AMENDMENT TO THE FINAL REPORT

STUDY TITLE

AN ORAL (GAVAGE) DEVELOPMENTAL NEUROTOXOCITY

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

DATE FINAL REPORT ISSUED

20 March 2009

DATE AMENDMENT ISSUED

19 March 2010

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

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AMENDMENT TO THE FINAL REPORT

Section 1.4. Conclusions

Original Report Page: 27

Amendment 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 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.

Revised sentence:

Thus, the no-observed-adverse-effect level (NOAEL) for both F0 systemic 

toxicity and F1 neonatal and developmental neurotoxicity of decabromodiphenyl 

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.

Section 5.2.7. Weaning and Selection (Developmental Neurotoxicity Phase)

Original Report Page: 49

Amendment to Final Report Page: 10

Change: The sentence regarding the nicotine challenge selections 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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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/group [PND 61, 120, and 

180]) following the initial 60-minute test session, and then subjected to a second 

60-minute test session immediately following 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

Amendment 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 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.

Reason for Change: The sentence was clarified.

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Section 5.2.8.4. Locomotor Activity (Subset A)

Original Report Page: 52

Amendment 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 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.

Revised sentence:

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 hydrogen 

tartrate/kg of body weight (equivalent to 0.175 mg nicotine base/kg) in 

0.9% saline. This dosage level of nicotine hydrogen tartrate was selected 

because it was consistent with the dosage 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 Examinations

Original Report Page: 88

Amendment 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 groups, respectively, and for 1 female each in 

the 1 and 1000 mg/kg/day groups.

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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, respectively, 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

Amendment to Final Report Page: 15

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 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.

Revised sentence:

Thus, the no-observed-adverse-effect level (NOAEL) for both F0 systemic 

toxicity and F1 neonatal and developmental neurotoxicity of decabromodiphenyl 

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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WIL-635002 

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Decabromodiphenyl oxide 

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: 

I%Tccln io 

Date 

Manager, Quality Assurance 

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REVISED PAGES

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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 found to be 

at least 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. 

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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 immediately following either a 

prepared nicotine or saline challenge. 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. 

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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, although they may have been 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. 

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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 hydrogen tartrate/kg of body weight (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 consistent with the dosage level used for positive 

control studies in the performing laboratory. 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 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 

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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, 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, 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. 

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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 found to be 

at least 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. 

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