AMENDMENT TO THE FINAL REPORT
STUDY TITLE AN ORAL (GAVAGE) DEVELOPMENTAL NEUROTOXOCITY STUDY OF DECABROMODI PHENYL OXIDE IN RATS

STUDY NUMBER
WI L

- 635002 DATA REQUIREMENT OPPTS Guideline 870.6300 OECD Guideline 426
STUDY DIRECTOR Melissa J. Beck, PhD
DATE FINAL REPORT ISSUED

20 March 2009
DATE AMENDMENT ISSUED 19 March 2010
PERFORMING LABORATORY
WI L Research Laboratories, LLC 1407 George Road Ashland, OH 44805

- 8946
SPONSOR Bromine Science and Environmental Forum

37 Square de Meeûs 1000 Brussel s, Belgium

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### A MENDMENT 

### T O 

### T HE 

### F INAL 

### R EPORT
Section 1.4
. Conclusions Original Report Page : 27 Am endment to Final Report Page : 9 Change : The sentence regarding the no - observed

- adverse

- effect

- level ( NOAEL) was changed as follows. Original sentence: Thus, the no - observed

- adverse

- effect level (NOAEL) for both F 0 sy stemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodipheny l oxide was considered to be 1000 mg/kg/day, the highest dosage level administered in this study

. Revised sentenc e: Thus, the no - observed

- adverse

- effect level (NOAEL) for both F 0 sy stemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodipheny l oxide was found to be at least 1000 mg /kg/day , the highest dosage level administered in this study

. Rea son for Change : The sentence was changed to indicate that the NOAEL is at least as high as the highest dose tested on study

.

Section 5.2.7
. Weaning and Selection (Developmental Neurotoxicity Phase) Original Report Page : 49 A mendment to Final Report Page : 10 Change : The sentence regarding the nicotine challenge sele ctions was changed as follows. Original sentence: 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

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Bromine Science and Environmental Forum 180]) following the initial 60

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

- minute test session.
Revised sentence: Animals were either administered nicotine (20 [PND 61] or 10

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

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

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

- minute test session immediately f ollowing either a prepared nicotine or saline challenge. Reason for Change : The sentence was changed to indicate that the second hour of testing occurred after challenge with nicotine or saline.

Section 5.2.8
. Developmental Landmarks, Sensory Function and Neurobehavioral Testing (Developmental Neurotoxicity Phase; Subsets A and B) Original Report Page : 51 A mendment to Final Report Page : 11 Change : The final sentence was changed as follows. Original Sentence: Cumulative litter values include pups that tested positive for the event but were found dead prior to the end of the testing period.
Revised Sentence: Cumulative l itter values include pups that tested positive for the event, although they may have been found dead prior to the end of the testing period. Reason for Change : The sentence was clarified.

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Section 5.2.8.4. Locomotor Activity (Subset A) Original Report Pa ge : 52 Am endment to Final Report Page(s): 12
- 13 Change : The sentence regarding the dose level of nicotine was changed as follows. Also, a second sentence was added. Original sentence: On PND 61, those animals d esignated for PND 72 neuropatho logy (10 ani mals/sex/group) and 10 other animals/sex/group (total o f 20 animals/sex/group) were administered 0.5 mg/kg nicotine.
Revised sentence: On PND 61, those animals d esignate d for PND 72 neuropathology (10 animals/sex/group) and 10 other animals/sex /group (tota l of 20 animals/sex/group) were administered 0.5 mg/kg nicotine hy drogen tartrate/kg of body

w eight (equivalent to 0.1 75 mg nicotine base/kg) in 0.9% saline. T his dosage level of nicotine hy drogen tartrate was selected because it was consistent with the d osage level used for positive control studies in the performing laboratory

. Reason for Change : The form of nicotine was clarified and the rationale for dose level selection for nicotine was provided.

Section 6.5.5.2.1. Macroscopic Examination s Original Re port Page: 88 Am endment to Final Report Page: 14 Change : The second sentence was changed as follows. Original sentence: Dark red material covering the entire brain was observed for 2, 2 and 1 males in the 10, 100 and 1000 mg /kg/day g roups, respectively , and for 1 female each in the 1 and 1000 mg/kg/day groups.

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Bromine Science and Environmental Forum Revised sentence: Dark red material covering the entire brain was observed for 2, 2, 2 and 1 males in the control, 10, 100 and 1000 mg/kg/day groups, respectivel y, and for 1 female each in the 1 and 1000 mg/kg/day groups. Reason for Change : The sentence was updated to indicate the number of control pups with dark red material covering the entire brain.

Section 7. Conclusions Original Report Page: 96 Am endment to Final Report Page: 15 Change : The s entence regarding the no - observed

- adverse

- effect

- level ( NOAEL) was changed as follows. Original sentence: Thus, the no - observed

- adverse

- effect level (NOAEL) for both F 0 sy stemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodipheny l oxide was considered to be 1000 mg/kg/day, the highest dosage level administered in this study

. Revised sentence: Thus, the no - observed

- adverse

- effect level (NOAEL) for both F 0 sy stemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromod ipheny l oxide was found to be at least 1000 mg /kg/day , the highest dosage level administered in this study

. Reason for Change : The sentence was changed to indicate that the NOAEL is at least as high as the highest dose tested on study

.

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The changes to this report had no impact on the scientific validity or interpretation of the results of this study.
The amendment did not entail generation of new data, revision of calculations or modification of previously submitted data and did not change the conclusion of the study. The revisions to the final report included in the amendment may have resulted in changes in pagination in the final report; any potential changes in pagination are not addressed in this amendment.
The signature below certifies that the revised pages have been reviewed and approved by the Study Director.

Assistant Director, Neurosciences Study Director

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Date(s) Date(s) Findings Findings Date(s) of Reported to Reported to Inspectiods) Phase InsDected Studv Director Management

19-Mar-201 0 Final Report Amendment 19-Mar-20 10 19-Mar-20 10 D ec abromodip heny 1 oxide

Auditor(s1

H.

Johnson 

### QUALITY ASSURANCE UNIT STATEMENT

This amendment 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. Quality Assurance findings, derived from the review of the amendment to the final report, are documented and have been reported to the Study Director. A status report is submitted to management monthly.
This amendment 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.
This amendment to the final report will be stored in the Archives at WIL Research Laboratories, LLC, or another location specified by the Sponsor.

Amendment Audited and Released By: 

# io I%Tccln Date Manager, Quality Assurance

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### R EVISED 

### P AGES

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There were no test substance-related effect s on brain weights, lengths or widths on PND 21 and 72. There were no hi stopathological 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 differe nces in morphometric measurements that were considered related to te st substance administration. 

### 1.4. C ONCLUSIONS There was no evidence of maternal toxicity at any dosage level of decabromodiphenyl oxide evaluated in this study. Additionally, there were no effects on offspring survival and growth, or on any of the neurobehavioral parameters evaluated in this study. Normal patterns of habituation were observed at a ll relevant ages tested for both locomotor activity and auditory startle re sponse. Therefore, under the conditions of this study, no evidence of developmental neurotoxicity was observed at any dosage level evaluated. Thus, the no-observed-adverse-eff ect level (NOAEL) for both F 0 systemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodiphenyl oxide was found to be at least 1000 mg/kg/day, the highest dosage le vel administered in this study. Based on the mean plasma concentration of the test substance in F 1 pups at 8 hours following maternal dose administration on lactation da y 4, the internal expos ure at dosage levels between 10 and 1000 mg/kg/day was determin ed in this study and a previous study (Beck, 2009; WIL-635001) to be 1998 ng/mL to 2535 ng/mL.

**Page 9 of 15**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 morpho metry 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 ni cotine 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]) followi ng the initial 60-minute test session, and then subjected to a second 60-minute te st session immediately following either a prepared nicotine or saline challenge. Indi cators of physical deve lopment (bal anopreputial separation and vaginal patency) were evaluated for F 1 animals assigned to Subsets A and B. All F 1 animals not selected for behavioral evalua tions were euthanized and necropsied on PND 21. F 1 animals selected for le arning and memory assess ment on PND 22, and not selected for PND 72 evaluation, and F 1 animals selected for loco motor activity assessment on PND 120 and 180 were ne cropsied following comple tion of these respective assessments.

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### 5.2.8. D EVELOPMENTAL 

### L ANDMARKS 

### , S ENSORY 

### F UNCTION AND 

### N EUROBEHAVIORAL 

### T ESTING 

### (D EVELOPMENTAL 

### N EUROTOXICITY 

### P HASE 

### ; S UBSETS 

### A AND 

### B) The various investigations described herein were used to assess the maturation and behavior of the selected F 1 pups. These procedures were co ncluded when the oldest pups were 180 days of age. Cumulative litter values include pups that tested positive for the event, although they may have been found dead prior to the end of the testing period. 

### 5.2.8.1. B ALANOPREPUTIAL 

### S EPARATION 

### (S UBSET 

### A) Each male pup was observed for balanoprep utial separation beginning on PND 35 (Korenbrot et al

. , 1977). The day on wh ich balanopreputial separa tion 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. V AGINAL 

### P ATENCY 

### (S UBSETS 

### 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 wa s first observed to open was recorded for each pup. Examination of the females wa s continued daily until vaginal patency was present. Individual body wei ghts were recorded on the day of attainment of this landmark. 

### 5.2.8.3. D ETAILED 

### C LINICAL 

### O BSERVATIONS 

### (S UBSET 

### A) Thirty pups/sex/group were observed outside of the home cage on PND 4, 11, 21, 35, 45 and 60 by the same technicians, whenever pos sible, without knowledge of the animal’s group assignment. The same animals were ex amined 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 (litte rmates were used preferentially when available). Parameters evaluated were identical to those evaluated for the F 0 dams (see Section 5.1.2.), except for piloerection, fur appearance, palpebral closure, eye color, eye prominence, pupillar y response, gait, backing and grooming. **Page 11 of 15**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 fu r growth or inability to complete sufficient moveme nt 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) incl uded forelimb and hindlimb grip strength measurement. 

### 5.2.8.4. L OCOMOTOR 

### A CTIVITY 

### (S UBSET 

### 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/gr oup) 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 Mo tor Monitor System (Kinder Scientific, LLC, Poway, CA). This personal computer-controlled system utilizes a series of infrared photobeams surrounding a clear, plastic recta ngular 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 di straction from extraneous environmental stimuli or activity by technician s or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere wi th the path of the b eams. Each animal was tested separately. Data were collected in 5-minute epochs (pri nt intervals) and the test session duration was 60 minutes. Thes e data were compiled as six 10-minute subintervals for tabulation. On PND 61, 120 and 180, rats were temporar ily 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 an imals designated for PN D 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 hydrogen tartrate/kg of body wei ght (equivalent to 0.175 mg nicotine base/kg) in 0.9% saline. This dosage level of nicotine hydrogen
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tartrate was selected because it was consiste nt with the dosage level used for positive control studies in the performing la boratory. The remaining animals (10 animals/sex/group) were administered 0.9% saline. On PND 120 and 180, only 20 animals/sex/group were assessed for locomotor activity, and therefore 10-11 animals/sex/group were admi nistered 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 volum e of 1 mL/kg. Followi ng administration of nicotine or 0.9% saline, animals were immedi ately 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 singl e photobeam) and ambulatory locomo tor activity (e. g., interruption of 2 or more consecutive photobeams). 

### 5.2.8.5. A COUSTIC 

### S TARTLE 

### R ESPONSE 

### (S UBSET 

### A) An acoustic startle response test was perfor med 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 hi gh-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 appropr iate size, which was then placed into the isolation cabinet. Each enclosur e was equipped with a motion sensor. Acoustic startle response testin g 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

**Page 13 of 15**WIL-635002 Decabromodiphenyl oxide Bromine Science and Environmental Forum The thickness of the pons was slightly smaller (p<0.05) fo r males in the 1000 mg/kg/day group when compared to males in the co ntrol 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 (S UBSET 

### D) 6.5.5.2.1. M ACROSCOPIC 

### E XAMINATIONS Summary Data: Table 52 Individual Data: A ppendix A - Table A54 Pathology Report: Appendix I The necropsies of F 1 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, 2 and 1 males in the control, 10, 100 and 1000 mg/kg/ day groups, respectively, and for 1 female each in the 1 and 1000 mg/kg/day groups. Additionally, abse nce 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 c ontrol group and/or in a manner that was not dose-related. Therefore, these fi ndings were not attributed to F 0 maternal test substance administration. 

### 6.5.5.2.2. B RAIN 

### W EIGHTS 

### /B RAIN 

### M EASUREMENTS Summary Data: Table 53 Individual Data: Appe ndix 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.
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### 7. C ONCLUSIONS There was no evidence of maternal toxicity at any dosage level of decabromodiphenyl oxide evaluated in this study. Additionally, there were no effects on offspring survival and growth, or on any of the neurobehavioral parameters evaluated in this study. Normal patterns of habituation were observed at a ll relevant ages tested for both locomotor activity and auditory startle re sponse. Therefore, under the conditions of this study, no evidence of developmental neurotoxicity was observed at any dosage level evaluated. Thus, the no-observed-adverse-eff ect level (NOAEL) for both F 0 systemic toxicity and F 1 neonatal and developmental neurotoxicity of decabromodiphenyl oxide was found to be at least 1000 mg/kg/day, the highest dosage le vel administered in this study. Based on the mean plasma concentration of the test substance in F 1 pups at 8 hours following maternal dose administration on lactation da y 4, the internal expos ure at dosage levels between 10 and 1000 mg/kg/day was determin ed in this study and a previous study (Beck, 2009; WIL-635001) to be 1998 ng/mL to 2535 ng/mL.

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