• { ,' ~ , ~ ~ ~ ~ ~ • l ' • • ~ • ,1( • .._ 1i' 1 .,.. .•~ - , f f ' • \ fl •, ~ • t' ' 

t"" :\. - • , '-<~ ';: <\O~.n • ·, ' , _....,'"" " • ., .,_ 

CHEMICAL MANUFACTURERS AssooF'tz- o3' 7·/.:l ?9 

March 18, 1997 

Dr. Lynn Goldman 

Assistant Administrator 

OfficiO' of Prevention, Pesticides and Toxic Substances TS-7101 

Environmental Protection Agency 

401 :.1 Sttet~, SW, Room 637, East Tower 

Washington, DC 20460 

Dear Or. Guldman: 

TI:e Chemical Manufacturers Association makes available to the public and 

approp1iate government agencies final reports of environmental, health and safety resea~ 

that it manages. In keeping with this policy, the following recently completed report is :: 

enclosed: 

A 28-Day Repeated Dose Oral Toxicity Study of HBCD in Rats 

Only volume 1 of 3 is enclosed with this letter. Volumes 2 and 3 contains 658 pages 

and only contain raw data. 

This report does not include confid~ntial information. 

If you have any questions, please call me at 703-741-5637. 

Enclosure 

Sincerely, 

Jfa./rn~~ ~ 

Hasmukh Shah, Ph.D. 

Director, CHF84970000012 

Contains No Cll 
FINAL REPORT 

VOLUME 1 OF 3 

(Text and Summary Tables) 

STUDY TITLE 

A ~f.-DAY REPEATED DOSE ORAL 

TOXICITY STIJDY OF HBCD IN RATS 

DATA REOUIIUMENT 

OECD Guideiine, Section 407 

STUDY DIRECTOR 

Christopher P. Chengelis, Ph.D., D.A.RT. 

STI.TDY INITIATED ON 

Aprii 1 (. 1996 

STUDY COMPLETION lJA TE 

February 13, 1997 

PERFORMITNGLABORATORY 

WIL Research Laboratories, Inc. 

i 407 George Road 

Ashland, Ohio 44805-9281 

LABORATORY STUDY NUMBER 

Wl:L-186004 

SPONSOR PROJECT NUMBER 

BFRIP 2. 0-WIL HBCD 

Chemical Manufacturers Association 

Brominated Flame Retardant Industry Panel 

1300 Wilson Blvd. 

Arlington, V sinia 22209 

Page 1 of 925 

WiL RESEARCH LABORATORIES, INC. A Subsidiary of Great !_akes Chem:cai Corporation, Ashland, OH 44805-!:!281 (419) 289-8700 
• 

• 

wn..-186004 

CMABFRIP 

A 28-Day Repeated Dose Oral 

Ioxjcity Study of HBCP in Rats 

COMPLIANCE STATEMENT 

, . 

• 

Ibis study, designated \Vn.-186004, was conducted in compliance with the United 

States Environmental Protection Agency (EPA) Good Laboratory Practice Regulations (40 

CFR Put 792), October 16, 1989, the Organization for Economic Cooperation anti 

Development (OBCD) Principles of Good Laboratory Practice [C (81) 30 (Fi.W)], the 

Standud Operating Procedures of wn. Research Laboratories, Inc., and the protocol as 

approved by the sponsor (Chemical Manufacturers Association Brominatcd Flame 

Retardant Industry Panel). 1be protocol was designed and the study was conducted in 

accordance with the OBCD Guidelines for testing of Chemicals, Health Effects Test 

Guidelines, Section 407, adopted May 12, 1981. 

Study ~..rector: 136~ Date 
WJL-186004 

CM.ABFR1P 

• A 28-Day Repeated Dose Oral 

Toxicity Stwfy of HBCILiJ.LRIU 

TABLE OF f:ONTENTS 

VOLUME I ~ 

I. Summary 14 

n. Objective 18 

m. Study Design 19 

IV. Experimental Procedures 20 

A. Introduction 20 

B. Test and Control Articles 20 

1. T.'!St Article Identification 20 

2. Control Article Identification 21 

3. Preparation 21 

4. Administration 21 

5. Sampling and Analyses 22 

• c. Animal Receipt and Acclimation/Pretest Period 23 

D. Animal Housing 23 

E. Diet, Drinking Water and Maintenance 24 

F. Environmental Conditions 24 

G. Assignment of Animals to Treatment Groups 24 

H. Clinical Observations and Survival 25 T Body Weights 25 .... 

J. Food Consumption 26 

K. Functional Observational Battery 26 

1. Home Cage Observations 26 

2. Handling Observations 26 

3. Open Field Observations 27 

4. Sensory Observations 27 

5. Neuromuscular Observations 27 

6. Physiological Observations 27 

• L. Locomotor Activity 27 

-3-
' . 

- . ' .,. ' ' ~ - ', ' '.;. . . ' .. . .._ ' 

WIL-!M004 

~...,. 

~~~~ 

.. .&"'~ 

• VO" ~VME 1 (eolltiaued) fiG 

M. Clinical Pathology 28 

1. Hematology 28 

2. Serum Cbemistry 29 

N. Pathology 29 

l. Macroscopic BYaminalion 29 

2. Organ Weights 30 

3. Microscopic Examination 30 

0. Statistical Methods 31 

P. Data Retention 31 

v. Results 32 

A. Clinical Observations and Survival 32 

B. Body Weights 32 

c. Food Consumption 32 

D. Functional Observational Battery 33 • 1. Home Cage Observations 33 

2. Handling ObseiVations 33 

3. Open Field Observations 33 

4. Sensory Observations 34 

5. Neuromuscular ObseiVations 34 

6. Physiological Observations 34 

B. Loc:omotor Activity 35 

F. Clinical Pathology 35 

1. Hematology 3S 

2. Serum Chemistry 35 

G. Pathology 37 

1. Macroscopic Examination 37 

2. Organ Weights 37 

3. Microscopic Examination 38 

VI . Discussion and Conclusions 39 • -4-
,.._ • ~ t ~ ' • a ". : • -/ • • r ' 1 ,-1 • ~ -

.. / ~ .. .. ~ .. ~ . 

\\"'L-186004 

CMABFRIP 

• VOLUME 1 (continued) 

• 

• 

vn. Key Personnel and Report Submission 

vm. Pathologist Summary .:;wement 

IX. Quality Assurance Unit Statement 

X. References 

-5-

41 

42 

45 
• 

• 

• 

WIL-186004 

CMABFRIP 

A 28-Day R!pated Dose Oral 

ToxicitY Study of HBCP in Rats 

INDEX OF TABLES 

VOLUME 1 (continued) 

1. Summary of Survival and Dispositico 

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

(Prior to Scheduled Necropsy/Dispositions) 

3. Summary of Clinical Findings: 

(At Tune of Dosing) 

Total Occurrence/No. of Animals 

4. Summary of Clinical Findings: 

(1-Hour Post-Dosing) 

Total Occurrence/No. of Animals 

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

(Recovery Period Observations) 

6 . Body Weights (Grams) - Summary of Means 

7. Body Weight Gains ·(Grams) - Summary of Means 

8. Weekly Food Cocsumption (Gram~Animal/Day)- Summary cfMean& 

9. Functional Observational Battery Summary Incidence - Home Cage 

Observations (Week -1 Pretest Evaluation) 

iU. Functionai Observational Battery Summary Incidence - Home Cage 

Observations (%] (Week -1 Pl'etest Evaluation) 

11. Functional Observational Battery Summary Incidence - Home Cage 

Observations (Week 3 Evalllation) 

12. Functional Observational Battery Summary Incidence - Home Cage 

Observations [%] (Week 3 Evaluation) 

13. Functional Observational Battery Summary Incidence - Home Cage 

Observations (Week 5 Recovery Evaluation) 

14. Functional Observational Battery Summary IncidC"~ce - Home Cage 

Observations [%] (Week 5 Recovery EvaluatiOI· 

-6-

~ 

47 

49 

51 

53 

55 

51 

61 

55 

69 

71 

73 

15 

77 

79 
. . ' . . ' , " . . . '. ·~: . . ~ . . . "'~"" ;·: ... ". - •, '. . . . . . . . ' ' .. 

WlL-186004 

-

CMABFRIP 

• VOLUME l (continued) 

15. Yiunctional Observational Battery Summary Incidence -

Handling Observations (Week -1 Pretest Evaluation) 81 

16. Functional Observational Battery Summary illcidence -

Handling Observations [%](Week -1 Pretest Evaluation) 87 

17. Functional Observatlonal Battery Summary Incidence -

Handling Observations (Week 3 Evaluation) 93 

18. Functional Observational Battery Summary Incidtmce -

Handling Observatiocs [%] (Week 3 Evaluation) 99 

19. Functional Observational Battery Summary Incidence -

Handling Observations (Week 5 Recovery Evaluation) 105 

20. Functional Observational Battery Summary Incidence -

Handling Observations [%] (Week 5 Recovery Evaluation) 111 

21. Functional Observatioi'al ~.ery Summary Incidence -

Open Field Observatioas (W (;ek -1 PretP..st Evaluation) 117 • 22. FunctioLal Observational Battery Summary Incidence -

~n Field Observations [%] (Week -1 Pretest Evaluation) 121 

23. Functional Observational Battery Summ.u)' Incidence -

Open Field Observations (Week 3 Evaluation) 125 

24. F!!!!ctiana! Obser--.::ticna! .Bar..er-; SumiJ.Jar. llic.~ 1ecce -

Open Field Observations [%] (Week: Evaluation) 129 

25. Functional Observational Battery Summary Incidence -

Open Field Observation~ (Week 5 Recoven· Evaluation) 133 

26. Functional Observational Battery Summary Incidence -

Open Field Observations [%] (Week 5 Recovery Evaluation) 137 

27. Functional Observational Battery Summary Incidence -

Sensory Observations (\Veek -1 PrP..-test Evaluation) 141 

28. Functional Observational Battery Summary Incidenc!!-

Sensory Observations [%] (Week -1 Pretest Evaluation) 145 

• -7-
~~ ~~~~~---~ ............ UV\Ar"W' 

CMABFRIP 

• VOLUME 1 (coatiaued) 

29. FullctiOIIII Observational Battery Summary Incidence -

Sensory Observati<D (Week 3 Evaluation) 149 

30. Functioaal Observational Battery Summary Incidence -

Sensory ObseiVations [%](Week 3 Evaluation) 1S3 

31. Functi'JDal ObseJVational Battery Summary Incidence -

Sensory ObseJVations (WeekS Recovery Evaluation) 1S7 

32. Functional ObseJVational Battery Summary Incidence -

Sensory ObseJVations [%] (Week S Recovery Evaluation) 161 

33. Functirnal ObseJVational Battery Summary Incic:leoce -

Neuromuscular Observations (Week -1 Pretest Evaluation) 16S 

34. Functional Observational Battery Summary Incidence -

Neuromuscular ObseJVations t.%] {Week -1 Pretest Evaluatio11) 167 

3S. Functional ObseiVational Battery Summary Incidence -

Neuromuscular Obselvations (Week 3 Evaluation) 169 

• 36. Functional ObseJVational Battery Summary Incidence -

Neuromuscular ObseJVations [II] (Week 3 Evaluation) 171 

37. Functional ObseJVational Battery Summary Incidence -

Neuromuscular Observations (WeekS Recovery Evaluation) 173 

38. Functional ObseJVational Battery Summary Incidence -

Nfllli'QIIlnscular ~QM [%] (W~ S ~v~ Rv2lwation) !75 

39. Functional Observational Battery Summary Incidence -

Physiological ObseJVations (Week -1 Pretest Evaluation) 177 

40. Functional Obserrational Battery Summary Incidence -

Physiological ObseJVations (Week 3 Evaluation) 179 

41. Functional Observational Battery Summary Incidence -

Physiological ObseJVations (Wee..1c S Recovery Evaluation) 181 

42. Group Mean Motor Activity Counts (Week -1 Pretest Evaluation) 183 

43 . uroup Mean Motor Activity Counts (Week 3 Evaluation) 187 

• -8-
wn.-186004 

CMABFRIP 

• VOLUME 1 (continued) .ew 

44. Group Meail Motor A<..1ivity Count'> (•.Veek 5 Recovery Evaluation) 191 

45. Hematology Values - Summary of Mero •1 195 

46. Leukocyte Differenili!l Count (%) - Summary of Means 203 

47. Leukocyte Counts - Summary of Means 207 

48. Serum Chemistry Values - Summary of Means 211 

49. Gross Necropsy ObservatiClns Incidence Summary 

(Week 4 Primary Necropsy} 223 

50. Gross Necropsy Observa~ons Incidence Summary 

0N eelr 6 Recovery Necropsy) 224 

51. Organ Weights (Gran&s)- Summary of Means 

(Week 4 Primary 1\ . .:ropsy) 225 

52. Organ Weights (tirams) -Summary of Means 

• (Week 6 Recovery Necropsy) 229 

53. Organ Weights Reh>tive to Final Body Weights (Grams/100 G~ams) -

Summary of Means (Week 4 Primary Necropsy) 233 

54. Organ Weights Relative to Final Body Weights (Grams/ ~00 Grams) -

Summary of Means (Week 6 Recovery Necropsy) 237 

55. , T~stomorphological Diagnosis - Summary Incidence 

, Week 4 Primary Necropsy) 241 

)6. Histomorphological Diagnosis - Summar} Incidence 

(Week 6 Reco.rery Necropsy) 255 

VOUJME2 

57. Individual Survival and Disposition 269 

58. Individual Clinical Observat:ons (Prior to Scheduled 

Necropsy/Dispositions) 273 

, 

59 . Individual Clinical Ot_:ervations (At Time of Dosing) 279 

• -9-
. '/' --~·- J; ·~·:·' :._:_ ... __ :.-.. :". -"' ,, ( - .. -~:· ~-~~ ·" ... ~·:·-; . -~---· ~ 

• - ....... - ~ ~ ' ' ::: :'~ ~. :. .,..._ ..; •• ... :.,;~ J~ ¥ ... ~ 1' .... t~ ' ~ ~~} -

rrrr 

WD..-186004 

CMABFRIP 

• VOLUME 2 (coatinued) fit&.~ 

60. Individual Clinical Observations (1-Hour Post-Dosing) 283 

61. Individual Clinical Observations (Recovery Period Observations) 287 

62. Individual Body Weights (Grams) 288 

63. Individual Body Weight Gains (Grams) 296 

64. Individual Food Consumption (Grams/ Animal/Day) 304 

65. Individual Functiooal Observational Battery Data -

Home Cage Observations (Week -1 Pretest Evaluation) 312 

66. lndividua' :-'· nctional Observational Battery Data - Homer"' · )bservations (Week 3 Evaluation) 320 

67. Individual Functional Observational Battery Data -

Home Cage Observations (Week 5 Recovery Evaluation) 328 

68. Individual Functional Observational Battery Data -

• Handling Observations (Week -1 Pretest Evaluation) ':\?~ • JL 

69. lndividua.l Functional Observational Battery Data -

Handling Ocservations (Week 3 Evaluation) 356 

70. Individual FuncHonal Observational Battery Data -

Handling Observations (Week 5 Recovery Evaluation) 380 

71. Individual Functional Observational Battery Data -

Open Field Observations (Week -1 Pretest Evaluation) 392 

72. Individual Functional Observational Battery Data -

Open Field Observations (Week 3 Evaluation) 408 

73. Individual Functional Observational Battery Data -

Oven Field Observations (Week 5 Recovery Evaluation) 416 

74. Individual Functional Observational Battery Data -

Sensory Observations (Week -1 Pretest Evaluation) 424 

75. Individual Functional Observational Battery Data -

Sensory Observations (Week 3 Evaluation) 440 

• -10-
... 

wn.-186004 

CMABFRIP 

• VOLUME 2 (contiuued) 

76. Individual Functional Observational Battery Data -

Sensory Observations (Week 5 Recovery Evaluation) 448 

77. Individual Functional Observational Battery Data -

Neuromuscular Observations (Week -1 Pretest Evaluation) 456 

78. Individual Fucctional Observational Battery Data -

Neuromuscular Observations (Week 3 Evaluation) 472 

79. Individual Functional Observational Battery Data -

Neuromuscular Ob[..!tvations (Week 5 Recovery Evaluation) 488 

Pn: T ~..:·~-., . --.u -~ • ~· ·i:J<. ~ ~· ··· "''at;onal Battery Data - ....... ~ ~ lro..~ 

- - ) ~ vCSe.vaL. ·; {Week -1 Pretest Evaluation) 496 

!'1. ludividual Functional Observational Battery DataPLysiological Observations (Week 3 Evaluation) 504 

32. Individual Functional Observational Battery Data -

Physiological Observations (Week 5 Recovery Evaluation) 512 

• 83. Individual Motor Activity Counts (Week -1 Pretest Evaluation) 516 

84. Individual Motor Activity Counts (Week 3 Evaluation) 532 

85. Individual Motor Activity Counts (Week 5 Recovery Evaluation) 548 

VOLUME3 

86. Individual Hematology Values (Week 4 Primary Necropsy) 557 

87. Individual Hematology V3.lues (Week 6 Recovery Necropsy) 565 

88. Individual Leukocyte Differenticl Count(%) (Week 4 

Primary Necropsy) 569 

89. Individual Leukocyte Differential Count (%) (Week 6 

Recovery Necropsy) 577 

90. Individual Leukocyte Counts (Week 4 Primary Necropsy) 581 

91. Icdividual Leukocyte Counts (Week 6 Recovery Necropsy) 589 

• -11-

,' ~ - - ' - -' ~-~'r---'-~- ___,___,.~·~~ : ~ --.... ~" • -. ·~ : -,. '__,. • '\ ~ - t ,, • 

" ~ . t" ·- • , . ~ . ' . . . ' ... I r ,. ~ . J' . ' . I . --"',..... ~ ~' • ' •' I ,·' ~ ' '' 1 • 

1 

·.., ~· J' ". ff'; , '' ..:tf ...f.,..:.._~ ' ~ -1:' J ·-
wn.-186004 

CMABFRIP 

• VOLUME 3 (coatinued) ~ 

92. Individual Serum Chemistry Values (Week 4 Primary Necropsy) 593 

93. Individual Serum Chemistry Values (Week 6 Recovery Necropsy) 609 

94. Individual Gross and Microscopic Description of Organs 

(Week 4 Prim2ry Necropsy) 617 

95. Individual Gross and Microscopic Description of Organ~ 

(Week 6 Recovery Necropsy) 669 

96. Individual C"'"p'l Weights and Final Body Weights (Grams) 

(Week 4 Primary Necropsy) 697 

97. Individual Organ Weights and Final Body Weights (Grams) 

(Week 6 Recovery Necropsy) 705 

98. Individual Organ Weights Relative to Final Body Weights 

(Grams/100 Grams) (Week 4 Primary Necropsy) 709 

99 . Individual Organ Weights Relative to Final Body Weights 

• (Grams/100 Grams) (Week 6 Recovery Necropsy) 717 

• -12-
• 

• 

l!l"'rmo· N&lliM 

···:Ii·l~~ 

CMA B-<'RIP 

ft. :.!~-fr.n Repe?red Dv.5-e Oral 

T!?~~:i~tdy of Tio~:::.;v i'l &w 

A. Analyses of Dosing Prepa.rntions {Sponso7'-Ge1le1"3.ted Data) 

B. Gravim<-.trk Analysis of T'tO.>iDg ?nrmulations 

(Wf'- P..ese:l!"ch I.abo:atories, Inc.) 

C. PrettS1 Climcal Cibs 1ativn'> 

D. Scoring Criteriz for Ft nctional Oos;~rvational Battery 

E. Summarieo> of V:tLdat'.:m S~dies (WIL-9S;J26, WJL-99C1?.. 

G . 

H. 

I. 

J . 

Wll.-99G34 <'- ' WIL· 1fC")35) 

WIL Motor Activity Ifu~ orical Control Data 

Clinical Pathology Historic o.i C ~nt·::>l Data 

(WIL Research Labora10ries, IGc..) 

Study Protocol 

-13-

----:: . -

721 

~29 

763 

766 

782 

813 

818 

873 

9u3 
' ... .,.. - ' ' ~ - . .f·' - ' . ~ ' • ~. • ... • I ' -:. :, ' • .. ' ·~- l- ..._ ... - - I.'- ·... I 

• 

• 

• 

WJL-186004 

CMABFRIP 

I. SJJMMAIY 

A 28-Day Repeated Dolle Oral 

Toxicity Study of UBCD in Rats 

1be test article, hexabromocyclododecane (HBCD), Lot #357/, in the vehicle, 

com oil, was administered orally by gavage to three groups of Sprague-Dawley Crl: 

CD BP rats for a period of 28 conset:utive days. Dose levels were 125 (low), 350 

(mid), or 1000 (high) mg/kglday, administered in a dosage volume of 5 ml/kg. The 

test groups consisted of 6 males and 6 females in the 125 and 350 mglkg/day groups 

and 12 males and 12 females in the 1000 mglkg/day group. A concurrent control 

group comprised of 12 males and 12 females received the vehicle, com oil, for 28 

consecutive days at a dosage volume of S mllkg. At the end of the dosing p'!riCid, 6 

animals/sex/group were eutbanized and necropsied. The remaining 6 animals/sex in 

the control and 1000 mg/kglday groups remained on-test untreated for a 14 day 

recovery period. At the end of the recovery period, all animals were eutbanized and 

necropsied . 

Animals were observed twice daily for mortality and moribundity. Clinical 

signs were recorded daily. Body weight and food consumption were measured 

weekly. Functional observational battery and motor activity evaluations were 

perfonned during weeks -1 (pretest), 3, and 5 (recovery). Samples for hematology 

(42 day) necropsies. Complete necropsies were perfonned on all rats. The brain, 

liver, kidney, heart, spleen, testes and epididymis or ovaries, adrenal glands, and 

thymus from all animals were weighed at each necropsy. Approximately 40 tissues 

were collected and p~rved at each necropsy from all animals. The following 

tissues were examined microscopically from the control and high dose animals: liver, 

kidney, heart, spleen, testes (males), prostate (males), seminal vesicles (males), 

epididymis (males), ovaries (females), adrenal glands, thymus, bone with marrow 

(sternebra), brain, stomach, cecum, duodenum, ileum, jejunum, lymph node, 

peripheral nerve (sciatic), spinal cord, lung, trachea, uterus (females), urinary 

-14-
• 

• 

• 

wn.-186004 

CMA BFR.IP 

bladder, and all gross lesions.) The lungs, liver, kidneys, stomach, gross lesions and 

target organs were examined in all dose levels. 

Survival was not affected by administration of the test article. All animals 

sunrived to the scheduled necropsy. Clinical signs observed during the study were 

nom .. pecific, low in incidence, non-dose-reJated and not considered related to test 

article. 

Body weights, weight gain and food consump~:on of treated animals were 

compared statistically by sex and treatment day to their respective control groups 

(p<0.05 or p<O.on. Body weight, weight gain and food consumption was not 

affected by treatment. No statistically significant differen 'eS in body weight between 

control and treated animals were detected with the exception of an increase in mean 

female body weight in the 350 mg/kg/day during week 2 of treatment. Mean female 

body weight at that time point was 19ti g vs 179 in u,~.- control group. No 

statistically significant differences in body weight gain between control and treated 

animals were detected with the exception of a decrease in mean male body weight gain 

in the 1000 mg/kg/day recovery group during week 1 of recovery. Mean male body 

weight gain at that time point was 21 g vs 31 in the control group; mean male body 

weight was not statistically different from the control mean. No statistically 

significant differences in food consumption between control and treated animals were 

detected with the exception of an increase in mean female food consumption in the 

350 mg/kg/day during weeks -1, I, and 2 of treatment. Mean female food 

consumption at that those time points were 18, 17 and 17 g vs 16, 15 and 15 gin the 

control group, respectively. 

Functional observation battery and motor activity results from treated animals 

were compared statistically by sex and treatment day to their respective control groups 

(p<0.05). These parameters were not affected by treatment with the test article. No 

statistically significant differences were observed between treated and control animals 

at any time p.:;int. 

No statistically significant differences between treated and control animals were 

found for hematology parameters with the exception of an increase in the mean 

-15-
• 

• 

• 

WJL-186004 

C'MABFRJP 

activared partial tbromboplastin time in tbe 1000 mglkg/day males on week 4 and a 

decrease in the mean prothrombin time in the 1000 mglkg/day females on week 4. 

These statistical differences were not of toxicological significance. 

No toxicologically significant effects on serum chemistry values related to test 

article administration were observed at the 28 day prima. y a~d 42 day recovery 

necropsies. Scattered instances of statistically significant differences between treated 

and control animals were detected for some serum chemistry parameters at the 28 day 

primary necropsy. These scattered statistical differences were not considered 

toxicologically significant because the statistical differences occurred: in the absence 

of a dose response, in the absence of the accompanying clinical chemistry changes 

expected, in the opposite direction from what occurs in a toxic state, in a direction 

which is without physiologic significance, or due to potential interference with the 

laboratory method. No statistically significant differences in serum chemistry 

parameters were detected between groups at the 42 day recovery necropsy. 

No gross lesions which could be attributed to the test article were detected at 

either necropsy. Gross lesions were nonspecific, low in iucidence, non-dose-related 

and considered incidental. 

No microscopic lesion which could be attributed to the test article were detected 

on histopathologic exam. Microscopic changes were nonspecific, low in incidence, 

non-dose-related and considered incidental. 

No statistical significant differences in organ weight or organ to body weight 

ratios were detected between control and trt:ated animals with one exception. 

Absolute liver weights were statistically significantly increased with respect to control 

means at the 28 day necropsy in males in the high dose and females in the mid and 

high dose. Liver to body weight ratios in mid and high dose males and low, mid and 

high dose females were statistically significantly increased at the 28 day necropsy. 

At the recovery necropsy, male absolute and liver to body weight ratio were 

statistically comparable to the control mean whereas female absolute liver weights and 

Hver to body weight ratio were statistically significantly increased with respect to 

control mean. The difference in absolute liver weight between control and treated 

-16-
• 

• 

• 

WIL-186004 

CMABPRIP 

females was less pronounced at the end of the recovery period, indicating the increase 

in liver weight was reversible in f~ as well as males. In the absence of test 

article related histopathologic and serum chemistry changes, increases in liver weight 

are considenc.: ~<Il adaptive, rather than a toxic response, are not uncommon in the rat, 

and are most likely the result of microsomal induction. 

In conclusion, no systemic toxicity was obseiVed at any dose 1evel. Based on 

the results of this study, the NOAEL (No ObseiVed Adverse Effect Level) of HBCD 

administered orally to male and female rats for 28 consecutive days was 1000 

mg/kg/day . 

-17-
• , r ,( ! )\r ' ' '- I • • - • .. ~ f' 1 • 

~ ' ,. ~ l_ r ' •? .~ • • / -l,.: >I: 

• 

• 

• 

WIL-186004 

CMABF'BJP 

n. OBJECTIY.E 

Tne objective of the study was to evaluate the possible toxic effects of 

HexabromocyclododecaDe (HBCD) when administered orally (gavage) to rats for 28 

days. 'Ibis included evaluation of poteDtia1 neurotoxicity by function.'ll observational 

battery (FOB) and motor activity (MA) assessment. Reversibility, persistence or 

delayed occurrence of toxic effects was evaluated by a 14-day post-treatment recovery 

period. Tbe selected route of administration was oral by gavage as this ro•tte is an 

acceptable and standard method for administering test article per OECD Guideline 

407. Tbe animal model, tbe Sprague-Dawley Crl:~R rat, is recognized as 

appropriate for subchronic toxicity studies and was se~ected based on the availability 

of historical control data . 

-18-
• 

• 

• 

WIL-l tsOllH 

CMABFRIP 

m. STIJDY DESIGN 

Acclimation and 

Pretest Period 

I 

Assignment of Animals 

to Study Groups 

I 

12 Males, 12 Females/Group (1 and 4) 

and 6 Males, 6 Few_>Jes/Group (2 and 3) 

Treated Daily for 28 Days 

I 

Body Weight!' and Food Intake 

Recorded Weekly, Clinical 

Observations Recorded Daily 

I 

Functional Observational Battery 

and Motor Activity Evaluations 

Petformed at Appropriate Intervals 

Clinical Pathology Evaluations on 

All Animals Prior to Scheduled Necropsy 

Necropsy Examinations Performed on 

6 Animals/Sex/Group (1-4) At the 

End of the Dosing PeriGd; Selected 

Organs Weighed; Selected Tissues 

Mi 

Necropsy Examinations Petfonned on 

6 Animals/Sex/Group (1 and 4) 

After a 14-Day Recovery Period; 

Selected Organs Weighed; Selected 

Tissu Mic 

-19-
• 

• 

• 

< ' - ~, . ~ ~ .. <" ; -

' . ' 

WIL-1Bti004 

CMABFRIP 

IV. EXPBRIMBNTAI. PR.OCEPURES 

A. IND,ODl]CDON 

1"be experimental SlUt date, initiating with test article administration, was May 

17, 1996 (week 0). 1"be primary necropsy was on June 14, 1996. The recovery 

necropsy was on June 28, 1996. 1"be experimental tennination date, concluding 

with the last pathology examination, was October 8, 1996. 

B. TSST AND CONIROL ARDCiffl 

1. TEST ARDCLE IDENTIFICA]JQN 

Hexabromocyclododecane (HBCD) was received from Chemical 

Manufacturers Association via Wildlife International Limited, Easton, 

Maryland, as follows: 

Label 

Identification 

#3577HBCD 

Approximately 300 g 

(Composite"' of #3462, 

3519, and 3551) 

Prep. date: 2/19/96 

Exp. date: 12/16/96 

[Wn. Log No. 3014A] 

HBCD 

Test substance no. 3577 

Composite* of no's 3462, 

3519, 3551 

Prep. date: 2/19/96 

Expires: 12/16/96 

[WIL Log No. 3014B] 

No. of 

Containers 

BFceived 

One Bottle 

Gross weight 

646.3 g 

One Bottle 

Gross weight 

430.1 g 

Description 

White 

powder 

White 

powder 

Date 

Received 

4/17/96 

617/96 

"' The test article was a composite sample of equal portions of commercial 

HBCD manufactured by CMA BFRIP members. 

The stability and purity of the bulk test article were the responsibility of 

the sponsor. For purposes of dose calculations, the te..q article was considered 

to be I 00% HBCD. The bulk test article was stored in a sealed container in 

a dry, well-ventilated area at room temperature (68°F ± 4°F) and was 

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considered to be stable under these conditions. C.1 April19, 1996, a 1.013 g 

sample was taken from tile first shipment of test arncie and stored in the 

archives at wn. Research laboratories, Llc. An approximate one gram 

reserve sample ;vas to be taken from tlle second sfa]-iment of test article in 

accordance with wn. standard operating procedures. However, after the 

remaining test article had been returned to the spo~sor, it was discovered that 

a retention sample was not collected from this shipment. This deviation from 

the protocol had no impact on the scientific validity of the study as both 

shipments were of the same lot number. 

2. CONTROL ARTICLE IDENI'IFICATION 

The vehicle control article utilized in preparation of the test mixtures and 

for adtrinistration to the control group was Mazola~ com oil manufactured by 

Best Foods, CPC International, Inc., Englewood Cliffs, New Jersey. 

3. PREPARATION 

A sufficient amount of com oil to dose the control grour ~·nimals was 

placed in a labeled storage container. The vehicle control article was stirred 

continuously throughout the sampling and dosing procedures using a magnetic 

stir bar and stir plate. 

The appropriate amount of test article, HBCD, for each group was 

weighed into a tared, precalibrated storage container. A st~ bar was added 

and the container was tared a second time. An appropriate volume of vehicle 

was added and the weight of the formulation was recorded. 'The formulations 

were stirred continuously throughout the sampling and dosing procedures using 

magr>etic stir bars and plates. 

All dosing formulations were prepared daily and stored at room 

temperature. Test article concentration and homogeneity of ·£he dosing 

formulations were verified using gravimetric determination. 

4. ADMINISTRJ\TION 

The test mixtures were administered ora 1ly by gastric intubation via a 16-

gauee stainless steel gavage cannula (Popper and Sons, Inc., Hyde Park, New 

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York 11040) as a single daily dose for 28 con~ve days through the day 

prior to tbe scheduled primary necropsy. A dose volume of S mllkg was used 

for all dosage levels. 1be concurrent control group animals teeeived the 

vehicle, com o:U, on a compuable regimen at a dose volume of S mllkg. 

Individual dosages were adjusted based on the most recent body weights, to 

provide tbe comet mglkg/doay dose. On May 28, 1996 (study day 11), all 

males and the first female in the 3SO mglkg/day group were inadvertently 

dosed using the dose volumes for all males and the first female in tbe 125 

mglkg/day group, and on June 7, 1996 (study day 21), tbe first four males in 

the 1000 mglkg/day group were inadvertently dosed using tbe dose volumes 

for the first four males in the 3SO mglkg/day group. All arumab that were 

underdosed received a conecting dose amount. In the 3SO mglkg/day group, 

three males (nos. S028S, S0286 and S031S) were overdosed by 0.1 u.l and one 

female (no. S0322) was overdosed by 0.03 mi. One male (no. S0283) in tbe 

1000 mglkg/day group was overdosed by 0.2 mi. 'Ibis deviation from tbe 

protocol bad no impact on the outcome of the study as all animals received tbe 

correct dosing &alution and only a limited number of animal!i received a 

slightly bigber than intended dosage. The following table presents tbe study 

group assignment. 

Dosage Do.qge Number 

Group Test Level Concentration Volume of Allimals 

-Number Artisle <mg/b/dayl (mglml) Cml/lsgl »!Is! ~ 

1 

2 

3 

4 

s. 

Vehicle 0 0 s 12 12 

HBCD 125 2S s 6 6 

HBCD 350 70 s 6 6 

HBCD 1000 200 s 12 12 

SAMPLING AJIIP ANALYSES 

Prior to the initiation of dosing, sample dosing suspensions were prepared 

for Groups 2 and 4, and duplicate 1 mi aliquots were withdrawn from tbe 

middle stratum of each suspension immediately after preparation. The 

suspensions were stilTed overnight using magnetic stir bars 8.i.ld plates, and 

duplicate 1 m1 aliquots were wit!tdrawn from the middle stratum of each 

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WIL-186004 

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su-;pension approximately 2_. hours following p:reparation. The wnples \Vere 

frozen lU soon as possible after collection and were shipped on dry ice to 

Albemarlt: Corporation Technical Cen·er, Baton Rouge, Louisiana, on May 9, 

1996, for stability analysis. Duplicate one-gram samples of the test article 

were collected ;md frozen on the first and last days of t~~ article 

administration, and were shipped on dry ice to Albemarle Cot¥->ration 

Technical Center on May 20, 1996 and Junt; 17, 1996, respectively, for 

stability analysis. The results of th~ <JJalySF;S are presented in Appendix A. 

Gravimetric assays of the dosing formulations for verification of test 

article concentration and homogeneity v1ere perfonr.ed '"t WIL Research 

Laboratories, Inc. Concentration Jetenninations v:ere performc..1 daily prior 

to d"se administration. Homogeneity determinations were perfunneci on study 

days 0, 13 and 27. The results of these analyses are presented in Appendix B. 

C. ANIMAL RECEIPr ANI.> ACCLIM<\TIQN/PRETEST PERIOD 

Forty male and forty female Sprague-Dawley Crl:~R rats were received 

on May 2, 1996, from Charles River Laboratories, Inc., Portage, Michigan. The 

animals were 28 days old upon receipt. Each animal \/as examiiJed by a qualified 

technician on the day of receipt. Animals were uniquely identified by Monel metal 

eartags displaying the pennanent identification nua;!:ler. Ail anim~.s were housed 

for a 15-day acclimation and pretest period; observations v<...re made twice daily for 

mortality and general changes in appearanc.e or behavi"r. 

Pretest data collection began on May 6, 1996. bdividual body weights we;e 

recorded and detailed physical examinations were perfoTJlled on the fu-n and last 

days of pretest week -1 (May 10 and 16, 1996, respectively). Jndividual food 

consumption was measured for the interval week -1 to week 0. For computer 

entry, pretest data were assigned to computer pro~ol number WIL-186004P. 

Pretest clinical observations ~ presented in Appendix C. 

D. ANIMAL HOUS!'NG 

Upon arrival, all animals were housed three per cage ~y sex for a minimum 

or three days. Thereafter, all animals were housed individually in clean, wire-mesh 

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cages suspended above cage-boaJd. All animals were maintained by the animal 

husbandry staff in accordance with the "Guide for the Care and Use of Laboratory 

Animals" 1

• The animal facilities at WlL Research Laboratories, Inc., are 

accredited by the American Association for Accreditation of Laboratory Animal 

Care (AAALAC). 

E. Dmr. DRINKING WATER. AND MAINTENANCE 

The basal ration used in this study was Purina• Certified Rodent ChowGD 

#5002. The diet utilized at WlL Research Laboratories, Inc., is a certified feed 

with appropriate analyses performed by the manufactun·1· and provided to wn. 

Research Laboratories, Inc. Municipal water supplying the facility is sampled and 

analyzed for contaminants according to Standanl Operating Procedures. The results 

of these analyses are main;.ained at WlL Research Laboratories, Inc. Contaminants 

were not present in animal feed or water at levels sufficient to interfere with the 

objectives of this study. The basal diet and drinking water d~livered by an 

automatic watering system were provided ad libitum throughout the study period 

e:1rc, ·:. •i"ring the period of fasting prior to blood collection when food but not 

W'~k ·. Na.: V ~thheld, 

F. ENVIRONMENTAL CQNDI110NS 

All animals were housed throughout the acclimation period and during the 

temperature at 72° ± 4°F (22° ± 2°C), relative humidity at approximately 30-

70% and a minimum of 10 air exchanges per hour. Room temperature ana relative 

humidity were recorded twice daily. Actual recorded ranges for temperature and 

humidity were 71.9° to 73.0°F (22.2° to 22.8°C) and 33.7% to 67.3%, 

respectively, during the study period. Light timers were set to provide a 12-hour 

ligbt/12-hour dark photoperiod. 

G. ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS 

On May 16, 1996 (the day prior to test article adminimation), all available rats 

were weighed and examined in detail for physical abnormalities. Animals judged 

suitable for testing, as determined by the study director, were selected for use in 

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the computerized randomization procedure. The individual body weights were 

entered into the wn. Toxicology Data Management System (WTDMS"'). A 

printout containing the animal numbers, co:rrezponding body weights an..i individual 

group assignments was generated based on computer-generated random number 

pennutations. The animals then were arranged according to the printout. The 

control and 1000 mg/kg/day groups each consisted of 12 males and 12 females. 

The 125 and 350 mg/kg/day groups each consisted of six malec: and six femaies. 

After randomization into study groups, the animals were then randomized into two 

study replicates to allow for the reasonable conduct of the FOB and locomotor 

activity assessments. Each dose group and sex were equally represented within 

each study replicate. Animal no. 50292 was replaced by animal no. 50289 orJ 

study day -1 as animal no. 50292 died shortly after being handled for pretest 

clinical obsetvations .?..nd weighing. The selected animals were approximately six 

weeks old at the initiation of dosing; body weight values ranged from 159 to 222 

grams for the males and from 121 to 161 grams for the females. Several animals 

weighed less than the protocol-specified minimum weight (175 g for males, 125 g 

for females) at the initiation of dosing. This deviation bad no impact on the 

outcome of the study as all animals were within the prot'"iCOl-specified age range 

(4-8 weeks) at the initiation of dosing. 

H. CI.Thi1CAL OBSERVATIONS AND StJRvTvAL 

The animals were observed twice daily, once in the m0ming and once in the 

afternoon, for mortality and moribundity. Clini':al observations were perfonned 

on all animal> at the time of dosing and approximately one to two hours following 

dosing (designated as one hour post-dosing tor reporting purposes). During the !4-

day recovery period, clinical observations were performed daily on the remaining 

six animals/sex in the control and high dose groups. Each animal received a 

detailed physical examination prior to the scheduled necropsy. 

I. BODY WEIGHTS 

Individual body weights were measured and recorded weekly beginning one 

week prior to test article administration (week -1). Mean body weight changes 

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were calculated for eacb study week. A final body weight was recorded for each 

animal on tbe day of scheduled necropsy. 

1.~ 

Individual food consumption was measured weekly beginning one week prior 

to test article administration (week -1). Food intake was calculated as g/animallday 

for the corresponding body weight intervals. 

K. FUNCUQNAI. OB$ERVADONAL BA'ITER.Y 

Functional observational battery (FOB) evaluations were performed on six 

animals/sex/group during the pretest week, the fourth week of test article 

administration and the last week of the recovery period (weeks -1, 3 and S, 

respectively). lbe same six animais/sex in the control and 1000 mg/kg/day groups 

were used for each evaluation intetval. Testing was performed by the same 

technicians without knowledge of the animal group assignment. lbe FOB was 

performed in a sound-proofed room equipped with a white noise generator set to 

operate at 70 ± 10db, with the following exception. Home cage obsetvations were 

performed in the animal room. All animals were observed for the following 

parameters as described below (refer to Appendix D for a detailed description of 

the scoring criteria used for eacb obsetvation): 

1. HOME CAGE OBSERV ATIQNS 

Posture 

ConwisionstTremors 

Feces consistency 

'2. HANDIJNG OBSERVATIONS 

Ease of removal from cage 

Lacrimation/Cbromodacryorrbea 

Piloerection 

Palpebral closure 

Red/Crusty deposits 

Eye prominence 

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Biting 

Paipebrai (eyelid) ciosure 

Ease of handling animal in band 

Salivation 

Fur appearance 

Respiratory rate/ character 

Mucous membranes/Eye/Skin color 

Muscle tone 
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3. OPEN f!F1 n OBSERVATIONS (evaluated over a 2 minute observatic.n 

period) 

Mobility 

Rearing 

Convulsions/Tremors 

Grooming 

Biza.rre/Stereotypic behavior 

Time to first step (seconds) 

4. SENSORY OBSERVATIONS 

Approach response 

Startle response 

Pupil response 

Forelimb extension 

Air righting refle.>.. 

5. NEUROMUSCULAR OBSERVATIONS 

Hindlimb extensor strength 

Hindlimb foot splay 

6. PHYSIOLOGICAL OBSERVATIONS 

Catalepsy 

Body temperature 

L. LOCOMOTOR ACTIVITY 

Gait 

Arousal 

Urination/Defecation 

G~tscore 

Backing 

Touch response 

Tail pinch re~ponse 

Eyeblink response 

Hindlimb extension 

Olfactory orientation 

Grip strength-hind and forelimb 

Rotarod performance 

Body weight 

Locomotor activity observations were performed on six animalsi sexi group 

during the pretest week, the fourth week of test article administration and the last 

week of the recovery period, (weeks -1, 3 and 5, rrspecti.'!ely). The same six 

animals/sex from the control and 1000 mg/kg/day groups were used for each 

evaluation interval. Locomotor Activity, recorded after the completion of the FOB, 

was measured automatically using the Digiscan 'Micro' Animal Activity System 

(Omnitech Electronics, Inc., Columbus, OH 43228). This is a personal computercontrolled system consisting of individual test chambers which utilize a series of 

infrared photobeams surrounding a clear plastic, rectangular cage to quantify an 

animal's motor activity. The testing of treatment groups was done according to 

replicate sequence. The activity system was operated in the Stagger Start mode of 

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operation. Data were collected in one-minute epochs and the test session was 41 

minutes in dlr.ration for each animal. Animal placement into the activity cage in the 

Stagger Start mode of operation initiated the data collection process. The first 

epoch was often incomplete due to the placement of the animal in the activity cage. 

For this reason, the first minute of data was deleted. The remaining 40 minutes 

of data collection (4, 10 minute subsessions) were compiled for tabular 

presentation. 

Locomotor Activity was divided into t:wo categories: total and ambulatory 

activity. Total motor activity was defined as a combination of fine motor skills 

(i.e. grooming; interruption of one or two adjacent photobeams) and ambulatory 

motor activity (interruption of tluee or more consecutive photobeam~). Refer to 

Appendix E for additional details on the motor activity methodology. 

M. CLINICAL PADIQLQGY 

Blood samples for clinical pathology evaluations were taken from the vena cava 

of all animals just prior to tile scheduled necropsy. The animals were fasted 

overnight prior to necropsy and the collection of blood samples. Clinical pathology 

methods, procedures and references are presented in Appendix F. 

1. IIBMATOLQGY 

1be following hematologic evaluations were conducted on blood samples 

collected from all animals at the scheduled necropsies: 

Total Leukocyte Count (White Cell) 

Erythrocyte Count (Red Cells) 

Hemoglobin 

Hematocrit 

Mean Corpuscular Volume (MCV) 

Mean Corpuscular Hemoglobin 

(MCH) 

Mean Corpuscular Hemoglobin 

Concentration (MCHC) 

Platelet Count 

Platelet Estimate• 

RBC Morphology-

• = Presented only on individual tables 

( ) = Designates tabular abbreviation 

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.Differential Leukocyte Count 

-Neutrophil 

-Lymphocyte 

-Monocyte 

-Eosinophil 

-Bascphil 

Prothrombin Time (Pro Time) 

Activated Partial Thromboplastin 

1ime (AP'IT) 
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2. SERIJM CHEMISTRY 

The following serum chemistry evaluations were conducted on blood 

samples collected from all animals at the scheduled necropsies. 

Glucose 

Blood Urea Nitrogen 

Blood Creatinine 

Total Protein 

Albumin 

Albumin/Globulin Ratio 

(A/G Ratio) 

Globulin 

Calcium 

Phosphorus 

Chloride 

Total Bilirubin (Total Bill) 

( ) = Designates tabular abbreviation 

N. PATIIOLQGY 

1. MACROSCOPIC EXAMINATIQN 

Gamma Glutamyltransferase 

(Glutamyl Transfer) 

Serum Aspartate Aminouansferase 

(Aspa.rtat Transfer) 

Serum Alanine AminotransferaSP. 

(Alanine Transfer) 

Serum Alkaline Phosphatase 

(Alkaline Phos'tse) · 

Sodium 

Potassium 

Total Cholesteroi (Cholesterol} 

A complete necrorsy was conducted on all animals that were euthanized 

at the end of the dosin6 period or following a two week recovery period. All 

unimals were euthanized by cartx:m dioxide asphyxiation and exsangumated. 

The necropsy included, but was not limited to, examination of the external 

surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities 

including viscera. At the time of necropsy, the following tissues and orga_n_s 

were collected and preserved in 10% neutral buffered formalin: 

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AdreDals {2)* 

Aona 

Bone witb marrow {sternebrae)* 

Bone marrow smear (from femur)• 

Brain (fonbrain, midbrain, 

hindbrain)* 

Byes witb optic nerve (2) 

GastrointestiDa tract 

Esophagus 

Stomach* 

Duodenum* 

Jejunum* 

Ileum* 

Cecum• 

Colon 

Rectum 

Heart* 

Kidneys (2)* 

Liver (sections of two lobes)* 

Lungs [including bronchi, fixed by 

inflation with fixative (2)]* 

Lymph node (mediastiul)* 

Lymph node (mesenteric)* 

Mammary gland (females only) 

Ovaries with oviducts (2)* 

Pancreas 

Peripherzl nerve (sciatic)* 

PituitaJy 

Prostate* 

Salivary glands [submaxillary (2)] 

Seminal vesicles (2)* 

Skeletal muscle (vas'rus medialis) 

Skin 

Spinal cord (cervical, midthoracic, 

lumbar)* 

Spleen* 

Testes with epididymides (2)*" 

1bym":Js* 

Thyroids [with parathyroids if 

present (2)] 

Trachea* 

Urinary bladder* 

Uterus with vagina* 

All gross lesions• 

• = Bone marrow smears were obtained at the scheduled necropsy but were 

not placed in 10~ neutral buffered formalin. 

" = Tbe testes and epididymides were preserved in Bouin's solution. 

2. ORGAN WElGHTS 

1be following organs from animals euthanized at the week 4 primary and 

week 6 recowry necropsies were weighed: 

Adrenals 

Brain 

Epididymides 

Heart 

Kidneys 

Liver 

Ovaries 

Spleen 

Testes 

Thymus 

Paired organs were weighed together. Organ to final body weight ratios 

were calculated. 

3. MICROSCOPIC EXAMiiiADON 

After fiXation, specified tissues (designated with an asterisk in Section 

IV.N.l.) were sent to Colorado Histo-Prep, Inc., Fort Collins, Colorado, for 

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trimming and staining (hematoxylin-eosin). All tissues from the control and 

high dose group animals designated with an asterisk in Section IV .N .1 and the 

lungs, liver, kidneys, stomach and gross lesions from the low and mid dose 

g:oup animals were examined microscopically. Microscopic examination of 

these tissues was conducted by Carney B. Jackson, D.V.M., B.S. An. Sci., 

D.A.C. V.P., D.A.C. V.P.M., Assistant Director of Pathology and Veterinary 

Medicin~. wn.. Research Laboratories, Inc. 

0. STATISTICAL ME'lliODS 

All analyses were conducted using two-tailed tests for minimum significance 

levels of I % and 5% comparing the treatment groups tc the vehicle control group 

by sex. All means were presented witb standard deviations (S.D.) and the numbers 

of sampling units (n) used to calculate the means. All statistical tests were 

performed by a Digital* MicroV ~ 3-'!rJ() computer with appropri.ate 

programming. Analysis of body weight, body weight cbange, food consumption, 

clinical pathology values, continuous functional observational battery (FOB) data 

and absolute and relative organ weight data were subjected to a one-way analysis 

of variance, followed by Dunnett's tesf. Discontinuous (ordinal or descriptive) 

FOB data were analyzeJ using Fisher's exact tesf. Statistical tests for locomotor 

activity data were performed using a personal computer installed with SAS/STAT 

statistical software3

• Clinical labo·'itory values for cell types that OCCI!r l'!t a !ow 

incidence (i.e., monocytes, eosinophils and basophils) were not subjected to 

statistical analysis. 

P. DATA RETENTION 

The sponsor will b..:-. title to all documentation records, raw data, specimens 

or other wor'· product ce:;er..ted during the performance of the study. All work 

product induding raw pt.per data and specimens will be retained in the archives at 

Wll.. Research laborn;ories, Inc., as per protocol. 

Raw <bta in m:>gnetic form, a retention sample of the test article and the 

originaJ final report will be retained in the archives at Wll.. Research Laboratories, 

Inc., in compliance with regulatory requirements . 

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V. Bffltn.TS 

A. CUNJCAL OBSJmVADONS AND SURVIVAL 

Summary Data: Tables 1, 2, 3, 4, 5 

Individual Data: Tables 57, 58, 59, 60, 61 

AU animals survived to the scheduled ~ies. 

No clinic:al signs wbich could be attributed tc the test a.-ticle were observed at 

the clinical examiutions performed at the time of dosing, one hour following 

dosing, during the recovery period or prior to the scheduled necropsies. Clinical 

findings in the treated groups occulTed similarly in the control group and/or in a 

limited number of animals. No relationship to the test article was evident. 

B. BQDY WEIGHTS 

Summary Data: Tables 6, 7 

Individual Data: Tables 62, 63 

Mean body weights in the treated groups were statistically comparable to the 

conttol group means throughout the dosing and recovery periods, with the 

following exception. Mean body weight in the 350 mglkg/day group females was 

significandy (p<O.OS) increased on week 2. Mean body weight gains in all treated 

groups were statistically comparable to the control group values throughout the 

dosing period. During the recovery period (weeks 4-5 and 5-6), mean body weight 

gain in tile 1000 mg/kglday group males was reduced (statistically significant at 

p<0.01 for week 4-5) in comparison to the male control group mean. However, 

these decreases were not attributed to treatment as no te.~ article-related effects on 

body weight were noted during the dosing period. In the 1000 mglkg/day group 

females, mean body weight gains were statistically <'.omparable to the control group 

values during the recovery period. 

C. FOOD CONSUMPUQN 

Summary Data: Table 8 

Individual Data: Table 64 

Food consumption, evaluated as g/animallday, was not affected by test article 

administtation at any dose leve!. The only statistically significant (p<O.OS or 

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p<O.Ol) differences from the control group were slight increases in food 

consumption in the 125 and/or 350 mglkg/day group females during the frrst two 

weeks of test article administration. However, the values ~·ere comparable to their 

respective pretest values and decreases, :rather than increases, in food consumption 

are generally considered to be toxicologically significant; no relationship to 

treatment was evident. All other food consumption values in tht> treated groups 

were similar to the control group values. 

D. FUNCTIONAL OBSERVATIONAL BATTERY 

1. HOME CAGE OBSERVATIONS 

Summary Data: Tables 9, 10, 11, 12, 13, 14 

Individual Data: Tables 65, 66, 67 

Historical Control Data: Appendix G 

No remarkable differences were apparent between the control and treated 

groups when the home cage observati..Jns were evaluated during study weeks 

-1 (pretest period), 3 and 5 (recrvery period) . 

2. HANDliNG OBSERVATIONS 

Sum'llary Data: Tables 15, 16, 17, 18, 19, 20 

Individual Data: Tables 68, 69, 70 

Historical Control Data: Appen<Hx G 

No remar'ICable differences were apparent between the control and treated 

groups when the handling observations were evaluated during study weeks ··1 

(pretest period), 3 and 5 (recovery petiod). 

3. QPEN FIELD OBSERVATIONS 

Summary Data: Tables 21, 22, 23, 24, 25, 26 

Jndividual Data: Tables 71, 72, 73 

Historical Control Data: Appendix G 

N.:. remarkable differences were apparent between the control and treated 

groups when the open field observations were evaluated during study weeks -1 

(pretest period), 3 and 5 (recovery period) . 

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4. SENSORY OISBIYA'DONS 

Summary Data: Tables 27, 28, 29, 30, 31, 32 

Individual Data: Tables 74, 75, 76 

Historical Control Data: Appendix G 

No remarkable differences wme apparent between the control and treated 

groups when the sensorimotor observations we~e evaluated during study weeks 

-1 (pretest period), 3 and S (recovery period). 

S. NBlJROMUSClJLAR OBSERYA'UONS 

Summary Data: Tables 33, 34, 35, 36, 37, 38 

Individual Data: Tables 77, 78, 79 

Historical Control Data: Appendix G 

No test article-~eJated diffeieDces were apparent between the control and 

treated groups when the neuromuscular responses were evaluated during study 

weeks -1 (pretest period), 3 and S (recovery period). 1be only statistically 

significant (p< 0.01) differences from the control group v.ere decreased 

hindlimb grip strengths in the 125 and 1000 mglkg/day group females at the 

week 3 evaluaf on. However, similar values were observed for these groups 

during the prer t evaluation and the 1000 mglkg/day group female mean at the 

week S recovery evaluation was comparable to the control group value; 

theref A'e, the decreases could not be attributed to the test article. 

6. PHYSIOLOGICAL OBSERVADQNS 

:Summary Data: Tables 39, 40, 41 

Individual Data: Tabl~ 80, 81, 82 

Histor;caJ Control Data: Appendix G 

No remarkable differences were apparent between the control and treated 

groups when the physiological parameters were evaluated during study weeks 

-1 (pretest period), 3 and S (recovery period) . 

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E. LOCOMOtOR ACDVlTY 

Summary Data: Tables 42, 43, 44 

Individual Data: Tables 83, 84, 85 

Histoncal Control Data: Appendix H 

No remarkable differences in mean ambulatory and total motor activity values 

in the 125, 350 and 1000 mg/kg/day group males and females were observed 

during study weeks -1 (pretest period), 3 and 5 (recovery period). 

F. CLUnCALPATHOLOGY 

1. IIEMATOLQGY 

Summary Data: Tables 45, 46, 47 

Individual Data: Tables 86, 87, 88, 89, 90, 91 

No test article-related effects on hematology parameters were observed at 

the week 4 primary or week 6 recovery evaluations. A slight increase in mean 

activatuf partial thromboplastin time (APTI) in the 1000 mg/kg/day group 

males was statistically significant (p<0.05) when compared to the control 

group value at the week 4 evaluation. However, APTT increases of this 

magnitude are not considered to be toxicologically significant. A statistically 

significant (p<0.01) decrease in mean prothrombin time occurred in the 1000 

mg/kgiday group femaies at the week 4 eval•Jation. However, increases, 

ratb~r than decreases, in prothrombin time are gener . .!ly considered to be 

toxicologically significant. No other remarkable differences in hematology 

parameters were observed. 

2. SERUM CHEMISTRY 

Summary Data: Table 48 

Individual Data: Table 92, 93 

Historical Control Data: Appendix I 

No adverse effects related to test article admini!.trati0n on ser._ .•. chemi~ 

parameters were observed at any dose level at the 28 day prim2.ry necropsy or 

the 42 day recovery necropsy . 

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1Un_1G~ 

~~~ ~~~-~"""~ 

CMABFRIP 

• 

• 

• 

Scattered instances of statistically significant differences between treated 

and control animals were detected for some serum chemisuy puameters at the 

28 day primary oecropsy. These scattered statistical differences were not 

ooosidered an adverse effect of the test article because the statistical differences 

occumd: in the absence of a dose response; in the absence of the 

accompanying clinical chemistry changes expected (i.e. t!te increase in total 

pJasma protein in mid and high dose males and high dose females was not 

accompanied by an increase in hematocrit and total red blood cell count 

numbers); in the opposite direction from what occurs in a toxic state (i.e. the 

increase in globulin and albumin/globulin ratio in mid dose males and the 

increase in albumin in high dose females where a decrease in these parameters 

is indicative of toxicity); in a direction which is without physiologic 

significance (i.e. the decrease BUN and creatinine in mid dose females, the 

decrease in serum alanine aminotransferase and :.lSp&Itate aminotransferase in 

low, mid and high dose males and mid and high dose males, respectively); due 

to interf~ with the laboratory method (i.e. the increase in ser .. m chloride 

in mid and high dose males and in low, mid and high dose females - free 

bromine in the test Miele can artificially elevate chloride by reacting in the 

laboratory method as free chloride anions). Additionally, although detected as 

statistically different from concurrent control values, the absolute change in the 

measured parameters were small and within the range of· historical ct'lgtrol 

values for this species, strain, sex and laboratory (i.e. total plasm~ protein and 

the albumin/globulin ratio in mid dose males, cholesterol in mid a."ld high .iose 

females, serum glucose in high dose females, serum calcium in high dose 

females). 

No statistically significant differences in serum chemistry parameters were 

detected between groups at the 42 day recovery necropsy . 

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• 

CMABFRIP 

G. PAlHOLOGY 

1. :MACRQSCOPIC EXAMINATiON 

Silmmary Da1a: Tables 49, 50 

Individual Data: Table-: 94, 95 

Nc test article-reJated gross lesions were obser-.~ed at the week 4 primary 

necropsy or at the week 6 recovery necropsy. Gross lesions in the controi and 

treated groups occurred with similar frequency or were observed in single 

animals. 

2. ORGAN WEIGHTS 

Summary Data: Tables 51, 52, 53, 54 

Individual Data: Tables 96, 97, 98, 99 

No statistical significant differences in organ weigh!: or organ to body 

weight ratios were detected between control and treated animals with one 

exception. Absolute liver weights were statistically significantly increased with 

respect t{'l control mearo~ at the 28 day primary necropsy in males in the high 

dose a..-td females in the mid and high dose. Liver to tody weight ratios in mid 

and high dose males and low, mid and high dose females were statistically 

significantly mcreased at the 28 day necropsy. At the recovery necropsy, maY: 

absolute and liver to bcly "';eight ratio were statistically comparable to the 

control mean at the recov<!ry n~rop~y whereas female absolute liver weights 

and liver tc oody weight ratio were statistically significantly increased with 

respo.,"t to control mean. The dll:erence in absolute liver weights between 

control and treated females wa.s less pronuunced at the end of the recovery 

period, indicating the increase in livex wei~bt was reversib!e in females as well 

as males. In the absence of test article related ilistopatho!ogic and serum 

chemistry chang~, increases in liver weight are considered an adaptive, rather 

than a toxic response, are not uncommon in the rat, and are most likely the 

result of microsoma.t induction . 

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• 

• 

• 

WIL-186004 

CMABFT.dP 

3. MICROSCOPIC EXAMINATION 

Summary Data: Tables SS, S6 

Individual Data: Tables 94, 9S 

No test article-related histopathological lesions were observed at 1\e week 

4 primary or week 6 recovery necropsies. Lesions noted in the treated groups 

occUlTed in a limited number of animals, in a manner wbicb was not doserelated, or with similar frequency in the control group . 

-38-

+ ~1.. ' • ~ • ~ - .... ~ - ' - l-1 - ' "- .... ....... 

t ,, ' ' '. ' I I . I j . /. I I' I ' . 1- ~ t ,._ ' b ~ - \ • • ~ • --:::- ~ ..:...-" ~ -
• 

• 

• 

WlL-186004 

CMABFRIP 

VI. DISCUSSION AND CONCLUSIONS 

All animals survived to scheduled necropsy. No clinical signs related to test article 

administration were observed. 

No test article-related effects on body weight, body weight gain, food consumption, 

functional observation battery or locomotor artivity were found in either sex at any dose 

during either the 28 day dosing interval or during the 14 dz.y recovery period. 

No test articJe-related adverse effects on serum chemistry or hematology parameters 

were detected during the study. No test article-related gross or microscopic lesions 

were found at any dose level in either sex at either necropsy. 

Absolute and relative organ weights were unaffected by test article administration 

with the exception of li·1er weight. Absolute liver weight was statistically increased at 

the 28 day necropsy in rna les of the high dose and in females at the mid and high dose; 

relative liver weights were increased ii1 the mid and high dose males and in low, mid 

and high dose females. At the day 42 recovery necropsy, male absolute and relative 

liver weight were statistically comparable to the control mean. Female absolute and 

relative liver weight was statistically increased relative to the control mean at the 

recovery necropsy. This difference was less pronounced than at the 28 day necropsy, 

which indicates the increase in female liver was reversible as was the male liver weight. 

Increases in liver weight in the absence of test article-related changes in serum 

chemistry and hlstopathoiogy are likely the resuit of microsomal induction, which is an 

adaptive response in tlte rat and not evidence of toxicity. 

In conclusion, no evidence of systemic toxicity was observed at any dose level or 

time point. Based on the results of this study the NOAEL (No Observed Adverse 

Effect Level) of HBCD administered orally to male and female rats for 28 consecutive 

days was 1000 mg/kg/day. 

engelis, Ph.D., D.A.B.T. 

tudy Director 

-39-

13 F;6 '!) 

Date 
-

• 

• 

~ ---........ ~ - ,...-,.~~ ! . • ~ .,.# ' ' 

' J - ''- ' -~- • ' t ' ' • ' .,.. 

/!"' > "-- "' "-.. I -

.- -~ 

WD.-186004 

CMABFRIP 

VB. ICBY PP$0NNBJ, AND REPQRT SUBMISSION 

Study Stmmisog: 

Kerin Clevidence, B.S. 

Sally A. Keets, A.S. 

Gary R. Kiplinger, B.S. 

Ken D. Miller, B.A., M.T.(ASCP) 

Daniel W. Sved, Ph.D. 

ieyiewed By: 

Group Supervisor of Gross Pathology and 

Developmental Toxicology Laboratory 

Maaager of Vivarium 

Mauager of General Toxicology 

Senior Groups Supervisor -

Clinical Pathology 

Director of Metabolism and Analytical 

Chemistry 

Carney B. , D.V.M., B.S. An. Sci., 

D.A C.V.P., D.A.C.V.P.M . 

Assistant Director of Pathology and Veterinary Medicine 

Stu logist 

' .s. 

lmecbllr'of Toxicology 

lis, Ph.D., D.A.B. T. 

·or Toxicologist 

Study Director 

d-l:.laz _ Date 

I) &6 1') 

Date 
• 

• 

• 

wn..-186004 

CMABFRIP 

vm. PAJHOLQGISI SUMMARY STATEMENT 

Study animals were pre-designated for eutbanization either at the end of the dosing 

period (week 4) or following a two-week recovery period (week 6). All animals were 

euthanized by carbon dioxide asphyxiation and exsanguinated. A complete macroscopic 

examination was perfonned on each study animal that included, but was not limited to, 

the external surfaces, all orifices and cranial, thoracic, abdominal and pelvic cavities 

including viscera. No test article-related external or internal findings were noted at the 

week 4 frlmary or the week 6 recovery necropsies. 

SpP..:cified tissues (refer to section IV .N .1.) were preserved in 10% neutral buffered 

formalin and prepared of microscopic evaluation by Colorado Histo-Prep, Inc., Fort 

Collins, Colorado. Microscopic examination of the tissues was performed by Carney B. 

Jackson, D.V.M., B.S. An. Sci., D.A.C.V.P., D.A.C.V.P.M., Assistant Director of 

Pathology and Veterinary Medicine, wn.. Research Laboratories, Inc. 

Findings (lesions) in the test aWcle-treated groups occurred either in a limited 

number of animals, in a manner which was not dose-related ar with similar frequency in 

the control group. 

In conclusion, under the conditions of this study, no test article-related macroscopic 

or microscopic fmdings (lesions) were apparent at any dose leve! at the week 4 primary 

and week 6 recovery necropsies. 

13~!7 Carney B. ackson, D.V.M., B.S. An. Sci., Date 

D .. C.V.P., D.A.C.V.P.M. 

Assistant Director of Pathology and Veterinary Medicine 

Study Pathologist 

-41-

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- .. . "'"; ' - . . . ' ~ I - ~ • 

' ' 

~ ! • I ~ ~ • I 

• 

• 

WD..-186004 

CMABPRIP 

. , "' ' 

IX. OUAI.lTY ASSJJIANCE UNIT SIATBMENI 

Date(s) of Pbue 

Ipsnestjgp<(l Ip=Jed 

5/17/96 Teat MaterW Prepanlioo mel 

Allalylis 

5/W96 ADimiJ c.e " Equipment 6/11/96 FUDCtioaal Oblervatioaal Battery 

6/14196 Blood Collectioo & Allalylis 

6/14/96 Necropsy 

9124196 Study Records Vol. 1-1 

9123, 24, 26, 27196 Study Records Vola. 1-2 & 1-3 

9124196 Study Records Vol. 1-4 

9125196 Study Records Vol. Rx-1 

9125196 Study Records Vol. N-1 

9126196 Study Records Vols. C-1, 2, 3 

10/10/96 Study Records Vol. HIP-I 

10/14/96 Draft Report (Appendix B ODly) 

9/26, 10/3, 4, 6, 7, 

14, 20, 21196 lmft Report (w/o AppeDdix B) 

Date(s) F'mdiop 

Reported to 

Study Directer 

5/17/96 

5128196 

6/11/96 

6/14/96 

5/14/96 

9126196 

9127196 

9126196 

9126196 

9126196 

9126196 

10/10/96 

10/14/96 

10/21/96 

Date(s) FiDdiDp 

Reported to 

M•p•geppt 

6120196 

6120196 

1130196 

1130196 

7130/96 

10/24/96 

10/24/96 

10/24/96 

10/24/96 

10/24/96 

10/24/96 

11/26/96 

11/26/% 

11126196 

1be study wu condo~ and inspected in accordance with the United States EPA Good 

Laboratory Pm.'tice Regulations, the OBCD Principles of Good Laboratory Practice, the 

Standatd Operating Procedures of wn.. Aesearcb Laboratories, Inc., and the protocol. 

Quality Assurance fmdings, derived from the inspections during the conduct !lf the study 

reponed to the study director. A status report is submitted to management monthly. 

1be raw data, retention sample(s), if applicable, and the final report in the Archives at 

wn, Research Laboratories, Inc.' or another location specified by the sponsor. 
• 

• 

• 

wn.-186004 

CMABFRIP 

Colorado Histo-Prep, Inc. 

Post Oflicc Box 270733 • Fon Collins. CO 80527 • 970-493-2660 

CH-P Study # 1948 

Client's Study #WIL-186004 

QUALITY ASSURANCE S·IATEMtNT 

This study has been conducted in accordance with the t~A Health Effects 

Test Guidelines (40 CFR Part 792) and the OtCD GLP's, C(81) 30 (Final) Annex 

2, including appropriate SOP's and designated recording re~~irements. 

The study •as inspected by a member of the Quality Assurance Unit and 

reported to the Project Director and Management as recorded below. 

Comp~risons were made between the raw data and the final report, and 

they were found to be in agreement. All records were reviewed to assure tha~ 

any problems found during the course of a Quality Assurance Inspection were corrected and properly documented. 

PHASE REVIEWED 

Tissue receipt in file 

Protocol reviewed 

Tissue list compared to 

protocol 

Animal numbers, ~Px, 

necropsy dates reviewed 

Trimming 

Embedding 

Microtomy 

Slides compared 

to blocks 

SliQ~5 CO!!!par~d 

to blocks 

Blocks compared 

to inventory 

Wet tissue compared 

REVIEWED BY 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

Aimee Kramer 

to inventory Aimee Kramer 

Handwritten forms checked 

for writeovers 

Computer forms compared 

to CH-P animal forms 

Necropsy sheets compared 

to animal forms 

Slides returned 

Slides returned 

Blocks returned 

Wet tissue finalized 

for shippment 

Ai:tr""e Kramer 

Aimee Kramer 

Aimee 

Aimee 

Aimee 

Ai.mee 

Kramer 

Kramer 

Kramer 

Kramer 

Aimee Kramer 

-43-

OAT£ 

REVI.EWED 

07-12-96 

07-11-96 

07-11-96 

07-12-96 

07-15-96 

07-19-96 

07-25-96 

07-25-96 

07-30-96 

07-30-96 

10··18-96 

07-29-96 

07-30-96 

07-2S.-96 

07-26-96 

07-30-96 

10-13-96 

10-18-96 

PROJECT 

DIRECIOR 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

07-15-96 

07-19-96 

0'/-25-96 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

MANAGEMENT 

10-18-96 

10-18-96 

10-18-96 

10-18-96 

07-15-96 

07-19-96 

07-25-96 

10-18-96 

10-18-96 

l0-18-96 

10-18-96 

10-18-96 

10-18-96 

10-18-96 
• 

• 

• 

WIL-186004 

CMABPIUP 

Colorado Histo-Prep, Inc. 

Pcllll Office Boa 270733 • Fort Collins. CO IO:o . 1 • 97o-493·2660 

The raw histology data consistinq of paper records related to this study 

are archived at Colorado Histo-Prep, Inc., located at 319 Lincoln court, Fort 

Collins, co 80524. Raw data of blocks, slides, and wet tissues are 

temporarily stored at Colorado Histo-Prep, Inc. until forwarded to client. 

All raw Quality Assurance data are archived at Colorado Histo-Prep, Inc., Fort Collins, co 80524. Storage is in accordance with the EPA and OECD quidelines • 

-44-

\-- - - I t \ • 0 • 
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-- , , f • • ' r - • ~- I - . • ~ ' I - • / 

WIL-186004 

CMA BFI{D) 

X. REFERENCES 

1. National Research Council (1996) Guide for the Care and Use of Laboratory 

Animals, Institute of Laboratory Animal Resourr---..s, Commission on Life Sciences. 

National Academy Press, Washington, D.C. 

2. BMDP (1979) Biomedical Computer Programs. (Dixon, W.J. and Brown, M.B., 

eds.) University of California Press, Berkeley, CA, pp. 612, 780, 781. 

3. SAS (1991) SAS/STAT User's Guide, Varsion 6, 4th Edition. SAS Institute, Cary, 

North Carolina, 1028 pages . 

-45-