

(F1 - Animals Selected for Neuropathology - PND 72 - Subset D)................3612

A55. Individual Final Body and Brain Weights and Brain Measurements 

(F1 - Animals Selected for Neuropathology - PND 72 - Subset D)................3761

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

(F1 - Animals Selected for Neuropathology - PND 72 - Subset D)................3771

A57. Individual Brain Morphometry [Cm] (F1 - Animals Selected for 

Neuropathology - PND 72 - Subset D)............................................................3781

B. Certificate of Analysis (Sponsor-Provided Data) ..................................................3809

C. Analyses of Dosing Formulations (WIL Research Laboratories, LLC)................3816

D. Nesting Material Analysis......................................................................................3874
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 11 (continued) Page

E. Food and Water Analyses.......................................................................................3881

F. Animal Room Environmental Conditions..............................................................3952

G. Bioanalytical Phase Animal Data...........................................................................3966

1. Individual Clinical Observations (F0 - Daily Examinations)...........................3967

2. Individual Body Weights during Gestation [G] (F0) .......................................3984

3. Individual Body Weight Changes during Gestation [G] (F0)..........................3986

4. Individual Body Weights during Lactation [G] (F0)........................................3988

5. Individual Body Weight Changes during Lactation [G] (F0)..........................3990

6. Individual Gestation Lengths [Days] (F0)........................................................3992

7. Individual Litter Viability (F1).........................................................................3993

8. Individual Pup Viability (F1)............................................................................3995

9. Individual Postnatal Survival [% Per Litter] (F1).............................................3997

10. Individual Pup Clinical Observations (F1).......................................................3999

11. Individual Offspring Weights [G] (F1).............................................................4025

12. Individual Offspring Weight Changes [G] (F1)...............................................4038

13. Individual Pup Necropsy Findings (F1 - 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)............................4265
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

VOLUME 12 (continued) Page

M. Statistical Analysis Report (BioSTAT Consultants, Inc.)......................................4299

VOLUME 13

N. WIL 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 Neurotoxicity 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. WIL 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 ........................................................................................................4736
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

1. SUMMARY

1.1. OBJECTIVE

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 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 

levels of 100, 300 and 1000 mg/kg/day.

1.2. STUDY DESIGN

Four groups of bred female Sprague Dawley Crl:CD(SD) rats (35/group; Groups 2-5)

were administered 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). F0 females were approximately 13 weeks 

of age at the beginning of test substance administration.

All animals were observed twice daily for appearance and behavior. Clinical 

observations were recorded daily (beginning on gestation day 0) and approximately 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

1 hour following dose administration during the treatment period. Body weights and 

food consumption were recorded at appropriate intervals during gestation and lactation. 

In addition, detailed clinical observations (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 F0 females were allowed to deliver and rear their offspring to lactation day 21, at 

which time the dams were euthanized and necropsied. F0 females that failed to deliver 

were necropsied on post-mating day 25.

Clinical observations, body weights and gender identities were recorded for the F1 pups at 

appropriate intervals. On postnatal day (PND) 4, litters were culled (reduced) 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 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 (Subset 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 pups/sex from the control and 1000 mg/kg/day

groups 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 

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 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 

during the PND 61, 120 and 180 locomotor activity assessments. Animals were 

administered nicotine (20 [PND 61] or 10-11 [PND 120 and 180] pups/sex/group) or saline
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

(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 F1 animals assigned to Subsets A and B. All F1 animals not selected for 

behavioral evaluations were euthanized and necropsied on PND 21. F1 animals selected for 

learning and memory assessment on PND 22, and not selected for PND 72 evaluation, and 

F1 animals selected for locomotor activity assessment on PND 120 and 180 were necropsied 

following completion of these respective assessments.

An additional bioanalytical 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 from 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 F0 females 

were recorded at appropriate intervals. Clinical observations, body weights and gender 

identities were recorded for the F1 pups at appropriate intervals. Blood samples were 

collected from 4 F0 females/group and their F1 litters at 8 hours after dose administration 

on lactation day/PND 4. The remaining 4 F0 females/group had milk collected 8 hours 

following dose administration on lactation day 4. All F0 dams and F1 pups were 

euthanized following sample collection and discarded. One F0 female in the bioanalytical 

phase that failed to deliver was euthanized on post-mating day 25 and discarded without 

examination.

1.3. RESULTS

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 in a previous preliminary range-finding study

(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 differences were apparent when 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

comparing plasma concentrations (internal exposures) between the external dosage levels 

of 10 to 1000 mg/kg/day. Mean 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 509 

and 1250 ng/mL, respectively. These values were similar to those noted in the 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 F0 maternal animals during the 

study. There were no test substance-related clinical findings noted during the daily 

examinations or at approximately 1 hour following dose administration. Maternal 

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 differences 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 all groups.

There were no test substance-related effects on F1 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 vaginal 

patency) were unaffected by maternal test substance administration. There were no test 

substance-related effects on locomotor activity, which was evaluated on PND 13, 17, 21, 

61, 120 and 180.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

There were no test substance-related effects on brain weights, lengths or widths on 

PND 21 and 72. There were no histopathological 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 morphometric measurements that 

were considered related to test substance administration.

1.4. CONCLUSIONS

There was no evidence of maternal toxicity at 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 

activity 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-effect level (NOAEL) for both F0 systemic toxicity and F1

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 F1 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

2. INTRODUCTION

2.1. RATIONALE FOR STUDY DESIGN

The study design combined the required elements of both the U.S. EPA OPPTS 870.6300 

(1998) and the OECD 426 (2007) 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

specifically covered the OECD element that proposes conducting all behavioral 

evaluations in the same 30 F1 offspring/sex/group, except the post-weaning learning and 

memory 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, locomotor 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 (purity not stated) on PND 3.

2.2. GENERAL STUDY INFORMATION

This report presents the data from “An Oral (Gavage) Developmental Neurotoxicity 

Study of Decabromodiphenyl Oxide in Rats.” Due to software spacing constraints, the 

study title is presented as “Oral Dev. Neurotoxicity 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 F0 generation were defined as the F1

litters; pups from the F1 litters were selected to constitute the F1 generation.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

The following computer protocols were used for data collection during the study:

Computer Protocol Type of Data Collected

WIL-635002 .....................................F0 maternal and F1 litter data (including F0

detailed clinical observations)

WIL-635002A ..................................F1 male post-weaning body weight, clinical 

observation, developmental landmark and 

necropsy (Subset B and Subset A not selected 

for PND 72 evaluation) data

WIL-635002D ..................................F1 female post-weaning body weight, clinical 

observation, developmental landmark and 

necropsy (Subset B and Subset A not selected 

for PND 72 evaluation) data

WIL-635002X ..................................F1 Subset A FOB, acoustic startle response, 

locomotor activity (PND 13, 17 and 21) and 

learning and memory (PND 62) data

WIL-635002W .................................F1 Subset B (PND 22) learning and memory 

data

WIL-635002N ..................................F1 Subset A locomotor activity data (PND 61, 

120 and 180)

WIL-635002U ..................................F1 Subset C (PND 21) necropsy and brain 

weight/measurement/morphometry data 

WIL-635002V ..................................F1 Subset D (PND 72) necropsy and brain 

weight/measurement/morphometry data

WIL-635002T...................................Bioanalytical phase animal data

2.3. KEY STUDY DATES1

Date(s) Event(s)

4 December 2007....................................Experimental starting date (animal receipt)

17-21 December 2007 ............................ F0 mating

24 December 2007..................................Experimental start date (initiation of F0 test 

substance administration)

24 December 2007 - 4 February 2008.... F0 dose administration (developmental 

neurotoxicity phase)

29 December 2007 - 18 January 2008.... F0 dose administration (bioanalytical phase)

12 January - 12 July 2008....................... F1 developmental indices and behavioral 

testing

29 January - 4 February 2008................. F0 lactation day 21/F1 PND 21 necropsies

(including F1 Subset C necropsies)

 

1 Date ranges represent the first and last dates of evaluation.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

KEY STUDY DATES (CONTINUED)

Date(s) Event(s)

9-15 February 2008 ................................ F1 necropsies (Subset B - following 

completion of PND 22 learning and 

memory assessments)

20-26 March 2008 .................................. F1 PND 72 (Subset D) necropsies

18 July 2008............................................Last F1 necropsy (following PND 180 

motor activity assessment)

20 November 2008 .................................Experimental termination/completion date 

(last morphological examination)
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

3. STUDY DESIGN SCHEMATIC (KEY PHASES)

Acclimation period

Developmental neurotoxicity phase Bioanalytical phase

F0 clinical observations, body 

weights and food consumption 

recorded at appropriate intervals

F0 clinical observations and body 

weights recorded 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

F0 FOB performed on gestation days 10 

and 15 and lactation days 10 and 21

All F0 females allowed to deliver

Female that 

failed to deliver 

euthanized and 

discarded

Gross necropsies 

performed on F0 animals 

that were found dead

during gestation

F1 offspring observed daily; 

detailed physical examinations 

and body weights recorded at 

appropriate intervals

All F0 females allowed to deliver

Gross necropsies

performed on F0 females

that were found dead

following parturition or 

failed to deliver

Gross necropsies 

performed on F1

pups that were 

found dead prior 

to weaning

F1 pups potentially exposed to 

test substance in utero and 

through nursing during lactation

F1 offspring observed daily; detailed 

physical examinations and body weights 

recorded at appropriate intervals

Blood samples for 

plasma analysis 

collected from 4 

F0 females/group 

and their litters at 

8 hours following 

dosing on 

lactation day 4; F0

females 

euthanized and 

carcasses 

discarded 

following blood 

collection

Milk samples 

collected from 

4 F0 females/

group at 8 hours 

following dosing 

on lactation 

day 4; F0 females 

and F1 pups 

euthanized and 

discarded 

following sample 

collection

F1 pups exposed to test 

substance in utero and through 

nursing during lactation 

F0 females necropsied on lactation day 21

Gross necropsies 

performed on F1 animals 

that were found dead 

following weaning
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

STUDY DESIGN SCHEMATIC (KEY PHASES) (CONTINUED)

Subset C

15 pups/sex/group 

selected for in situ 

perfusion; brain weights 

and brain measurements 

on PND 21

50 pups/sex/group selected for 

post-weaning developmental 

landmarks and/or 

neuropathological evaluation

Nonselected pups 

necropsied on 

PND 21

Neuropathological and 

morphometric 

evaluations conducted 

for 10 pups/sex in 

control and high-dose 

groups

Subset A

30 rats/sex/group selected for startle 

response evaluations on PND 20 

and 60; locomotor 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 F1 Subset B 

rats/sex/group

20 rats/sex/group

selected for locomotor 

activity evaluations on 

PND 120 and 180; 

nicotine challenge 

followed 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 conducted 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 185-191
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

4. EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS

4.1. TEST SUBSTANCE AND VEHICLE

4.1.1. TEST SUBSTANCE IDENTIFICATION

The test substance, decabromodiphenyl oxide, was received on behalf of the Sponsor 

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 

Sponsor and WIL Research Laboratories, LLC. A Certificate of Analysis for the test 

substance was provided by the Sponsor 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 conditions. 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. VEHICLE IDENTIFICATION

The vehicle used in preparation of the test substance formulations and for administration 

to the control group was corn oil (lot no. 126K0117, exp. dates: 2 November 2008, 

26 December 2008, 7 January 2009, and 15 January 2009, received from Sigma Aldrich, 

Inc., St. Louis, MO).
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

4.1.3. PREPARATION OF VEHICLE AND DOSING FORMULATIONS

For the 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. Aliquots 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 indicated 

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 bring 

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 light 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

The test substance formulations were visually inspected by the study director on 

21 December 2007, and were found to be visibly homogeneous and acceptable for dose 

administration.

4.1.4. SAMPLING AND ANALYSIS

Prior to the initiation of dose administration, non-dosing test substance formulations were 

prepared on 10 December 2007 at concentrations spanning the range of concentrations to 

be used on study (0.2 and 200 mg/mL). Duplicate samples (1 mL each) for homogeneity 

determination were collected from the top, middle and bottom strata of these 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. Following 10 days of refrigerated storage, the aliquots 

were remixed for at least 30 minutes and then duplicate samples (1 mL each) for stability 

and resuspension homogeneity were collected on 20 December 2007 from the top and 

bottom strata of these aliquots. Duplicate samples (1 mL each) for concentration analysis 

were collected from the middle stratum of each dosing formulation (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 

Laboratories, LLC, using a validated gas chromatography 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 results are summarized in 

Section 6.1.

4.2. NICOTINE CHALLENGE

All F1 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

(-)-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 nos. C719583, C729301, C732651, 

C736066 and C740993, exp. dates: 1 March 2009, 1 July 2009, 1 August 2009, 

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% sodium 

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 within a laminar flow hood.

For preparation of the nicotine formulation, 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 the 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 stirred 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 room temperature.

4.3. TEST SYSTEM

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 recognized 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 effects of 

developmental neurotoxicants.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

4.4. ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT 

REGIMEN

The test and vehicle control substance formulations were administered orally by gavage

to F0 females, via an appropriately sized flexible, Teflon®-shafted, stainless steel 

ball-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; total of 37-39 doses). A dosage volume of 5 mL/kg was used. 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 F0 maternal 

animals were assigned to study groups as follows:

Developmental 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. JUSTIFICATION OF DOSAGE LEVELS AND VEHICLE SELECTION

Dosage levels were selected based on 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 corn oil as the delivery vehicle from gestation day 6 

through lactation day 21 at dosage levels of 100, 300 or 1000 mg/kg/day. An additional 

2 groups of females received either 0 or 1000 mg/kg/day of the test substance in soya 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

phospholipon:Lutrol (SPL) to compare 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 clinical findings of 

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

substance-related changes in mean maternal body weights, body weight 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 internal exposure when dams 

received 100, 300 or 1000 mg/kg/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 administered the test 

substance in corn 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/day were selected for use in this study with the intent of 

establishing an internal dose-response curve between 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 study 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

was the minimum required to meet the objectives. The F1 animals were exposed to the 

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

4.6. F0 MATERNAL GENERATION

4.6.1. ANIMAL RECEIPT AND ACCLIMATION

Two hundred forty-one sexually mature female Sprague Dawley Crl:CD(SD) rats were 

received from Charles River 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 following day. Each rat was uniquely 

identified by a Monel metal 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 of 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 November 

2007 or 20 November 2007.

4.6.2. ANIMAL HOUSING

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, OH). 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 the 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 are accredited by the Association 

for Assessment and Accreditation of Laboratory Animal Care International (AAALAC 

International).

4.6.3. DIET, DRINKING WATER AND MAINTENANCE

The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent 

LabDiet®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 each 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 maintained 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. ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an 

environmentally 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 recorded 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 to 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 

10 fresh air changes per hour.

4.6.5. ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING 

PROCEDURES

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 record 

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 copulatory plug 

or the presence of sperm in a vaginal lavage and verified by a second technician. Each 

mating pair was examined daily. The day on which evidence of mating was identified 

was termed gestation day 0, and the animals were separated.

The experimental 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 of 8 rats per group. Following completion of the mating period, the 

bred females were assigned to groups using a WIL Toxicology Data Management System 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

(WTDMSTM) computer program which randomized the animals based on stratification of 

the gestation day 0 body weights in a block design. Body weight values ranged from 

224 g to 299 g on gestation day 0.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

5. PARAMETERS EVALUATED

5.1. F0OBSERVATIONS

5.1.1. CLINICAL OBSERVATIONS AND SURVIVAL (BOTH PHASES)

All rats were observed twice daily, once in the morning and once in the afternoon, for 

moribundity and mortality. Individual clinical observations were recorded daily (prior to 

test substance administration during the treatment period) for each F0 female from 

gestation day 0 until necropsy. Females in the developmental neurotoxicity 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 was

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. DETAILED CLINICAL OBSERVATIONS (DEVELOPMENTAL

NEUROTOXICITY PHASE)

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

Lacrimation/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

Mucous 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

1991; and O’Donoghue, 1989). Testing was performed by the same technicians, 

whenever possible, without knowledge of the animal’s group assignment.

5.1.3. BODY WEIGHTS (BOTH PHASES)

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 

calculated 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 were 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 calculated 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. FOOD CONSUMPTION (DEVELOPMENTAL NEUROTOXICITY PHASE)

Individual 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 

for 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 an unscheduled death, weighing error, food spillage, obvious erroneous value, 

etc.), group mean values 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

5.1.5. PARTURITION (BOTH PHASES)

All females were allowed 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. MACROSCOPIC EXAMINATIONS (DEVELOPMENTAL NEUROTOXICITY 

PHASE)

A gross necropsy was performed on F0 females that died during the course of the study. 

The necropsy consisted of examination of the external surface, all orifices and the cranial, 

thoracic, abdominal and pelvic 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 F0 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. F1 LITTER PARAMETERS

5.2.1. LITTER VIABILITY AND DEATHS (BOTH PHASES)

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. Intact offspring 

dying or sent to necropsy due to the death of the dam during PND 0 to 4 were examined 

externally and sexed; the stomachs were examined 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. LITTER REDUCTION (DEVELOPMENTAL NEUROTOXICITY PHASE)

To reduce variability among the litters, 8 pups/litter, and when possible 4/sex, were 

randomly selected on PND 4. Culled pups were weighed, euthanized by intraperitoneal 

injection of sodium pentobarbital on PND 4 and discarded. 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. CLINICAL OBSERVATIONS (BOTH PHASES)

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 bioanalytical 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

phase received a detailed physical examination on PND 1 and 4. Any abnormalities in 

nursing behavior were recorded.

5.2.4. BODY WEIGHTS (BOTH PHASES)

Pups were individually weighed on PND 1, 4, 7, 11, 14, 17 and 21, and thereafter at 

weekly intervals until necropsy. Pups were also weighed whenever they were removed 

from their cages for behavioral testing. Mean pup weights were presented by sex 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 this report. Pups in the 

bioanalytical phase were individually weighed on PND 1 and 4. When 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 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.

5.2.5. SEX DETERMINATION (BOTH PHASES)

Pups in the developmental neurotoxicity phase were individually 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

5.2.6. CALCULATION OF LITTER PARAMETERS (BOTH PHASES)

Litter parameters were defined as follows:

Mean Live Litter 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-Selection)-7, 

7-14, 14-21 or 4 (Post-Selection)-21

5.2.7. WEANING AND SELECTION (DEVELOPMENTAL NEUROTOXICITY 

PHASE)

Each dam and litter remained together until weaning on lactation day 21. Following 

standardization of litters (culling) on PND 4, one male and one female 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 identification 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 (Subset 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 pups/sex from the control and 1000 mg/kg/day groups 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 brain weight evaluations (PND 72). 

Within Subset D, 10 pups/sex from the control and 1000 mg/kg/day groups were selected 

for neuropathology and morphometry evaluations (PND 72). In addition, 10 males each 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

from the 1, 10 and 100 mg/kg/day groups that were assigned 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 

during the PND 61, 120 and 180 locomotor activity assessments. Animals 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 session, 

and then subjected to a second 60-minute test session. Indicators of physical development 

(balanopreputial separation and vaginal patency) were evaluated for F1 animals assigned to 

Subsets A and B. All F1 animals not selected for behavioral evaluations were euthanized 

and necropsied on PND 21. F1 animals selected for learning and memory assessment on 

PND 22, and not selected for PND 72 evaluation, and F1 animals selected for locomotor 

activity assessment on PND 120 and 180 were necropsied following completion of these 

respective assessments.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

The following table summarizes the allocation of offspring to subsets for behavioral tests, 

brain weights, neuropathology and morphometry evaluations.

No. Selected (Subset) Age Evaluation

15/sex/group (C)

a

PND 21 Brain weights, Neuropathology,

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 activityb

20/sex/group (B) PND 22 Early Learning and Memory**

30/sex/group (A)* PND 62 Late Learning and Memory**

15/sex/group (D)***; aPND 72 Brain weights, Neuropathology,

and Morphometry

 * - The same pup subset was used for DCO, auditory startle, locomotor activity, and 

PND 62 learning and memory assessment. Except as directed for learning and 

memory, the same pups 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/group 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 evaluations 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 designated 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 

assessed for locomotor activity; 10-11 animals/sex/group were administered 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 PND 120 and 180.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

5.2.8. DEVELOPMENTAL LANDMARKS, SENSORY FUNCTION AND 

NEUROBEHAVIORAL TESTING (DEVELOPMENTAL NEUROTOXICITY 

PHASE; SUBSETS A AND B)

The various investigations described herein were used to assess the maturation and 

behavior of the selected F1 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. BALANOPREPUTIAL SEPARATION (SUBSET A)

Each male 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 separation was present. Individual body weights were recorded on the 

day of attainment of this landmark.

5.2.8.2. VAGINAL PATENCY (SUBSETS A AND B)

Each female pup was observed for vaginal perforation beginning on PND 25 (Adams et 

al., 1985). The day on which the vaginal 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. DETAILED CLINICAL OBSERVATIONS (SUBSET 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 of the animal’s 

group assignment. The same animals were examined at each interval, except when an

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 evaluated 

for the F0 dams (see Section 5.1.2.), except for piloerection, fur appearance, palpebral 

closure, eye color, eye prominence, pupillary response, gait, backing and grooming. 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

These parameters were not recorded for pups on PND 4 and/or 11 due to the early 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. LOCOMOTOR ACTIVITY (SUBSET A)

Locomotor activity testing was performed in a room 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 at each interval. Locomotor activity was measured 

automatically using the Kinder Scientific Motor Monitor System (Kinder Scientific, LLC, 

Poway, CA). This personal computer-controlled system utilizes a series of infrared 

photobeams surrounding a clear, plastic 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 technicians 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 administered 0.5 mg/kg nicotine. The remaining animals (10 animals/sex/group)

were administered 0.9% saline. On PND 120 and 180, only 20 animals/sex/group were 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 

administration 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. ACOUSTIC STARTLE RESPONSE (SUBSET A)

An acoustic startle response test was 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 enclosure 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 decibels (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 analyzed in 5 blocks of 10 trials each. Startle response 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

measurements obtained were maximum response amplitude (MAX), average response 

amplitude (AVE) and latency to MAX (TMAX).

5.2.8.6. BIEL MAZE SWIMMING TRIALS (SUBSETS A AND B)

Swimming ability, as well as learning 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 the 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. Testing for 

Subset B animals was initiated when each animal was 22 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. For 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, animals were given 2 minutes to 

complete the task successfully. Upon completing the trial, 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 learning. 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 Path B maze (reverse of Path A).
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 to 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 removed 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. MACROSCOPIC AND MICROSCOPIC EXAMINATIONS

(DEVELOPMENTAL NEUROTOXICITY PHASE)

5.2.9.1. OFFSPRING NOT SELECTED FOR BEHAVIORAL EVALUATIONS 

All F1 animals that were not selected 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 pelvic cavities, including viscera. Tissues were retained only as

deemed necessary by the gross findings.

5.2.9.2. OFFSPRING EUTHANIZED FOLLOWING COMPLETION OF THE 

PND 22 LEARNING AND MEMORY ASSESSMENTS (SUBSET B) OR 

PND 180 LOCOMOTOR ACTIVITY ASSESSMENT (SUBSET A) AND

NOT SELECTED FOR NEUROPATHOLOGY/MORPHOMETRY/BRAIN 

WEIGHT MEASUREMENTS

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 on PND 180 (Subset A), but not allocated to Subset D for 

neuropathology, morphometry and brain weight measurements, were euthanized by 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

carbon dioxide inhalation and subjected to a gross pathology examination. A final 

clinical observation and body weight were 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 pelvic 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. NEUROPATHOLOGY (SUBSETS C AND D)

5.2.9.3.1. MACROSCOPIC EXAMINATION

On PND 21, 15 rats/sex/group assigned to Subset C were macroscopically examined for 

neuropathology. On PND 72, 10 animals/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 startle response and/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 

Procedures (SOPs). The whole brains were removed (including olfactory bulbs), 

weighed and the dimensions (length [excluding the olfactory 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 necropsied 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. MICROSCOPIC EXAMINATION

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 brain 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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-dose groups that met the 

criteria for measurement. The criteria for measurement were based on the quality of the 

perfusion, processing and sectioning, and the degree of homology with tissue sections 

from other selected animals. In addition to the qualitative histopathological evaluation, a 

simple morphometric analysis, as described in Section 5.2.9.3.3., was performed. 

Specific measurements taken were defined in the WIL SOPs, but included at least 

2 measurements from the neocortical, hippocampal and cerebellar 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 C3 - C7 and at lumbar swellings T13 - L4

Trigeminal ganglion/nervesa

Lumbar dorsal root ganglion at T13 - L4*

Lumbar dorsal root fibers at T13 - L4*

Lumbar ventral root fibers at T13 - L4*

Cervical dorsal root ganglion at C3 - C7*

Cervical dorsal root fibers at C3 - C7*

Cervical ventral root fibers at C3 - C7*

Sciatic nerves (2)+

Sural nerves (2)+

Tibial nerves (2)+

Peroneal nerves (2)+

Optic nervesa

Eyesa

Skeletal muscle (gastrocnemius)

a

= Both processed and evaluated microscopically

+

= 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 future evaluation.

* = 4-6 tissues were collected at necropsy; 2 tissues were evaluated 

microscopically.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. MORPHOMETRIC ANALYSIS

A quantitative examination of the brain was conducted on the 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 between males in the control and 

1000 mg/kg/day groups on PND 72, a quantitative 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 specific levels that were analyzed, designated as Levels 1, 3 and 5, were defined as 

follows: Level 1 was a coronal section of rostral 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 visualization 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 sectioning of tissues. As a result, 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. BIOANALYTICAL ANALYSES (BIOANALYTICAL PHASE)

5.3.1. BLOOD COLLECTION

Maternal blood samples (approximately 0.5 mL each) were collected from the lateral tail 

vein of 4 F0 females/group on lactation day 4 at 8 hours post-dose administration. The F1

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 blood samples from the F1 pups were pooled by litter. All blood 

samples were collected into chilled and uniquely labeled tubes containing lithium heparin 

as the anticoagulant. Samples were maintained on wet ice and protected from light until 

centrifugation.

5.3.2. MILK COLLECTION

On lactation day 4, milk samples were collected from 4 F0 females/group (different 

females than those used for blood collection) at 8 hours post-dose administration. 

Females were separated from their litters approximately 5.5 hours prior to milk 

collection. Food jars were maintained with the dams during separation. A 0.05-mL 

injection of oxytocin was subcutaneously administered to each dam prior to milk 

collection. Milk samples (approximately 0.4 mL each) were collected 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. SAMPLE PREPARATION AND ANALYSIS

Plasma was isolated 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 Department at WIL Research Laboratories, LLC for the presence and 

concentration of the test substance using a validated GC/MS method presented in 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. EUTHANASIA

Except for 1 female in the 10 mg/kg/day group that failed to deliver, all surviving F0

females in the bioanalytical phase were euthanized by carbon dioxide inhalation 

following sample collection. The bioanalytical 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 F1 pups (not used for blood collection) 

were euthanized by an intraperitoneal injection of sodium pentobarbital. All carcasses 

were discarded.

5.4. STATISTICAL METHODS

All statistical tests were performed using appropriate computing devices or programs. 

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum 

significance 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 slightly. 

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 recommended 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 separation 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 F1
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

pups and brain morphometric data were analyzed by Dunnett's test (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 consumption 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. LOCOMOTOR ACTIVITY

5.4.1.1. LOCOMOTOR ACTIVITY ACROSS SESSIONS PND 13, 17 AND 21

Cumulative total and ambulatory counts were analyzed with a repeated 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 was 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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. LOCOMOTOR ACTIVITY WITHIN SESSIONS 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 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 (random effect), fixed effects for TRT and TIME and the 

interaction 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 significance 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 the 

pooled time intervals only.

5.4.1.3. LOCOMOTOR ACTIVITY WITHIN SESSIONS 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 

from six 10-minute time intervals were analyzed with a RANOVA. Factors in the model 

included 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 the model for PUP (random effect), 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 RANOVA model, Dunnett’s test was conducted, at 

the 0.05 significance level, to compare each test substance-treated group with the control. 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 the 

pooled time intervals only.

5.4.2. ACOUSTIC STARTLE RESPONSE

Each analysis endpoint was analyzed with a RANOVA. Factors in the model included

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 analysis was conducted, 

by sex, with a RANOVA including factors in the 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 

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*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. BIEL WATER MAZE

Latency and the number of errors for Learning Paths A and B and Memory Path A were 

statistically analyzed individually.

Analyses were conducted with a RANOVA. Factors in the model included 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. 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 TRIAL and the interaction terms TRT*TRIAL. 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 RANOVA model, 

Dunnett’s test was conducted, at the 0.05 significance level, to compare each test 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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.

5.4.4. PUP BODY WEIGHTS

Pup body weights for the pre-cull (PND 1 and 4), post-cull (PND 4, 7, 11, 14, 17 and 21),

and post-weaning (weekly from PND 28 through 181) phases were analyzed separately 

for each phase with a RANOVA. Factors in the model included PUP and LITTER 

(random effects), fixed 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 (random effect), 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 RANOVA 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 at 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. MATERNAL BODY WEIGHTS AND FOOD CONSUMPTION

Body weights and food consumption for gestation days 6 to 20 and lactation days 1 to 21 

were analyzed separately with a RANOVA. Factors in the model included ANIMAL 

(random effect), fixed effects 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 denominator degrees of freedom was applied. In the 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 of 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 above for body weights: gestation days (0-6, 6-9, 9-12, 12-15, 

15-18 and 18-20) and lactation days (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. DATA RETENTION

The Sponsor 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 Archives 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 WIL 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6. RESULTS AND DISCUSSION

Data tables from the 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. ANALYTICAL CHEMISTRY

Analytical Chemistry Report: Appendix C

Results of the analyses 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

Based on these results, the analyzed test substance dosing formulations (preparations)

were within the 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, these

results confirm that the protocol-specified dosages of test substance were administered to 

the animals.

6.2. BIOANALYTICAL RESULTS (BIOANALYTICAL PHASE)

Individual Animal Data: Appendix G

Plasma Bioanalytical Report: Appendix K

Milk Bioanalytical 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. Therefore, milk samples were only collected from 3 females in 

the 10 mg/kg/day group on lactation day 4. All other females in the bioanalytical phase 

were gravid and survived to the scheduled euthanasia.

The measured concentrations of the test substance in maternal and pup plasma on 

lactation day/PND 4 following maternal 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 4
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

Text Table 4. Concentrations of the 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 analyzed 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-635001), 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 concentrations 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 external dosage levels of 

10 to 1000 mg/kg/day. Mean concentrations 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 concentrations 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). Maternal milk 

concentrations 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. However, there were milk samples from 2 dams in the 1 mg/kg/day 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

group with concentrations that were below the lower limit of quantitation (250 ng/mL). 

These milk samples were not included in the calculation of the mean.

6.3. F0 MATERNAL GENERATION (DEVELOPMENTAL NEUROTOXICITY 

PHASE)

6.3.1. CLINICAL OBSERVATIONS AND SURVIVAL

Summary Data: Tables 1, 2, 3

Individual Data: Appendix A - Tables A1, A2

There were no test substance-related mortalities among the F0 maternal animals. Six 

non-test substance-related deaths occurred during the study. 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 found dead on gestation 

day 17. Female nos. 90551, 90508 and 90502 in the 1000 mg/kg/day group were found 

dead on gestation day 18 and female no. 90503 in this same group was found dead on 

lactation day 1. No remarkable clinical findings were noted for any 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. Evidence of possible 

intubation 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 considered test substance-related. There 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 F0 animals in the control, 1, 10, 100 and 1000 mg/kg/day groups 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 test substance-treated groups, 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

including hair loss on various body surfaces, mucoid feces and red or clear material 

around the eyes, 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. PREGNANCY STATUS

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. DETAILED CLINICAL OBSERVATIONS

Summary Data: Tables 4, 5

Individual Data: Appendix A - Tables A3, A4

Positive Control Data: Appendix O (Study nos. 99199, 99263, 99310)

No remarkable differences were apparent between the control and test substance-treated

groups when the detailed clinical 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 F0 females was significantly (p<0.05) higher than that in the control group on 

gestation day 15. However, 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 F0

females regarding the detailed clinical observations.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.3.4. BODY WEIGHTS 

6.3.4.1. GESTATION

Summary Data: Tables 6, 7; Figure 1

Individual Data: Appendix A - Tables A5, A6

Mean F0 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. LACTATION

Summary Data: Tables 8, 9; Figure 2

Individual Data: Appendix A - Tables A7, A8

Mean F0 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 statistically significant.

6.3.5. FOOD CONSUMPTION

6.3.5.1. GESTATION

Summary Data: Tables 10, 11

Individual Data: Appendix A - Tables A9, A10

Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, was 

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.

6.3.5.2. LACTATION

Summary Data: Tables 12, 13

Individual Data: Appendix A - Tables A11, A12

Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, was 

unaffected by test substance administration during lactation. Differences between the 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

control, 1, 10, 100 and 1000 mg/kg/day groups were slight and not statistically 

significant.

6.3.6. GESTATION LENGTH AND PARTURITION 

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 F0 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, respectively, 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. F0 MACROSCOPIC EXAMINATIONS

6.3.7.1. UNSCHEDULED DEATHS

Summary Data: Table 15

Individual 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 macroscopic 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 found 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 the 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

macroscopic examination, this female was determined to have 4 dead 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. No macroscopic findings were 

observed for either of these females that revealed the cause of death. Therefore, these

deaths at 10 and 1000 mg/kg/day were not considered test substance-related.

6.3.7.2. FEMALES THAT FAILED TO DELIVER

Summary Data: Table 16

Individual Data: Appendix A - Table A15

One female 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 noted at necropsy for females that failed to 

deliver.

6.3.7.3. FEMALES - LACTATION DAY 21

Summary Data: Tables 17, 18

Individual Data: Appendix A - Tables A16, A17

Reproductive Historical Control Data: Appendix N

No test substance-related internal findings were observed at any dosage level during the 

scheduled necropsy on lactation day 21. The only recorded internal macroscopic findings 

observed in the 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 clear 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

fluid and dark red contents in the uterus for female no. 90699 and a firm liver for female 

no. 90571 in the control group. There were no test substance-related effects on the 

number of former implantation sites and the number of unaccounted-for sites. 

Differences between the control and test substance-treated groups were slight and not

statistically significant.

6.4. F1 LITTER DATA (DEVELOPMENTAL NEUROTOXICITY PHASE)

6.4.1. PND 0 LITTER DATA AND POSTNATAL SURVIVAL

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, percentage 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 PND 21 were unaffected by F0 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. GENERAL PHYSICAL CONDITION

Summary Data: Table 21

Individual Data: Appendix A - Table A21

The numbers of F1 pups found dead and/or missing, as well as the general physical 

condition of all F1 pups in this study were unaffected by F0 maternal test substance

administration. The numbers of pups 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 groups, respectively, were missing and presumed 

to have been cannibalized.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.4.3. OFFSPRING BODY WEIGHTS

Summary Data: Tables 22, 23; Figure 3

Individual Data: Appendix A - Tables A22, A23

Reproductive Historical Control Data: Appendix N

Mean F1 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 F0

maternal animals 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 control 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 groups were not attributed 

to F0 maternal test substance administration.

6.4.4. NECROPSIES OF PUPS FOUND DEAD

Summary Data: Table 24

Individual 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. No internal findings that could be attributed to F0 maternal test substance 

administration were noted at the necropsies of the aforementioned pups that were found 

dead.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.4.5. NECROPSIES OF PND 21 PUPS NOT SELECTED FOR 

NEUROPATHOLOGICAL EVALUATIONS OR PUPS EUTHANIZED DUE 

TO DEATH OF DAM

Summary Data: Table 25

Individual Data: Appendix A - Table A25

No internal findings were found that could be attributed to F0 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 

maternal test substance administration were dilated renal pelves for 3, 1, 2 and 4 pups in 

the 1, 10, 100 and 1000 mg/kg/day groups, respectively, dark red discoloration of the 

right mandibular 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. F1 GENERATION

6.5.1. CLINICAL OBSERVATIONS AND SURVIVAL

Summary Data: Tables 26, 27

Individual Data: Appendix A - Tables A26, A27

Following weaning of the F1 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 F1 animals in the 10 and 

100 mg/kg/day groups. All other F1 animals survived to the scheduled necropsies.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

There were no test substance-related clinical findings at the weekly examinations of the 

F1 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. These findings are commonly observed for laboratory rats of this 

strain. A soft mass in the lateral facial area was noted for female no. 90524-11 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. BODY WEIGHTS

Summary Data: Tables 28, 29; Figure 4

Individual Data: Appendix A - Tables A28, A29, A30, A31

Mean weekly body weights and body weight changes in the F1 males and females 

following weaning were unaffected by F0 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 group values. The only significant (p<0.05) difference in F1

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, mean weekly body weights were slightly lower than the control group at the 

beginning of the post-weaning period (PND 28) and remained lower (decrease ranging 

from 3.8% to 5.4%) through PND 63. The differences in mean weekly body weights

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. Mean 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 differences between external dosage levels 

of 10 to 1000 mg/kg/day, and because weekly mean body weights at 10 and 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

100 mg/kg/day were generally similar to or slightly higher than the control group, the 

slight transient differences in mean body weights and body weight gains noted in the 

1000 mg/kg/day group were not attributed to maternal test substance administration.

6.5.3. DEVELOPMENTAL LANDMARKS, SENSORY FUNCTION AND 

BEHAVIORAL TESTING

6.5.3.1. BALANOPREPUTIAL SEPARATION

Summary Data: Table 30

Individual Data: Appendix A - Table A32

Reproductive Historical Control Data: Appendix N

Mean ages of attainment of balanopreputial separation and mean body weights at the age 

of attainment were unaffected by F0 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 compared 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. VAGINAL PATENCY

Summary Data: Table 31

Individual Data: Appendix A - Table A33

Reproductive Historical Control Data: Appendix N

Mean ages of attainment of vaginal patency and mean body weights at the age of 

attainment were unaffected by F0 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 at 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

the age 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 significant 

from the concurrent control group.

6.5.3.3. DETAILED CLINICAL OBSERVATIONS

Summary Data: Table 32; Figures 5, 6, 7, 8

Individual Data: Appendix A - Table A34

Positive Control Data: Appendix O (Study nos. 99199, 99263, 99310)

Historical 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 the 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 groups and on PND 35 for the 10 mg/kg/day group 

females. Because all values were within the range of the WIL historical control data the 

lower urination counts were 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. In 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 historical 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 coarse tremors (4.5 mm) on PND 35. Because tremors 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 occurrence of tremors 

in the 1 female on PND 35 was not considered test substance-related.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.5.3.4. LOCOMOTOR ACTIVITY

Summary Data: Tables 33, 34, 35, 36; Figures 9 through 34

Individual Data: Appendix A - Tables A35, A36, A37, A38

Statistical Analysis Summary: Appendix M

Positive Control Data: Appendix O (Study nos. 99435, 99441)

Historical Control Data: Appendix Q

Nicotine Challenge Figures: Appendix R

Locomotor activity was assessed with a personel computer-controlled system that utilizes 

a series of infrared photobeams to quantify ambulatory locomotor activity (e.g., 

interruption of 2 or more consecutive photobeams) and total locomotor activity (a

combination of fine locomotor skills [i.e., grooming; interruption of a single photobeam]

and ambulatory locomotor 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 F1

litters were unaffected by F0 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 minutes, 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 were only slightly lower than that of the concurrent control group, 

the difference was significant (p<0.05). However, 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 F1 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 and
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

100 mg/kg/day groups were determined to be similar statistically to the concurrent 

control group value on PND 180, which further supports that slightly greater habituation 

was the cause of the lower mean cumulative total counts for F1 litters in the 

1000 mg/kg/day group on PND 180. Other slight differences 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-related 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 adult rats, which is characterized by high levels of activity 

during the first 10-20 minutes of the hour and then decreasing over successive 10-minute 

subintervals to low levels of activity 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 second 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 

activity 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 ambulatory activity during the first subinterval 

(66-75 minutes) of the second 60-minute test session. This activity did decrease with 

successive subintervals for both nicotine-administered and saline-administered control 

animals, and therefore demonstrated the expected habituation response, but also 

suggested that the dose of nicotine was ineffective in control animals. The increased 

activity during the 66-75 minute subinterval, when compared to the 51-60 minute 

subinterval for the same group of animals, 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 not 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

necessarily respond as expected, it is difficult to fully evaluate the impact of the 

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. Group mean data 

for nicotine-administered animals did not decrease as rapidly over the second 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, despite a 

somewhat slower, although expected, habituation in nicotine-administered animals 

compared to saline-administered animals, it 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 animals 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, when 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 animals dosed with saline in the 

1000 mg/kg/day group on PND 61, 120 and 180 compared to the control group. 

However, saline is not 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 demonstrated 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 animals 

dosed with nicotine, it is not biologically plausible that saline administration prior to the 

second hour of testing would manifest an effect. Finally, no differences in locomotor 

activity patterns for animals dosed with saline in the 10 and 100 mg/kg/day groups were 

observed, and internal 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 decreased activity in animals dosed with saline in the

1000 mg/kg/day group was not attributed to maternal test substance administration.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.5.3.5. ACOUSTIC STARTLE TEST

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 nos. 99418, 99440)

Historical Control Data: Appendix S

The acoustic startle response habituation paradigm was conducted as a longitudinal 

assessment with selected F1 animals evaluated on PND 20 and again at sexual maturity 

(PND 60). Administration of the test substance at 1, 10, 100 and 1000 mg/kg/day to the 

F0 maternal animals had no significant effect on auditory startle responsiveness. At 

PND 20 and 60, the MAX, TMAX, and AVE values for all trials combined were similar for

the F1 litters in the control and test substance-treated groups. On PND 60, the mean 

overall TMAX value in the 1 mg/kg/day group was significantly (p<0.05) lower than the 

control group mean using repeated measures analysis of variance. However, because the 

mean overall TMAX 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 trial block, and the response gradually decreased with each successive block of 

trials.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.5.3.6. BIEL MAZE SWIMMING TRIALS

Summary Data: Tables 38, 39, 40, 41; Figures 41 through 48

Individual Data: Appendix A - Tables A40, A41, A42, A43

Statistical Analysis Summary: Appendix M

Positive Control Data: Appendix O (Study nos. 99438, 99199)

Historical Control Data: Appendix T

Swimming ability 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 groups, 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 between the F1 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 committed in Path A (trials 1-4). When 

Dunnett’s test was conducted for each of the trials 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 significantly (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 1000 mg/kg/day groups for trials 1 and 3, 

respectively, was not considered test substance-related. Significant (p<0.05) treatmentby-sex interactions were noted when the mean time to escape and the mean number of 

errors committed for Path B (trials 5-10) were evaluated on PND 22. Therefore, 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

On 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 the 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-treated 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. MACROSCOPIC EXAMINATIONS (UNSCHEDULED DEATHS AND 

ANIMALS NOT SELECTED FOR NEUROPATHOLOGY)

6.5.4.1. UNSCHEDULED DEATHS

Summary Data: 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 considered test substance-related.

6.5.4.2. ANIMALS EUTHANIZED FOLLOWING PND 22 LEARNING AND 

MEMORY ASSESSMENTS (SUBSET B)

Summary Data: Tables 44, 45

Individual Data: Appendix A - Tables A46, A47

F1 pups in Subset B were euthanized following completion of the PND 22 learning and 

memory assessments. Hydrocephaly was noted for 1 male in the 1000 mg/kg/day group,

and dilated renal pelves were noted for 1, 2 and 1 males in the 10, 100 and 

1000 mg/kg/day groups, respectively. In addition, a cystic kidney was noted for 1 male

each in the control, 100 and 1000 mg/kg/day groups and 1 female each in the control and 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 all 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 for 

the other internal findings noted, these occurred in single animals, at similar incidences in 

the control group and/or in a manner that was not dose-related, and therefore were also 

not attributed to F0 maternal test substance administration. No other internal findings 

were noted.

6.5.4.3. ANIMALS EUTHANIZED FOLLOWING PND 180 LOCOMOTOR 

ACTIVITY ASSESSMENT (SUBSET A)

Summary Data: Tables 46, 47

Individual Data: Appendix A - Tables A48, A49

F1 pups in Subset A were euthanized following completion of the PND 180 locomotor 

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 these 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 F0 maternal test substance administration.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.5.5. NEUROPATHOLOGY

6.5.5.1. PND 21 (SUBSET C)

6.5.5.1.1. MACROSCOPIC EXAMINATIONS

Summary Data: Table 48

Individual Data: Appendix A - Table A50

Pathology Report: Appendix I

The necropsies of F1 animals selected for brain weights on PND 21 revealed no gross 

observations that 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. BRAIN WEIGHTS/BRAIN MEASUREMENTS

Summary Data: Table 49

Individual 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. 

However, because there were no effects on brain weights relative to final body weights in 

females at this dosage level, or in males and females at 10 and 1000 mg/kg/day, the 

difference was not considered test substance-related.

6.5.5.1.3. QUALITATIVE HISTOPATHOLOGY AND BRAIN MORPHOMETRY

Summary Data: Tables 50, 51; Figures 49 through 54

Individual Data: Appendix A - Tables A50, A53

Pathology 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

The thickness of the pons was slightly smaller (p<0.05) for males in the 1000 mg/kg/day 

group when compared to males in the control group. The 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. These 

statistically significant reduced measurements fell within the WIL historical control data 

ranges, and thus the differences were considered spurious.

6.5.5.2. PND 72 (SUBSET D)

6.5.5.2.1. MACROSCOPIC EXAMINATIONS

Summary Data: Table 52

Individual Data: Appendix A - Table A54

Pathology Report: Appendix I

The necropsies of F1 animals selected for brain weights on PND 72 revealed no gross 

observations that were considered associated with administration of the test substance. 

Dark 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 macroscopic findings were noted in single 

animals, at a similar incidence in the control group and/or in a manner that was not 

dose-related. Therefore, these findings were not attributed to F0 maternal test substance 

administration.

6.5.5.2.2. BRAIN WEIGHTS/BRAIN MEASUREMENTS

Summary Data: Table 53

Individual 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 females at any dosage level on 

PND 72 when compared to the control group.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.5.5.2.3. QUALITATIVE HISTOPATHOLOGY AND BRAIN MORPHOMETRY

Summary Data: Tables 54, 55; Figures 55 through 60

Individual Data: Appendix A - Tables A54, A57

Pathology Report: Appendix I

Historical Control 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 histopathologic changes in the 

brains, spinal cord, spinal ganglia and nerve roots, trigeminal ganglion and nerve, eyes or 

optic nerves, skeletal muscle or peripheral nerves (sciatic n., sural n., tibial n., 

peroneal n.) were all considered to be incidental findings or related to some aspect of 

experimental manipulation (i.e., including perfusion rate/pressure and/or processing 

technique). Because the prevalence, severity and/or histological character of these

incidental tissue alterations were similar between control and 1000 mg/kg/day group 

animals, no qualitative test substance-related differences were found.

The initial morphometry evaluation on PND 72 included Level 1 measurements of the 

height of the hemisphere and the vertical thickness of the cortex in F1 animals from the 

control and 1000 mg/kg/day groups. 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 historical 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 historical control data. There 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.

Because of the statistically significant differences between the control and 

1000 mg/kg/day group males, the Sponsor requested that the morphometry evaluation be 

performed with F1 males from the 1, 10 and 100 mg/kg/day groups on PND 72 to 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

determine how the results might compare with that of the study control and 

1000 mg/kg/day group males. Upon evaluating the PND 72 mean values for all 

morphometric measurements from males in all groups, the following was noted:

1. mean values for the Level 1 height of the hemisphere measurement in males of 

the 1, 10 and 100 mg/kg/day groups 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 the study control group value;

2. mean values for the Level 1 vertical thickness of the cortex in males of the 1, 10 

and 100 mg/kg/day groups were similar to each other and were not statistically 

significantly 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 control 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 final 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 does 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 group
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

on PND 72. Yet, no statistically significant differences were reported in any treatment 

group, including females, in regards to functional activity 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 summary 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 statistically 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 test 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 F0 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<0.01
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

6.6. DISCUSSION

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 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 

levels of 100, 300 and 1000 mg/kg/day.

There were no test substance-related mortalities in the F0 maternal animals during the 

study. There were no test substance-related clinical findings noted during the daily 

examinations or approximately 1 hour following 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 differences 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 F1 animals at any dosage level. In addition, attainment of sexual 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

developmental landmarks (balanopreputial separation and vaginal patency) were 

unaffected by maternal test substance administration.

Administration of the test substance to the F0 maternal animals had no significant effect 

on swimming ability or learning and memory. 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. Auditory startle responsiveness was also unaffected by maternal test substance 

administration, and normal 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 substance-related effects on locomotor activity when evaluated during 

a 60-minute test session on PND 13, 17 and 21. The F1 pups in the current study were 

also evaluated for locomotor activity on PND 61, 120 and 180 because changes in 

locomotor 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 F1 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 substance are higher in pups that 

receive 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 F1 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 locomotor activity, as well as auditory startle response (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 histopathological alterations related to test substance 
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

administration in PND 21 or PND 72 brains, or in PND 72 central or peripheral 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 males 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 of the test substance-treated groups were within the range of 

values in the WIL historical 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 greater than the maximum mean values 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, there 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 differences in the test 

substance-treated males were considered adverse or associated with F0 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 following maternal dose administration. In a previous preliminary range-finding 

study, similar maternal and pup plasma concentrations of the test substance were found 

for dosage 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 levels 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

respectively. Mean concentrations of the test substance in maternal milk samples at 1 

and 10 mg/kg/day on lactation day 4 were 509 and 1250 ng/mL, respectively; these 

values were 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

7. CONCLUSIONS

There was no evidence of maternal toxicity at 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 

activity 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-effect level (NOAEL) for both F0 systemic toxicity and F1

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 F1 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

8. KEY STUDY PERSONNEL AND REPORT SUBMISSION

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, Neurosciences
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

KEY STUDY PERSONNEL AND REPORT SUBMISSION (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 Analytical 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 

Laboratory
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

9. QUALITY ASSURANCE UNIT STATEMENT

9.1. PHASES INSPECTED

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-2007 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 28-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.Stauffer
WIL-635002 Decabromodiphenyl oxide

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 24-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 Test Article Administration 

(Nicotine Challenge) 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 Records (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.Tooman
WIL-635002 Decabromodiphenyl oxide

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-2008, 

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-2008 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-2008 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-Nov-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-Sep-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.Daniels
WIL-635002 Decabromodiphenyl oxide

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-Sep-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-Nov-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, Plasma 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-2008, 

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.Tooman
WIL-635002 Decabromodiphenyl oxide

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 Draft 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) 04-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-2009 J.Tooman

05-Feb-2009 Protocol Amendment X 

Review 05-Feb-2009 18-Mar-2009 K.Mentzer
WIL-635002 Decabromodiphenyl oxide

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 Principles of Good Laboratory Practice, the

standard operating procedures of WIL Research Laboratories, LLC, and the Sponsor's 

protocol and protocol amendments with the following exceptions. The data located in 

Appendix B (Certificate of Analysis) were the responsibility of the Sponsor. Quality 

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 another location specified by the Sponsor.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

9.2. APPROVAL

This study was inspected according to the criteria discussed in Section 9.1.

Report Audited 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 Assurance
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

10. REFERENCES

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 

design 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-635001). 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 determination 

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 tables 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 neurobehavioral 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 and techniques appropriate for developmental neurotoxicity testing: A report 

from the ILSI Risk Science Institute Expert Panel on neurodevelopmental 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 external 

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.; McCormick, 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 comparisons 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 Animal 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 implantation 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, 2002-2003.

Steel, R.G.D.; Torrie, J.H. Principles 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 decabrominated diphenyl ether (PBDE 209) 

during a defined period of neonatal brain development. Toxicological Sciences

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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

11. DEVIATIONS FROM THE PROTOCOL

This study was conducted in accordance with the protocol and protocol amendments, 

except 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 time 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 (developmental 

neurotoxicity phase) would be observed approximately 1 hour following dose 

administration. In 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 these 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. 90714 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 to 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 on 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-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

Protocol Section 8.10.4.2 states that locomotor activity would be monitored using

30 rats/sex/group on PND 13, 17, 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, the animals were unloaded and placed in their home 

cage after approximately 5 minutes because the test session had not started. On 

PND 21, female nos. 90533-14 and 90641-13 in the 1 mg/kg/day group, no. 90620-09 

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; however, 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 time 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 female no. 90657-14 in the 

100 mg/kg/day group for trial 6. During the PND 62 learning and memory 

assessments, the 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 for 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. 90508 and 90551 in 

the 1000 mg/kg/day group.
WIL-635002 Decabromodiphenyl oxide

Bromine Science and Environmental Forum

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 % neutral-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, the fetuses of this dam were 

also sexed and crown-rump lengths were measured.

Protocol Section 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 examined 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

Protocol 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 quality or integrity of the data or the 

outcome of the study.