### NATIONAL TOXICOLOGY PROGRAM Technical Report Series

No. 383



# TOXICOLOGY AND CARCINOGENESIS

STUDIES OF

1-AMINO-2,4-DIBROMOANTHRAQUINONE

(CAS NO. 81-49-2)

IN F344/N RATS AND B6C3F 1 

# MICE

(FEED STUDIES)

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

National Institutes of Health

## FOREWORD

The National Toxicology Program (NTP) is made up of four charter ag encies o f the U.S. Departm ent of
Health and Hu man S ervice s (DHH S): the National Cancer I nstitute (NCI), National Ins titutes of Health; the
National Ins titute o f Env ironm ental H ealth Sc iences ( NIE HS), N ational Inst

itu tes of Heal th; the National
Center for Toxicological

Research (NCTR), F ood and Drug Administration; and the National In stitute for Occupational Safety and He alth (NIOSH ), Centers for Disease C ontrol. In July 1981, the Carcino genes is Bioassay Tes tin g Progr am, NCI, was transferred to the NI EHS. The NTP coordinates the rele vant program s, staff, and res ources from these P ublic Health Servi ce age

ncies re lating

to basi c and ap

plied re search a

nd to biological assay developmen t and validation.

The NTP devel ops, ev

aluates, and dis semin ates sci entific i nform ation ab out pot entially toxic and hazardo

us chem icals. This k

nowled ge is u sed for protecti

ng the health of the A

meric an peop le and f

or the p rimar

y prevention of disease.

The studies

describ ed in th

is Techni cal Rep ort

were performed under

the dir ection o f the NI

EHS an d were
conducted

in com pliance with N TP labor

atory health a

nd safe ty requirements and must

meet or exce ed all applica ble fede ral, state, and local

health and safety regulations. Anim al care and use were in

accordance
with the Public Health Se

rvice Poli cy on

Hum ane Car

e and U se of A

nimal s. The p rechron

ic and c hronic
studies were conducted in com pliance with Food and

Drug Administration (F DA) Good Labo ratory Practice Regulations, and

all asp ects of the c

hronic s tudies w ere sub

jected to retrospe ctive quality assuran ce audi ts before being prese nted for public review.

These studies

are designed and co

nducted to characterize and evaluate the toxicolog ic potential, including
carcino genic

activity, of selected chemicals in laborat ory an imals (usuall y two species , rats a

nd mi ce).
Chem icals selected for NTP toxicol ogy and carcinogenesis studies are chosen primarily on the bases of
human exposu re, lev el of pr

oductio n, and chem

ical stru cture. The inter

pretiv e concl

usions present ed in th

is
Technical Report are based only on the results of these

NTP studies. Extrapolatio n of these results to other species and quantitative risk analyses for

humans require wider analyses beyond the pu rview of these stud

ies.
Selection per se is not an indicator of a chemical' s carcinogenic potential.

These NTP Technical Reports are availab le for sale from the Na ti onal Technical Inform ation Service,
U.S. Department of Comme rce, 5285 Port Roy

al Road, Sp ringfi eld, VA 22161 (703- 487- 4650). Sing le copies of this Techni cal Repor t are av ailable withou

t charg e while

supplie s last fr

om N TP Centr

al Data Manage ment, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Par k, NC 2770 9 (919- 541- 3419).
Listi ngs of all p

ublishe d NTP re

ports an d ong oing s tudies a

re also availa

ble fr om NT P Centr

al Data Manage ment. The Abstracts and other study inform

ation for 2 -year s tudi es ar e als o avai lab le at the N TP's World Wide W eb sit e: http ://nt p-server .niehs .nih.go v.

## NTP TECHNICAL REPORT

ON THE



# TOXICOLOGY AND CARCINOGENESIS

STUDIE S OF

1-AMINO-2,4-DIBROMOANTHRAQUINONE

(CAS N O. 81-49- 2)



# IN F344/N RATS AND B6C3F 1 

# MICE

(FEED STUDIES)

NATIONAL TOXICOLOGY PROGRAM

P.O. Box 12233
Researc h Triangl e Park , NC 27 709

August 1 996

NTP TR 383

NIH Publicat ion No. 96- 2838



## U.S. DEP ARTMENT OF HEALTH AND H UMAN SERV ICES
Public Health Service
National Institutes of Health2 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



# CONTRIBUTORS

National Toxicology Program

Evaluated and i nterpreted results and reported fin dings

J.E. H uff, Ph.D

., Study Scientist
G.A . Bo or man , D. V.M. , P h. D.
D.A. Bri dge, B.S.

J. R. Buc he r, Ph. D.

M.R. El we ll , D. V.M. , P h. D.

T.J . Go eh l, Ph. D.

J. K. H as ema n, Ph. D.

G.N. Rao , D. V.M. , P h. D.

G. S. Tr av l os , D. V. M.

D.B. Wal te rs , P h. D.

K.L . W itt, M.S

., Oak Ri dge Associ ated Un iversit ies



## EG&G Mason Research Institute

Conducted st udies, evaluated pat hology findings

H.S . Lilja, P h.D., Principal Investigator
A. J. Blo ck , P h. D.

H.J . E sb er , P h. D.

R. W. Fl ei s ch ma n, D. V. M.

M. Ha gop ia n, Ph. D.

C. F. Moy er , D. V. M.

L. E. Se nde lb ac h, Ph. D.



## Experimental Pathology Laboratories, Inc.

Provided pathol

ogy quality

assurance

J.F. H ardisty, D .V.M

., Principal Investigator
S. Bo t t s , D. V. M.

H.R. Brown , D.V. M., M.S.



## Dynamac Corporation

Prepa red qual ity as surance

audits

S. B reche r, Ph.D

., Principal Investigator



## Analytical Sciences, Inc.

Provided stati stica l analyses

R.W . M orris, P h.D., Principal Investigator
N.G. Mint z, B.S

. S. Rose nblu m, M.S. 

## NTP Pa thology Wo rking Gro up

Evaluated slide s, prepared pathol ogy re port on rats (25 Janua ry 199 1)

R.M . Ko vatch, D .V.M

., Chair Pathology

Associate s, Inc. T. A nde rs on, D.V .M. , P h. D.

Hoffmann-L

aRoche, I nc. H.R. Brown , D.V. M., M.S. Experimental

Pathology

Laborato ries, I nc. J. Ei gh my , D. V. M.

USUHS , Bethe sda, MD
J. R. Ha i l ey , D. V. M.

National Tox

icology Program
M. P. Jo ki ne n, D. V. M.

National Tox

icology Program
M.M. McDon al d, D.V .M. , P h. D.

National Tox

icology Program

Evalua ted slid es, prep ared pa thology repor t on mic e (23 Janua ry 199 1)

R.M . Saue r, V.M .D., Chair PATHCO, Inc. S. Bo t t s , D. V. M. Experimental

Pathology

Laborato ries, I nc. R. Cat tl ey , V. M.D. , P h. D. Chemical Industry Institute of Toxicology
C.K .S. Ela ngba m, P h.D., Observer
Oklaho ma State Unive rsity
J. R. Ha i l ey , D. V. M.

National Tox

icology Program
M. P. Jo ki ne n, D. V. M.

National Tox

icology Program
M.M. McDon al d, D.V .M. , P h. D.

National Tox

icology Program



## Biotechnical Services, Inc.

Prepared Technical Report

D.D . Lam brigh t, Ph.D

., Principal Investigator
S.R. Gunne ls , M.A.
M.J. Nic holl s, B.S.

K.L. Shaw , B. A.3



# CONTENTS

ABSTRACT

................. ................. ................. ................. ......... 5

EXPLANATION O F LEVELS OF EVIDENCE OF C ARCINOGEN IC ACTIVITY

................. . 13

TECHNICAL REPO RTS REVIEW SUBCO M M ITTEE

................. ................. ...... 14

SUM M ARY OF TEC HNICAL REPO RTS REVIEW SUBCO M M ITTEE COM M ENTS

.............. 15

I NTR ODUCT I ON

................. ................. ................. ................. .... 17

MATERIALS AND METHODS

................. ................. ................. .......... 21

RESULTS

................. ................. ................. ................. ........... 31

DIS CUSSI ON AND CONCLUSI ONS

................. ................. ................. ..... 81

REFERENCES

................. ................. ................. ................. ....... 85

A PPE ND IX A Summary of Les ions in Male Rats i n the 2- Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

................. ................. ...... 93

A PPE ND IX B Summary of Les ions in Femal e Rats i n the 2- Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

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

A PPE ND IX C Summary of Les ions in Male Mice in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

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

A PPE ND IX D Summary of Les ions in Femal e Mice in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

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

A PPE ND IX E Summary of Les ions in Male Rats i n the St op-Exposu re Evaluat ion

of 1-Ami no-2,4- dibromoant hraquinone

................. ................. ...... 289

A PPE ND IX F Summary of Les ions in Femal e Rats i n the St op-Exposu re Evaluat ion

of 1-Ami no-2,4- dibromoant hraquinone

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

A PPE ND IX G Geneti c Toxic ology

................. ................. ................. ..... 325

A PPE ND IX H Organ W eight s and Organ-Weight -to- Body-Weight Rati os

................. ...... 335

A PPE ND IX I Chemical Characterizati on and Dose Formu lati on Studies

................. ....... 343

A PPENDIX J Feed and C omp ound Con sumptio n in the 2 -Yea r Feed Studies

of 1-Ami no-2,4- dibromoant hraquinone

................. ................. ...... 353

A PPE ND IX K Ingr edien ts, Nu trien t Co mpo sition, a nd C ont amin ant Lev els

in NIH-07 Rat and Mouse Rati on

................. ................. ........... 361

A PPE ND IX L Senti nel Ani mal Program

................. ................. ................. 3674 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 3835



# ABSTRACT

1-AMINO-2,4-DIBROMOANTHRAQUINONE

CAS No. 8 1-49-2

Chemic al Formul a: C 14 H 7 Br 2 NO 2 Molecular Weight: 381.04

Synonym: ADBAQ



### 1-A mino- 2,4- dibro moanthraquinone is an anthraquinone

derived vat dy e, a mem

ber of a class of inso luble dye s that are impreg

nated into t extile fiber s. Five anthraquinone-d

erived dyes with repre senta tive and
diver se s tructure s, as w ell as th e paren t chem ical,

an thraquinone, were sel ected for toxic ology and carcino

genes is evaluat ion

. Sim

ilar to the benzidine dye initia

tive, the rational e for selecting these vat dyes was to

gener ate suffici ent toxicol

ogic data to permi

t more reliab l e predictions of carcino

genic ity to be made on other ch emi

ca ls in this class, thereby elim inating or reducing the nee d

to study every anthraquinone dye. 1-Amino2,4-dibrom

oanthraquinone is the last anthraquinonederived dye in this group to be studied.

Groups of male a

nd femal e F344/N rats and B6C3F 1 

### mice were exposed to 1-amin o-2,4- dibromoanthraquinone (87% to 97% pure) for 13 weeks or for 9, 15

, or 24 months. Because 1-amin o- 2,4- dibrom

oanthra

quinone was predicted to be carcino genic

, these s tudie s

were desig ne d to evalu ate the potential for tumor

progr ession and regression. Absorption a nd excretio n studies were c arried out in male F344/N rats. Geneti c toxicity was determined in vitro using Salmon ella typhim urium and cultured Chines e hamster ovary cells. Extens ive chemical analyses wer e performed to id entify and chara cteri ze impurities of th e 1-am ino- 2,4- dibrom oanth raquinone used in t

hese studies

.



# 13-W EEK 

# S T U DY IN 

# R A T S Groups of 10 m

ale and 10 femal

e rats were given 0, 2,500

, 5,000, 10,000, 25,000 , or 50,000 ppm 1-amino2,4-dibrom

oan thraquinone in feed for 13 wee ks. Thes e levels corresp ond to approx imate ly 150 t o 3,2 00 mg
1-am in o-2,4- dibromoan

thraq uinone /kg body weight pe r d ay for males and to approxi matel y 170 to 3,200 mg/

k g f or fema les. Chemical-

related morta

lity was limited t o

one male and one fe male in the 50, 000 ppm groups. Fina l

mean bod y weigh ts and body weight gains of a ll expose d

groups of rats were signif

icantly lower than those of t h e controls. Feed consumption by all ex p osed groups wa s less than th at by the con

trols throughout

the study and

gener ally decreased with increasing e xposure concent ra

tion. Pink-red staining of the fur and ta il was observed i n all exposed groups. Absolu

te and relat ive liver weigh

ts o f
all exposed groups were gener ally signif icantly greate

r than those of the controls.6 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### Chemical- related

lesions were p resent i

n the li ver, kidney, and spleen of m

ale and female rats. Nonneoplastic les ions in th e liver included foci of hepatoc ellula r alteration, diffuse hepatocellular hypertrop hy(cytomeg aly), hepatoc ellu

lar cytop lasmi c vacuolation, bil e duct hyperplasia, inflam

mation, a n d pigmenta

tion.
These difference s w ere observed prima rily in the 25,00 0 and 50, 000 ppm grou

ps of males and

females; the spectrum of proli ferat ive lesions of the bile ducts (hyper

plasi a, fibrosis, and necroti

zing cholangitis) in the 25,00 0 and 5 0,000 ppm groups was morph ologic ally consisten

t with the lesion descri

bed as cholangiofibrosis. Pigm en

tation was presen

t in the renal t ubule epithelium of al l

groups of exposed rats; nuclear enlargement (karyomegal y) was also pr es ent in t he rena l tubule epithelium in some of the exp osed rats. A ccumulation o f hyaline droplet s in the cytoplasm of the renal tubule epithelium

and tubule lum

ina was present in 2,50 0, 5,000, 10,000, and 25,000 ppm males. I ncidences of hematop oiesis o f the spleen in exposed groups of male s and females were in

creased comp ared to those in

the
controls.



# 13-W EEK 

# S TUDY IN 

# M ICE Groups of 10 male and 10 female mice were given

0, 2,500, 5,000, 10,000, 25,000, or 50,000 ppm 1-am in o

2,4- dib romoanthraqui

none in feed for 13 weeks. Thes e levels correspon d to approximately 500 to 10,600 m g 1-amin o-2,4- dibromoanthraquino

n e/kg bo dy weight pe r day for males and ap p roxim ately 660 to 11,700 mg/

k g
per day for fem

ales. There was no che

mical-related
mortali ty. Feed

c onsumption and final mean body weigh ts of exposed groups were similar to those of th e controls. Red staining of

the fur was ob served

in all exposed groups. Absolu

te and re lative liver we ights o f
the exp osed groups we

re greater than those of the
control s except for the absolu te live r weigh t o f
2 ,500 ppm males. Absolu

te a nd relat ive kidney weight

s of 25,000 and 50,000 ppm males w ere lowe r than thos e of the controls.

Chemical- related

lesions were limited to the l ivers of
males a nd consisted of pigmentation of hepatocytes at al l

exposu re con centrations and centrilo bula r hepatoc ellula r hypertrophy at 10,000, 25,0 00, and 50,000 ppm. Mini

mal pigment wa s present in the liver of o ne femal e in th e 25,0 00 ppm group and in one femal e in the 50 ,000 pp m
group. 

# 2-Y EAR 

# S TUDY IN 

# R ATS Groups of 70 male and 70 f

emale rats were given 0, 5,000

, or 10,000 ppm 1-amin

o- 2 ,4-dibrom

oanthraquinone in fee d
for 103 weeks. I

n addition, group s of 50 male a nd 50
femal e rats were giv en 2,00 0 ppm 1- amino2,4-dibrom

oanthraquinone in f

eed for 104 weeks. Thes e
exposu re concentrations were approximat ely equal to 90

, 240, or 490 mg 1-amin

o-2,4- dibromoanthraqui

none/k g body weight for m

ales and 110, 285, or 600 mg /kg for
females. Ten anima

ls from each g

roup we re evaluated fo r histo patho logy at 9 months. Additional grou ps of
10 animals from

the 0 and 10,000

ppm g rou ps we re evaluated for histopatho logy at 15 months.

Survival, Body W

eights, Feed Consum

ption, and Clinical Findings

In the 2-year study

, survival of the 10 ,000 ppm males an d
females was signif

icantly l ow er than that of the control s

. Survival of the 2,000 a nd 5,000 ppm groups was simila r
to that of the controls. Duri ng the last y ear of the study

, the mean body

weights of expo sed males were 8 0% to
91% those of

controls, and the mean

body weig

hts of exposed females were 6 7% to 84% those of controls.
Feed cons ump tion among exposed groups was gen erall y simila r, but w as le ss than that by co ntrols. The fur and
urine of all exposed male and femal e groups were dis col

ored.

Pathology Findings

In the 2-year st

udy, 1-amin

o-2,4- dibromoanthraquino n e

was a ssociated with significant c hemical-re lated increase s in the incidences of benign and malignan t neop las ms i n the liv er, larg e intestine, kidney, and urinary bladder o f
males and

fema les. C hemic al-re lated no nneopla stic proli ferat ive and

degenerative lesions occurred in the
liv er, kidney, urinary bladder

, and forestomach of male s and females.

The inciden ces of foci of hepatoce llular alteration an d pigmentation in the liver of males and femal es were increased at the 9-m onth interim evaluation, and a hepatocellular

ade noma was present in one 5,000 pp m male. At the 15- month interim evaluation, hepatocellular aden oma or car cin oma (combined)

occ urred in all males and nine females in the 10,000 ppm groups. By the end of the 2-year study, hepatocellular

a d enoma, carcinom

a, cholangiom

a, o r chol angi ocar cin oma were observed in males and1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383 7



### females in the 5,000 and 10,000 ppm groups. In

the
2 ,000 ppm groups, similar liver neop las ms were presen t in 63% of the males and in 83% of the f emales. Of th e hepatocellular carcinom

as in the 5, 000 and 10,000 pp m
group s of males and females, 31 % to 49% were metastatic to the lungs or

other s ites. I

ncrease s in the

incidences of foci of hepatocellular alterati on (basoph il ic

, eosinophilic, and clear cell) and pigmentat ion of the live r

were also observed

in exposed grou

ps of males and
females.

Ade nomatous polyps (adenoma) of the large intest ine

were present in six 10,000 ppm males at th e 15-mo nt h
interim evaluation. Incidence s of ad enomatous poly p (adenoma) and carcinoma of the large intestine were signif icantly inc reased in exposed groups of males an d
females after 2 years ; mul tiple benign and m

alignant
intestinal neoplasms we

re observed

in many of these

rats.

In the kidney, incidences of renal tubul e adenoma an d

car cin oma were significantly increa sed in exposed

g roup s of males and females after 2 years. Renal tubul e adenomas were present in two 10,0 00 ppm m ales at
15 months. There were also chemica l-rel ated increase s in the i ncidences and severities of re nal tubule epitheli a l hyperplasia, pigm entation, and transitional c ell hyper pla

sia in the ki dney of males and females. Hyaline drop le t accumulation

was presen

t in all exposed

male rats at 9 months.

Inc idences of transitional cell papi llo ma an d carcinom a of the urina

ry bladder were incr eased at 2 years in male s and females in the 10,000 ppm groups. Tra nsitional cel l hyper plasia was obs erved in exposed males and femal e s
at the 15-m

onth interim eva lua

tion. Other nonneo

plasti c lesions observ

ed in the urinary bladder

at 2 years included meta plasia of the transitional epithelium and
submucosal strom

al tissue.

In the forestomach, the incidences and sev erities of
inflam mation, ulceration, hyper

kerato sis

, and hyper

pla

sia of the squamous mucosa we re increas ed in al l exposed groups of m ales a nd females at 2 years, but no t at the 9- or 15-mo

nth interim ev aluations.

In exposed m ale s and females, the incidences of
mononuclear ce ll leuke

mia were signif

icantly decreased

. The incidences of atrophy of the s eminal vesicl e wer e incre ased in expose d male rat s in the 2-year stu dy. 

## Stop-Exposure Evaluation in Rats

G roups of 40 male a

nd 40 female ra

ts wer e give n 20,000 p pm 1-amin

o-2,4- dibromoanthraquino ne in f ee d
for 9 or 15 mo

nths. At 9 m

onths, 10 ma

les and 10 female s were evaluated for histopat hology (9-m

onth interi m evaluation

groups)

. After 9 month s of exposure, an
additional 10 ma les and 10 females were fed control die t until t he e nd of th e 15- month evalu ation (9 -m

onth stop- exposu

re group s), and 20 males

and 20 females
continued to recei ve 20,000 ppm 1-amin o-2,4 -d ibr omo

anth raquinone unt il the end of the evaluation (15-mo nt h

exposu re group s). The approxi

mate daily consumption o f
1-amin o-2,4- dibr omo anthraquinone was 1,335 mg/kg fo r males and 1, 790 mg/kg for females in the 9-mon th stop

exposu re group s and 1 ,115 m

g/k g f or males and 1,435 mg /kg f

or fem ales in the 15-

month exposu re
groups.

Surv ival wa s similar among groups except for the femal e s in the 15-m

onth exposu

re group; th e surviv

al of this grou

p

was lower than that of the controls. Lower m ean body weigh ts were related to increased exposu re duration. Th e

mean b ody weigh

ts of e xposed males were 76% to 82 %
that of controls, and the mean bod y wei ghts o f e xpose d females were 73% to 84% that of controls.

For the stop-ex

posure evaluation, simila

r chem ical- relate d

neop las ms and nonn

eoplast ic lesions were obser ve d in th e

same sit es a s in the 2-y

ear study: liv er, large intestine, kidney, urin

ary bladder, and forestomach.

Af ter 9 months of dietar

y exposu re to a concentration o f 20,000 ppm 1-am

ino- 2,4- dibro m oanthraquinone,
h epatocellular aden oma and car cin oma occurred in m ale s and femal es. Nonneoplast ic chemical- related lesions i n the liver of exposed rats included pigmentation , focal hepatocellular

alte ra tion, and bile duct hy perplasia.
Ne opla sms at other sites i n males included one aden oma

tous polyp (adenom

a) in the large intestine and one
transitional cell p a

pil loma in the urinary bladder

. Hyali n e droplet accum ulation was pr esent in the kidney

of exposed males at 9 months.

In the stop- exposu

re groups examined at 15 months, hepatoce llular aden oma and car cin oma were presen t i n
most males and females. Adenomatous polyp (ade noma) of the colon, renal tubule cell adenoma, a n d urinary bladder transitional cell papi llo ma and8 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### car cin oma also occurred in m ales and females.
Nonneo plastic chemical-

related lesi ons included foci o f hepatocellular a lteration in the liver and hyper plasia o f
the renal tubule e pit

helium and urinary bladder transi

tional epi the liu m. Hyper

plasia, hyperker

atosis, inflam

mation, and ulc eration were ob serv ed in the
for est oma chs of some male and femal e rats continuousl

y exposed for 15 m

onths.



# 2-Y EAR 

# S TUDY IN 

# M ICE Groups of 60 m

ale and 60 fema le mice were g

iven 0, 10,000, or 20,000 ppm 1-am ino

- 2,4-di bromoanthraquino

ne in feed for 104 weeks. Th e daily compou

nd consumption was approxi matel y 1,69 0

or 3,470 mg 1-amino-2,4-di bromoan thraq uinone /kg bo d y weight for males and 1,950 or 4,350 mg /kg for females

. T en animals from

each group w ere evaluat ed fo r histopathology at 15 months.



## Survival, B ody Weight

s, Feed Con sumption, and Clinical Findings

In the 2-y ear study, surv

ival of exposed m

ales was signif icantly lower than that of the controls. Sur vival o f exposed females wa s similar to that of the controls. Th e final mea n body wei ghts of exposed males were 83% t o 85% that of controls, a

nd the final mean body weigh ts o f exposed fema les wer e 81% to 86% that of controls.
Feed consumption b

y exposed groups was gener ally
similar to that by control s. Dis colorati on of th e fur, urine, and feces was observed in all exp osed groups.



## Pathology Findings

In the 2-year stu

dy, 1-ami no-2,4-

dibromoanthraquino

n e

was associated

with significant chem

ical-related increases in the incidences of benig n and malig

nant

ne opla sms in the li ver, forestomach, and lung of male s and females.

Incidences of hep atocellular ad enoma and carcinom a

were incre ased in exposed group s at the

15- month
interim eval

uation an d at 2 years. At 2 years, there wer e significant

increase s in the i

ncidenc es of m

ultiple hepatocellular

aden oma an d car cin oma in males and
females and in the inci dences of hepa toblasto ma in
males. Centrilobular

hypertrophy of

hepato cytes in
males an d foci of hepatocellular alteration and p igm enta

tion in the liver of m

al es and females were also
chemical- related changes. Squamous cell papilloma of the forestomach m ucosa

occurr ed in 10,000 ppm females and 20,000 ppm males
and femal e s at the 15-m

onth interim evaluat ion, and th e incidences of squamous cell pa pil loma and carcino ma

were si gnific antly increased in exposed groups of male s and fema les at 2 years. Chemica l-related hyper

plasia o f
forestomach epithelium was also pre sent at 15 months an d at 2 years.

Al veolar/bronchiolar adenomas were present only in th e
expo sed group s of males and females at 15 months, an d
the incidences of

alveo lar/bron

chiolar adenom

a were signif icantly increased in e xposed males and f emales a t 2 years. The inc idences of m ultiple alveo lar/bron

chiola r adenoma were also increased in exposed males.

In the k idney, pigm

entation was present in the renal
tubules of most m

ice after 2 years of exposure.



# D ISPOSITI ON

AND 

# M ETABO LIS M 

# S TUDIES
Adult male F3 4 4/N rats were giv en [ 14 

### C]-labeled
1-am ino-2,4- dibromoanthraquino

ne as a single intra ve

nous dose o f 0.4 m

g/kg body weig ht or as a sing le oral
dose of 2, 23, 118, 8 14, or 1,473 mg /kg. A 6- hour bi le
cannul a tion study was also pe rformed . From day 0
throu gh day 3 aft

er intravenous admin

istrati on, about 50 % of the 14 

### C was excreted in the feces, 1 5% in the urine, an d
6% in expired air. Un metabolized 1 -amino

2,4-d ibromoanthraquinon

e accounted for les s than 3% o f
the excreted 14 

### C af

ter intrav enous administration. For ora l doses administered, the amount of the dose that

was absorbed fit the equatio n: absorbed dose = 6.6 log( dose )

. After intravenous admi nistration, the metabo

lites of 1-amin o-2,4 -d

ibromoanthraqu

inone in blood wer

e prim arily in the pla sma fraction (bl ood:plasma ratio of approxi matel y 0.5:1). The h igh est concentrations of 14 

### C
in tissues 15 minu

tes after in

traven ous dos ing w ere in

excreto ry organs, lung, kidney, small intestine, liver, adipose tissue, and adren al gland.



# G ENETIC 

# T OXICOLOGY

1-Am ino-2,4- dibromoanthraquino ne w as mutag

enic
in Sal monella typhimurium strains TA9 8 and TA1537 i n the absence of S9; with S9, an equivocal response was observed in TA1537. 1-

Amin o-2, 4-di bromo anth raquinon e

resu l ted in an equivocal response i n1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383 9



### strain TA100 with and with

out S9, and no muta

genic
activi ty was detecte d wit

h strain TA1535. In culture d
Chin ese h amster ovary cells, 1-am ino

2,4-d ibromoanthraquinon e induced sister chrom ati d
excha nges with and without S9; chromosom al aberra

tions were induced only in the absence of S9

.



# C ONCLUSIONS
Under th e conditions of these 2-year feed studie s, ther e

was clear evidence of ca rcinogenic activity* of
1-amin o-2,4 -d

ibrom oanthra quinone in male and femal e F344/N rats based on increased inci d ences of neoplasm s in the liver, large inte st ine, kidney, and urinary bladder

. There was clear ev idence of carci nogenic activity of
1-amin o-2,4- dibromoant

hraquinone in male and femal e B6C3F 1 

### m ice based on increased inciden ces of neopl asms in th e liver , fores tomach, and lun g. Exposu re of male and fem ale rats to 1-amino2,4-dibrom

oanthraquinone for 2 years was associated wit h
baso phil ic focus (males on ly), clear cell focus,

eos inophil ic foc us, and pigmentation in the liver; rena l
tubule hyper

plasia

, renal tubule pigme ntation, and transi

tional cell hyperplasia in th e kidney; transiti onal ce ll hyperplasia, squamous metaplasia, and stromal metaplas i a (females only) in the urinary bladd er; squamous hyp erpla

sia, hyperkeratosis, ulcerat ion, and inflam mation of th e forestomach

mucosa; and sem inal ves icle a troph y. Exposu re of male and female mice to 1-amino2,4-d ibr omo anthraquinone for 2 years was associated wit h
centrilobular hepatocellular hypertrophy (males onl y), basoph ilic focus, cl ear cell foc us (fem ales onl y), eosinop hilic focus, and pigmen

tati on in the liver; pigmen

tatio n in the kidney; and hyperplasia, basa l cell hyper pla

sia, hyper k eratosis, and inflam

matio n of the forestomac

h

mucosa.

__________

* Explan ation of Levels of Eviden ce of Carc inogeni c Activi ty is on p age 13. A summary of t he Techn ical Rep orts Revi ew

Subcom

mittee comme nts and the

public di scussio

n on this Techn

ical Re port app ears on page

15.10 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

Font S ize: 11pt Summ ary of the 2-Year Carcinogenesis and Gen etic Toxicology Studies of 1-Amino-2,4-dib romoanthraquinon e

Male
F344/N Rats Fema le
F344/N Rats Male
B6C3F 1 Mice Fema le
B6C3F 1 Mice

Doses 0, 2,000 , 5,000, or 10,000 ppm [ap proxi mat ely 90, 240, or 490 mg/k g/da y] 0, 2,000 , 5,000, or 10,000 ppm [ap proxi mat ely 110, 285 , or 600 mg/k g/da y] 0, 10,000, o r 20,000

ppm [ap proxi mat ely 1,690 or 3,4 70 m g/kg /da y] 0, 10,00 0, or 20,000 ppm [ap proxi mat ely 1,950 or 4,3 50 m g/kg /da y]

Body weights Exposed groups lower
than controls Exposed groups lower
than controls Exposed groups lower
than controls Exposed groups lower
than controls

2-Year survival

rates 26/50, 24/40, 21/60,
10/50 38/50, 32/40, 38/60,
12/49 40/50, 22/51, 23/50 39/50, 34/50, 33/50

Nonne oplas tic
effects Liver : baso philic focus ( 9/50, 12/40, 24/59, 22/50); clear
cell focus (3/50, 26/40, 3 9/59, 27 /50);
eosin ophili c focus (1/50, 13/4 0, 14/59, 6/50); pi gmentation (3/50, 19/4 0, 48/59, 39/50) Kidney : renal tubule hype rplas ia (9/50, 30/40, 2 5/59, 19 /50);
renal tubule pigme ntation ( 5/50, 40/40, 5 8/59, 49 /50);
transitiona l cell hype rplas ia (30/50, 40/40, 51/59, 35/50)
Urinar y bladder : transitiona l cell hype rplas ia (1/50, 5/38, 17 /58, 30/ 50);

squamous metapl asia (0/50, 0/38 , 0/58, 3/50) Liver : cle ar cel l foc us (3/50, 28/4 0, 39/60, 17/48); e osinop

hilic focus ( 7/50, 23/40, 12/60, 1 /48); pigme ntation ( 1/50, 19/40, 51/60, 45/48) Kidney : renal tubule hype rplas ia (1/50, 12/40, 2 3/60, 27 /48);
renal tubule pigme ntation ( 0/50, 40/40, 6 0/60, 48 /48);
transitiona l cell hype rplas ia (10/50, 16/40, 44/60, 21/48)
Urinar y bladder : transitiona l cell hype rplas ia (1/50, 2/40, 41 /60, 41/ 46);

squamous metapl asia (0/50, 1/40 , 4/60, 8/46); stromal metapl asia (0/ 50, 0/40, 4/60, 2

/46) Liver : centrilo bular hepato cyte hype rtrop hy (0/ 50, 17/51, 1 3/50); basophil ic focu s (0/50, 4/5 1, 3/50);
eosin ophili c focus (0/50, 6/5 1, 1/50); pigme ntation ( 1/50, 50/51, 47/50) Kidney : renal tubule pigme ntation ( 0/50, 42/51, 43/50)
Fores tomach : hype rplas ia (1/50, 9/50, 4/ 50); basal cell hype rplas ia (0/50, 0/50, 2/ 50); hype rkera tosis ( 1/50, 7/50, 6/ 50);
inflam mation ( 2/50, 6/50, 13/50) Liver : baso philic focus ( 0/50, 4/50, 5/50); cl ear ce ll f ocus (0/50, 10 /50, 9/5 0);
eosin ophili c focus (0/50, 4/5 0, 2/50); pigme ntation ( 0/50, 44/50, 49/50) Kidney : renal tubule pigme ntation ( 0/50, 43/50, 43/50)
Fores tomach : hype rplas ia (9/48, 15/50, 19/50); basal
cell hype rplas ia (0/48, 7/50, 3/ 50); hype rkera tosis ( 10/48, 14/50, 1 7/50);
inflam mation ( 7/48, 10/50, 21/50)

(cont inu ed)1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383 11

Summ ary o f the 2-Y ear C arcino gene sis and G enetic T oxicolog y Stud ies

of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

Male
F344/N Rats Fema le
F344/N Rats Male
B6C3F 1 Mice Fema le
B6C3F 1 Mice

Nonne oplas tic
effects (contin ued) Fores tomach :
squamous hype rplas ia (3/49, 19/3 9, 25/59, 26/49); hy

perke ratos is (5/49, 18/3 9, 21/59, 20/49); ul cer ( 3/49, 10/39, 1 5/59, 16 /49);
inflam mation ( 3/49, 12/39, 1 1/59, 11 /49);
Seminal vesicle : atrophy (1/49, 30/

40, 35/59, 23/50) Fores tomach :
squamous hype rplas ia (2/49, 7/40 , 26/60, 33/47); hy

perke ratos is (2/49, 7/40 , 23/60, 28/47); ul cer ( 1/49, 2/40, 7/ 60, 17/4 7);
inflam mation ( 0/49, 1/40, 13/60, 10/47)

Neopla stic eff ects Liver : hepatocel lular
adenom a (1/50, 20/40, 4 0/59, 34 /50); hepatocell

ular
carcino ma (1/50, 12/40, 5 5/59, 46 /50);hepatocholangiocarcino ma (0/50, 0/40, 6/59, 2

/50) Large intestine (all sites) : adeno

matous polyp (ade

noma) (0/50, 13/4 0, 51/59, 40/50); carcinoma (0/50, 1/40 , 11/59, 17/50) Kidney (rena l tubu le) : adenom a (2/50, 10/40, 1 1/59, 14 /50);
carcino ma (0/50, 0/40, 2/59, 1 /50)
Urinar y bladder : transitiona l cell papill oma (0/

50, 1/38, 2/58, 8/ 50);
transitiona l cell
carcino ma (0/50, 0/38, 1/58, 4

/50) Liver : hepatocel lular
adenom a (0/50, 28/40, 4 7/60, 29 /48); hepatocell

ular
carcino ma (0/50, 12/40, 5 7/60, 45 /48);hepatocholangiocarcino ma (0/50, 0/40, 11/60, 13/48) Large intestine (all sites) : adeno

matous polyp (ade

noma) (0/50, 28/4 0, 53/60, 43/49); carcinoma (0/50, 2/40 , 21/60, 8/49) Kidney (rena l tubu le) : adenom a (0/50, 3/

40, 16/60, 1 6/48);
carcino ma (0/50, 0/40, 0/60, 2

/48)
Urinar y bladder : transitiona l cell papill oma (0/

50, 2/40, 7/60, 9/ 46);
transitiona l cell
carcino ma (0/50, 0/40, 8/60, 1 6/46) Liver : hepatocel lular
adenom a (10/50, 38/51, 3 9/50); hepatocell

ular
carcino ma (9/50, 18/51, 2 1/50); hepato blasto ma (0/50, 3/51, 5/50)
Fores tomach : squamous cell papill oma (0/

50, 13/51, 16/50) ; squamous cell
carcino ma (0/50, 12/51, 13/50) Lung : alveolar/
bronchiolar adeno

ma (7/50, 26/5 1, 24/50) Liver : hepatocell

ular
adenom a (6/50, 45/50, 4 9/50); hepatocell

ular
carcino ma (0/50, 23/50, 27/50)
Fores tomach : squamous cell papill oma (2/

50, 16/50, 2 7/50); squamous cell
carcino ma (0/50, 12/50, 11/50) Lung : alveolar/
bronchiolar adeno

ma (4/50, 17/5 0, 13/49)

(cont inu ed)12 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

Summ ary o f the 2-Y ear C arcino gene sis and G enetic T oxicolog y Stud ies

of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

Male
F344/N Rats Fema le
F344/N Rats Male
B6C3F 1 Mice Fema le
B6C3F 1 Mice

Level of eviden ce of car cinogen

ic
activit y Clear ev idence Clear ev idence Clear ev idence Clear ev idence

Geneti c toxicolo gy Salmonella

typhimur ium gene mutatio n: Equivoc al in st rain TA 100 with and with out S9; n egati ve in s train T A1535 w ith and w ithout S 9; positiv e in str ain TA 1537 with

out S9, e quivoca l wit h S9; pos itive

in strai n TA9 8 witho

ut S9, negati ve w ith S9
Sister c hromat id exch anges Cultured Chinese hamste r ovary cells in vitro : Positive with and withou t S9 (combined results from testi ng in two laboratories)
Chromo somal

aberr ations
Cultured Chinese hamste r ovary cells in vitro : Weakl y pos itive witho ut S9, ne gative

with S 91-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383 13



## EXPLANA TION OF LEV ELS OF EVID ENCE OF C ARCINOG ENIC ACTIV

ITY

The National Toxicology Program describes the results of indi vidual experiments on a chemical agent and notes the strength of t he
evidence for conclusions regarding each study. Negati ve results, in which the study animals do not have a greater i ncidence of neopla sia tha n cont rol ani mals, do no

t neces sarily m

ean th at a ch emica l is not a carc inogen , inasm uch as the ex perim ents ar e conducted un

der a limited set o f conditions. Po sitive results demo nstrate that a chem

ical is carcinogenic for laboratory anim als under
the conditions o f the study and indicate that expo sure to the chem ical has the poten

tial for hazard to hu

mans. Othe

r organizations,
such a s the In ternatio nal Ag ency f or Res earch o

n Can cer, ass ign a s trength of evid ence fo r conc lusion s base d on a n exam ination of all
availab le evid ence, i ncludi ng ani mal st udies s uch as those condu

cted b y the N TP, ep idemi ologic studie s, and estima

tes of e xposu re. Thus, the actual determination o f risk to huma ns from chem

icals found to be

carcinogenic in laboratory anim als requires a wid

er analysis that extend

s beyond the purview of the se studies.

Five categories o f evidence of ca rcinogenic activity a

re used in the T

echnical Repo

rt series to summ

arize the strength o f the evidence
observed in ea

ch experimen

t: two categories for positive results ( clear evidence and some evidence ); one c ategor y for u ncerta in findings ( equivocal evidence ); one category fo r no observab

le effects ( no evidence ); and one category for experi ments that cannot
be evaluated b ecause of m ajor flaws (

inadequat e study ). These categories of i nterpretati ve conclusions were first adopted in June
1983 and the

n revised in M

arch 1986 for use in the Tech nical Report series

to incorporate m

ore specifically the c

oncept of actua

l weight of evidence of carcinoge nic activity. For each separate experiment (male r ats, female rats, male mice, female mice), one of the following five categor ies is selected t o describe the findings . These categories ref er to the strength of t he experimental evidence and
not to po tency or mechanis m.

• Clear evidence of carcinogenic acti vity is demonstrated by studi es that are inter preted as showing a dose-rela ted (i) increase of ma

lignant neoplasm

s, (ii) increase of a com

bination of m

alignant and be

nign neoplasm

s, or (iii) marked
increa se of b enign neopla

sms if there i s an in

dicatio n from this or other s tudies of the ability

of suc h tum ors to p rogres s to malignancy.

• Some evidence of carcinogenic acti vity is demonstrated by studi es that are inter preted as showing a
chemical-relate d increased incidence of neopl asms (malignant, benign, or combined) in which the strength of the response is less tha

n that required for clear evidence.

• Equivocal evidence of carcinogenic activity is demo nstrated by studies

that are interpreted a

s showing a marginal increase of neoplasms that may be chemical related.

• No evidence of carcinogenic activity is demo nstrated by studies

that are interpreted a

s showing n

o chemical-related
increases in ma

lignant or benign

neoplasms.

• Inadequate

study of carcinogenic acti vity is demonstrated by studi es that, because of major qual itative or quanti tative l imitati ons, ca nnot b e interp

reted a s valid for sho wing either t

he pre sence or abs ence o f carcin ogenic activity.

When a conclusion statement for a part icular experiment is selected, consider ation must be given to key factors that would extend the actual boundary of an indivi dual category of evidence. Such consideration should all ow for incorporation of sci entific experi ence and
current understandi ng of long-term carcinogenesi s studies in laborat ory animals, especial ly for those evaluati ons that may be on the
borderline betw

een two adjac

ent levels. These

considerations sh

ould include:

• adequ acy of the ex perim ental d esign a nd con duct;

• occurrence of c

ommon versus uncom

mon neop lasia;

• progression (or lac

k thereof) from

benign to m

alignant neoplasia

as well as from

preneoplastic to n

eoplastic lesions;

• some benig n neop lasms have t he cap acity to

regres s but o

thers (o f the sa

me m orpho logic ty pe) pro gress. At pre sent, it is impossible to i dentify the diffe rence. Therefore, where pr ogression is known to be a possibil ity, the most prudent

course is to as sume

that be nign n eoplas ms of those types h

ave th e pote ntial to becom e mali gnant;

• combinin g benign a nd malign ant tumor incide nce known or thought to repr esent s tages of progres sion in the same organ or tissue;

• latency in tumor inducti on;

• multiplicity in site-spec ific neoplasia;

• metastases;

• suppo rting in forma tion fro m pro liferativ e lesio ns (hy perpla sia) in t he sam e site o f neop

lasia o r in oth er exp erime

nts (same lesion in ano ther se x or sp ecies);

• presence or abs ence of dose re lationships;

• statistical significance of th e observed tum

or increase;

• concurr ent cont rol tumo r inci dence as well as t he hist orical control rate an d varia bilit y for a sp ecifi c neopla sm;

• survival-adjusted an

alyses and false p

ositive or false neg

ative concerns;

• structure-act ivity correlat ions; and

• in some cases, genetic toxi cology.14 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTI FI C COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOM MITTEE

The members of the Technical Reports Review Subcommittee who evaluated the dr aft NTP Technical Report on 1-amino2,4-dibromoa

nthraquinone o

n June 21, 19 94, are listed below . Subcomm ittee membe

rs serve as indep endent scientists, no t as representative s of any instituti on, company, or governmental agency. In thi s capacity, subcommittee members have fi ve major responsibilities in review

ing NTP s tudies:

! to ascertain that all relevant li terature data have been ade quately cited and inte rpreted,

! to determine if th

e design and conditions of the NTP studies were approp

riate,

! to ensure that the Technica l Report presents the exper imental results and concl usions fully and clear ly,

! to judge the signifi cance of the experimental re sults by scientif ic criteria , and

! to assess the evalu

ation of the evide

nce of carcinog

enic activity and o

ther observed to

xic responses.



## Arnold L . Brown , M.D., Cha ir

University of Wi sconsin Medical S chool

Madison , WI



## Paul T. Bailey, Ph.D.

Environ mental and He alth Scie

nces La borato ry Mobil Oil Cor poration Princeton, NJ



## Meryl H. Ka

rol, Ph.D.

Departm ent of E nviron mental Occup ationa

l Health University of Pi ttsburgh

Pittsbur gh, PA



## Curtis D. Klaassen, Ph.D.

Department of Phar macology and Toxicology University of Kansas

Medical Center Kansas City, KS



## Claudia S. Miller, M.D.

University of Texas He alth Sciences Center

San Ant onio, T X



## Janardan K. Reddy, M.D., Principal Reviewer Department of Pat hology

Northwestern Uni versity Medical School Chicag o, IL 

## Irma Russo, M.D.

Fox Chase Cancer C enter
Philade lphia, P

A



## Louise Ry

an, Ph.D.

Division of Bio statistics
Harva rd Scho ol of

Public Heal

th and

Dana-F arber

Cancer Ins titute
Bosto n, MA


## Robert E. Taylor, M.D., Ph.D.

Department of Pharmacology
How ard Univ ersit y Col leg e of Medicine Washington, DC



## Matthew J. van Zwieten, D.V.M., Ph.D. , Princ ipal R eviewer Depar tment o f Safe

ty A ssess ment Merck R esearch Lab oratori es West Point, P

A


## Mary Jo Vodicnik, Ph.D.

Lilly MSG Developmen t Cent er Belg ium



## Jerrold Ward, D.V.M., Ph.D. , Princ ipal R eviewer

Nationa l Cance r I nstitute Frederick, MD1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383 15



## SUMMARY

OF TECHNI CAL REPO RTS REVIEW SU BCOMMITTEE COMMENTS

On June 21, 1994, the dra ft Technical Report on the
toxic ology and c arc inog enes is st udie s of 1 -amino

-- 2,4-dibrom

oanthraquinone received pu blic review b y th e National Toxicolo gy P rogram 's B oard of

Scienti fic
Coun selors' Techni cal Reports Review Subcomm ittee

. The review mee tin

g was held at the National Inst itute o f
Environmen

tal Health Scien ces, Researc h Triangl e Park

, NC.

Dr. J.E. Huff, NI

EHS, introduced

the toxicology

and
carcino genes is studies of 1-am ino- 2,4- dibro

moan thra

q u inone by discussing the uses and rational e for study

, incl uding it being a part of a class study of ant hraquinon e derivatives. He de scribed the exp eri m ental design, report ed on survival and body w eight effects, and com

mented on chemical-

related neoplas

ms and non

neoplas ti c lesions in m ale and fe male rats and mice. The propose d
conclusions fo r the studies were clear evidence of

carcino genic activity in ma le and f

emale F344/N rats and in male an

d female B6C 3F 1 

### mice.

Dr. Huff r eviewed t

he carcino genic responses in othe r anthraquinone deriv

ative s that had been studied, notin g that the liver seemed to be a m ajor site and that 1-amin

o

2,4-dibr omo anthraquinone was the most active as far a s the number of sites. Inte

rpre tive conclusions that coul d
be drawn fr om the cumul ative Na tiona l Toxi cology

Pr ogram studies o n this class of insolub le dyes were tha t anth raquinones are typical ly mutag enic and clastog

enic

, they are carcino genic to male and fema le rats and mice

, and they are predicted to represent l ikely ca rcinogeni c hazards to humans ex posed t o these age nts, especia ll y

oc cupat ional ly. Dr. J.R. Bucher

, NIEHS, r eported tha t th e to xicology and

carcinogenesis studies on anthraquinone were in p

rogress.

Dr. van Zwi eten, a principal rev iewer, agreed with th e proposed conclus ions. He thought t here should be mor

e discussion of th e findi ngs from the stop- exposu re group s of rats. (Stop- exposu

re groups were evaluated a t 9 an d
15 m onths a s part o f an atte mpt t o gain in sight into th e progression or re gression of chemical- induced lesions. )
Dr. van Zwieten noted the high impur ity leve ls in the firs t lot of the chemical used for the 13-we ek studies and fo r the first 2 mont

hs of th e 2- year s tudies a

nd said that a stat ement indicating that this did not affect the integr it y of the stu dies migh t be helpful

. Dr. Huff responded tha t the im purities had been chara

cterized (page 20; Arneso

n et al. , 1996 ).

Dr. Ward

, the second principal review er, agree d with th e proposed concl usions. He commente d that no hyalin e drop le ts were reported in the k idney of rats after
9 months, and sinc e 1-amin

o-2,4- dibromoanthraquino

n e might cause a ccum ulation of " 2 : 

### -g lobulin , the re

port should indicate that droplet s were sea rched for bu

t no t found or found but not reported (pag e 83). Dr. War d objec ted to characterizing cholang iofibros is f ound in th e liver of rats in

a 13- week study

as "prem aligna nt." H e stated th at this lesion is usually induced by li ver carcino

gens but does not ty

pically progress to bile d

uct neoplasms. Dr. M.R. Elwell

, NIEHS

, said this interpre tatio n

was from the liter at

ure and the wo rding on neop lastic potential would be rev

ised to also re flect Dr

. Ward 's experience.

Dr

. Reddy, the third principal revi ewer, also agreed wit h

t he proposed conclusions. He said it would have been
useful to chara cteri ze t

he chemical nature of th e pigmen t that accumulated in the liver, kidney, and other o r gans, a s well as in the fur and tai l. Dr. Huff re sponde d that, logica lly, the pigment was either th e chemical or one o f
its metabolites, but the feasibility of g oing back and defining it better wou

ld have to be d

etermined.

Dr. Russo had ob served evidence of chronic infla mma

tion in one of the plate s and wondered whether t he live r le sions were associated with hepatitis. Dr. Karol aske d
if there was inflammation of the eosi nophili c foci, whic

h
would suggest a hypersensitivity -type reac tion.
Dr. Elwe ll said there was som e inflamma tion with the cholangiofibrosis, but this was r eally limited to the foca l
lesions w here there was fibrosis and to cystic bile ducts

, and there w as not an eosinop hilic inflammation; the ter

m "eo sinophi lic foci" referred to a focal cellular alteratio n of hepatocytes.

Dr

. Bailey c ited a statement from the use, production,
and human exposure sections that

"no individualiz ed
information was located regarding am ounts produce d16 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### or spec ific uses of 1-amin o-2,4 -dibr omo

anthraquinone," leading

him t o wond er if th is c hemi

cal is currentl y used. Dr. Huff said this was a valid

ques tion for 1- amino -2,4 -dib ro moanthraquinone an d
the other anthraquinone der ivatives. H e said pr opri

etary inform ation w as diffic

u lt to obtain, althoug

h h e

was hopeful that a request to the American Phar ma

ceutical Association concerning anthra quinone dyes i n over-the- counter or prescription ite ms might yield

some data on human exposure. There was some di scussion that prim ary exposu

re to these dyes would b e from topical application

or exposure.

Dr. van Zwieten m

oved that the Technical Report on
1-a mino-2 ,4-dibrom

oanthr aquinone be accepted with th e revisions discussed and the conclusions as written for
male and female rats and mice, clear ev iden ce of

carcino genic activity. Dr. Reddy seconde d the motion

, which was accepted u

nanimously with eleven v otes.17



# I NTRODUCTI ON

1-AMI NO-2 ,4- DIBRO MOANTH RAQUI NONE

CAS No. 8 1-49-2

Chemical Form

ula: C 14 H 7 Br 2 NO 2 Mole cu lar Weig ht: 381.04

Sy n on ym: ADBAQ



## C HEM I C AL AND 

## P HYSI CAL 

## P ROPERTI ES



### 1-A mi no- 2,4- dibromoanthraquino ne, a reddish brow n

to orange powder, is a

n anthraquinone- derived va t dye

. Anthraqui none (9,10-anthraquinone: CAS No. 84 -65

- 1), which does not occur nat ura lly, was firs t synthesize

d by Laurent i n 1835 as an oxi dati

on product of ant

hracene and nitric acid (C hung, 197 8). "Anth ra" comes

from the Greek word for coa l, fr om which anthracene

wa s ori gi nal ly

obta in ed.



## U SE



## , P RODUCTI ON



## ,
AND 

## H UMAN 

## E XPOSUR E



### Anthra quin one is an important and widely used start in g

ma terial for the manufa

cture of vat dyes (

Mer ck Index

, 1 989). Class homologu es of anthraquinone comprise a greater number of dyes having outst anding "fastness " properties than any

othe r gr oup o f commercial dyes (Chung, 1978; Chung a nd Far ris, 1979). No informa

ti on was located

regarding amounts produce d or spe

cif ic uses of 1-ami

no-2,4- dibromoant

h raquinone. Th e
2-a lkyl derivat

i ves of anthraquinone wi

th alkyl chain s ranging from one to five carbons are most often used i n the dye industr y (Chun g, 1978). Vat dyes a re a class of water -inso luble dyes that can b e easily redu ced ( i.e., vatted) to a water-solubl e and

usua lly colorless leuco form in w hich they can read il y im pregnat e fibers and tex tiles. Subsequent oxid atio n then produces the insolu ble colored fo rm that is remark

ably "fast" to washi

ng, li ght, and chemical s. The reducing ag ents are usually alkali ne solutions of sod iu

m hydrosulfite ; ox ida

ti on ta kes pl ace in

the pre sen ce of air, perborate, d ic hrom ate , a nd ot her agents (Hawley

, 1981 ). Vat dyes are us ed typically for cotton, woo l, an d

cellu lose acetate. Produc

ti on of vat dyes in

th e Unite d

States tota le d 14, 000,000 kg (30.8 mi ll ion pounds) i n
1991 (U SITC, 1993); these figures do not account fo r the "large" amounts e xtracted from botani cal specie s containing

naturally occurring anthraquinones used therapeuticall

y and for other purposes.



## A BSORPTI ON



## , D I STRI BUTI ON



## , M ETAB OLISM



## , AND 

## E XCRET I ON



### No in for ma ti on on the absorption, distri

bution, me tabo

lism, and exc ret ion

of1-amino-2,4-dibromoanthraquinon e in experime ntal anima ls or in humans w as found in a search of the avail able li terature.18 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



## T OXI CI TY



### No inform at ion

on t he t oxi ci ty

of 1- ami

no- 2,4- dibromoanthraquino ne in ex perim ental

anim als or i n humans was found in a sear

ch of the availa ble lit

erature

.



## R EPRODUCTI VE

AND 

## D EVELOPMENTAL 

## T OXI CI TY



### No in for ma ti on on the repro duc tiv e and development a l

toxici ty of 1 -amino-2 ,4-dib romo anthr aquino ne in experimental

ani mal s or in humans was found in a

search of the avail able li

terature.



## C ARCI NOGENI CI TY 

## Experimenta l Animals

Chem icals belonging to the ant hraquinone clas s of dye s

are car cino genic to rodents (IA RC

, 1987; Sendelbach

, 1989) and consistently induce ne oplasms of the l iver (Huff et al. , 1991). However

, each anthraquin one derivative appea

rs to in duce canc er i n other organs o r tissu e site s as w ell

(Huff et al. , 1991).

For t he five anthraquinones evaluated and reported b y NCI/NT P, the 2-year exp osure concentrations in th e feed var ie d from a l ow of 300 ppm (0.03%)

for 2-m eth yl -1- ni troa

nth raq ui none to a hi gh of
20,00 0 ppm (2%) for 1-ami no-2,4- dibromoant

h raqui

none (Tabl e 1).



## Humans

No information on the potenti al ca rcinoge nici

ty of 1-amino-2,4-dibr om oanthraquinone in humans was found in a search of the avail able li terature.



## G ENETI C 

## T OXI CI TY



### All five anthraqui

nones evaluated and reported by

NCI/ N TP induced mutations in Salmonella

typ h imuri um (Brown a

nd Brown, 1976; Haworth

et al

. , 19 83 ; Dunk el et al. , 1985; Zeige r et a l.

, 1988)

. Each also ca

used sis te r ch romatid exchanges and

chromoso mal abe rrations in cultured Chinese hamste r

ovar y c el ls (An ders on et al. , 1990; Love day et al.

, 1990; NTP, unp ubl is hed) . S9 a ctiva ti on w as n ot required for 1-amino-2,4-dibromoanthraquinone to

p rodu ce these effects. The parent com pou nd, anthraqui

none, is also mutag enic in S. ty phimur ium ; 

### signi fica

nt
increases in mutant col onies were observed with strains T A98 and TA100 wi th and wi thout S9 (Zei ger

et a l

., 1988 ). In addition, anthraqui none and

1-ami noanthra quinone (250 mg/kg) were reported t o induce DNA strand breaks in live r and kidney tissue o f

male Swiss (C D-1 ®) mi ce followi ng intraperi

toneal injection (Cesarone et al. , 1982).



## S TUDY 

## R ATI ONALE



### The NCI selected and evaluated se ver al of the anthraquinone- derived dyes for a class stu dy to deter

mine whether these dye s have any inherent po tential fo r carci nogenic ity

in labora tor y rod ents an d, if so , in

humans as well. The first three studies were conducte d

wi th 2-amin oanthraquinone (NCI, 1 978a)

, 1-amino-2-methy

lanthraquin one ( NCI, 1978 b), and 2-methyl- 1-nitroanthraquinone (NC I, 1978c). A fourt h substance, 1,4,5,8-te traami noanthraq

uinone (C.I. Disp erse Blue 1) was selected and evaluated for carci

nogeni city by the NTP (NTP, 1986 a) . This Tec hnica l Report addresses the fifth chemic al in th is class,

1-a mi no- 2,4- di bromoanth

raquinone. In addition, th e parent chem ical, anthraquinone, has been selected fo r study to complete the overall effort on these dyes.

Anthra quinone and the five subs tit uted anthraquinone s (Fig ure 1), repre senta tive of

a large gr oup o f amino -, alkyl-, and nit ro-, o r halog en-co ntaining a nthra quinones, w ere chosen for tox icolo gic ch ara cte ri za ti on a n d

to esta blis h some predictive structur

e-ac tivi ty r el ati on

sh ips that could be used on other dyes in this cat egor y rather than testing each and every one. Other ch emica l

classes that have been l ikewi se eva luated by the

NCI/N TP to reduce the need for "o ne-b y- one" testin g in cl ude benzidine-

based dyes (Morgan et al., 1 9 94), phthalates (K luwe et al.

, 1982 ; Huff and Kluw e, 1984 ; Kluwe et al. , 1985), benzene and meta bolit es (Huff, 1992), dioxins (Huf

f, 199 2), a n ili

nes (Weisburger

et al.

, 1984 ; Lamb et al. , 19 86 ), natu rally occurring "gu ms" (Melnick et al. , 1983), chl orinated p araffi n s (Bucher et al. , 1987), 1,3-butadiene and derivatives (Melnick and Huff, 1992), pesticides (Yang et al

., 1989; Huff and Has eman, 1991) , and penici lli ns and

tetra cycl ines (Dunnick et al.

, 1989; Dietz et al.

, 19 91)

.

The bases f

or se lectio n of an thraq uin ones (and o ther

chemical classes as w

ell) centered mai

n ly on four

cri teria: 1) l ack

of av ai la bl e or a dequ ate data

on

car cino genic ity, 2) ma gni tude of producti on and useIntroduction 19



### T ABLE 

### 1 Exposure Co ncentrations in the N CI/NTP 2-Year Feed Studies o f Anthraquinone Deriv atives a

Anthraquin one Derivat ive Low Dose Hig h Do se Ca rcinog enic R espon se (ppm) (ppm)

Rats

M ale

2-Aminoanthraquinone b 3,500 6,900 liv er 1-Amino-2,4-dibromoanthraquinone 2,000 10,000 liver, large int estine, kidney, uri nary bladder 1-Amino-2-methylanthr aquinone b 1,000 2,000 liv er, ki dney 2-Methyl-1-nit

roanthraquinone 600 1,200 liver, skin 1,4,5,8-Tetraaminoanthraquinone 1,250 5,000 urinar y bladder, pancreas

Fem ale

2-Aminoanthraquinone c 2,000 1-Amino-2,4-dibromoanthraquinone 2,000 10,000 liver, large int estine, kidney, uri nary bladder 1-Amino-2-methylanthr aquinone 1,000 2,000 liv er 2-Methyl-1-nit

roanthraquinone 600 1,200 skin 1,4,5,8-Tetraaminoanthraquinone 1,250 5,000 urinar y bladder

M ice

M ale

2-Aminoanthraquinone 5,000 10,000 liv er 1-Amino-2,4-dibromoanthraquinone 10,000 20,000 liver, forestomach, lung 1-Amino-2-methylanth r aquinone 600

_ d

2-Methyl-1-nit

roanthraquinone 300 600 hema ngi osar coma 1,4,5,8-Tetraaminoanthraquinone 600 2,500 liver, lun g e

Fem ale

2-Aminoanthraquinone 5,000 10,000 li ver , l ymph oma 1-Amino-2,4-dibromoanthraquinone 10,000 20,000 liver, forestomach, lung 1-Amino-2-methylanth r aquinone 600

_ d li v er 2-Methyl-1-nit

roanthraquinone 300 600 hema ngi osar coma 1,4,5,8-Tetraaminoanthraquinone 600 2,500

a Data f rom NCI, 1978a , 1978b, 1978c; NTP, 1986a b Exp osure co ncentrations in this study were time-weighted averages.
c Inadequ ate stu dy d Two dosage regimens were use d, but t he ti me-weig hted aver age concent rati ons were t

he same.

e "Equivocal evidence" for both organs20 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



### patterns, 3) awareness of pote ntial and actual human

exposure, a

nd 4) rep res ent ati on of as broad a spec-

tru m of structur al diversity

withi n this cl ass as po ssibl e. 1

- Am in o-2, 4-di bromoanthraq uinone was selected fro m a
group of 36 environmental

ly signi ficant aryl bromides

. Because every other anthraquinone deri vative tested s o far for carci nogenic activ ity had been shown to be

car cino genic in ro den ts, 1 -amin o-2, 4-

- dibromoanthraquinone was al so e xpected to be
carcino genic in labo rat or y a nim als. Thus, th e experimental

design, whi le be ing c onsiste nt wi th a "c ore pro tocol" (Huff et al. , 1988), contains several modifications such a s "stop-exposure" groups t o better char act eriz e this chem ical. Ad dit iona lly,
chemical dis

pos it ion studies were accomplished pr ior t o the 2-year

exposure s to per

mi t opt im al sel ect ion of

exposure co ncentrations for this wa ter -i nsol uble dye.

Because these chemical s may and often do c onta in

con sider able quantities of the parent c hemical and other anthraqu i none derivatives, a n extensive chem

ical analysis was undertaken on these f ive c hemi cals t

o quanti tate their puri ty an d t o ide nti fy

the ma jor imp u rities of 1-amino-2,4-dibromoanthraquinone (Arneson et al. , 1996).

1-Ami no- 2,4- dibr omoa

nthra quinone 2-Ami noant hraqui none

1-Ami no- 2-m ethy

lant hraqui none 2-Met hyl -1- nit roa

nthra quinone

1,4,5, 8-T etr aam inoa

nthra quinone

F IGURE 1

Fiv e An th ra qu in on e De ri vat ive s Eva l ua te d by th e NC I/ NTP21



# MATERIALS AND METHO DS

P ROCUREMENT

AND 

## C HARACTERI ZATI ON OF 

## 1-A MINO 

## -2,4- DIBR OMOANTHRAQU I NON E 

### 1-Amino- 2,4-di bromoa nthraquinone wa s obtained from A m erican Color and Chemi cal Corpor ati on (Charlotte, NC; lot 1076-C) and Mobay Corpor ati on (Pittsbu rgh, PA). The

secon d lot was procured from Moba y Cor porati on since A merican Color and Chemi cal Corpora tion had stopped product ion

. Lot 1076-C was used in the 13-week studies and
for 2 months of the 2-year s tudies. The l ot from Mobay Corporation was assig ned lot number
M06 1583 and was used throughout the remainder of the 2-year studi es. Identity, purity, and s tabil ity anal

yses were conducted b y the analytica l chemistry l abora tory, Midwes t Res earch Insti tute (Kansas C ity , MO ) (App endix I). Reports on analyses performed in

suppo r t of the 1-ami n o

- 2,4-dibromoanthraqu inone studies are on f ile at the Nat io nal Institute of Environmental Health Sciences (NIEHS).

The two lots of the chemical, a reddish brown to

orange powde r, were identif ie d as 1-am ino2,4-dibr omoanthraquinone by infrared

, ultra viol et/visi

ble, a nd nuclear magneti c

resonance spec trosco py. T he puri ty of each l ot

was determined b

y elemental

analyses, Karl
F i scher water an alysis, thin -laye r chromatograph y, and high- perfo rmanc e liquid chromatograph y.

For lot 1076-C, eleme ntal analy ses for carbon, hydrogen, nitrogen, and bromine were in general agreement with

theore tica l val

ues for 1 -amino

2 ,4-dibromoanthraq

uinone. Karl Fischer wat er

ana ly sis indicated approximately 0.21% water.
Thi n -layer and high-performance liqui d

c hro matography

indicated a major peak and eight impuriti es. Five of the impuri ties had peak

areas of les s than 0.3%

. The t hree ma jor impuriti es we re identifi ed as anthraqui none, 1-amino-2-bromoanthraqui none, a nd 2-ami

no1,3- dibromo anthraquinone. By high

performance liq uid ch rom atog raphy

, anthraquinone w as found to be present at a

concent ratio n of approximately 5.0%. 1-Amino2 -bromoa nthraq uinon e and 2-amino-1,3

- dibromoanthraqui

none were found to be present

at concentrat i ons of approximate l y 4.3% and 2.2%, respect ivel y. Th e overa ll pu rity of lot 1076-C was de t ermined to be approximately 87%.

For lot M061583, element al ana l yses for carbon, hydrogen, nitrogen , and bro mine wer e in general agreement with theoretical v alu es for 1- amino2,4-dibromoanthraqu inone. Karl Fis che r water analysis indicated approximately 0.32% water. Thin-layer and high-performance l iq ui d ch romatography

indi cated a major peak an d si x impuriti es with the same retention t imes as found
for lot 1076-C. A total impurity area of 3% of the total chromatographic p eak area was found. The overall purity of lo t M061 583 was deter mined to be approximatel

y 97%.

Stabilit y studies perfo rmed us in g high

perform a nce liquid

chromatography i

ndicated that 1-amino-2

, 4-dibromoanthraqui none, when stored protected fr o m light, was stable as a bulk chemical for at least 2 weeks at te mperat ures up
to 60 " 

### C. To en sure stabi lity, the bulk chemical was
stored in the dark at 4 " 

### ± 3 " 

### C thro ughout the
studies. During the 2-year studies, the stability of the
bulk chemica l w as mo nitor

ed per iodic ally by the study la borator

y using hig

h-performa

nce liquid
chromato gr aphy; no degradation of 1-amino2,4-dib romoa nthraq uinone was observed

throug hout
the studies.



## P REPARAT ION AN D 

## A NALYSIS

OF 

## D OSE 

## F ORMULATI ONS 

### The dose formulations were prepared weekly by mixing 1-amin o-2,4-d ibrom oa nth raquinone wit h feed (Tab le I1). Homogeneity and at least 2- week stability at 2 5 

## B C were confirm ed by the analytica l chemi st ry labora tory usin g spectropho tom etry and high-performance liqui d chromatograph y, respectively. D u ring the
13 -wee k and 2-year feed studi es, the dose formulations were stor e d in the dark for no longer than 2 weeks.

The stu d y laboratory conducted period ic

a n alyses o f the 1-amin o

- 2,4-dibromoanthraqu inone dose formula t ions using a spectr ophotom etric method. For the 13-week feed studi es, dose formulations were22 1-Am ino-2, 4-dibromoanthraquinone, N TP TR 383



### analyzed at the beginni ng, midpoint , and en d of the

stu die s ( Ta ble I2). During the 2-year feed studies, t he
dose f ormulati ons were analyzed every 6 to 10 weeks (T ab le I3). All dose fo

rmulations for r

ats and mice

were withi n 10% of the target concentrat ions during the
13-week and 2-year studies. Result s of pe rio dic referee analyses perfor

m ed by the a

nalytical chemistry
lab or ato ry in dic ate d g oo d a gr ee me nt w ith t he re su lts obtained by the s

tudy laboratory (T

able I4).



## 13-W EE K 

## S TUDI ES


### The 13-week studies were cond uc ted to eval uate the

cu mu lati v e tox ic e ff ec ts o f r ep ea ted ex po su re to
1- am ino -2 ,4- dib

ro mo an thr aq uin o ne and to determine
the appropriate exposure concentrati ons to be us ed i n the 2-year studies.

Ma le and female F344/N rats and B6C3F
1 

### mice were

o btained fr

om Ch ar les Riv er Br ee din g L ab or ato rie s (Portage, MI). Upon

receipt, the animals were 5 weeks
old. The rats and mice were quar antined for 15 days
bef ore the s tudies beg an.

Groups of 10 male and 10 female rats and 10 male and

10 female mice received

0, 2,500, 5,000, 10,000, 25,000, or 50,000 ppm 1-amino

- 2,4-dibromoanthraquinone in feed for 13 weeks. Males
and females were housed five per cage; water and feed
were available ad libi tum , and feed consumption was
measured wee k ly. Cli nical fi ndings were recorded
twice daily

. Animals were weighed at study ini tiat ion, weekly, an

d a t the en d o f th e s tud ies . F ur the r d eta ils of study design and animal mai n tenance are summarized
in Ta ble 2

.

A necropsy was p erfo rmed on all animal s. The brain,
he ar t, right ki dney, liver, lungs, right testi s, and thymus
of all ani mals were weighed. Tissues for microsco p ic
examinati on

were fixed and preserved in 10% neutral
buffe r ed fo rm alin , p ro ce ss ed an d tr imm ed , e mb ed de d in paraffin, sectioned t o a

thickness of 4 to 6 : m, and
stained with hematoxyl in an d eosi n. A c om ple te his top ath olo gic ex am ina tion wa s p er fo rm ed on all

an ima ls that di ed

pr ior to the end of the studies, control animals, and animals administ e red 50,000 ppm.
Table 2 li sts the t issues and organs examined. 

## 2-Y EAR 

## S TUDI ES 

## Stu dy Desi gn Groups of 70 ma le a nd 70 fe ma le r ats received 0, 5,000,

or 1 0,0 00 pp m 1 -a min o- 2,4 -d ibr om oa nth ra qu ino ne in feed, and a gr

ou p o f 5 0 m

ale an d 5 0 f em ale ra ts received 2,000 ppm 1 -amino-2,4-dibromoant hraquinone
in feed for 104 weeks. Te

n male an d 1 0 f

em ale ra ts from the 0 , 2,000, 5,000 , and 10,00 0 ppm groups were designated for an interim eval uation af ter 9 months.

Ten ma le and 10 female rats from

the 0 and 10,000 ppm groups were designated

for an interim eval uation after
15 months. Groups of 60 m ale and 60 female mice
received 0, 10,0 00, or 20,000 ppm 1-amino2,4-dibromoanthraquinone in feed for 104 weeks. Ten

ma le and 10 female mice per

group were evaluated after
15 months.



## Stop-Exposure Evaluation Groups of 40 male and 40 female rats received 20,000 p pm 1-am ino-2,4-dibromo

anthraquinone

in fe e d
for 9 months, when 10 males a nd 10 females we

re evaluated. At 9 months, the dosed feed was replaced
with a c on tro l d iet for 1 0 male and 10 female rats,
which w e re then necropsied and evaluated at
15 months. Twenty male and 20 female rats conti nued

t o r eceive 20, 000 ppm 1-amino

- 2,4-dibromoanthraquinone in f eed and were also

eva luated at 15 mo nths

. 

## Source and Specification of Animals

Ma le and female F344/N rats and B6C3F
1 

### mice were
ob tain e d fr om Fr ed er ick Ca nc er Re se ar ch Fa cilit y (Frederick, MD). Ra

ts were quarantined 12 to 14 days (males) or 9

days (females) and mice were quarantined

12 days (males) or 15 da y s (females) before the beginning of the studies. Five male and five fe ma le r ats and mice were s elect ed and evaluated for evidence of parasites and

gro ss o bse rva tion o f dis eas e. Sero logy samples were collected for vi ral screening. Rat s an d
mice were approximately 6 weeks old at the beginning
of the 2-y ear studies. The healt h of the animals was
monitored du rin g th e s tud ies ac co rd ing to t he pr oto co ls
of the NTP

Sentinel Animal Progra

m (App endix L).



## Animal Maintenance
Males and females were housed five per cage. Feed and

water were available ad libi tum . Feed consu mpt ion

was measured mo n

thly (Appendix J). Cages andM

ater ial s and M

ethods 23



### racks were rotated every 2 week s during the studi es. Further detail s of

anima l ma

inte na nc e a re giv en in

Ta ble 2. Information on feed compositi on and
contami nants i s provide d in Appendi x K.



## Clinical Examinations and Pathology

All anima ls were observed twice dail y. Clini cal findings were recorded weekly for 14 w ee ks then

mo nth ly until the end of the studi es . Anima ls were weighed at study ini tiat ion, weekly for 14 weeks, and monthly thereafter.

An ima ls were kill ed wit

h CO
2 

### , a

nd a c om ple te

necropsy was performed on all an i mals. The right kid ne y a nd live r o f r ats and mice were weighed at
the interim evaluations (Appen dix H). At necropsy, all

organs and tissues were examined for gross lesions, and
all ma jor tiss ue s w er e f ix ed and preserved in 10%
neutral buffered formalin, process e d and trimmed,
embedded in p ar af fin , s ec tion ed , a nd sta in e d w ith
he ma tox ylin and eosin for microscopic examinati on. His top ath olo gic ex am ina tion s w er e p er fo rm ed on all
tis sues with grossly vi sible l esions. For all pa ire d

organs (i.e., adrenal gland, ki dney, ovary) , sam ples
from each organ were exa

mined. Tissues examined

are list ed in Table 2.

Microscopi c e valuati ons were completed by the study laboratory pathologist, and the pathology d ata were entered into t he Toxicology

Da ta Management Sys tem. The microscope

slides, paraffin blocks, and

residual wet
tissues were se

nt to the NTP Arc

hives for i nventory, slide/ block

match, and wet-ti ssue audit. The s lides

, indivi dual animal data records, and pathol ogy tables

were evaluated by

a n in de pe nd en t pa tho log y q ua lity

assess men t laboratory. The indi vidual ani mal records
and tables were compared for accuracy, the slide and
tissue co un ts were verified, and the hi stotechni que was evaluated by the qualit y assessment laboratory. The

qu ality assessmen

t pathol ogi st m

icr os co pic all y

re vie we d s ele cte d n eo pla sm s o r n on ne op las tic lesions.

The quality assessme

nt report and s

lides were submitt ed

to the NTP P

athology W

orking Gro

up (PW G) chair,
who reviewed the selected slides and any other tissues
for which a disagreement in di agnos i s between the laboratory an d q ua lity as se ss me nt p ath olo gis ts existed.

Re pr es en tativ e histopathol ogy slides cont aining examples of lesions related to chemical admi nistrat ion, examples of disagreements in diagnoses b etwe en the
la boratory and qualit y assessment pathologi sts, o r lesions of general int ere st w er e p re se nte d b y th e c ha ir

to the PW G fo r re view

. Tissues examined included t he
adrenal cortex (fem

ale rats), ear (rats),

kidn ey (rats), large intest ine (rats), liver, lung (mice)

, skin (rats), forestomach, thyroi d gl and (rats), and urinary bladder (rats). The PWG

consisted of the qual ity assessment pathologi st

and other pathologi sts experience d in rodent

tox ico log ic pathology. This group examined the t issues
wit hout any knowledge of exposure g roups or

pr ev iou sly rendered diagnoses. When the PWG

consensus dif fe re d f ro m th e o pin io n of the laboratory

pa tho log ist, the dia gn os is was changed. Thus, the final diagnoses repres en t a consensus of contractor patholog ists

and the PWG. Details of these review procedures

have been descr ibed by Maronpot and

Boorman (1982) and Boorm

an et al

. (1985). For subsequent

analysis of pathol ogy data, the di agnosed
lesions for each

tissue t ype were evaluated separately or
combined acc or din g to the gu ide line s o f M

cC on ne ll
et al. (1986)

.



## S TATI STI CAL 

## M ETHODS 

## Survival Analyses
The pr ob ab ility of su rv iva l wa s e stim ate d b y th e

pr od uc t- limi t procedure of Kaplan and Me ier (1958) and is pres ented in the form of gr ap hs . A nim als found
dead of othe r th an

na tur al c au se s o r m iss ex ed an ima ls

were ce ns or ed fr om the su rv iva l an aly se s; a nim als dying fr om na tural causes were not censored.
Stati stical analyses for

po ss ible do se -r ela ted ef fe cts on survival used Cox's (1972) met hod fo r test ing t wo
groups fo r e qu ality and Ta ro ne 's (1 97 5)

life tab le te st to identify dos e-r elated tren ds. A ll reported P values for
the survival analyses are two sided.



## Calculation of Incidence
The inc ide nc es of ne op la sm s or nonneoplastic l esions

as pr es en ted in Tables A1, A5, B1, B5, C1, C5, D1, D5,

E1 , E 3, F1 , a

nd F3 ar e g ive n a s th e n um be r o f a nim als bearing such

les ion s a t a specific anat omic sit e and the
number of an im als wit h that si te examined microscopical ly. For calculati on of stati stical signifi cance, the inci dences of most neoplasms (Tables A3, B3, C3, D3, E2a, E2b, F2a, and F2 b) and o f a

ll

no nn eo pla stic lesions are given as t he n um bers of

an ima ls affected at each sit e examined microscopicall y. Howev er, wh en ma cr os co pic examinati on

was required

to detect neoplasms in cer tain t issues (e.g., harderi an gland, intestine, mammary g land, and sk

in) before microscopic evaluat ion or when neoplasms24 1-Am ino-2, 4-dibromoanthraquinone, N TP TR 383



### had multi ple potent ial si tes o f oc cur ren ce ( e.g., leu ke mia or lymphoma), the denominators consist of
the number of animals on which a necrops y was performed. Tables A3, B3, C3, D3, E2a, E2b, F2a, and
F2b als o give the survival-adj usted neoplasm rate for
each group an d each site-specifi c neoplasm, i.e. , the

Ka pla n- Me ier es tima te of the neoplasm incidence that

wo uld have been observed at the end of the study in the
absence of mortali ty from all other competing ri sks (Kaplan an

d Meier, 195 8).



## Analysis of Neoplasm Incidence
The majorit y of neoplasms in these stud i es were considered

to be inc ide nta l to the ca us e o f d ea th or not

ra pid ly lethal . Thus, the primary stat isti cal method
used was logisti c regression analysis, which assumed
that the diagnosed ne o plasms were discovered as the

re su lt of death from an unrelated cause an d th us did not affect th e risk of death. In thi s approach, neoplasm prevalence was modeled as a logisti c function of
chemical exposure and time. Bo th line ar an d q ua dr atic

t erms in time

wer e inc orp ora

ted in itially, a nd th e

qu ad ra tic ter m w as eli minated if the fi t of the model

was not signi ficantl y enhanced. The neoplasm
incidences of exposed and con t ro l groups w

ere compared on the basis of the li kelihood sc ore test for
the regress

ion coefficient of dose

. This method of adjusti ng for intercurrent mortali ty is t he prev al ence

an aly sis of Dinse and Lagakos

(1983), further described
and ill ustrated by Dinse and Haseman (1986). When neoplas ms are inciden t al, t his comparison of the
time -s pe cif ic neoplasm prevalences also provides a comparison

of t h e time-speci fic neoplasm inci dences (McKn ight and Crow

ley, 1984).

In ad ditio n to log is ti c regression, other methods of
stati stical a

nalys is were used, and the result s of these

tes ts are sum mar ized in the app end ixes

. These methods
include the li fe tabl e tes t (Cox, 1972 ; Tarone, 19 75),

ap pr op ria te for rapidly let hal neoplasms, and the Fi sher

exact test and the Co ch

ra n- Ar mita ge tre nd tes t (A rmitag e, 19 71 ; Ga

rt et al. , 1979), procedures based on the overall proporti on of lesion-bearing ani mals.

Te sts of signifi cance included pairwi se comparisons of
each do se d g ro up wit h c on tro ls a nd a te st f or an ov er all
dose-related trend. Co n tinuit y-correc ted tests were
used in the analysis of neoplasm inci dence, and reported P v alue s ar e on e sid ed. The p rocedures
described in the preceding paragraphs were a lso used to e va lua te selected nonneoplast ic lesi ons. For further
discussion of thes e stati stical method s, refer to

Haseman (198 4).



## Analysis of Nonneoplastic Lesion Incidences

Because all nonneoplast ic lesi ons in thi s study we re considered to be inci dental to the cause of death or not

ra pid ly lethal , the primary st atist ical anal ysis used was

a logist ic re gr

es sio n a na lys is in wh ich no nn eo pla stic
lesion prevalence was modeled as a logisti c function of
chemical exposu

re and time. For lesi ons detected at the
inte rim ev alu atio n, th e F is her exa

ct test, a procedure
based on the ov er all proportion of affected animal s, was used.



## Analysis of Continuous Variables Organ and body weight data, whi ch have ap pr ox ima tely

no rmal distri butions, were anal yzed using t h e

pa ra me tric multi ple comparison

pr oc ed ur es of Du nn ett (1955) an d W illia ms (19 71, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the signi ficance
of the dose-r

el ated trends an

d to determine wh

ether a
trend-sensit ive tes

t (W ill ia ms' test) was m

ore

ap pr op ria te for pairw ise co mparisons than a test that
do e s not assume a monotonic dose-related t rend (Du nne tt's test). Prior to analysi s, extreme values
iden tifi ed by the out li er test of Dixon and Ma sse y (1951) were examined by NTP per sonne l, and imp lau sib le values wer e eli minat ed from t he analysis. Average se ve rity values were analyzed for

signifi cance using the Mann-Whitney U test (Holl ander and Wolfe, 1973).



## Historical Control Data Although the concurren

t co ntrol group is al ways the
fi rst and most appropriate control group used for
evaluati on, histori cal control dat a can be helpful in the

ov er all assessme nt of

les ion inc ide nc e in ce rta in
instances. Consequently, neoplasm inci dences from the

NTP histori cal control database (Haseman et al ., 1984, 1985) a re inc luded i n the NTP repor ts for neo plasms appearing to show compound-related effects.



## Q UALI TY 

## A SSURANCE 

## M ETHODS


### The 13-week and 2-year studies were co nd uc ted in compliance wit h Food and Drug Administrat ion Good Laboratory

Practice Regulati ons (21 CF R, Part 58). In
addi t ion, as reco

rds from the 2-year

studies were
submitted to the NT P Archives, the s e

### Materials and Methods 25

studie s were audited retro s pectively by an independent quality assura nce contract or. Separate audits covering completen ess and

acc ur acy of the pathology data, pathology specimens, final pathology tables, and a draft of this NTP Technical Repo r t were conducted. Au d it procedure s and findings are present ed in the reports and are on file at NIEHS. The audit findin gs were reviewed and asses sed by NTP staff, s o al l discrepan cies had been resolve d or

we re otherwise address ed during the preparation of this Technical Report.



## G ENE TIC 

## T OXI COLOGY 

### The genetic t oxicity o f 1-amino

2,4-d ibromoant

hra q uinone was asses sed by testi ng the ability of the chemical to in duce mutati ons in various strains of Salmonella typhimurium cells and siste r chromati d exchange s and chromoso mal aberratio ns in cultu red Chinese hamster ovary cells. The prot ocols for these studie s and the results ar e given in Appendix G.

The genetic toxicity studi e s of 1-amino2,4-dibromoa n thraquin one are part of a larger effort by the NTP to develop a database that would permit the evalua ti on of carcino genicity in experimen tal animals from the struct ure and

respons es of the chemical in short-t erm in vitro and in vivo genetic toxicity tests. These genetic toxicity tests were originally de v eloped to study mechanisms of chemical ly induced DNA d amage and to predict carcino genicity i n animals, based on th e ele ctrophilic theory of chemical carcinogenesis and the somatic mutation theory (Miller a nd Miller, 1977; Straus, 1981; Crawford, 1985 ).

There is a s tr ong correlation between a chemical's pote nt ial electroph ilicity (struc tural alert to DNA reactivi ty), mutage n i city in


### Salmonell a , and carcino genicity in rodents. The combination of electroph ilicity and 

### Salmonella mutagenicity is highly correlat e d with the induction of carcino genicity in rats and m i ce and/or at mult iple tissue sites (Ashby and Tennant, 1991 ). Other i n vitro genetic toxicity

tests do not correlat e w ell with rodent carcino genicity (Tennant et a l

., 1987; Zeiger

et al

., 1990 ), although these other tests can provide information o n the types of DNA and chromosome eff ects that can be induced by the chemical being investigated. Data from NTP studie s sh ow th at a positive response in


### Salmonella is currently the most p r edictive in vitro test for rodent carcino genicity (89% of the
Salmonella mutage ns were rodent carcinoge ns), and that there is no compleme ntarity among the

in 

### vitro genetic toxicity tests. That is, no battery of tests that included the 

### Salmonella test improved the predictivi ty of the 

### Salmonella test alone.26 1-Amino-2, 4-dibromoanthraquinon e, N TP TR 383

T ABLE 2 Experimental Design and Materia ls and Methods in the Feed Stud ies of 1-Amino-2,4-dibr

omoanthraquinone

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Study Laboratory

EG&G Mason Research Institut e (Worces

ter, MA) Same as 13-week studies Same as 13-week studies

Strain and Sp

ecies

Rats: F344/N

Mice: B6C3F 1 Rats: F344/N

Mice: B6C3F 1 Rats: F344/N

Animal Source Charles River Breeding Labor atories (Portage, MI) Frederick Cance r Research Facility (Frede rick, MD) Same as 2-year studie s

Time H eld Before Studies 15 days Rats: 12-14 days (males) or 9 days (females)

Mice: 12 days (males) or 15 days (females) 12-14 days (males) or 9 days (females)

Average Age When Studies Began

7 weeks 6 weeks 6 weeks

Date of First Dose

Rats: 22 April (males) or 29 April (females) 1982

Mice: 6 May (males) or 13 May (females) 1982 Rats: 13 July (males) or 4 August (females) 1983

Mice: 20 June (males) or 30 June (females) 1983 13 July (males) or 4 August (females) 1983

Durati on of Dosi ng 13 weeks 104 weeks 9-Month stop-exposure group: 39 weeks (males) o r 40 weeks (fem ales) followed by control fee d for remainder of study 15-Month exposure group:

66 weeks

Date of Last Dose

Rats: 21-23 July (males) or 28-30 July (females) 1982

Mice: 4-6 August (males) or 11-13 August (females) 1982 Rats: 3 July (males) or 25 July (females) 1985

Mice: 10 June (males) or 20 June (females) 1985 9-Month stop-exposure group: 10-13 April (m ales) or 8-10 May (females)

1984 15-Month exposure group: 10-12 October (males) or 7- 9 November (females) 1984Materi als and Methods 27

T ABLE 2 Experimental Design and Materia ls and Methods in the Feed Stud ies

of 1 -Amino -2,4 -di bromo ant hraq uin one (continued)

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Necropsy Dates Rats: 21-23 July (male s) or 28-30 July (females) 1982

Mice: 4-6 August (males) or 11-13 August (females) 1982 Rats:
9-Month interim evaluation: 10-13 April (males) or 8-10 May (females) 1984

15-Month inter im evaluation: 10-12 October (males) or 7- 9 November (females) 1984

Terminal: 10-16 July (males) or 1-8 August (females) 1985

Mice:

15-Month interim evaluat ion: 19-21 September (males) or 26-28 September (females) 1984

Terminal: 17-20 June (m ales) or 27 June - 2 Ju ly (females) 1985 9-Month interim evaluat ion: 10-13 April (m ales) or 8-10 May (females)

1984

15-Month term inal: 10-12 October (males) or 7- 9 November (females) 1984

Average Age at Necr

opsy 20 weeks 9-Month interim evaluat ion:

45-46 weeks
15-Month interim evaluat ion:

72 weeks

Terminal:

110-112 weeks 9-Month interim evaluat ion:

45-46 weeks
15-Month term inal:

72 weeks

Size of St udy Groups

10 males and 10 females Rats: 70 males and 70 females in t he 0, 5, 000, and 10, 000 ppm groups; 50 males and 50 females in the 2,

000 ppm group

Mice: 60 males and 60 females 40 males and 40 females

Method of Animal Di stribution Animals were caged by 1-gram wei ght

classes and the

n distributed i

nto treatment groups such that wit

hin a given sex,

all cage weights were approximat ely equal (± 2 g). Animals were distribu ted randomly in to groups of approximately equa l initial mean body weight. Same as 2-year studie s

Animals per Cage

5 5 5

Method of Animal Identificat ion Ear punch Ear punch Ear punch28 1-Amino-2, 4-dibromoanthraquinon e, N TP TR 383

T ABLE 2 Experimental Design and Materia ls and Methods in the Feed Stud ies

of 1 -Amino -2,4 -di bromo ant hraq uin one (continued)

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Diet NIH-07 open formula meal diet ( Zeigler Brothers, Inc., Gardners, PA), Available

ad li bit um , changed weekly Same as 13-week studies Same as 13-week studies

Water Tap water (City o f Worcester) available

ad li bit um via automatic water

ing system (Edstrom Industries , Inc. , W aterford, WI) Same as 13-week studies Same as 13-week studies

Cages

Po ly ca rb on ate ca ge (L ab Pr od uc ts, I nc ., Rochelle Park, NJ), changed twice w eekly Same as 13-week studies Same as 13-week studies

Bedding Aspen Bed® heat-t

reated hardwood chips (American Excelsior,

Baltimore,

MD), changed twice w eekly Same as 13-week studies; BetaChips®

hardwood ch ips (Northeastern Pro ducts,

Warrens burg, NY) were use d if necessary. Same as 2-year studie s

Cage Filters

Nonwoven f iber filters (Lab Produ cts, Rochelle Park,

NJ; or Snow Filtra

tion, Cincinnati,

OH); changed every 2 weeks No nw ov en fib er fil ter s (S no w F ilt ra tio n C o., Cincinnati,

OH), changed every 2 we eks Same as 2-year studie s

Racks

Sta in les s st ee l ra ck s (L ab Pr od uc ts, I nc ., Maywood, NY),

changed every 2 weeks Same as 13-week studies Same as 13-week studies

Anima l Room Environm ent Average tempe rature:
22° to 2 6° C Relative humidity : 24% to 66% (rats), 28% to 66% (mice) Fluorescent light: 12 hours/day

Room air: 12 to 15 changes/hour Average tempe rature:
19° to 26° C Relative humidity : 16% to 76% Fluorescent light: 12 hours/day

Room air: 12 to 15 changes/hour Same as 2-year studie sMateri als and Methods 29

T ABLE 2 Experimental Design and Materia ls and Methods in the Feed Stud ies

of 1 -Amino -2,4 -di bromo ant hraq uin one (continued)

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Doses
0, 2, 500, 5, 000, 10, 000, 25, 000, or 50,000 ppm in feed,

available ad li

bit um Rats: 0, 2, 000, 5, 000, or

10,000 ppm in feed,

available ad li

bit um Mice: 0, 10, 000, or 20,000 ppm in feed,

available ad li

bit um 20,000 ppm in feed, available ad li

bit um

Type and Freq

uency of Observation

Observed twice dail

y; animals weighed initially, weekly, and at end of studies;

clinical obser

vations recorded twi

ce daily; feed consump tion measured we ekly Observed twice dail y; animals weighed initially, weekly for 14 weeks, and mo nthly thereafter; cl

inical observat

ions recorded weekly for 14 weeks,

then monthly until end of the studies; feed consumption measured monthly Same as 2-year studie s

Method of Sacrifice

CO 2 asphyxiation CO 2 asphyxiation CO 2 asphyxiation

Necropsy Necropsy performed on all ani mals. Organs weighed were brain,

heart, r ight kidney,

liver, lungs, right testis, and thymus. Ne cr op sy wa s p er fo rm ed on all an im als

. Organs weighed at the 9- and 15-m onth

interim evaluati

ons were right kidney and

liver. Ne cr op sy wa s p er fo rm ed on all an im als

. Organs weighed at 9 months and 15 mont hs were right kidney and li

ver.

Histopathology Complete histopathol

ogic examinations were performed on all animals that died during the study, cont

rol animals,

and 50,000 ppm animals. In addition to tissue masses, gross lesions, and associated l

ymph nodes, the

tissues examine d included: adrenal gl and, bone and marrow, brai

n, clit oral gland, epididymis,

esophagus, he art, ki dney, large intestine (ce cum, colon,

rectum), l

iver, l ung, ly mp h n od es (m an di bu lar an d m es en ter ic)

, mammary g land, nose, ovary, pancreas, parathyroid gland,

pituitary gland,

preputial gland, pros

tate gland,

salivary gland,

seminal

vesic le, skin, small intes tine ( duodenum, jejunum, ileum), spleen, stomach, testis, thymus, thyr

oid gland, t

rachea, uri

nary bladder, a

nd uterus. The kidney, l

iver, s pleen (rats), thymus (rats), and uterus (rats) of all other exposed animals

were examined. Complete histopathol ogic examinations were performed on all animals. In addition to

tissue masses , gross l

esions, and associated lymph nodes, t

he tissues exami

ned included: adrenal gland,

bone and marrow, brai

n, clitoral gland, epididymis, esophagus, gallbladder (mice) , heart, kidney, la

rge intestine (ce cum, colon,

rectum), l

iver, l ung, lymph nodes (mandibular and mesent eric),

mammary g land, nose, ovary, pancreas, parathyroid gland,

pituitary gland,

preputial gland, pros

tate gland,

salivary gland,

seminal

vesic le, skin, small intes tine ( duodenum, jejunum, ileum), spleen, stomach, testis, thymus, thyr

oid gland, t

rachea, uri

nary bladder, and uterus. Complete histopathol ogic examinations were performed on all animals. In addition to

tissue masses , gross l

esions, and associated lymph nodes, the tissues exam ined included: adrenal gland,

bone and marrow, brai

n, clitoral gland, epididymis, esophagus, heart, kidney, la rge intestine (

cecum, colon, rectum), l iver, l ung, lymph nodes (mandi bular and mesenteric),

mammary gland, nose,

ovary, pancrea

s, parat hyroid gland,

pituitary gland, preput

ial gland, prostate gland,

salivary gland, se minal vesicle,

skin, s mall intesti

ne (duodenum, jej unum, ileum), spleen, stomach, testis, thymus, thyr

oid gland, t

rachea, uri

nary bladder, and uterus.30 1-Amino-2, 4-dibromoanthraquinon e, N TP TR 38331



# RESULTS

RATS

13-W EEK 

# S TUDY

One male (week 13) and one femal e (week 8) in the 50,000 ppm groups died during the study (T able 3).
The dea ths of one 5,0 00 ppm male (week 4) and tw o addition a l 50,000 ppm males (week 13) were not chemical related. Th e final mean body weigh ts a n d body weight gains of all exposed rat groups were si gnific antly lower tha n those of the controls. Feed

consum ption by al l expos ed gro

ups was less th an th a t by the control s throughout the study and gener ally

decreased with increasing exposu re concentration (Tab le 3). The greatest differe nces in feed co nsump

tion from that by the control s occurr ed in the 25,00 0 and 50,000 ppm males and females. Feed co nsump

tion by these g

roups ran ged from 45% to 79% that b y the control s at week 1 and from 64% to 82% that by
the control s at week 1 3. Diet ary levels of 2,500, 5,000, 10,000, 25,000, and 50,000 ppm delive red dail y doses of approxi matel y 150, 300, 620, 1,600, and 3,2 00 mg 1-amino-2,4-di bromoan thraq uinone /kg bod y

T ABLE 3

Sur viva l, M ean Body W eights, and Feed Consumpti on of Rats in the 13-Week Feed Study

of 1-Amino-2, 4-dib romoanthraqui none

Final Weight
Mean Body Weight b (g) Relative Feed
Dose Survival a Initial Final Change to Contro ls Consumption c (pp m) (%) Week 1 Week 13

Male

0 10/10 180 ± 3 358 ± 3 179 ± 3 14.9 18.1 2,500 10/10 180 ± 3 325 ± 3** 145 ± 3** 91 14.3 16.7 5,000 9/10 d 180 ± 3 328 ± 3** 148 ± 6** 92 13.9 17.1 10,000 10/10 181 ± 3 310 ± 3** 129 ± 3** 86 13.5 17.0 25,000 10/10 181 ± 3 232 ± 3** 52 ± 4** 65 11.7 14.9 50,000 7/10 e 180 ± 3 164 ± 6** -17 ± 6** 46 10.3 11.6

Fema le

0 10/10 140 ± 2 211 ± 3 71 ± 2 13.0 15.7 2,500 10/10 140 ± 2 197 ± 3** 57 ± 3** 93 10.5 12.7 5,000 10/10 140 ± 2 188 ± 3** 47 ± 3** 89 10.2 12.1 10,000 10/10 140 ± 2 185 ± 2** 45 ± 2** 88 9.6 11.9 25,000 10/10 140 ± 2 159 ± 2** 19 ± 2** 75 7.0 10.6 50,000 9/10 f 140 ± 2 130 ± 4** -10 ± 4** 61 5.9 11.5

** Significantly diffe

rent (P # 0.01) from the control group by Williams' or Dunnett's test

a Number of animals surviv ing at 13 weeks/nu mber initially in group b Weig hts and weig

ht chan ges are given as mean ± standard error. Subsequ ent calcul ations are based on animals surviv ing to the end of the stu dy.

c Feed consumption is expressed as grams of feed consu med per animal per day. d Week of death: 4

e Week of death: 13, 13, 13 (2 were accidental deaths) f Week of death: 832 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### weight to males a nd 170, 340, 660, 1, 500 , and 3,200 mg /kg to fe ma les. Pink-red

stai ning of the f

ur and
tail was obs erv ed in a ll exposed groups of rat s. The bedding of all exp ose d groups except t he 2,500 ppm

groups was stai ned pink-re d from day 2 of the st udy.
Lethar gy and ema ci ati on were noted in al l 50,000 ppm
males. Fe m ale r ats i n the 25,000 and 50,000 ppm groups

were let hargic and stagger ed, and 50,000 ppm f emales
exhibited hu

nched posture.

The rela tive liver we igh

ts of exposed groups of males
and the ab so lute and rela tive liver we igh ts of exposed

groups of females were sig nif ican tly great er than those
of the co ntr

ols (T ab le H1 ). The ab so lute and rela tive thymus w e igh

ts of exposed males and females were sig nif ican tly lower than those of controls. The lower
ab so lute brain, he art, kidney, lung, and testi s we igh ts of exposed m ale and fem

ale rats were pr

ob ab ly related

to the lower final mean body we igh ts of these group s.

Observati ons at necropsy included red or pink stai ning
of the gastr oi nte stinal tract co nte nts and/or muco sa, kidneys, and urine. In addit ion, regional lymph nodes
and livers were dark in color, and capsular surfaces of
the livers were granular in appearan ce. These findi ngs

were most common in the 25,000 and 50,000 pp m
group s.

Chem ical-related lesi ons were pre sent in the liver, kidney, and spleen of m ale and fem ale rats. In the liver, a spec tru m of no

nn eo pla stic degenerat ive and proli ferat ive lesi ons occurred in males and females in
the 25 ,000 and 50,000 ppm groups (T ab le 4). Hepato cellular cy

tom eg aly (hypert rophy) was present in all rats in the 25,000 and 50,000 ppm groups and in

most females in the 10,000 ppm group. Th is lesi on

cons ist ed of enlarged hepatocy tes wi th eo sin op hilic cytoplasm and marked varia tion in nuclear size. In the
centrilobular areas of a few rats from exposed group s, there was a minim

al to m ild cy top lasm ic vacuolat ion (vacuolar degen

era ti on). The incidence of vacuolar degenerat ion was not dose-re lat ed, but at the higher

expo sure concent ra tions, minim al hepatocellular

ne cro sis was s omet imes associated wi th vacuolar change. Focal he

pa toc ellu l ar alt erat ions incl uding basoph ilic, eosinoph

ilic, or clear cell foci were also present in the 25,000 and 50,000 ppm group s. I n the periportal region of the he pa tic lobules, there was an increased num

ber of inflam

mato ry cells around the
bile d u cts

. Bile duct hyperp lasi a consisted of proli ferat ion of oval cells in the periportal

area as we ll as proli ferat ion of larger bile du cts lined by hyperch

romatic, ple om

or ph ic bilia ry epi th el iu m. Focal ne cro sis of biliary epit heli um and acu te in fl ammation (necrot izi ng cholang itis) in some hy

pe rp lasti c bil e du cts were associated wi

th periportal fibrosi s

. The spect rum of proli ferat ive bile duct lesi ons (hyp erplasia, necrot izi ng cholang itis, and fibrosis) wa s

m or ph olo gic all y c onsist ent wi

th the lesi on described

as cholang

iofibrosis. A bro wn pigment was present in
the cytoplasm of hepatocy tes. Th e pigment was negati ve for iron, PAS, bile, and acid-fast stai ning; did

not polarize light or fluoresce; and was considered to

repres ent 1-am ino-2 ,4-d ib romo anthra quinone and/or it s metabo lites.

In the kidney of exposed groups of males and females,
there were che mi cal -related increases in the inc idences of a brown, gra

nular pigment in the tub

ule epit heli um (T ab le 4). Th is bro wn pigment had the

sa me stai ning features as the pigment that was present in the liver. In bot h males and females, there were renal tub ule cells wi

th enlarged nu

clei. In males, there

was a hyali ne droplet nephropathy characterized by an increase i n eo sin

op hilic pr ote in dr op lets (hyali ne droplet accumulat ion) in the cy to pla sm of the renal

tu bu le epit heli um as we ll as in the lumen of the
tubules. The

re was no evidence of increased sev erity
of tub ule regener ati on in males or females.

Chem ical-related eff

ects in the spleen of a ll exposed

groups of males and females consisted o f a sl ight
increase in th e am ount of he m ato po iesis rela tive to that no rm ally present in controls.

Other no ns pe cific changes included lym ph oid depletion in the thymus and decreased uterus si ze. Th ese findi ngs were a t tributed to the m ark ed ly lower body weight ga in in rats from the higher expo sure group s.

Do se Selecti on Rationale: Based on chem ical dis po sition studies, mean body weights, and chem ical-related
lesi ons of the liver, kidney, and spleen present m ain ly
in the 2 5,000 and 50,000 ppm group s, expo sure concentr ati on s sel ected for the 2-year feed study of 1-amino-2,4- dibromoanthra quinone in rats were 0, 2,000, 5,000, and 10,000 ppm. Muc h of th e differences in mean body we igh ts recorded for the 13-w

eek

st ud ies were mo

re like ly due to decreased feed palatab ility than to any over

t toxic ity. None t heless, ifResu lts 33



### T ABLE 

### 4

Inci dence s of

Selected

Nonne opl astic Lesi ons in Rats in t he 13-Week Feed Stu dy of 1-Amino-2, 4-dibromoanthr aquinone

0 ppm 2,500 ppm 5,000 ppm 10,000 ppm 25,000 ppm 50,000 ppm

Male

Liver a 10 10 10 10 10 10

Basop hilic Focus b 0 0 0 0 4* (1.5) d 9** (2.3)
Clear Cell Focus 0 0 0 0 6** (1.0) 0 Eosinophilic Focus 0 0 0 0 4* (1.5) 0 Cytomegal

y 0 0 0 0 10 ** (3.2) 10 ** (4.0)
Bile Duc t Hyperplasia 0 0 0 0 8** (2.3) 10 ** (3.1)
Inflammation 0 0 0 0 10 ** (2.1) 10 ** (3.0)
Fibrosis c 0 0 0 0 10 ** (1.9) 10 ** (3.1) Necrotizing Cho langitis c 0 0 0 0 7** (1.3) 10 ** (2.8) Vacuolar Degeneration c 0 0 4* (1.5) 5* (1.8) 3 (1.3) 4* (1.5) Pigmentation 0 0 0 0 10 ** (1.5) 9** (1.0)

Kidney 10 10 10 10 10 10

Renal Tubule Pigmenta tion 0 10 ** (1.0) 9** (1.0) 10 ** (1.1) 10 ** (2.2) 10 ** (1.8) Hyaline Droplet Accumulation 0 10 ** (1.7) 9** (1.7) 10 ** (2.0) 2 (1.0) 0

Female

Liver 10 10 10 10 10 10

Basop hilic Focus 0 0 0 0 1 (1.0) 9** (2.6) Eosinophilic Focus 0 0 0 0 1 (1.0) 0 Cytomegal

y 0 0 0 8** (1.0) 10 ** (2.2) 10 ** (4.0)
Bile Duc t Hyperplasia 0 0 0 4* (1.0) 9** (1.6) 10 ** (2.4)
Inflammation 0 0 0 0 10 ** (1.6) 9** (2.3)
Fibrosis c 0 0 0 0 2 (2.0) 9** (2.7) Necrotizing Cho langitis c 0 0 0 0 0 9** (2.6) Vacuolar Degeneration c 0 0 0 1 (1.0) 2 (2.0) 8** (1.4) Pigmentation 0 0 1 (1.0) 7** (1.0) 9** (1.2) 10 ** (1.5)

Kidney 10 10 10 10 10 10

Renal Tubule Pigmenta tion 0 10 ** (1.0) 9** (1.0) 10 ** (1.2) 10 ** (1.7) 10* * (1.9)

* Significantly diff

erent (P # 0.05) from the control group by the F isher exact test

** P # 0.01

a Nu mber of anima ls with organ ex amined microscopically b Number of animals with lesion

c Data fr om Fl eis chma

n et al ., 1986 d Average severity

of lesions in aff

ected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked



### ex posure selection were based on mean body wei ght s alone for male rats, the 10,000 ppm exposure concentra

ti on coul d have been considered sl ightly high. More

over, cons idering the lack of live r toxici ty at exposur e s of 10

, 000 ppm and be l ow, t h is e x posure conc en

tra ti on wa s predicted not to adv ers ely affect th e healt h or surviva l of the se ani

mals . Hig her exp osure
concentr ations (20, 000 ppm) were chosen for the start

stop, pr og res si on/r egr ess ion e xperi ments (stop-exposur e evalu ation).34 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



## 2-Y EAR 

## S TUDY 

## Survival

Estimates of 2-year survival probabili ties for male an d
female rats are shown i n Table 5 an d i n the Kaplan- Meier surv ival curves in Figu re 2. Survival o f
male and fem al e ra

ts i n th e 10, 000 ppm

groups was signifi cantly l ower than that of the controls.



## Body Weights, Feed and Compound Consumption, and Clinical Findings

Mean b ody

wei ghts of ex

posed mal e and fem ale ra

ts were low er than those of the controls after week 2 (Tables 6 and 7, Figu

re 3). Final mea

n body weight

s o f exposed m a l e s w ere 14% to 30% lower than th a t of the controls; final

me an b ody

weig hts of exposed
females were 20% to 46% lower than that of the
controls. Feed consumption by ex posed males and
fema le s was si

milar am ong exposed groups and was slight ly lower than that by

the cont rol s (Ta bles J3 an d J4). Dietary l evels o f 2,00 0, 5,000, and 1 0,000 ppm
delivered average daily

doses of app roxi mat el y 90, 240, and 49 0 mg 1- amino-2 ,4-dib romo anthr aquinone/kg body w eight to males and 1 10, 285, and 60 0 mg/k g to femal es. Di scolorati on of the fur and urin e

w as ev ide nt in all exposed groups as ear ly as day 8 an d

wa s ob ser

ved t hrou ghou t th e st udy . Em aci ati on

occur red in a dose-related manner in male and fem ale

rats and occurred in over 50% of the rats exposed to 10,0 00 ppm.

T ABLE 5

Surviv al of Ra ts in the 2-Yea r Feed Study o f 

## 1-Amino-2,4-dib romo anthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

Animals initia lly in study 70 50 70 70

9-Mont h interim evaluation a 10 10 10 10

15-M onth interim evaluat ion a 10 0 0 10

Moribund 19 15 34 33

Natural deat hs 5 1 5 7

Animals sur viving to st udy termination 26 e 24 21 10 Percent prob abilit y of survival at end of study b 53 60 35 20

Mea n su rvi val (da ys) c 586 618 615 547

Survi val ana lyses d P<0.001 P=0.467N P=0.141 P<0.001

Female

Animals initia lly in study 70 50 70 70

9-Mont h interim evaluation a 10 10 10 10

15-M onth interim evaluat ion a 10 0 0 10

Mis sexed a 0 0 0 1

Moribund 8 5 15 29

Natural deat hs 4 3 7 8

Animals sur viving to st udy termination 38 f 32 38 12 Percent prob abilit y of survival at end of study 76 80 63 25

Mea n su rvi val (da ys) 610 626 643 569

Survi val ana lyses P<0.001 P=0.857N P=0.216 P<0.001

a Censo red fr om sur vival a nalyses b Kaplan -Meier determina tions bas ed on the numb er of an imals alive on f irs t day of termina l sacr ifice

c Mean of

all deat hs (u ncensor ed, censor ed, and t erminal s acrif ice) d The r esul t of the life table tren d test (Ta rone , 1975) is i

n the con trol c olu mn, and the r esul ts of the life table pairw ise c ompa rison s (Cox, 1972)
with the controls are in the expos ed columns. A lower mortality in an exposur e group is indicat ed by N.

e Incl udes th ree m ale s t hat died dur ing th e la st week of th e st udy. f Incl udes one f

emal e th at died dur ing th e la st week of th e st udy.

### Results 35



### FIGURE 

## 2

Kaplan-Meier Survival Curves for Rats Administ ered l-Amino-2,4-dibromoanthraquinone in Feed

for 2 Years36 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



### T ABLE 

### 6 Mean Body Weight s and Survival of Male Rats in t he 2-Year Feed Stud y of 1-Amino-2, 4-dibromoanthr aquinone

Weeks 0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Study (g) Survivors (g) con trol s) Survivors (g) con trol s) Survivors (g) con trol s) Survivors

1 139 70 136 98 50 136 98 70 134 97 70

2 161 70 165 103 50 164 102 70 155 97 70

3 206 70 203 99 50 198 96 70 184 89 70

4 235 70 225 96 50 222 95 70 204 87 70

5 240 70 233 97 50 230 96 70 215 90 70

6 269 70 258 96 50 257 96 70 237 88 70

7 287 70 271 94 50 269 94 70 248 86 70

8 302 70 285 94 50 280 93 70 260 86 70

9 312 70 295 95 50 287 92 70 268 86 70

10 325 70 308 95 50 303 93 70 281 86 70

11 333 70 316 95 50 313 94 70 293 88 70

12 338 70 323 96 50 318 94 70 300 89 70

13 332 70 321 97 50 309 93 70 298 90 70

14 356 70 339 95 50 330 93 70 314 88 70

17 387 70 363 94 50 350 91 70 335 87 70

21 406 70 382 94 50 368 91 70 356 88 70

25 423 70 398 94 50 383 90 70 369 87 70

29 435 70 403 93 50 384 88 70 376 86 70

33 445 70 413 93 50 398 89 70 383 86 70

37 453 70 420 93 50 401 89 70 388 86 70

41 a 468 60 433 92 40 415 89 60 397 85 59

45 473 60 440 93 40 424 90 60 403 85 59

49 479 60 452 94 40 428 89 60 409 84 57

53 489 60 457 93 40 439 90 60 415 85 57

57 486 60 453 93 40 430 88 60 409 84 57

61 484 59 445 92 40 430 89 60 405 84 57

65 484 57 448 93 40 426 88 60 408 84 55

69 a 479 47 442 92 40 417 87 59 398 83 44

72 472 46 429 91 40 407 86 58 389 82 42

77 460 46 412 90 40 392 85 56 381 83 41

80 462 44 418 91 40 390 84 56 373 81 38

85 467 40 419 90 40 388 83 52 365 78 34

89 455 40 405 89 39 378 83 49 357 79 29

93 445 39 396 89 36 363 82 43 347 78 24

97 432 35 380 88 32 355 82 37 332 77 22

101 422 30 380 90 26 343 81 28 301 71 18

103 406 29 349 86 24 341 84 22 283 70 13

Mean for weeks

1-13 268 257 96 253 94 237 89

14-52 433 404 93 388 90 373 86

53-103 460 417 91 393 85 369 80

a Interim eva luations occurre d during week 39 for all groups and week 66 for the 0 and 10,000 ppm groups.Resu lts 37

T ABLE 7 Mean Body Weight s an d Su rvival of Femal e Rats in th e 2-Ye ar Feed Stu dy

of 1 -Am ino-2 ,4-di bro mo anthra quino ne

Weeks 0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

on Av. W t. No. of Av. W t. Wt. ( % of No. of Av. W t. Wt. ( % of No. of Av. W t. Wt. ( % of No. of

Stu dy (g) Surviv ors (g) controls) Surviv ors (g) controls) Surviv ors (g) controls) Surviv ors

1 93 70 93 100 50 94 101 70 94 101 70

2 114 70 110 96 50 107 94 70 102 90 70

3 133 70 128 96 50 121 91 70 116 87 70

4 147 70 141 96 50 136 93 70 128 87 70

5 157 70 151 96 50 146 93 70 137 87 70

6 166 70 159 96 50 152 91 70 145 87 70

7 173 70 169 97 50 161 93 70 150 87 69

8 180 70 174 97 50 167 93 70 158 88 69

9 186 70 178 96 50 173 93 70 167 90 69

10 191 70 185 97 50 179 94 70 173 90 69

11 196 70 189 96 50 184 94 70 177 90 69

12 203 70 194 95 50 188 93 70 181 89 69

13 208 70 195 94 50 192 93 70 185 89 69

14 212 70 201 95 50 196 93 70 189 89 69

17 222 70 209 94 50 205 92 70 200 90 69

21 228 70 216 95 50 213 93 70 205 90 69

25 237 70 219 93 50 216 91 70 208 88 69

29 246 70 223 91 50 219 89 70 212 86 69

33 251 70 227 90 50 222 88 70 213 85 69

37 258 70 233 90 50 230 89 70 218 85 69

41 a 265 59 233 88 40 227 86 60 217 82 59

45 272 59 239 88 40 231 85 60 221 81 59

49 284 59 246 87 40 237 84 60 227 80 59

53 299 59 257 86 40 245 82 60 232 78 59

57 311 59 264 85 40 249 80 60 238 77 58

61 315 59 267 85 40 251 80 60 238 76 57

65 328 59 275 84 40 257 78 60 242 74 57

68 a 333 49 277 83 40 257 77 60 241 72 47

73 343 49 286 83 40 263 77 60 245 71 46

77 347 49 289 83 40 269 78 60 243 70 45

81 351 49 296 84 40 270 77 60 239 68 43

85 354 49 295 83 40 268 76 58 234 66 39

89 354 47 298 84 37 262 74 55 229 65 34

93 356 45 299 84 37 251 71 53 224 63 28

97 358 43 298 83 33 250 70 46 213 60 23

100 361 42 293 81 32 243 67 43 202 56 20

103 362 38 290 80 32 234 65 39 194 54 13

Mean for weeks

1-13 165 159 96 154 93 147 89

14- 52 248 225 91 220 89 211 85

53-103 341 285 84 255 75 230 67

a Interim ev

aluations occurred du ring week 40 for all groups and week 66 for the 0 and 1 0,000 ppm groups.38 l-Ajnino-2,4-dibromoan thraquinone, NTP TR 383

FIGURE 

## 3

Growth Curves for Rats Administered 1-Amino- 2,4-dibromoanthraquinone in Feed for 2 YearsResu lts 39



## Pathology and Statistical Evaluation
This sec ti on desc ribes th e stat istically si gni fi can t or biologica lly note wor thy cha nge s i n th e i nci de nces o f
mononuclear

cel l l euk emi a; n eopl asm s of the l iver, large intestine, kidney, urinary b ladder, and other

organs; and non neo plast ic lesions of th e liver, k idney

, urin ary bladder, foresto ma ch, and seminal vesicl es o f

rats. Summaries of the i

ncidences of

neoplasms and

nonn eopla stic lesions , individ ual animal tu mor diagnoses, statist ical analy ses of primary neoplasms
that occurred wi

th an inci dence of at l east 5% in at lea s t

one exp os ure group, and hi stor ical i ncidences for the

neo plasms menti oned in th is sec tion are pre sented i n Append ix A for male rats and Ap pend ix B fo r female

rats.

Liver: 

### At the 9-month inter im evalu at ion, the absolut e and re lativ

e live r wei ghts of e xposed groups of males
and females were signi

fican tl y greater than those of th e

con tro ls (Tabl e H2). One hepatocellul ar adeno ma wa s observed in a 5,000 ppm male at 9 months (Table s 8
and A1). Incidences of foci of h epatocellular altera tio n

w ere in cre ase d in males in the 10,000 ppm group, an d

a mini mal accumu

lati on of pigment in he

patocytes wa s
prese nt in males and femal es from the 10,000 ppm
groups and in femal es fr om th e 5,000 ppm group (Tables 8, A5, and B 5). At th e 15 -month interim evaluation, the

abs olu te a nd re la ti ve l iv er w

ei ght s of exposed groups of f

emale s were signif

icant ly greate

r than those of the

controls ( Table H3). Inc ide nce s of single and multipl e hep ato cellul ar adenomas and

car ci nom as were increased at 15 months in 10, 000 pp m males and fe m ales (Tables 8, A1, and B1). Incidence s of foci of hepatocellul

ar al ter ati on and accumu la

ti on o f pigment in hepatocy

te s were also i

ncreased in expose d groups of m ales a

nd femal es (Tabl es 8, A5, and B5).

At the end o f the 2-year stu dy, the i ncidences of hepato

cellul ar adenoma, hepatocel lul ar c a rcinoma, and hepatocellular ad enoma or carci noma (co mb in ed) wer e signifi can tly increased in all exposed group s of males and females (T ables 8, A3, and B3). The inciden ces o f mult iple hepa tocellular adenomas
and multiple hepa

to cellul ar carcinomas in exposed mal e and female groups w

ere great er than those of the

cont rols (Tables 8, A1, and B1). Inc i denc es of
h epatocellul

ar ade noma or carcinoma (co mb in ed) in al l exposed groups of male s and females exceeded the NT P
hist ori cal range s (males: 0%-10%; females: 0%- 6% )
for feed stu

dy cont rol s (Tables 8, A4 a, and B4a). Th e majori ty of the benign and mal ignant live r neoplasm s consisted of wel

l-di fferentiated neo plast ic hepato cyte s wi th cellu lar atypia and increased number s o f mitoses

. Carcinomas had trabecu

lar, glan dular, or solid g

ro wt h
patterns (Plate 1) wit

h areas of necrosis, ca

vitation, an d
fibrosis. Met astases were common i n the lungs (Plates 2 and 3), bu t metast atic foci were also present i n the stoma ch, pancreas, adrenal gl and, ly mph node, an d spleen.

The inci dences of single and multiple hepatocholan gio

carcinoma wer

e signifi cantly i

ncreased in 5,000 pp m males and females and in 10,000 ppm females (Ta ble s 8, A3, and B3). The se n eop lasms co nsist ed of a
mixt ure of mal igna nt hepa toc ytes and welldifferentiated

cuboidal epitheli

um forming distinct
ductul ar str

uct ure s (Pl ate 4). Both hepatocellul ar an d
bil iary compo nents of th is neop lasm were pr esent in

metast atic foci. In add ition, s eve ral other benign (cholangioma and hep atoc hol ang iom

a) and malig

nan t (cho la ngiocarcinoma) liver neoplasms occur red only i n exposed groups of males and females (Tab le s 8, A1,
and B1 ).

During the 2-year stud

y, the inci dences of pigmenta tio n

a n d foci o f hepat ocellula r altera tion ( clear ce ll, basophilic, and eos inoph ilic ) were increase d in expose d

groups of males and female s (Tables 8, A 5, and B5).
Cells in some foci had in

ten sely eos inop hilic c ytoplas m and hepato cellula r atypia simi lar to the appearan ce of
cells in the hepatocellul

ar neoplasms. The pigment wa s considered to be 1-a

mi no- 2, 4-dib romoanthraquinone o r

a metabolite based on the resul ts of the histochemi cal

pr oc e dures performed during the 13-week stu dy and th e 15-month interim eval

uation.40 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 8

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Liver in Rats in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

9-Month Interi m Evaluation

Number E xamin ed 10 10 10 10

Basophil ic Fo cus a 0 0 1 (1.0) b 1 (1.0)
Clear Cell Focus 0 0 0 4* (1.0)
Eosino philic F ocus 0 0 0 1 (1.0) Pigmentation 0 1 (1.0) 0 6** (1.0)

Hepatocell

ular Adeno

ma 0 0 1 0

15-M onth Interim Evaluation

Number E xamin ed 10

- c

- 10

Basophil ic Fo cus 1 (1.0) 5 (1.0)
Clear Cell Focus 0 7** (1.0)
Eosino philic F ocus 1 (1.0) 0 Pigmentation 0 10** (1.0)

Hepatoc ellular Aden oma (Mu ltiple) 0 2 Hepatocell

ular Adeno

ma (Single or M ultip le) 0 4* Hepatoc ellular Ca rcinom a (Mult iple) 0 3 Hepatocell

ular Carcinoma (Single or M ultip le) 0 7** Hepatocell

ular Adeno

ma or Carcinoma 0 10**

2-Year Study

Number E xamin ed 50 40 59 50

Basophil ic Fo cus 9 (1.1) 12 (1.7) 24** (1.8) 22** (1.5)
Clear Cell Focus 3 (1.0) 26** (1.8) 39** (1.8) 27** (1.8)
Eosino philic F ocus 1 (1.0) 13** (2.9) 14** (2.1) 6 (2.6) Pigmentation 3 (1.0) 19** (1.1) 48** (1.1) 39** (1.1)

Hepatoc ellular Aden oma (Mu ltiple) 0 10** 23** 24**

Hepatoc ellular Aden oma (Sin gle or Mult iple)
Over all r ate d 1/50 (2%) 20/40 (50%) 40/59 (68%) 34/50 (68%)
Ter minal r ate e 1/26 (4%) 16/24 (67%) 18/21 (86%) 9/10 (90%) Adjust ed rate f 3.8% 71.3% 92.3% 97.0% First incidence (

days) 729 (T) 675 521 435 Logistic r egress ion test

g P<0.001 P<0.001 P<0.001 P<0.001

Hepatoc ellular Ca rcinom a (Mult iple) 0 1 43** 37**

Hepatoc ellular Ca rcinom a (Sing le or Multi ple)
Over all r ate 1/50 (2%) 12/40 (30%) 55/59 (93%) 46/50 (92%)
Ter minal r ate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%) Adjust ed rate 2.7% 43.5% 100.0% 100.0% First incidence (

days) 666 650 465 436 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

(cont inu ed)Results 41

T ABLE 8

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Liver in Rats in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm

Male (continued)

2-Year Study

(conti nued)

Hepatocell

ular Adeno

ma or Carcinoma

h

Over all r ate 2/50 (4%) 25/40 (63%) 57/59 (97%) 47/50 (94%)
Ter minal r ate 1/26 (4%) 19/24 (79%) 21/21 (100%) 10/10 (100%) Adjust ed rate 6.4% 83.1% 100.0% 100.0% First incidence (

days) 666 650 465 435 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Number E xamin ed 50 40 59 50

Hepatocholangio

ma 0 0 1 1 Hepatocholangio carcinoma 0 0 6* 2 Cholangioma 0 0 2 0 Cholangiocarcinoma 0 0 0 1

Fema le

9-Month Interim

Evaluati on

Number E xamin ed 10 10 10 10

Basophil ic Fo cus 1 (1.0) 0 0 1 (1.0)
Clear Cell Focus 0 0 0 1 (1.0) Pigmentation 0 2 (1.0) 6** (1.0) 6** (1.0)

15-M onth Interim Evaluation

Number E xamin ed 10

- - 10

Basophil ic Fo cus 8 (1.0) 9 (1.4)
Clear Cell Focus 0 5* (1.6) Pigmentation 1 (1.0) 10** (1.1)

Hepatoc ellular Aden oma (Mu ltiple) 0 5* Hepatocell

ular Adeno

ma (Single or M ultip le) 0 8** Hepatoc ellular Ca rcinom a (Mult iple) 0 3 Hepatocell

ular Carcinoma (Single or M ultip le) 0 6** Hepatocell

ular Adeno

ma or Carcinoma 0 9**

2-Year Study

Number E xamin ed 50 40 60 48

Basophil ic Fo cus 39 (1.3) 15** (1.6) 22** (1.7) 16** (1.4)
Clear Cell Focus 3 (1.3) 28** (1.6) 39** (2.0) 17** (1.6)
Eosino philic F ocus 7 (1.4) 23** (2.0) 12 (2.5) 1 (4.0) Pigmentation 1 (1.0) 19** (1.1) 51** (1.4) 45** (1.1)

(co nti nued)42 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 8

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Liver in Rats in t he 2-Year Feed Study

of 1-Amino-2, 4-dib romoanthraqui none (contin ued)

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm

Fema le (conti nued)

2-Year Study (conti

nued)

Number E xamin ed 50 40 60 48

Hepatoc ellular Aden oma (Mu ltiple) 0 18** 39** 22**

Hepatoc ellular Aden oma (Sin gle or Mult iple)
Over all r ate 0/50 (0%) 28/40 (70%) 47/60 (78%) 29/48 (60%)
Ter minal r ate 0/38 (0%) 23/32 (72%) 29/38 (76%) 8/12 (67%) Adjust ed rate 0.0% 75.5% 83.7% 83.6% First incidence (days)_

- i 600 575 418 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Hepatoc ellular Ca rcinom a (Mult iple) 0 7** 51** 41**

Hepatoc ellular Ca rcinom a (Sing le or Multi ple)
Over all r ate 0/50 (0%) 12/40 (30%) 57/60 (95%) 45/48 (94%)
Ter minal r ate 0/38 (0%) 12/32 (38%) 37/38 (97%) 12/12 (100%) Adjust ed rate 0.0% 37.5% 98.3% 100.0% First incidence (

days)

- 729 (T) 575 460 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Hepatocell

ular Adeno

ma or Carcinoma

j

Over all r ate 0/50 (0%) 33/40 (83%) 59/60 (98%) 47/48 (98%)
Ter minal r ate 0/38 (0%) 28/32 (88%) 38/38 (100%) 12/12 (100%) Adjust ed rate 0.0% 89.1% 100.0% 100.0% First incidence (

days)

- 600 575 418 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Hepatocholangio

ma 0 0 2 0 Hepatocholangio carcinoma 0 0 11** 13** Cholangioma 0 0 0 1

* Significantly different (P # 0.05) fr om the contro l gro

up by the Fishe r exa ct test (9-mo nth and 15-m

onth inte rim ev aluatio ns) or the lo gistic

regres sion test (2-yea r stu dy)

** P # 0.01 (T)Te rminal sacrifice

a Number of animals with lesion b Average severit y grade of lesion s in aff ected ra ts: 1 = mi nimal; 2 = mild ; 3 = mod erate; 4 = marked

c Liv er no t micro scopica

lly exami ned in thi s gro

up d Number of animals with neoplasm per number of animals with liver examined m icroscopically

e Obser ved inc idence

in animal s surv

iving un til the end of the stud y f Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. g In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l. h Histori cal inc idenc e for 2-year NTP feed studi es with u ntreat ed cont rol groups (mean ± s tand ard devi ation ): 45/1 ,350 (3.3% ± 3.6% ); range,
0%-10% i Not appl

icable ; no neo

plasms in animal

group j Histor ical inc idence : 9/1,351 (0.7% ± 1. 5%); ran ge, 0% -6%Results 43



### Large Intestine: Adenomatous poly ps (adenom as)

were observ ed in the large i ntesti ne of 10,000 ppm
males and females at the 15-month interim evaluation (Tables 9, A1, and B1). At 2 year s, the incidences of adenomatou s poly ps (adenom as) in the rectum were significantly
increased in all exposed gr oups of males and
females (Tables 9, A3, and B3). The incidence of
carcinom a of the



## T ABLE 

## 9 Incidences of Neoplasms of the Large Intestine in Rats in the 2-Year Feed Study
of 1-Ami no-2,4-dib romoa nthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

15-M onth Interim Evaluation

Rectum

a 9

- c

- 10

Adenomatous P olyp (A

denoma) b 0 6**

2-Year Study

Colon
Adenomatous P olyp (A

denoma)
Over all r ate d 0/50 (0%) 1/40 (3%) 1/59 (2%) 3/50 (6%)
Ter minal r ate e 0/26 (0%) 1/24 (4%) 0/21 (0%) 1/10 (10%) Adjust ed rate f 0.0% 4.2% 4.3% 19.9% First incidence (

days)

- h 729 (T) 720 590 Logistic r egress ion test

g P=0.027 P=0.484 P=0.494 P=0.081

Carcinoma i

Over all r ate 0/50 (0%) 0/40 (0%) 1/59 (2%) 4/50 (8%)
Ter minal r ate 0/26 (0%) 0/24 (0%) 1/21 (5%) 0/10 (0%) Adjust ed rate 0.0% 0.0% 4.8% 20.4% First incidence (

days)

- - 729 (T) 590 Logistic r egress ion test P=0.003

- P=0.457 P=0.046

Rectum Adenomatous P olyp (A

denoma)
Over all r ate 0/50 (0%) 13/40 (33%) 51/59 (86%) 40/50 (80%)
Ter minal r ate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%) Adjust ed rate 0.0% 45.8% 100.0% 100.0% First incidence (

days)

- 659 478 352 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Carcinoma i

Over all r ate 0/50 (0%) 1/40 (3%) 10/59 (17%) 15/50 (30%)
Ter minal r ate 0/26 (0%) 0/24 (0%) 5/21 (24%) 4/10 (40%) Adjust ed rate 0.0% 3.8% 32.4% 63.0% First incidence (

days)

- 718 608 493 Logistic r egress ion test P<0.001 P=0.480 P=0.003 P<0.001

Large Intestine (All Sites) 50 40 59 50

Adenomat ous Polyp (Ad enoma) (M ultip le) 0 1 34** 32** Adenomatous Polyp (Adenoma) (Single or M ultip le) 0 13** 51** 40**
Carci noma (M ultip le) 0 0 0 3 Carci noma (Si ngle or Mu ltiple) 0 1 11** 17**

(cont inu ed)44 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 9

Inci dences of Neoplas ms of t he Large I ntest ine i n Rats i n the 2- Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (contin ued)

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm

Fema le

15-M onth Interim Evaluation

Rectum 10

- - 10 Adenomatous Polyp (Adenoma) 0 2

2-Year Study

Colon 50 40 60 49

Adenomatous P olyp ( A

denoma) 0 1 2 2 Carcinoma

j 0 1 2 1

Rectum Adenomatous P olyp (A

denoma)
Over all r ate 0/50 (0%) 27/40 (68%) 53/60 (88%) 43/49 (88%)
Ter minal r ate 0/38 (0%) 23/32 (72%) 38/38 (100%) 12/12 (100%) Adjust ed rate 0.0% 75.0% 100.0% 100.0% First incidence (

days)

- 616 582 512 Logistic r egress ion test P<0.001 P<0.001 P<0.001 P<0.001

Carcinoma

j

Over all r ate 0/50 (0%) 1/40 (3%) 19/60 (32%) 7/49 (14%)
Ter minal r ate 0/38 (0%) 1/32 (3%) 13/38 (34%) 4/12 (33%) Adjust ed rate 0.0% 3.1% 41.7% 41.9% First incidence (

days)

- 729 (T) 606 625 Logistic r egress ion test P<0.001 P=0.466 P<0.001 P=0.001

Large Intestine (All Sites) 50 40 60 49 Adenomat ous Polyp (Ad enoma) (M ultip le) 0 18** 46** 32** Adenomatous Polyp (Adenoma) (Single or M ultip le) 0 28** 53** 43**
Carci noma (M ultip le) 0 1 1 1 Carci noma (Si ngle or Mu ltiple) 0 2 21** 8**

** Significantly different (P # 0.01) from the co ntrol gro up by the Fishe

r exact test (15-month interim ev aluation) or the

logistic re

gression te st (2-yea r stu dy) (T)Te rminal sacrifice

a Number of animals with large intesti ne examined microscopic ally b Number of animals with lesion

c Lar ge inte stine no

t micro scopica

lly exami ned in thi s gro

up d Number o f anim als with neoplas m per nu mber of an imals n ecropsi ed

e Obser ved inc idence

in animal s surv

iving un til the end of the stud y f Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. g In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l. h Not appl

icable ; no neo

plasms in animal

group i Histori cal inc idenc e for 2-year NTP feed studi es with u ntreat ed cont rol groups (mean ± s tand ard devi ation ): 1/1, 353 (0 .1% ± 0 .4%); range, 0%-2% (inclu des all ca rcinom as of th e large int estine) j Histori cal inc idenc e: 0/1,3 51 (in cludes a ll carci nomas of the large i ntesti ne)Results 45

colon was sign ific an tly increased in 10,0 00 p pm m ales,

and the i ncidences of rect al carcinoma were significantly

increa sed in 5, 000 and 1 0,000 ppm males and f emales (Tables 9, A3, and B3). The i ntes ti nal neopl asms th at

occurred in the dis tal colon and rectum of rat s were morphologically simil ar.

Adenom atous polyps ( adeno mas) con si ste d o f
peduncu lat ed, exophyti c masses (Plat e 5) of well- different iat ed, columnar epithelium with prominent, hyperchro matic nuclei. C arcino ma (a den oca rcin o ma) was

gene rally si mi lar to adenoma, except that i nvasion of the

s tromal stalk of the neop lasm a nd ex tension into th e submucosa, and o ccasio nally into the mus cular wall, were

evident mic ros copi cal ly . I n t he ma ligna nt neoplasm s, irregula r gland ular s truct ures or c ords of at

ypical epith elia l

cells were present in t he submucosa (Plate 6) and were frequ ently associ ated with a scirrhous respo nse. In males,

metas tat ic c olon carcin oma was observed in the lung and mesenteric lymp h node s, and metast atic rectal car cin oma

was observed in

the l umbar ly mph nodes an d pancreas (T able A1 ). Incidences of carcinoma (colon and rectum

combine d) in exp osed grou ps of males and femal es
exceeded the NTP hi s t or i ca l r an ge s ( ma l es : 0%- 2%;

females : 0%) for f

eed study

control s (Tables 9, A4b, and

B4b ).



### Kidney : At the 9-month in t erim evaluat i on, the rela t ive

k idney weig hts of ex posed gr oups of males and f

ema le s

were significantly great er tha n those o f the con trols (T able
H2). Pigmen tat ion and hyal ine dropl et accumulation were

present in the kidne ys of all exposed males (Tab les 10 and
A5). Th e sizes o f some renal tu bule nuclei w ere m inimally increased in males a nd females from all exposure

g roups, and th e seve rity of the ne phro pathy (tu bule epithe lial

regen eratio n, mo nonu clear in flamm atio n , and

renal tubu le di lat ion with protein cas ts) was sl ightly more

severe than that observed in the controls (T able 10). At

the 15-m onth inte rim ev aluatio n, th e relat ive ki dney weigh ts of 10,000 ppm males and f emal es were significantl y great er tha n those o f the con trols (T able H 3). The

severi ty o f ne phropat

hy wa s i ncrea sed i n 10,000 ppm

males and fe male s com pare d to c ontro ls (Ta ble 10). Th is

wa s chara cterize d by a n incre ase in the foci of tub ule
epithe lial

regeneration; nucl ear enl arg emen t i n so me
tubule epi thel ium, s imil ar to t hat i n the 13- week st

udies

and at the 9-m onth inte rim ev aluati on, was also prese nt.

At 15 mont

hs i n th e 10,000 ppm groups , pi gment ati on of

renal tubul e epith eliu m was present in all rats; ren al tubu le epith e lial hyperp l asia w a s observed i n two mal e s and thr ee f emale s; and aden omas wer e obser ved in two males (Tables 10, A1, A5 , and B5).

At 2 years , th ere was a significan t dose -related increa se in

the inc idenc es of

renal tubu le a denoma i n expos ed grou ps
of males and f

emale s (Tabl es 10, A3, an d B3)

. M ultiple

adenomas were ob served i n all exposed groups of males

and in t he 5,000 and 10,000 ppm females . Adenomas

were expansile lesi ons involving one or m or e adjacent

tubul es and were generally f ive or more times the di ameter

of the no rmal re nal tub ule. T he ce lls within the adenomas

were gener ally similar in m orp holo gy to tho se in the f ocal
hyp erpla stic lesi ons. Carci nomas were l arger t han ad e

nomas and freque ntly had more cellular atypia, necrosis,

and local invasi on. One carci noma in a male rat metastasi zed to th e lung, and one in a female rat metas tasized to

th e adrenal gland. Renal tubule car cinomas occurr ed i n

two 5,000 pp m males, one 10,000 ppm male, and t wo 10,000 ppm females. The comb ined incidenc e s of re nal

tubule adenoma or carci noma were si gnif icant ly i ncreased

in expose d males and fem al es an d e xc ee de d t he NTP

hist orical ranges ( males : 0%-6%; f emale s: 0%-2%) for
feed study controls (Tables 10, A4c, and B 4c).

Incidenc es of r en al tubu le hype rplasia were sig nificantly

increa sed in expos ed ma l es an d fema les (Tables 10, A5,

and B5). Hyperplas ia c onsi ste d of a tu bule l ined by t

wo

or more la yer s of

ren al tu bul e epi th eli

um; these were mo st

often loca ted i n th e cort ex or ou ter st ripe of

the ou te r
medulla. Foci of hyperplasia were dis ti ngui shed f rom the

more baso philic fo ci of tubu le e pit heli al r egene rat ion typically assoc iated w ith nep hrop athy. There was no cl ear
d ose-re late d inc reas e in the inc idenc e or s everi ty of nephropathy in rat s at 2 years (Table 10). The inci dences

and s everit

y of t ransi tional

cell hyperpl asia i n the r enal pelvis were incr eased in e xposed g roups of m ales and

females (Tables 10, A5, and B5); ther e were no i ncreases
in th e inci dences of t

ransi tional

cell papill oma or carci

noma of the renal pel vi s (Tables 10, A1, and B 1). The

inci dence of a reddi sh brown pig ment wi thi n the r enal

tubule epitheli um and lumina of exposed rats was increased at t

he 9- and 15-month i nteri m evaluat ions and at

2 years. The p igment was c haract er ized in the 13-w eek

stu dy and at the 15-month interim eval uati on as PASnegati ve; resi stant

to diges tion by

diast ase; i sotropi c; and negative for melani n, hemosi derin, hematoidi n, bil e, lipofusc in, or ce roid staining m ethod s. The pigment was presumed to be 1-a min o-2, 4-di bromo anthraquinone or one

of its metabolites.46 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 10

Incidences of Neopl asms and No nne opla stic Lesi ons of the Kidney in Rats in the 2-Year Feed Study

of 1-Amino-2, 4-dib romoanthraqui none

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

9-Month Interi m Evaluation

Number Examin ed 10 10 10 10 Renal Tubule Hyal ine Droplet
Accumulation a 0 10** (2.0) b 10** (2.0) 10** (1.9)
Renal Tubule Pigmentation 0 10** (1.1) 10** (1.4) 10** (1.9)

15-Month Interi m Evaluation

Number Examin ed 10



### _ c



### _ 10 Neph r opathy 10 (2.0) 10 (2.5)
Renal Tubule Hype

rplas ia 0 2 (1.0)
Renal Tubule Pigmentation 0 10** (2.0)
Transitional Cell Hype

rplas ia 0 4* (1.3)

Renal Tubule Adenoma 0 2

2-Year Study

Number Examin ed 50 40 59 50 Nephr opathy 50 (2.9) 40 (3.6) 59 (2.9) 49 (2.7)
Renal Tubule Hype

rplas ia 9 (2.2) 30** (2.9) 25** (2.4) 19** (2.9)
Renal Tubule Pigmentation 5 (2.0) 40** (1.9) 58** (2.0) 49** (1.9)
Transitional Cell Hype

rplas ia 30 (1.4) 40** (2.1) 51** (1.9) 35* (1.6)

Transitional Cell Papilloma 0 0 1 0 Renal Tubule Adenoma (Multi ple) 0 4* 4 5*

Renal Tubule Adenoma (Single or Multi ple)
Overall rate d 2/50 (4%) 10/40 (25%) 11/59 (19%) 14/50 (28%)
Terminal rate e 2/26 (8%) 6/24 (25%) 4/21 (19%) 5/10 (50%) Adjust ed rate f 7.7% 33.6% 34.6% 68.3% First incidence (days) 729 (T) 618 636 588 Log istic regression test g P<0.001 P=0.007 P=0.014 P<0.001

Renal Tubule Carcinoma 0 0 2 1

Renal Tubule Adenoma or Carcinoma h

Overall rate 2/50 (4%) 10/40 (25%) 13/59 (22%) 15/50 (30%)
Terminal rate 2/26 (8%) 6/24 (25%) 4/21 (19%) 5/10 (50%) Adjust ed rate 7.7% 33.6% 39.4% 69.1% First incidence (days) 729 (T) 618 636 497 Log istic regression test P<0.001 P=0.007 P=0.005 P<0.001

(cont inu ed)Results 47

T ABLE 10

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Kidney i n Rats i n the 2- Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm

Fema le

15-M onth Interim Evaluation

Number E xamin ed 10

- - 10

Nephr opathy 10 (1.7) 10 (2.5)
Renal Tubule

Hype rplas ia 0 3 (1.0) Renal Tubule Pigmentation 0 10** (2.0)
Trans itional

Cell Hype rplas ia 3 (1.0) 4 (1.8)

2-Year Study

Number E xamin ed 50 40 60 48

Nephr opathy 50 (1.9) 39 (2.6) 60 (2.7) 46 (2.7)
Renal Tubule

Hype rplas ia 1 (3.0) 12** (2.5) 23** (2.7) 27** (2.7) Renal Tubule Pigmentation 0 40** (2.0) 60** (2.0) 48** (2.0)
Trans itional

Cell Hype rplas ia 10 (1.2) 16* (1.8) 44** (1.5) 21** (1.6)

Transitional Ce

ll Papill oma 0 0 0 1 Transitional Ce

ll Carcinoma 0 1 0 0 Renal Tu bule Aden oma (Mu ltiple) 0 0 5* 5*

Renal Tu bule Aden oma (Sin gle or Mult iple)
Over all r ate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%)
Ter minal r ate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%) Adjust ed rate 0.0% 8.0% 36.0% 69.7% First incidence (

days)

- i 600 601 625 Logistic r egress ion test P<0.001 P=0.049 P<0.001 P<0.001

Renal Tubule Carcinoma 0 0 0 2

Renal Tubule Adenoma or Carcinoma

j

Over all r ate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%)
Ter minal r ate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%) Adjust ed rate 0.0% 8.0% 36.0% 69.7% First incidence (

days)

- 600 601 625 Logistic r egress ion test P<0.001 P=0.049 P<0.001 P<0.001

* Significantly different (P # 0.05) fr om the contro l gro

up by the Fisch er e xact te st (9-m

onth and 15-

month int erim e valuat ions) o r the l ogist ic regression test (2-year study)

** P # 0.01 (T)Te rminal sacrifice

a Number of animals with lesion b Average severit y grade of lesion s in aff ected ra ts: 1 = mi nimal; 2 = mild ; 3 = mod erate; 4 = marked

c Kidne y not micro scopica

lly exami ned in thi s gro

up d Number of animals with neoplasm per number of animals with k idney examined microscopic ally

e Obser ved inc idence

in animal s surv

iving un til the end of the stud y f Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. g In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l. h Histori cal inc idenc e for 2-year NTP feed studi es with u ntreat ed cont rol groups (mean ± s tand ard devi ation ): 15/1 ,350 (1.1% ± 1.7% ); range,
0%-6% i Not appl

icable ; no neo

plasms in animal

group j Histor ical inc idence : 1/1,348 (0.1% ± 0. 4%); ran ge, 0% -2%48 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### Urinary Bladde r: Hype rplasia of the tr ansitional cel l epithelium of the urinary bladder was observed in
10 ,000 ppm fe males at 9 months and in 10,000 ppm males an d females at 15 months (Tables 11, A5, an d B5). Transitional cell hype rp lasia was present in mos t 5,000 and 10,000 ppm females at 2 years, and the
incidences of this lesion were si g n ificantly increase d
in 5,00 0 and 10,000 ppm males. Hype rp lasia consist

e d
of a diffuse or focal increa se in thickness of the
transiti on al epi the liu m; minimal cellula r pleomorphism and increased numb ers of mitot ic cell s

we re sometimes present

. Other nonneop lastic lesion s that occurred o nly in exposed ra ts include d squamou s metap lasia of the transitional epithelium and fatty metap lasia (fat proliferation) of the str oma of the
bladder wall. Tw o trans itional cell carcinomas and one papillom a occurred in 10,000 ppm f emales at the 15-m onth
in terim evaluation. At 2 years, incidences o f transitional cell papilloma, carcinoma, and papi llo ma or

car cin oma (comb i ned) were signif icantly increased i n 10,000 ppm males an d 5,000 and 10, 000 ppm fem ale s (Tables 11, A3, a nd B3) and exceed ed the NTP histori

cal ranges (males: 0%- 2%; femal es: 0%-2 %) for fee d study control s (Tables 11, A4d, and B4d). Transitional c ell papi llo ma consisted of a pedunc ulate d or broad-based mass of tr a nsitional epithelium with a central fibrovascular stroma; there was squamous metap lasia of the surface epi thelium in some papillomas. Tr ansition

al cell car cin oma was chara cteri ze d by an exophy

tic or endoph

ytic growt

h pattern and
invasion of the lamina propria or muscu laris of the
bladder wall (Plates 7 and 8). There was cellular atypia an d squ amous or m

ucous metaplasia o f transi

tional epithelium in some carcinomas.Results 49

T ABLE 11

Incidences of Neopl asms and No nne opla stic Lesi ons of the Urinary Bladder in Rats

in the 2-Year Feed Study of 1-Amino-2, 4-dib romoanthraqui none

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

15-Month Interi m Evaluation

Number Examin ed 10

_ b

_ 10
Transitional Cell Hyp e

rpla s i a a 0 3 (1.3 ) c Metaplasia, Squamou s 0 1 (2.0)

2-Year Study

Number Examin ed 50 38 58 50
Transitional Cell Hyp e

rpla s ia 1 (2.0) 5 (2.0) 17** (1.9) 30** (2.1) Metaplasia, Squamou s 0 0 0 3 (1.7)

Transitional Cell Papilloma
Overall rate d 0/50 (0%) 1/38 (3%) 2/58 (3%) 8/50 (16%)
Terminal rate e 0/26 (0%) 0/22 (0%) 2/21 (10%) 2/10 (20%) Adjust ed rate f 0.0% 3.7% 9.5% 40.3% First incidence (days)

_ h 700 729 (T) 493 Log istic regression test g P<0.001 P=0.459 P=0.192 P=0.004

Transitional Cell Carcinoma
Overall rate 0/50 (0%) 0/38 (0%) 1/58 (2%) 4/50 (8%)
Terminal rate 0/26 (0%) 0/22 (0%) 0/21 (0%) 1/10 (10%) Adjust ed rate 0.0% 0.0% 4.3% 24.5% First incidence (days)

_ _ 720 674 Log istic regression test P=0.001

_ P=0.491 P=0.022

Transitional Cell Papilloma or Carcinoma i

Overall rate 0/50 (0%) 1/38 (3%) 3/58 (5%) 12/50 (24%)
Terminal rate 0/26 (0%) 0/22 (0%) 2/21 (10%) 3/10 (30%) Adjust ed rate 0.0% 3.7% 13.5% 56.2% First incidence (days)

) 700 720 493 Log istic regression test P<0.001 P=0.459 P=0.096 P<0.001

Fema le

9-Month Interi m Evaluation

Number Examin ed 10 10 10 10
Transitional Cell Hype

rplas ia 0 0 0 2 (1.5)

15-Month Interi m Evaluation

Number Examin ed 10

_ _ 10
Transitional Cell Hyp e

rpla s ia 0 9** (2.6)

Transitional Cell Papilloma 0 1 Transitional Cell Carcinoma 0 2 Squamou s Cell Carcinoma 0 2

(cont inu ed)50 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 11

Incidences of Neopl asms and No nne opla stic Lesi ons of the Urinary Bladder in Rats

in the 2-Year Feed Study of 1-Amino-2, 4-dib romoanthraqui none (contin ued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Fema le (contin

ued)

2-Year Study

Number Examin ed 50 40 60 46
Transitional Cell Hyp e

rpla s ia 1 (1.0) 2 (3.0) 41** (2.0) 41** (2.3) Metaplasia, Squamou s 0 1 (1.0) 4 (2.3) 8** (2.9)

Fat Proliferation 0 0 4 (2.3) 2 (2.5)

Transitional Cell Papilloma
Overall rate 0/50 (0%) 2/40 (5%) 7/60 (12%) 9/46 (20%)
Terminal rate 0/38 (0%) 2/32 (6%) 6/38 (16%) 1/12 (8%) Adjust ed rate 0.0% 6.3% 17.6% 39.5% First incidence (days)

_ 729 (T) 691 637 Log istic regression test P<0.001 P=0.201 P=0.012 P=0.003

Transitional Cell Carcinoma
Overall rate 0/50 (0%) 0/40 (0%) 8/60 (13%) 16/46 (35%)
Terminal rate 0/38 (0%) 0/32 (0%) 6/38 (16%) 4/12 (33%) Adjust ed rate 0.0% 0.0% 19.5% 55.8% First incidence (days)

_ _ 670 367 Log istic regression test P<0.001

_ P=0.008 P<0.001

Transitional Cell Papilloma or Carcinoma j

Overall rate 0/50 (0%) 2/40 (5%) 17/60 (28%) 26/46 (57%)
Terminal rate 0/38 (0%) 2/32 (6%) 14/38 (37%) 6/12 (50%) Adjust ed rate 0.0% 6.3% 40.9% 78.1% First incidence (days)

_ 729 (T) 670 367 Log istic regression test P<0.001 P=0.201 P<0.001 P<0.001

Squamou s Cell Papilloma (Single or Multi ple) 0 0 1 2 Squamou s Cell Carcinoma 0 0 1 0

** Significantly diffe

rent (P # 0.01) from the control group by the Fisher exact test (9-mo nth and 15-mont h interi m evaluations) or the logi stic regression

test (2-year stu dy) (T)Te rminal sacrifice

a Number of animals with lesion b Urinary bladd er not microscopically examin ed in this group

c Average seve

rity grade of lesi ons in affected rats: 1 = minimal; 2 = mild; 3 = moderat e; 4 = marked d Number of animals with neoplasm per numb er of animals necrops ied or examin ed microscopically

e Observed incidence in animals surviv ing until the end of the study f Kaplan- Meier estima ted neoplasm incidence at the end of the study after adjustme nt for interc

urren t mort alit y. g In the control column are the P values associ ated with the trend test. In the exposed group colum ns are the P values corre sponding to pairwise

compar isons between the controls and that exposure group. The logi stic regression test rega rds these lesi ons as nonfatal. h Not appli cable; no neoplasms in animal group i Historical incidence for 2-year NTP feed studi es with untrea ted control groups (mean ± standard deviation): 3/1,329 (0.2% ± 0.6%); range, 0%-2% j Historical inci dence: 3/1,334 (0.2% ± 0.6%); range, 0%-2%Results 51



### Forestomach : Several prolifer ative and degen erativ e lesions occurred with increas ed incidences in the fore

stomach of exposed males and females (Table 12).
These mucosal lesi ons freque ntly occurred togethe r and consisted of thickening (hyperplasia) o f the

sq uamous epithelium and an increas e in the surface ker a tin layers (hyperkeratosis) (Tables 12, A5, and B5). Focal areas of hyper plasia

were some

ti mes adjacent to ulceration an d inf lamma

t ion of the squamous mucosa. There was no significant increase in incidenc es of neop las ms of the
forestomach (Tables 12, A1, and B1).

T ABLE 12

Incidences of Neopl asms and No nne opla stic Lesi ons of the Forestomach in Rats

in the 2-Year Feed Study of 1-Amino-2, 4-dib romoanthraqui none

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Male

Number Examin ed 49 39 59 49 Hyp e rker a tosi s a 5 (2.4 ) b 18** (1.8) 21** (2.0) 20** (2.0) Hyperpl asia, Squamou s 3 (1.7) 19** (3.0) 25** (3.2) 26** (3.0)
Inf lammat ion, Chronic Active 3 (2.0) 12** (2.4) 11 (2.0) 11* (2.2)
Ulcer 3 (2.7) 10** (3.2) 15** (2.7) 16** (2.6)

Squamou s Cell Papilloma c 0 2 0 1 Squamou s Cell Carcinoma 0 0 0 1 Squamou s Cell Papilloma or Carcinoma d 0 2 0 2

F em ale

Number Examin ed 49 40 60 47 Hype rkera tosis 2 (2.0) 7* (1.4) 23** (2.1) 28** (1.9) Hyperpl asia, Squamou s 2 (2.0) 7* (1.9) 26** (2.9) 33** (3.0)
Inf lammat ion, Chronic Active 0 1 (2.0) 13** (2.2) 10** (2.2)
Ulcer 1 (2.0) 2 (2.5) 7 (1.7) 17** (2.9)

Squamou s Cell Papilloma 0 0 0 1 Squamou s Cell Carcinoma 0 1 1 1 Squamou s Cell Papilloma or Carcinoma e 0 1 1 2

* Significantly diffe

rent (P # 0.05) from the control group by the logi stic regression test

** P # 0.01

a Number of animals with lesion b Average seve

rity grade of lesi ons in affected animals: 1 = minimal; 2 = mild; 3 = moderat e; 4 = marked

c Number of animals with neoplasm per numb er of animals necrops ied d Historical incidence for 2-year NTP feed studi es with untrea ted control groups (mean ± standard deviation): 4/1,353 (0.3% ± 0.7%); range, 0%-2%

e Historical inci dence: 2/1,351 (0.2% ± 0.5%); range, 0%-2%



### Miscell aneo us Neoplasms and Nonneo plastic Lesions : In exposed mal es a nd females, incidences of
mononuclear

cell leukem

ia occurred with sign ifican t negati ve trends (Tables 13, A3, and B3). The incidences of pituitar

y gland aden oma in males and
females (males: 0 ppm, 21/48; 2,000 ppm, 14/40; 5,000 ppm, 10 /56; 10,000 ppm, 10/49 ; females: 32/50

, 19/ 39, 32/60, 13/47; Tables A3 and B3) and th e incidence of mam

mary g land fib road enoma (21/50

, 10/40, 9/60, 5/49; Table B3) in fema les also occurre d
with significant negati ve tre nd s. These decreases ma y have been related to lower body weights.52 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



## T ABLE 

## 13
Inc id en ce s o f M on on uc le ar Ce ll Leu ke mi a i n R at s i n t he 2- Ye ar Fe ed St ud y of 1-Amino-2,4-dibromoanthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm

Male

15-M onth Interim Evaluation

Mononuc l

ear C e ll L euke m i a a 0/10

_ b

_ 2/10

2-Year Study

Mononucl

ear Ce ll L eukem ia
Over all r ate a 25/50 (50%) 5/40 (13%) 3/59 (5%) 1/50 (2%)
Ter minal r ate c 9/26 (35%) 4/24 (17%) 2/21 (10%) 0/10 (0%) Adjust ed rate d 59.0% 18.8% 11.7% 2.9% First incidence (

days) 514 604 650 590 Life tabl e test e P<0.001N P<0.001N P<0.001N P<0.001N

Logistic r egress ion test P<0.001N P<0.001N P<0.001N P<0.001N

Fema le

2-Year Study

Mononucl

ear Ce ll L eukem ia
Over all r ate 9/50 (18%) 1/40 (3%) 5/60 (8%) 1/49 (2%)
Ter minal r ate 6/38 (16%) 0/32 (0%) 1/38 (3%) 0/12 (0%) Adjust ed rate 21.5% 3.0% 10.0% 3.7% First incidence (

days) 620 689 601 662 Life tabl e test P=0.112N P=0.026N P=0.177N P=0.162N

Logistic r egress ion test P=0.011N P=0.023N P=0.111N P=0.024N

a Number

of anim als w ith neo plasm p er numb er of

animals necro psied b Animal s not mi crosc opical ly e xamine d in this g

roup

c Obser ved inc idence

in animal s surv

iving un til the end of the stud y d Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y.

e In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairw ise co mpariso ns betw een the contro ls and th

at expo sure g

roup. T he lif e tabl e anal

ysis rega rds neo plasms in animal s dyin

g prio r to terminal kill as being (direct ly or indirectly) the cause of death. The logistic regression test regards these lesions as non fatal. A negative trend or a lower incidence in an exposu re group is indicat ed by N.



### In males, th ere w a s a chemical- related i ncreased
incidence of atr ophy of the seminal ve sicles (1/49, 30/40, 35/59, 2 3/50; T able A5). This was not
evident at th e 9- or 15 -month inter im evaluati on, but was presen

t in most males at the end of the 2- year study. Atrophy of the seminal ve sicles of expos ed males

was characteri zed by a r eduction i n the si ze of the
secretory epi th eli

um f ro m a tall columnar shape to

a low cuboidal

shape and by

an incr ease in the

amou nt of connec tive tissue strom a in the gland

.Resu lts 53



## S TOP 

## -E XPOSURE 

## E VALUATI ON 

### Stop-exposu

re groups of male and

femal e rats were included in the NTP 2-year

study to evaluate the potential for p rogr essio n or r egres sion o f chemica lrelate d li ver, large inte stine, kid ney, urina ry bladder, an d
fo restomach lesions during a recovery per iod. Ten mal e and 10 female rats were exposed to 20 ,000 ppm
1-amino -2 ,4-dibromoanthraquinone in feed for 9 mon th s followed by adm

in is tra ti on of un dosed fee d until the en d
of the 15-month pe

ri od (9-month st op- exposure groups)

. In add ition, 3 0 males and 3 0 fema le s were exposed t o 20,000 ppm 1-ami n

o-2,4-dibromo anthraquinone in fee d
for 15 mon

ths (15-month exposure groups). Te n male s and 10 females from the 15-month exposure groups

were evaluated at the 9-month i n ter im eva lu ati on (9-month inter im evaluation groups)

.



## Survival

Estimates o f 2-y ear surv ival probabili ties for male an d
female rats are shown i

n the Kaplan- Meier survival

curves in Figure 4. Al l mal es survived 9 months; thre e males from the 9-month stop-exposu re group an d thre e males fro m the 15 -m

onth exposure group died before th e end of the 15-month eva

lu ati on (Ta ble 14). All femal e s in the 9 -month stop-ex

posure group su

rvived until

the

en d of the 15-month eva luation. One fem ale from the
15-month exposure group died during the first 9 months ;

an add itiona l seven females died between month 9 an d
the end of th e 15-month evalu ation (Tab le 15). 

## Body Weigh ts and Feed and Compound Consumption

The mean body wei

ghts of male and femal e rat s in th e 9-month sto

p- ex pos ure and 15-month exposure group s

are compared w

ith th e controls from the 2-yea r core
stu dy in Tables 14 and 15, and the growth curves for exposed ra ts in the 15-mont h exposur e groups a

re sho wn in Fi gur e 5. The mean body wei ghts of males
and females in the 9-m onth stop-exposur e groups wer e 20% to 22% lower than th ose of the controls at the
9-month interim ev

al ua ti on of the 2-year cor e stu

dy an d

were 20% to 21% lower than t hose of the contro ls at th e 15-month inter im evaluat

ion of the 2-ye ar core stu

dy

. The me an b ody

weights o f males an d fema les in the
15-month exp osur e gr

oups we re 19% to 21% lower
than those of the controls at th e 9-mo nth inte rim

eva lu ati on of the 2-year

core study

and we re 25% to
33% lower than those of the cont rol s at the 15-month
interim evaluation of the 2-y ear core st udy. F eed

cons ump ti on by 9-month sto

p- ex pos ure and 15-mont h

exposure males and femal es was gene rally lower tha n that by the con tro ls throughout the st udy (Tables J1 an d J2). The die tar

y l eve l of 20

, 00 0 ppm d elivered daily
doses of ap

prox im ate ly

1,300 m

g 1- amino2,4- dib rom oanthraquinone/kg body weight to males a n d 1,800 mg/ kg to femal es in the 9-month stop-exposur e
groups and daily dose s of appr oxim ate ly 1,100 mg/k g
to ma les and 1,4 00 mg/kg to fema les in the 15-mont h

exposure groups.

### 54 l-Amino-2,4-dibromoanthraquinone, NTP TR 383

FIGURE 

## 4
Kaplan-Meier Survival Curves for Rats Administ ered l-Amino-2,4-dibromoanthraqninone in Feed
in the 15-Month Stop-E xposure Evaluation

### Results 55

FIGURE 

## 5

Growth Curves for Rats Administered l- Amino-2,4-dibromoanthraquinone in Feed

in the 15-Month Stop-E xposure Evaluation56 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383

T ABLE 14

M ea n Bo dy W eig hts and Survi va l of M ale R ats in the Stop -Ex po sure E va luatio n of 1 -Am ino-2 ,4-di bro mo anthra quino ne

Weeks 0 ppm 20,000 ppm (9-Month Stop-Exposure) 20,000 ppm (15-Month Exposure)

on Av. Wt. Num

ber of Av. Wt. Wt. (% of Num ber of Av. Wt. Wt. (% of Num ber of Study (g) Survivors (g) con trol s) Survivors (g) con trol s) Survivors

1 139 70 130 94 10 136 98 30
2 161 70 144 90 10 149 93 30
3 206 70 160 78 10 167 81 30
4 235 70 169 72 10 173 74 30
5 240 70 169 71 10 177 74 30
6 269 70 193 72 10 201 75 30
7 287 70 204 71 10 211 74 30
8 302 70 211 70 10 217 72 30
9 312 70 225 72 10 226 73 30
10 325 70 239 73 10 247 76 30
11 333 70 255 76 10 264 79 30
12 338 70 267 79 10 277 82 30
13 332 70 267 81 10 271 82 30
14 356 70 284 80 10 292 82 30
17 387 70 305 79 10 318 82 30
21 406 70 327 81 10 334 82 30
25 423 70 341 81 10 351 83 30
29 435 70 338 78 10 355 82 30
33 445 70 351 79 10 364 82 30
37 453 70 352 78 10 364 80 30
41 468 60 a 374 80 10 b 377 81 20 a

45 473 60 384 81 10 381 81 20
49 479 60 395 83 10 383 80 20
53 489 60 398 81 10 389 80 20
57 486 60 393 81 10 377 78 20
61 484 59 379 78 9 370 77 19
65 484 57 388 80 7 364 75 17

Mean fo r we eks
1-13 268 203 76 209 78
14-37 415 328 79 340 82
41-65 480 387 81 377 79

a Inte rim eva luati on o cc

urre d du ring we ek 39. b Animals switched t o undos ed feedResu lts 57

T ABLE 15

M ea n Bo dy W eig hts and Survi va l of Fe ma le Ra ts in the Sto p-E xpo sure E va luatio n of 1 -Am ino-2 ,4-di bro mo anthra quino ne

Weeks 0 ppm 20,000 ppm (9-Month Stop-Exposure) 20,000 ppm (15-Month Exposure)

on Av. Wt. Num

ber of Av. Wt. Wt. (% of Num ber of Av. Wt. Wt. (% of Num ber of Study (g) Survivors (g) con trol s) Survivors (g) con trol s) Survivors

1 93 70 90 96 10 94 101 30
2 114 70 93 82 10 97 85 30
3 133 70 103 78 10 104 78 30
4 147 70 113 77 10 114 78 30
5 157 70 117 75 10 121 77 30
6 166 70 119 72 10 129 78 30
7 173 70 129 75 10 141 81 30
8 180 70 141 78 10 150 84 30
9 186 70 146 79 10 157 84 30
10 191 70 159 83 10 163 85 30
11 196 70 163 83 10 166 85 29
12 203 70 168 83 10 171 84 29
13 208 70 172 83 10 175 84 29
14 212 70 177 84 10 179 85 29
17 222 70 188 85 10 190 85 29
21 228 70 194 85 10 196 86 29
25 237 70 198 84 10 200 85 29
29 246 70 200 82 10 203 83 29
33 251 70 201 80 10 203 81 29
37 258 70 207 80 10 211 82 29
41 265 59 a 207 78

10 10 b 210 79 19 a

45 272 59 213 78 16
49 284 59 231 81 10 213 75 15
53 299 59 248 83 10 219 73 15
57 311 59 252 81 10 220 71 15
61 315 59 217 69 15
65 328 59 258 79 10 220 67 12

Mean fo r we eks
1-13 165 132 80 137 83
14-37 236 195 83 197 84
41-65 296 239 81 216 73

a Inte rim eva luati on o cc

urre d du ring we ek 40. b Animals switched t o undos ed feed58 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



## Pathology and

Statistica l Analysis

of Results

Summaries of the incid ences of n eoplasms and nonneo

p la stic lesions are sh own in Tables E1 and E3 for mal e
rats and Tables F1 and F3 for female r ats. For

sta tistic al analyses

, the inci dences in the 9-month stop

exposure groups and t he 15 -mo nth exposure groups a t the end of 15 months are c o mpared wi th the 15-mont h
inter im eva

lu ati on con tro ls of the 2-yea

r core stu dy fo r male rats (Ta ble E 2a) and fema le rats (Ta ble F2a). T h e incidences in th e 15-month exposure groups are

co mpared wi

th the 9-month stop-exp

osure groups afte

r 6 months of r ecovery

for ma le rats (Table E2b) and
fem ale rats (Table F2b)

. 

## Progression or Regress ion of Chemical-Induced Lesions

Liver: Rats in

the stop- exposure study e xposed to 20,000 ppm 1-a mino-2,4-dibromoanthraquinone in t h e feed for 9 or 15 months had chemica l -r e l a ted effects simi lar to those observed i n rats e xposed to
concentrations up to 10, 000 ppm in th e 2-year core
stu dy. The absolute and rela ti ve l iv er w

ei ght s of exposed males and females were s ignifi cantly g rea te r than those of the

contro ls at both the 9-month interim
and 15- month evaluations of the 15-month exposur e
gro ups (Table s H2 and H3). Wi th respe ct to both neoplasms and non neo plast ic lesions in the l iver

, ther e

was no e vi denc e of r

egr ess ion in the in ci denc e or

sev erity of chemical

-related pigmentati

on, focal hepat

o

cellul ar alteration, or hepatoce ll ul ar a denom a or carcinoma when admin istrat ion o f 1-amino

-- 2,4-dibromoanthraquinone was discontinu ed after
9 months. The inci dences and severi ty of live r lesions
after 9 months of exposu re we re simil ar with and
witho ut a 6-month recovery per iod. The inci dences of
liver lesions in the 15-month e xposure g roups w ere greater than in the 9-month stop-exposur e groups but
the severities were comparabl e (Tables 16, E2a, and F2a).Resu lts 59

T ABLE 16

Incidence s of Ne oplas ms a nd No nneoplastic Les ions of the L iver in Ra ts
in the 9-M onth, 9-M onth w ith 6-M onth R eco ve ry, a nd 15 -M onth E xpo sure E va luatio n of 1 -Am ino-2 ,4-di bro mo anthra quino ne



### 9-M onth E val uation a 15- M onth E val uation b

0 ppm 20,0 00 ppm 0 ppm 20,0 00 ppm 20,0 00 ppm

(9-M onth (9-M onth (15-M onth Expo sure) Exposure Plu s Expo sure)

6-Month Recovery)

Male

Number E xamined 10 10 10 10 20

Baso phil ic Fo cus c 0 6** (1.5) d 1 (1.0) 4 (1.0) 13** (1.5)

Clea r Cel l Foc us 0 4* (1.0) 0 6** (1.6) 13** (1.2)

Eosi nophi li c Foc us 0 0 1 (1.0) 0 2 (1.5)

Bile D uct Hyperplas ia 1 (1.0) 7** (1.3) 10 (1.1) 7 (1.0) 19 (1.3)

Ch ron ic an d C hr on ic A ctive Periportal I

nflammation

9 (1.0) 10 (1.0) 10 (1.1) 7 (1.7) 18 (1.9) Pigmentation 0 10**( 1.0) 0 8** (1.0) 18** (1.0)

Hepatoc ellular

Adenoma 0 2 0 7** 8* Hepatoc ellular

Carcinoma 0 2 0 7** 19** Hepatocellular Adenoma or

Carcinoma 0 2 0 9** 20**

Female

Number E xamined 10 10 10 10 18

Baso phil ic Fo cus 1 (1.0) 3 (1.3) 8 (1.0) 6 (1.2) 13 (1.5)

Clea r Cel l Foc us 0 1 (1.0) 0 5* (1.0) 13** (1.2)

Eosi nophi li c Foc us 0 0 2 (1.5) 2 (1.0) 3 (1.7)

Bile D uct Hyperplas ia 1 (1.0) 9** (1.1) 7 (1.1) 10 (1.5) 18* (1.6)

Ch ron ic an d C hr on ic A ctive Periportal I

nflammation 6 (1.0) 10* (1.7) 10 (1.0) 9 (1.6) 18 (1.7) Pigmentation 0 10**( 1.2) 1 (1.0) 10** (1.0) 17** (1.2)

Hepatoc ellular

Adenoma 0 2 0 6** 10** Hepatoc ellular

Carcinoma 0 1 0 6** 15** Hepatocellular Adenoma or

Carcinoma 0 2 0 8** 16**

* Significantly diff

erent (P # 0.05) from the control group by the F isher exact test

** P # 0.01

a Contr ols fr om t

he 9- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on. b Contr ols fr om t

he 15- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on.

c Number of animals with lesion d Average severity

grade of lesions in aff

ected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked



### La rge Intestine: The stop-exposure regimen had no
effect on the de velopment of adenomatous polyps (adenomas) of the large inte stine. An adenomatous

po lyp was observed in t he col on of one exposed male a

t 9 months. After 15 months, the in cidenc e o f
adenomatous polyps of the rectum was significan tly
increase d i n the 9-month stop-exposur e f emales (Tab les 17 and F2 a). At th e 15 -m ont h
evaluati on, aden omatous po ly ps were observed in th e
rectums of th

ree ma le s i n th e 9- month sto p-e xposure
group, seven males in the 1 5-month e xposure group, five f emales in the 9-month stop-ex posure group, and
three females i

n the 15-month

expos ure g

roup. No

car ci nom as of

the col on or rec tum

we re ob ser

ved.60 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



### Kidney: At the 9-month and 15-month e valuati ons, th e relative kidney w

eights of males and females in the
15-month exposure groups were s ignifi cantly g reater tha n those of the controls (Tables H2 and H3). In the exposed males and fema les at 9 months, kidney change s included pi gme nta ti on and minim al enlarge ment of

some renal tub ule cell nuclei ( karyom egaly ). In exposed males , the re was also hya line droplet

acc umu la ti on and a sl ig ht i

ncr eas e i n th e se ver

it y of nephr opathy

. At 15 months, renal tubul e epitheli al pigm entati on, kar yom ega ly, and increased

sever ity of nep hropathy and transitional cell hyperplas i a of the renal pelvis were observed in exposed groups o f
males and females. Th e severiti es of these lesions w er e si mila r or sli ghtly

less severe i

n the st op-exposu

re
gro ups tha

n i n th ose e xpos ed con ti nuou sl y f

or 15 months. At the 15-month evaluat ion, re nal tubule
adenomas wer

e observed in the 9-month stop-exp osur e and 15-month exp osure groups of mal es and females (Tables 17, E1, and F1). In the 9- month



## T ABLE 

## 17 Incidences of Neoplasms and Nonneop

lastic Le sions of the Large Intestine a nd Kid ney in Rats in the 9-Month, 9-M onth with 6-M onth Recovery, and 15-Month Exp osure Evaluation
of 1-Amino -2,4-dibrom oanthraquinone

9-Mont h Eval uat ion a 15-Mont h Eval uat ion b

0 ppm 20,000 ppm 0 ppm 20,000 ppm 20,000 ppm

(9-M onth (9-M onth (15 -M onth

Exposur e) Expo sure Plus Exposur e)

6-Mont h Rec overy)

Male

Large I ntestine, Colon c 10 10 10 10 20

Aden omatous Polyp (Aden oma) d 0 1 0 0 0

Lar ge I ntes ti ne, Re ctum 10 10 9 10 20

Aden omatous Polyp (Aden oma) 0 0 0 3 7*

Kidney 10 10 10 10 20

Nephr opa

thy 10 (1.0) e 10 (1.8) 10 (2.0) 10 (2.0) 20 (2.5)

Transition al Cell Hyperplas ia 0 0 0 1 (1.0) 11** (1.3) Hyaline Droplet Accumu lation 0 10** (2.1) 0 0 0 Pigmentation 0 10** (2.0) 0 9** (1.2) 20** (2.0)

Ren al Tubu le Hyperplasia 0 0 0 1 (1.0) 1 (1.0)

Renal Tubule Adenoma 0 0 0 3 2

Female

Lar ge I ntes ti ne, Re ctum 10 10 10 10 17

Aden omatous Polyp (Aden oma) 0 0 0 5* 3

Kidney 10 10 10 10 18

Nephr opa

thy 4 (1.0) 7 (1.0) 10 (1.7) 10 (2.2) 18 (2.1)

Transition al Cell Hyperplas ia 0 0 3 (1.0) 1 (1.0) 5 (1.6) Pigmentation 0 10** (2.0) 0 10** (1.9) 18** (2.0)

Ren al Tubu le Hyperplasia 0 0 0 2 (2.0) 2 (1.5)

Renal Tubule Adenoma 0 0 0 3 2

* Significantly diff

erent (P # 0.05) from the control group by the F isher exact test

** P # 0.01

a Contr ols fr om t

he 9- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on. b Contr ols fr om t

he 15- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on.

c Nu mber of anima ls with organ ex amined microscopically d Number of animals with lesion

e Average severity

grade of lesions in aff

ected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = markedResu lts 61



### stop-ex posure and 15 -month exp osure groups, renal
tubule hyperpl asia and

adenomas were obs erved in
mal es and females. A

t 15 months, renal tubu l e adenomas were observed in three mal es and three
fem ale s in the 9-month stop-exp osure groups and in tw

o males and tw o fem al es i n th e 15 -m onth exposu

re
grou ps. No re nal

tub ul e ca rci nom as w ere foun d.

Urinary Bladder: A transitional cell papi lloma w as
presen t in the urinary bladder o f one expo sed ma le a t 9 months ( Tables 18 and E1). When ex posure was discontinued at 9 months, no chemical -related

nonn eopla stic lesions or neoplasms were present a t 15 months. With co ntinuous treatmen t, transitional cel l hyperplas ia and neopl as ms of the urinary bladder developed by 15 months. In females, a min imal to mil d
transiti onal cell hyper plasia was obs erved at 9 month s and did not completely reg ress wi th t he c ess ati on of

exposure; a squamous cell papill oma wa s obs erv ed i n

one fe m a l e at 15 m onths (Ta b l e s 18 a nd F1). In contrast, continuous expo sure for 15 months
resulted in an in creas ed i nci denc e an d sev eri ty of
transitional

cell hype rp las ia, as w

ell as sq uamous metapla sia of the tra

nsiti onal ep ithel ium and a fatty metapla sia (fat proliferation) of the stroma o f the
bladd er wall.

Fore stomac h: C hem ical- related lesions of the forestomach were not present in mal es or females at the
9-month inter

im evaluation. H yperpl

asia, hyp erkera

tosis, inflam

mation, and ul cer ati on were pres ent in

appro ximately 20% of exposed males, but not in th e
cont rols at 15 months (T able 18). A squamo us cell papilloma was present in one male from th e 15-month

exposu re group (Ta ble E1). In female rats, forest oma c h
lesions were not present in the co ntr ol or 9-month stop

exposure g rou

ps. Hyperplasi

a, hyperkeratosis, and/o r ulcer a ti on we re observed in a few females i n the
15-month exposure group; no neoplasms w ere prese n t in the forestomach.62 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



### T ABLE 

### 18
Incidence s of Neoplasms and Nonneoplasti c Lesions of the Urin ary Bladd er and Foresto mach in Rats in the 9-Month, 9-M onth with 6-M onth Recovery, and 15 -Month Exp osure Evaluation
of 1-Am ino-2,4-dibrom oanthraquinone

9-Mont h Eval uation

a 15-Mont h Eval uation b

0 ppm 20,000 ppm 0 ppm 20,000 ppm 20,000 ppm

(9-Month (9-M onth (15-Month Exp osure) Exp osure Plus Exp osure)

6-M onth Recovery)

Male

Urinary Bladd er c 10 9 10 10 19

Fat Proliferation d 0 0 0 0 1 (3.0) e
Transitional Epithelial Hyperplasia 0 0 0 0 9** (1.9)

Squamous Cell Carcinoma 0 0 0 0 1

Transiti onal Epitheli al Papill oma 0 1 0 0 3

Transiti onal Epitheli al Carcinoma 0 0 0 0 1

Forestomach 10 10 10 10 20

Hyperkeratosis 0 0 0 2 (2.5) 1 (1.0) Hyperplasia 0 0 0 2 (2.0) 3 (1.3)

Infl ammation 0 0 0 1 (3.0) 1 (3.0)

Ulceration 0 0 0 2 (2.5) 0

Female

Urinary Bladd er 10 10 10 10 18

Fat Proliferation 0 0 0 0 2 (3.0) Transitional Epithelial Hyperplasia 0 4* (1.5) 0 4* (1.8) 17** (2.6) Transitional Epithelial Sq uamou s M etaplasia 0 0 0 0 3 (2.7)

Squamous Cell Papilloma 0 0 0 0 1 Squamous Cell Carcinoma 0 0 0 1 4

Transiti onal Epitheli al Papill oma 0 0 0 0 1

Transiti onal Epitheli al Carcinoma 0 0 0 0 1

Forestomach 10 10 10 10 18

Hyperkeratosis 0 0 0 0 1 (1.0) Hyperplasia 0 0 0 0 6* (1.0)

Ulceration 0 0 0 0 1 (3.0)

* Significantly diff

erent (P # 0.05) from the control group by the F isher exact test

** P # 0.01

a Contr ols fr om t

he 9- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on. b Contr ols fr om t

he 15- mont

h int eri m ev alua

ti on of the 2- ye ar c ore study

were used f

or s tat ist ica l co mpa ris on.

c Nu mber of anima ls with organ ex amined microscopically d Number of animals with lesion

e Average severity

grade of lesions in aff

ected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = markedResu lts 63



# MICE

13-W EE K 

## S TUDY


### One 25,000 p pm ma le (wee k 11) and one 5,00 0 ppm

m ale (wee k 13) died duri ng the s tudy (Tab le 19).
Neither death was chemical rel ate d. On e 10, 00 0 pp m female was accid ent ly ki ll

ed. Final mean b ody weight s of exp osed g

roup s of m al e an d fema le mi ce w ere simil ar to those of the controls. Mean bod y w eight gains of exposed groups of males and femal es we re
gener ally greater than th o se of the controls. Feed

con s um p t i on by ex p osed m i c e w as s i m i l ar t o that by the cont rol s. Die tar

y l eve ls of

2,500, 5,000, 10,000, 25 ,000, and 50,0 00 ppm delivered average dail y doses of appr oxim ate ly 500, 1,0 80, 1 ,850

, 6,200, an d 10,6 00 mg 1-a

mi no- 2,4- dib rom oa nthraquinone/kg bod

y wei ght to males and appr oxim ate ly 660, 1,150, 2, 600

, 5,9 00, and 1 1,700 mg/kg to females. Redd ened fur wa s

o bserved in 10,000, 25, 000, and 5 0,000 ppm mice as early as day

4 in males and day 5 in females and was
observed through out th e study. No other clini cal
observations wer e attr ibuted to 1-amino

2,4-dibromoanthraquinone.

T ABLE 19 Survival, Mean Body W

eights, and Feed Consu

mption of Mice in the 13-Week Feed S tudy

of 1- Amino-2 ,4- dibro moanthra quinone

Final W eight
Mean B ody Weig ht b (g) Relat ive Feed
Dose Survi

val a Initi

al Fin al Chan ge to Controls Consu mpti on c (pp m) (%) Week 1 Week 13

Male

0 10/10 23.7 ± 0.4 30.5 ± 0.6 6.9 ± 0.7 5.8 6.0

2,500 10/10 23.5 ± 0.3 30.6 ± 0.6 7.1 ± 0.6 100 5.3 5.6

5,000 9/1 0 d 23.5 ± 0.4 30.7 ± 0.7 7.3 ± 0.5 101 6.1 5.6

10,000 10/10 23.7 ± 0.5 32.1 ± 0.4 8.4 ± 0.4 105 5.5 4.8

25,000 9/10 e 23.4 ± 0.3 30.9 ± 0.6 7.5 ± 0.5 101 6.3 7.1

50,000 10/10 23.4 ± 0.3 31.5 ± 0.4 8.1 ± 0.4 103 6.1 5.5

Female

0 10/10 18.2 ± 0.3 24.0 ± 0.2 5.8 ± 0.2 4.8 6.2

2,500 10/10 18.1 ± 0.3 24.7 ± 0.6 6.6 ± 0.4 103 5.0 6.4

5,000 10/10 18.4 ± 0.3 25.0 ± 0.6 6.5 ± 0.4 104 4.3 5.7

10,000 9/1 0 f 18.3 ± 0.3 25.0 ± 0.4 6.5 ± 0.3 104 4.5 6.7

25,000 10/10 18.2 ± 0.2 23.6 ± 0.3 5.4 ± 0.3 98 3.8 6.1

50,000 10/10 18.4 ± 0.3 24.7 ± 0.4 6.3 ± 0.2 103 4.0 6.1

a Number

of anim als surv

iv ing a t 13 wee ks/

numbe r ini

ti all y i n gro up b W eigh ts an d w eigh t ch an ges are g iven as m ean ± st an da rd erro r. S ub seq ue nt c alcu latio ns are b ased on an imal s su rvivin g to t he en d o f the stu dy.

c Fe ed con su mpt ion is ex pres sed as g ram s of fee d c on su med per a nim al pe r d ay. d Week of

deat h: 13

e Week of

deat h: 11 f W eek of death : 2 (accid ental)64 1-Am ino-2, 4-dibrom oanth raqui

non e, NT P TR 383



### Absolu te and relativ

e live r w ei ght s of 5 ,000, 10,000, 25,000, and 50,00 0 ppm male and femal e mice w ere signi fi cantly great er tha n tho se of the co ntro ls (Tab le H4) . Absolut e and relative kid ney wei ght s of 25,000 and 50,0 00 ppm males were signi fican tly lo we r than those o f the contr ols. Obs ervations at necropsy
incl uded red staini

ng of the gastrointestinal tr

act and it s
contents in all

exposed male mice

ex cept th ose in the 2,500 ppm group and

red staining i

n the kidney

and
urine. These findings were observed less frequen tl y i n females than i

n males. Chemic al- relat ed lesions w ere present in the liver (Ta ble 20). There were increased inci dences of ce ntril

obular h yp ertrophy in the 10,000, 25,000 , and 50,000 ppm males wi

th a do se-rel ated increased sever

ity. M inim al go ld t o b ro wn pigm ent gran ules were present in the cyt oplasm o f hepatocytes of all expose d

groups of males. Pigmen

t was gene rally located in th e centrilobular

portion of the hepatic lobul e. Pigment
simil ar to that in mal e mice w as present in just a few hepatocytes in the li

ver of on e fe mal e i n th e 25, 000- ppm group and one 50,000 ppm femal e.

T ABLE 20 Incidences of Nonneoplastic Lesions of the Liver in Mice i n the 13-Week Feed S tudy

of 1- Amino-2 ,4- dibro moanthra quinone

0 ppm 2,5 00 ppm 5,0 00 ppm 10, 000 ppm 25, 000 ppm 50, 000 ppm

Male

Number E xamined 10 10 10 10 10 10

Centr il obul ar H yp ert

rop hy a 0 0 0 8** (1.8) b 8** (2.0) 10** (2.8) Pigmentation 0 5* (1.0) 8** (1.0) 8** (1.0) 8** (1.0) 10** (1.1)

Female

Number E xamined 10 10 10 10 10 10

Pigmentation 0 0 0 0 1 (1.0) 1 (1.0)

* Significantly diff

erent (P # 0.05) from the control group by the F isher exact test

** P # 0.01

a Number of animals with lesion b Average severity

grade of lesions in aff

ected mice: 1 = minimal; 2 = m ild; 3 = mod erate; 4 = marked



### Dose Selection Rationale: As n o 1-a mi no2,4-dibromo anthraquinone-rel ated adverse effects wer

e observed in feed con sump tio n , mean body wei ghts, o r surviv al, exposure concent rations chosen for the 2-yea r
stu dy were based mai

nly on the freq

uency and especially the sev erity of

centr il obular hypertroph

y of the
liver in mal e mice. Beca use only lesio ns of mild

sever it y were observed in the 10,000 and 25,0 00 ppm

groups, and lesions of m oderate severi ty were observe d in the 50,000 ppm group, and these were not life- jeopardizing lesions, exposure conc ent rat ion s selected for th e 2- ye ar s tudy of 1- ami

no2,4-dibromo anthr

aquinone in mice w ere 0, 10,000, an d 20,000 ppm. Other considerations incl ude consistenc y
among males and femal es (since femal es c ould have
been given higher exp osure concen trations) and corre

spondence to the exposure conc entrations selected fo r the start-stop, pr

ogr es si on/ reg res si on expe riment s (st op

exposure evaluation) i

n rats.Resu lts 65



## 2-Y EAR 

## S TUDY 

## Survival

E stimates of 2-year survival probabili ties for male a n d
fe male mice are sh own in Tab le 21 and in the KaplanMe ier survival curves in Figu re 6. Survival of expose d
male mice w

as signifi cantly l ower than that of the controls; survival of exposed femal e mice w as si mila r to that of the controls.



## Body Weights, Feed and Compound Consumption, and Clinical Findings

Mean body weigh

ts of exposed grou ps of

male mice

wer e lower than that of the con tro ls after week 9; mea n body wei ghts of ex

posed gro ups of femal es we re lower tha

n th at of the con tro ls after week 17 (Fig ure 7

, Tables 22 and 23). Fi

nal m ean body wei ghts of exposed grou

ps of ma le s w ere 15% to 17% lower than
that of th e controls; final mean body wei ghts of expose d
females we re 14% t o 19% lowerthan that of the controls. Feed consumpti

on by exposed male s and female s

was gene rally simil ar t o that by the con tro ls (Tables J 5
and J6). Dietary levels of 10, 000 and 20,000 ppm wer e estimated to de liver daily doses of appro ximately 1 ,70 0
and 3,500 mg 1-a min

o-2,4-di bromoanthra

quin one/k g body weig ht to males and 2,000 and 4,400 mg/kg t o females. Dis col orat

ion of th e fur, urine, and feces wa s evi dent in al l e xpos ed gr

oups as e arl

y a s day 8.

T ABLE 21

Survi val of Mic e in t he 2-Ye ar Fe ed Study of 1-Amino- 2,4 -dibr omoanthr aquinone

0 ppm 10, 000 ppm 20, 000 ppm

Male

Anima ls i

nit ial ly in st udy 60 60 60

15-M onth interim evaluation a 10 9 10

Accid ental d eath a 0 1 0

Morib und 7 23 21

Natur al de aths 3 5 6 Animals surviving to study termination 40 22 23

Perc ent p rob abi li ty

of surv iv al a t end o f s tudy

b 81 45 47

Mean survival (days) c 656 620 609

Survival analyses d P=0.001 P<0.001 P<0.001

Female

Anima ls i

nit ial ly in st udy 60 60 60

15-M onth interim evaluation a 10 10 10

Morib und 5 11 11

Natur al de aths 6 5 6 Animals surviving to study termination 39 e 34 e 33

Perc ent p rob abi li ty

of surv iv al a t end o f s tudy 78 69 66

Mean survival (days) 659 649 657

Survival analyses P=0.234 P=0.381 P=0.259

a Censored from survival analyses b Kaplan-Meier determinations based on the number of animals alive on first day of terminal sacrifi ce

c Mean of all deaths (uncensored, censored , and terminal sacrifice) d The resu lt of the life table trend test (Tarone , 197 5) is in the con trol column , and the resu lts of the life table pairw ise comparison s (Cox, 1 972 ) with the con trols

are in th e exposed column s.

e Inclu de s on e fema le th at d ied du rin g th e las t w eek o f the stu dy.

### 66 l-Amino-2,4-dibromoanthraquinone, NTP TR 383

FIGURE 

## 6
Kaplan-Meier Survival Curves fo r Mice Administered l-Amino-2,4- dibromoanthraquinone in Feed

for 2 Years

### Results 67

FIGURE 

## 7

Growth Curves for Mice Administered l-Amino- 2,4-dibromoanthraquinone in Feed for 2 Years68 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 22

M ean Body Weights and S urvival of Male Mice in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

W eeks 0 ppm 10,000 ppm 20,000 ppm

on Av. W t. Number of Av. W t. Wt. (% of Number of Av. W t. Wt. (% of Number of Study (g) Survivors (g) contr ols) Survivors (g) contr ols) Survivors

1 21.7 60 22.0 101 60 21.2 98 60
2 23.5 60 23.2 99 60 23.9 102 60
3 25.2 60 24.6 98 60 25.5 101 60
4 26.3 60 25.2 96 60 26.2 100 60
5 27.3 60 26.9 99 60 27.3 100 60
6 28.7 60 28.1 98 60 28.1 98 60
7 29.5 60 29.2 99 60 29.6 100 60
8 29.2 60 29.3 100 60 29.9 102 60
9 30.2 60 30.1 100 60 30.5 101 60
10 31.7 60 30.7 97 60 31.4 99 60
11 32.6 60 31.1 95 60 31.9 98 60
12 33.3 60 32.1 96 60 32.2 97 60
13 33.7 60 32.5 96 60 33.0 98 60
14 34.1 60 33.0 97 60 33.1 97 60
17 36.3 60 34.4 95 60 34.3 95 59
21 38.8 60 34.0 88 60 34.6 89 58
25 39.2 60 37.0 94 59 35.6 91 58
29 39.9 60 35.6 89 59 36.2 91 58
33 41.3 60 38.5 93 59 36.4 88 58
37 42.1 60 39.6 94 59 37.9 90 56
41 42.8 60 39.8 93 59 38.2 89 56
45 42.1 59 39.5 94 59 37.4 89 56
49 43.5 59 40.1 92 59 38.9 89 56
53 44.4 59 41.8 94 59 39.6 89 56
57 44.0 59 41.6 95 59 39.3 89 56
61 44.8 58 42.3 94 58 40.2 90 56
65 45.2 57 41.6 92 58 39.4 87 56
69 a 43.6 47 40.7 93 46 37.7 87 46
73 42.1 46 39.8 95 45 39.2 93 45
77 44.2 45 40.0 91 44 38.7 88 42
81 43.0 45 39.5 92 43 38.4 89 41
85 44.1 45 38.9 88 39 38.0 86 41
89 44.0 45 37.7 86 36 37.1 84 38
93 42.3 44 38.1 90 32 36.2 86 34
97 43.8 44 37.4 85 29 36.8 84 30
101 43.7 44 37.4 86 26 36.7 84 27
104 43.6 42 37.0 85 22 36.3 83 26

Mea n fo r w eeks
1-13 28.7 28.1 98 28.5 99
14-52 40.0 37.2 93 36.3 91
53-104 43.8 39.6 90 38.1 87

a Inte rim ev aluatio n occurr ed durin g we ek 66.Results 69

T ABLE 23

M ean Body Weights and S urvival of Femal e Mice in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone

Weeks 0 ppm 10,00 0 ppm 20,00 0 ppm

on Av. Wt. Number of Av. Wt. Wt. (% of Number of Av. Wt. Wt. (% of Number of Study (g) Survivors (g) controls) Survivors (g) controls) Survivors

1 17.1 60 17.4 102 60 17.1 100 60
2 18.1 60 18.4 102 60 18.0 99 60
3 19.3 60 19.8 103 60 19.6 102 60
4 19.9 60 20.7 104 60 20.4 103 60
5 20.8 60 21.3 102 60 20.8 100 60
6 21.6 60 22.1 102 60 21.1 98 60
7 21.8 60 22.5 103 60 22.4 103 60
8 21.9 60 22.9 105 60 22.8 104 60
9 22.9 60 22.9 100 60 23.3 102 60
10 23.2 60 23.6 102 60 24.0 103 60
11 23.6 60 24.0 102 60 24.3 103 60
12 24.4 59 24.4 100 60 24.7 101 60
13 24.8 59 24.8 100 60 25.0 101 60
14 25.2 59 25.5 101 60 25.4 101 60
17 26.0 59 26.6 102 60 26.0 100 60
21 27.7 59 27.3 99 60 27.3 99 60
25 29.2 59 28.6 98 60 28.1 96 60
29 30.2 59 28.8 95 60 28.9 96 60
33 32.1 59 30.7 96 59 28.8 90 60
37 32.9 59 31.9 97 59 30.5 93 60
41 34.2 59 33.0 97 59 30.1 88 60
45 35.2 59 32.8 93 59 31.2 89 60
49 36.6 59 34.5 94 59 32.5 89 60
53 36.8 59 35.0 95 59 32.5 88 60
56 38.2 59 36.3 95 58 33.1 87 60
61 39.1 59 36.2 93 58 33.8 86 60
65 38.4 59 36.1 94 57 33.5 87 60
69 a 37.6 49 35.4 94 47 32.4 86 50
73 37.3 48 35.7 96 46 32.7 88 49
77 39.3 48 35.8 91 46 33.1 84 49
81 39.9 48 36.4 91 45 33.8 85 47
85 39.5 48 36.7 93 44 33.6 85 45
89 41.1 46 36.1 88 44 33.7 82 44
93 41.4 45 35.3 85 44 33.3 80 41
97 40.0 44 33.6 84 41 31.9 80 39
101 38.5 43 34.1 89 40 31.5 82 37
104 39.2 39 33.6 86 35 31.9 81 35

Mea n fo r w eeks
1-13 21.5 21.9 102 21.8 101
14-52 30.9 30.0 97 28.8 93
53-104 39.0 35.5 91 32.9 84

a Inte rim ev aluatio n occurr ed durin g we ek 65.70 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



## Pathology and Statistical Evaluation

This sec tion desc ribe s the stat istic ally si gnif icant

or biologically noteworthy changes in th e incidences of neopl asms and/or nonneoplast ic lesi ons of the liver, forestom ach, lung, kid ney, uterus, and pitui tary g land of mice. Summaries of the incidences of neopl asms and nonneopl astic l esi ons, indivi dual animal tumor di agnoses , stat istical analys es of pr imary neop lasms that occurred with

an incidence of

at least 5% in at least one exposure

group, and hist orical i ncidences f or the neo pl as ms mentioned in this section are presente d in Appendix C for male mice and Ap pend ix D for female
mice.

Liver : At the 15-month interim evaluation, absolute and relat ive li ver weig hts of exposed g roups of females were significantly greater than thos e of
the controls (Table H5). Hepatocellu lar adenomas
and carcinomas were observed in exposed g roups of males and f

emales; none wer e present in the
controls (Tables 24, C1, and D1). At the e n d of the 2-y ear study , the i ncidences of hepat ocellular
ade noma and hepatocell ular ca rcinoma in expos ed

groups of males and females were significantly
incr eased (Tables 24, C3, and D3). The incidences of multi

ple hepatocel lular aden omas or multiple hepatocell ular carci nomas in expos ed

groups of males and females were also i ncreased (Tables 24, C1 , and D 1). In cide nces of hepa t o cellular ad e nom a s o r c ar c i n omas ( c om bined) in exposed g roups of males and f

emales exceeded
the NTP historical ra n ges for f eed study controls (Tables 24, C4a, an d D4a ). In addition t o hepatocellu lar

adenom as and car cinom as, a small
numb er of hepat oblastomas o ccurred in exposed

groups of males an d female s (Tab les 24, C1, and D1). These malign ant hep ato cel lu lar neo pl as ms contained

areas resembling hepatocellular c arc inoma; in addit ion, th ere were pro minent lobules or nodular foci separated by v ascular c h annels and composed of undifferentiated cells (Plates 9 and

1 0). The neoplast ic cells were elong ated w ith a
scant a mou nt of d arkly

staini ng cy

toplasm and

ov al hyper chrom atic nuclei. Cellula r pleomor phism and mit otic figures were commonly
present.

At the 15-month interim evaluati on and at t he end
of the 2-y ear study , the i ncidences of cent rilobul ar hypert rophy of hepatocytes i n exposed g roups of

m ales were significantlyincreased, and the inciden ces of hepat ocellular

pigmentati on w ere signif

icantly increased

in exposed

groups of males and
f emales (Tables 24, C5, and D5). This bro wn, granula r pig-ment r esembled that found i n the
13-week studi e s of 1 -amino-2,4- dibromoanthra

quinone; histoch emical procedur e s were not repeated during this 2-year study. The incidences
of c lea r c ell fo cus in exp os ed g

ro ups of fe ma l e mice were sig

nific antly

increased

at the end of t

he 2-ye ar st udy.Results 71

T ABLE 24

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Liver in Mice i n the 2- Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,00 0 ppm 20,00 0 ppm

Male

15-Month Interim

Evaluati on

Number E xamin ed 10 9 10

Centri lobul ar He

patocy te Hy pertr ophy a 0 9** (2.9) b 8** (2.9) Pigmentation 0 9** (1.1) 10** (1.2)

Hepatocell

ular Adeno

ma 0 2 4* Hepatocell

ular Adeno

ma or Carcinoma 0 3 4*

2-Year Study

Number E xamin ed 50 51 50

Basophil ic Fo cus 0 4 (1.0) 3 (1.0)
Centri lobul ar He

patocy te Hy pertr ophy 0 17** (2.0) 13** (2.0)
Clear Cell Focus 4 (1.3) 4 (1.0) 2 (1.0)
Eosino philic F ocus 0 6** (1.5) 1 (1.0) Pigmentation 1 (3.0) 50** (1.1) 47** (1.4)

Hepatoc ellular Aden oma (Mu ltiple) 6 29** 31**

Hepatoc ellular Aden oma (Sin gle or Mult iple)
Over all r ate c 10/50 (20%) 38/51 (75%) 39/50 (78%)
Ter minal r ate d 9/40 (23%) 20/22 (91%) 21/23 (91%) Adjust ed rate e 24.3% 94.7% 95.0% First incidence (

days) 723 451 484 Logistic r egress ion test f P<0.001 P<0.001 P<0.001

Hepatoc ellular Ca rcinom a (Mult iple) 1 3 9**

Hepatoc ellular Ca rcinom a (Sing le or Multi ple)
Over all r ate 9/50 (18%) 18/51 (35%) 21/50 (42%)
Ter minal r ate 7/40 (18%) 10/22 (45%) 9/23 (39%) Adjust ed rate 21.1% 58.1% 58.4% First incidence (

days) 445 505 535 Logistic r egress ion test P=0.002 P=0.017 P=0.003

Hepatocell

ular Adeno

ma or Carcinoma

g

Over all r ate 18/50 (36%) 43/51 (84%) 42/50 (84%)
Ter minal r ate 15/40 (38%) 21/22 (95%) 22/23 (96%) Adjust ed rate 41.7% 97.7% 97.7% First incidence (

days) 445 451 484 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Hepatoblastoma 0 3 5*

(cont inu ed)

* Significantly different (P # 0.05) from the co ntrol gro up by the Fishe

r exact test (15-month interim ev aluation) or the

logistic re

gression te st (2-yea r stu dy)

** P # 0.01 (T)Te rminal sacrifice

a Number of animals with lesion b Average severit y grade of lesion s in aff ected mi ce: 1 = mi nimal; 2 = mild ; 3 = mod erate; 4 = marked

c Number of animals with neoplasm per number of animals with liver examined m icroscopically72 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 24

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Liver in Mice i n the 2- Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone (continued)

0 ppm 10,00 0 ppm 20,00 0 ppm

Fema le

15-Month Interim

Evaluati on

Number E xamin ed 10 10 10

Pigmentation 0 10** (1.0) 9** (1.0)

Hepatoc ellular Aden oma (Mu ltiple) 0 1 4* Hepatoc ellular Aden oma (Sin gle or Mult iple) 0 2 7** Hepatocell

ular Adeno

ma or Carcinoma 0 2 8**

2-Year Study

Number E xamin ed 50 50 50

Basophil ic Fo cus 0 4 (1.3) 5* (1.2)
Clear Cell Focus 0 10** (1.2) 9** (1.6)
Eosino philic F ocus 0 4* (1.5) 2 (2.5) Pigmentation 0 44** (1.1) 49** (1.6)

Hepatoc ellular Aden oma (Mu ltiple) 0 40** 45**

Hepatoc ellular Aden oma (Sin gle or Mult iple)
Over all r ate 6/50 (12%) 45/50 (90%) 49/50 (98%)
Ter minal r ate 6/39 (15%) 32/34 (94%) 33/33 (100%) Adjust ed rate 15.4% 95.7% 100.0% First incidence (

days) 729 (T) 442 501 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Hepatoc ellular Ca rcinom a (Mult iple) 0 13** 13**

Hepatoc ellular Ca rcinom a (Sing le or Multi ple)
Over all r ate 0/50 (0%) 23/50 (46%) 27/50 (54%)
Ter minal r ate 0/39 (0%) 17/34 (50%) 16/33 (48%) Adjust ed rate 0.0% 57.2% 60.8% First incidence (

days)

) h 503 538 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Hepatocell

ular Adeno

ma or Carcinoma

i

Over all r ate 6/50 (12%) 46/50 (92%) 50/50 (100%)
Ter minal r ate 6/39 (15%) 33/34 (97%) 33/33 (100%) Adjust ed rate 15.4% 97.9% 100.0% First incidence (

days) 729 (T) 442 501 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Hepatoblastoma 0 0 2

d Obser ved inc idence

in animal s surv

iving un til the end of the stud y

e Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. f In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l.

g Historical incid ence for 2-year NTP feed studies with untreated control groups (mean ± standard d eviation): 531/1 ,466 (36. 2% ± 14.1 %);

range , 10%-68% h Not appl

icable ; no neo

plasms in animal

group i Histor ical inc idence : 247/1,46 2 (16.9% ± 1 0.7%); r ange, 3%-42%Results 73



### For estomach: S quamous cell papillomas were obser ve d
in 20,000 ppm

males and

in 10,000 and

20,000 ppm females at the 15-m

onth int erim evaluation (Tabl es 25

, C1, and D1). At th e end

of the 2-y ear study, the

incidences of squamous cell papi llo ma and squa mo us cell

car cin oma in expose d groups of males and femal es wer e signif icantly increased (Tables 25, C3, and D3). The
incidences of mu

ltip l e squam

ous cell papillo ma in 20,000 ppm male s and fe

males were also signif

icantl y increased in the 2 -y ear study (Tables 25, C1, an d D1).
Incidences of

squa mous c ell papi llo ma or c arc inoma (combined ) in exposed groups of males and fem ales wer e signif icantly greater than those in the controls and
exceeded the NTP histor ical r ange s for feed study

con trols ( Tables 25, C4b, and D4b). Com pared to the exophy tic mas ses with well- differentiated squamou s epithelium typic al of the squamous cell papillomas, th e
squamous cell carc inoma s we re loc ally in vasive

neop las ms that

sometime s resulted in perforation of the forest omach (Plate 11). Freque ntly, a squa mo us cel l

car cin oma appeared to arise at the base of a squamous cell papilloma. Metasta

tic neoplasms arising

from

squamous cell car

cinom as of t

he foresto mach

were

o bserved in the coagulating glands, colon , duo denu m, epididymis, gallbl adder, gl andular stomac h, jejunum

, kidney, liver, lung, ovary, p ancreas, prostat e gland, spleen, testis, and thym

us of expo sed mi ce (Tables C 1 and D1 ).

Non ne oplastic

lesions of the forestom

ach included

aca nthosis, hyperkeratosis, an d basal cell hy perplas ia (Tables 25, C5, and D 5). At the 15- mont h interi m evaluation, t here were exposure-related increases in th e incidences and severities of ac a nth osis and hyper kerato sis in exposed groups of males and f ema les

. At the end o f the 2-y

ear study, the incid

ences and
severities of these lesions in ex pose d groups of males an d
females w ere gener

ally greater than those in the contro ls

.74 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE 25

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Forest omach in Mice i n the 2- Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,00 0 ppm 20,00 0 ppm

Male

15-M onth Interim Evaluation

Number E xamin ed 9 9 10

Acanthosis (Hy

perplasia) a 0 2 (1.0) b 3 (2.0)
Basal C ell

Hype rplas ia 0 1 (1.0) 1 (1.0) Hype rkera tosis 1 (2.0) 2 (1.0) 3 (2.0)
Inflammation 0 0 3 (1.2)

Squamous Cell Papilloma 0 0 5*

2-Year Study

Number E xamin ed 50 50 50

Acantho sis 1 (1.0) 9** (1.1) 4 (2.0)
Basal C ell

Hype rplas ia 0 0 2 (1.5) Hype rkera tosis 1 (1.0) 7* (1.0) 6 (1.8)
Inflammation 2 (1.5) 6 (1.2) 13** (1.5)

Squam ous Cell Pa pilloma (Multi ple) 0 2 5*

Squam ous Cell Pa pilloma (Single or M ultip le)
Over all r ate c 0/50 (0%) 13/51 (25%) 16/50 (32%)
Ter minal r ate d 0/40 (0%) 10/22 (45%) 11/23 (48%) Adjust ed rate e 0.0% 51.0% 55.6% First incidence (

days)

) g 613 606 Logistic r egress ion test f P<0.001 P<0.001 P<0.001

Squamous Cell Carcinoma
Over all r ate 0/50 (0%) 12/51 (24%) 13/50 (26%)
Ter minal r ate 0/40 (0%) 4/22 (18%) 4/23 (17%) Adjust ed rate 0.0% 36.5% 37.7% First incidence (

days)

) 505 523 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Squamous Cell Papilloma or Carcinoma

h

Over all r ate 0/50 (0%) 19/51 (37%) 27/50 (54%)
Ter minal r ate 0/40 (0%) 11/22 (50%) 14/23 (61%) Adjust ed rate 0.0% 61.2% 73.9% First incidence (

days)

) 505 523 Logistic r egress ion test P<0.001 P<0.001 P<0.001

(cont inu ed)

* Significantly different (P # 0.05) from the co ntrol gro up by the Fishe

r exact test (15-month interim ev aluation) or the

logistic re

gression te st (2-yea r stu dy)

** P # 0.01 (T)Te rminal sacrificeResults 75

T ABLE 25

Inci dences of Neoplas ms and Nonneopl asti c Lesi ons of t he Forest omach in Mice i n the 2- Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone (continued)

0 ppm 10,00 0 ppm 20,00 0 ppm

Fema le

15-M onth Interim Evaluation

Number E xamin ed 10 10 10

Acanthosis (Hy

perplasia) 0 1 (1.0) 8** (2.0)
Basal C ell

Hype rplas ia 0 0 2 (1.5) Hype rkera tosis 0 2 (1.0) 7** (2.0)
Inflammation 0 1 (1.0) 5* (1.6)

Squamous Cell Papilloma 0 4* 2

2-Year Study

Number E xamin ed 48 50 50

Acanthosis (Hy

perplasia) 9 (1.7) 15 (1.7) 19* (1.6)
Basal C ell

Hype rplas ia 0 7* (1.4) 3 (1.7) Hype rkera tosis 10 (1.4) 14 (1.4) 17 (1.5)
Inflammation 7 (1.4) 10 (1.4) 21** (1.7)

Squam ous Cell Pa pilloma (Multi ple) 0 4 14**

Squam ous Cell Pa pilloma (Single or M ultip le)
Over all r ate 2/50 (4%) 16/50 (32%) 27/50 (54%)
Ter minal r ate 2/39 (5%) 12/34 (35%) 23/33 (70%) Adjust ed rate 5.1% 41.7% 72.4% First incidence (

days) 729 (T) 671 538 Logistic r egress ion test P<0.001 P<0.001 P<0.001

Squamous Cell Carcinoma
Over all r ate 0/50 (0%) 12/50 (24%) 11/50 (22%)
Ter minal r ate 0/39 (0%) 8/34 (24%) 5/33 (15%) Adjust ed rate 0.0% 30.9% 27.3% First incidence (

days)

) 587 501 Logistic r egress ion test P=0.002 P<0.001 P<0.001

Squamous Cell Papilloma or Carcinoma i

Over all r ate 2/50 (4%) 25/50 (50%) 34/50 (68%)
Ter minal r ate 2/39 (5%) 18/34 (53%) 25/33 (76%) Adjust ed rate 5.1% 60.7% 80.5% First incidence (

days) 729 587 501 Logistic r egress ion test P<0.001 P<0.001 P<0.001

a Number of animals with lesion b Average severit y grade of lesion s in aff ected mi ce: 1 = mi nimal; 2 = mild ; 3 = mod erate; 4 = marked

c Number o f anim als with neoplas m per nu mber of an imals n ecropsi ed d Obser ved inc idence

in animal s surv

iving un til the end of the stud y

e Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. f In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l.

g Not appl

icable ; no neo

plasms in animal

group h Histori cal inc idenc e for 2-year NTP feed studi es with u ntreat ed cont rol groups (mean ± s tand ard devi ation ): 22/1 ,474 (1.5% ± 2.0% ); range,
0%-6% i Histor ical inc idence : 33/1,470

(2.2% ± 3. 1%); ran

ge, 0% -14%76 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



## Lung: Alveolar/bronchiolar adenomas were
observed in exposed g roups of

males and

females

at the 15-month interim ev aluation (Tables 26, C1, and D1). During the 2-y ear study , the i ncidences
of alveol ar/ bronchiol ar adenomas in expose d

groups of males and females wer e signifi cantly
increased (Tables 26, C3, and D3). I n male mice,
the incidences of multi ple alveo lar/br onchi ola r adenomas in exposed groups were signif icantly
greater than that

in the con

trols (Tables 26, C1, and D1). The incidences of alveolar/ bronchiol ar adenoma in expos ed groups of males

a nd fe males exceeded th e NTP histor ical rang es for feed study controls (Tables 26, C4c, and D4c).
The alveolar

/bronchio l ar adenomas were g ene rall y well-cir cumscribed, expansile masses th a t slightly

comp ress ed the surrounding normal pulmonary
alveolar tiss ue (Plate 12). Wel l-dif ferent iat ed
cuboidal to columnar ep ithelia l cells formed papillary struct

ures or solid f

oci that f illed al veolar

spaces.

T ABLE 26

Inci dences of Neoplas ms of t he Lung in M ice in t he 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,00 0 ppm 20,00 0 ppm

Male

15-M onth Interim Evaluation

Number E xamin ed 10 9 10

Alve olar/bronchiol

ar Adenoma

a 0 3 5 *

2-Year Stud y

Number E xamin ed 50 51 50

Alv eol ar/bro nchiol ar Hy

perpl asia 1 (1.0) b 0 4 (1.3)

Alveolar/bro nchi olar Adenom a (Mult iple) 0 6** 9**

Alveolar/bro nchi olar Adenom a (Sing le or Multi ple)
Over all r ate c 7/50 (14%) 26/51 (51%) 24/50 (48%)
Ter minal r ate d 6/40 (15%) 12/22 (55%) 12/23 (52%) Adjust ed rate e 16.8% 71.0% 66.5% First incidence (

days) 445 578 248 Logistic r egress ion test f P<0.001 P<0.001 P<0.001

Alve olar/bronchiol

ar Carcinoma
Over all r ate 3/50 (6%) 4/51 (8%) 1/50 (2%)
Ter minal r ate 2/40 (5%) 2/22 (9%) 0/23 (0%) Adjust ed rate 6.9% 15.9% 3.0% First incidence (

days) 393 673 648 Logistic r egress ion test P=0.259N P=0.512 P=0.251N

Alve olar/bronchiol

ar Adenoma o

r Carcinoma g

Over all r ate 10/50 (20%) 28/51 (55%) 25/50 (50%)
Ter minal r ate 8/40 (20%) 13/22 (59%) 12/23 (52%) Adjust ed rate 23.3% 75.0% 67.5% First incidence (

days) 393 578 248 Logistic r egress ion test P<0.001 P<0.001 P=0.002

(cont inu ed)Results 77

T ABLE 26

Inci dences of Neoplas ms of t he Lung in M ice in t he 2-Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone (continued)

0 ppm 10,00 0 ppm 20,00 0 ppm

Fema le

15-M onth Interim Evaluation

Number E xamin ed 10 10 10

Alve olar/bronchiol

ar Adenoma 0 3 2

2-Year Study

Number E xamin ed 50 50 49

Alv eol ar/bro nchiol ar Hy

perpl asia 0 0 1 (1.0)

Alveolar/bro nchi olar Adenom a (Mult iple) 0 2 1

Alveolar/bro nchi olar Adenom a (Sing le or Multi ple)
Over all r ate 4/50 (8%) 17/50 (34%) 13/49 (27%)
Ter minal r ate 3/39 (8%) 14/34 (41%) 9/33 (27%) Adjust ed rate 9.8% 45.6% 33.5% First incidence (

days) 685 587 538 Logistic r egress ion test P=0.017 P=0.001 P=0.015

Alve olar/bronchiol

ar Carcinoma 0 0 2

Alve olar/bronchiol

ar Adenoma o

r Carcinoma

h

Over all r ate 4/50 (8%) 17/50 (34%) 15/49 (31%)
Ter minal r ate 3/39 (8%) 14/34 (41%) 10/33 (30%) Adjust ed rate 9.8% 45.6% 37.9% First incidence (

days) 685 587 538 Logistic r egress ion test P=0.006 P=0.001 P=0.005

* Significantly different (P # 0.05) from the co ntrol gro up by the Fishe

r exact test (15-month interim ev aluation) or the

logistic re

gression te st (2-yea r stu dy)

** P # 0.01

a Number of animals with lesion b Average severit y grade of lesion s in aff ected mi ce: 1 = mi nimal; 2 = mild ; 3 = mod erate; 4 = marked

c Number of animals with neoplasm per number of animals with lun g examined microscopica lly d Obser ved inc idence

in animal s surv

iving un til the end of the stud y

e Kapla n-M eier es tim ated neop lasm inc iden ce at the en d of t he st udy a fter a dju stme nt fo r int ercu rrent mort alit y. f In th e contr ol co lumn ar

e the P value s asso ciated w

ith the t rend te st. I

n the e xpose d grou p colum ns are the P v alues corr espond ing to pairwise comparisons between the con trols and that exposu re group. The logistic regression test regards these lesions as nonfata l. A negative

tren d or a lower in cid ence in a n exp osur e grou p is ind ica ted b y N

. g Historical incid ence for 2-year NTP feed studies with untreated control groups (mean ± standard d eviation): 265/1 ,469 (18. 0% ± 7.6%);

range , 4%-32% h Histor ical inc idence : 110/1,46 9 (7.5% ± 5. 0%); ran ge, 2% -26%78 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### Kidney: Pigmentation was p resent in the kidneys of mos t mice af ter 2 years of exposu re to 1-am

ino2,4-dibrom

oanthraquinone (males: 0 ppm , 0/50; 10,000 ppm, 42/ 51; 20,000 ppm, 43/50; females: 0/50

, 43/50, 43/50; Tables C5 and D5). This brown, gra nula r pigment in the renal tubule epithelium and tubule l umin a resembled the pigment described in the liver. T here wer e no othe r chemic al-rela ted le sions in the ki dney.

Uteru s: There was a significant, but not exposurerelated, increas e in

the incidence of uterine p olyps or

sarcomas (combined) (0 /50, 5/50, 0/50; Table D1) in th e 10,000 ppm females. Althou gh this incidenc e (10%) wa s above the av erage for hist orical c ontrols (3.5%), the

com bin ed incidence was within the historical control

range (0%-1 6%; Table D4d). This ma rginal increase wa s
not considered

to be c hemic al relate d. There were no

c hemical-re

lated nonneo

plastic lesions in the reprodu c

tive tr act (Tabl e D5).

Pituitary Gl

and: A signif

icant, but no t exposure-related

, increase in the incidence of aden oma o f the pi

tuitary gland (pars distalis) was observed in the 10,000 ppm females (1/43, 9/

45, 4/43; Table D1). The incidence o f
aden oma (20%) in

the 10, 000 ppm

fema les is sl ightly
above the average for historical c ontrols (15. 2%), but i s wi thin the historical range (2%-3 6%; Table D4e). Thi s marginal increase was no t consid

ered to be chemical
related

. The inciden ce of hyper plasia of the pars distali s of the pituitar

y gland was als o inc reased in the 10,000

- ppm femal es (7/43 , 22/45 , 7/43; Table D5 ).



# D ISPOSITI ON

AND 

# M ETABO LIS M 

# S TUDIES
Ad ult male F344/N rats received [ 14 

### C] -labeled 1-amin o

2,4- dibro m oanthraquinone as a single intravenous dos e of 0.4 mg 1-am

ino- 2,4- dibrom

oanth raquin one/kg bod

y weight or as a single o

ral dose of 2, 23, 118, 814, or 1,473 mg/kg. After excreta were collected for 72 ho u rs , the an imals were kil led, and tissues were re moved for

an alysis. Additional ani ma ls that received intravenou s doses of 1-amin

o-2,4- dibromoant

hraqu inone were kille

d
0.25

, 0.75, 2, 6, or 24 hours after chemical admin istra

tion, and their tissues were an

alyz ed. A 6-ho

ur bile cannulation study w as also performed. From day 0 throug h day 3 after intravenous admini stra

tion of [ 14 

### C]-1- am

ino-2,4- dibromoanthraqui

none, abou

t 50% of the 14 

### C was excreted in the feces, 1 5% in the urine, and 6% in expired air. Unmetabo lize d 1- amino

2,4- dib romoa nthra quinone accounted for less than 3% o f
the excreted 14 

### C aft er i ntravenous administration. Th e
amount of an oral dose that was abs orbed was calculat e d
f rom the percent o f the dose that was excreted in expire d
air or urine after oral admini stration versus the percent o f
the dose ex creted a fter intr a veno us administration.
Excretion of 14 

### C in e xpired air yielded the most consis

ten t res ults. F or oral doses g reater th an or eq

ual to
2 mg/kg, the amount of the dose t hat was ab sorbed fitte d
the equatio n: absorbed dose = 6.6 log( dose ), with
both doses expressed in m

g/kg. W hil e 90% of the
2 mg/kg dose was abs orbed, only 2% of the 814 mg/ k g dose was absorbed.

Two hours after intravenous administration, less than 3 % of the circ ulatin g 14 

### C was attributed to the parent c ompound

. The metabolites of 1- amino

2,4-dibrom

oanthraquinon

e in blood were pri

marily in th e plasm a fraction (bl ood: pla sma ratio o f appro ximat ely 0.5:1). The highest concentration s of 14 

### C in tissues
15 minu tes after intraveno us dosing were in excretor y
organs, lung, kidney, s mall intestin e, liver, adipose tissue

, and adrenal glan d. Tissue:blood ratios (TB R) for thes e tissues were greater than or equal to 3:1

. Only the live r and kidne y had TBRs si g nific antly greater than 1:1 at
72 hours. The terminal half- life of 14 

### C was approximatel y 40 hours in the l iv er and approxi matel y 90 hour s in the kidne

y. A dipose tissue c ontaine d prim arily
unmetabolized 1-amin

o-2,4- dibr omoanthraquino ne a t 24 hours; liver, m

uscle, and skin con ta ined m ostly
metabolites of 1-amin

o-2,4- dibromoanthraquino

ne. Th e elimination half -l i fe of 1-am ino

- 2,4- dib romoanthraquinone in adipose tissue was approxi

mately 11 hours.



# G ENETIC 

# T OXICOLOGY
1-Ami no-2,4- dibromoanthraquino ne (100 t o 10,000 : g /plate) was tested for induction of gene mut a

tions in four

strains of Salmon ella typhimurium in a prei ncubation protocol with a nd withou t Aroclor 1254-induce d male Sprag ue-Dawley

rat or Syrian hamst e r liver S9 (Table G1; Haworth et al.

, 1983). 1-Am

ino-2,4- dibromoanthraquino ne wasResults 79



### positive in the abs ence of S9 in the frame shift
strain s TA98 and TA1537; wit h S9, an equ ivocal

response was obtain ed in TA1537, and TA98 was negativ e. In TA100, 1-amino-2, 4-dibromoanthraquinone gave equivocal

respons es with and

without S9, and all trials were negative with TA1535.
The equivocal

calls were the results of positive or weakly positi ve r espo nses tha t were not duplicated
in a second trial. Precipitation of 1-amin o

2,4-dib romoanthraqui none occurred at concentrations of 100 : g/plate and above, and this may have
been a facto r in the nonreprodu cibilit y of the
results.

1-Amino-2,4-dibromoanthra quinone wa s tested in
two laborat ories for

induction of sister

chromatid exchang es a nd chromosoma l aberrations in cultured Chinese hamste r ovary cells, with and
without Aroclor 12 5 4- induced male Spr agueDawley rat li ver S9. In the sis ter chromatid exchange tes t, one laboratory observ ed a sig nific ant increa se in s ister

c hrom atid exchanges only i n the absenc e of S9,
while the second laborator y recor ded p ositive

responses with and wit

hout S9 (Tabl e G2; Lov eday et al. , 1 990). This dis crepancy cannot be explained

by a di ffer ence in dose levels employed

at the two laboratories because the positiv e r esponses with S9 were observ ed at 3, 1 0, 15, and

30 : g/mL at t

he second la

boratory , whereas
negat ive trials resulted f rom testing doses up to

100 : g/mL at t

he first laborato ry. In t

he chromos om al aberrati ons test , one labo ratory observ ed a weakly positive response only in the absence of S9 (Table G3). Another laboratory obtained a positive re spons e in the first trial without S9 but did

not duplicat e the posi tive r esponse i n the secon d
tri al, and the ov erall cal l withoutS9 was concluded to be equivo c al (L ove day et al. , 1990).
Neither laborato ry obs erved an i ncrease in ch romosomal aberrations with 1-amin o

- 2,4-dib romoanthraqui none in the presence of S9.80 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383PLATE 

### 1

An hepatocellular carcinoma in a female F344/N rat exposed to 10,000 ppm 1- amino-2,4-dibromoanthraquinone in feed for 2 years. Note compression of normal liver (small arrows) by neoplastic hepatocytes. Carcinoma embolus (large arrow) is in an hepatic vein. H&E; 90x PLATE 

### 2

Multiple metastatic foci (*) of an hepatocellular carcinoma in the lung of a female F344/N rat exposed to 5,000 ppm l-amino-2,4-dibromoanthraquinone in feed for 2 years. H&E; 15x

PLATE 

### 3

Detail of a metastatic focus of the hepatocellular carcinoma shown in Plate 2 shows the solid and acinar growth patterns of the well-differentiated neoplastic hepatocytes. H&E; 90x PLATE 

### 4

An hepatocholangiocarcinoma in a female F344/N rat exposed to 10,000 ppm lamino-2,4-dibromoanthraquinone in feed for 2 years. Note the welldifferentiated hepatocyte (solid areas) and biliary components within the neoplasm.
H&E; 140xPLATE 

### 5 An adenoma (adenomatous polyp) in the colon of a female F344/N rat exposed to 10,000 ppm l-amino-2,4-dibromoanthraquinone in feed for 2 years forms an exophytic mass (a rrows) that partially occludes the intestinal lumen (L). H&E; 25* PLATE 

### 6 Detail of a carcinoma in the colon of a female F344/N rat exposed to 10,000 ppm l-amino-2,4-dibromoanthra quinone in feed for 2 years. Note the disruption of the muscularis mucosa (M) layer at right with formation of irregular-shaped neoplastic glands (a rrows), inflammation, and fibrosis in the submucosa. H&E; 160x

PLATE 

### 7
A transitional cell carcinoma in the urinary bladder of a female F344/N rat exposed to 10,000 ppm l-amino-2,4-dibromoanthraquinone in feed for 2 years. Note the thickened neoplastic mucosal surface and a papillary projection of the neoplasm extending into the bladder lumen (L). The mucosal surface consists of a thickened layer of neoplastic transitional cells (arrows); a large nodule of transitional epithelium invades the wall of the urinary bladder. H&E; 160x PLATE 

### 8

Detail of the transitional cell carcinoma shown in Plate 7 shows cellular atypia and an increased number of mitotic cells. H&E; 320xPLATE 

### 9 An hepatoblastoma in the liver of a female B6C3F 1 mouse exposed to 20,000 ppm l-amino-2,4-dibromoanthraqui none in feed for 2 years consists of prominent neoplasm lobules separated by vascular channels. H&E; 80x PLATE 

### 10 Detail of the hepatoblastoma shown in Plate 9 shows closely packed undifferentiated neoplastic cells with scant cytoplasm, oval nuclei, and numerous mitotic cells (arrows). H&E; 320x

PLATE 

### 11 A squamous cell carcinoma of the forestomach in a male B6C3F 1 mouse exposed to 20,000 ppm l-amino-2,4-dibromoanthraquinone in feed for 2 years. Note the thickened keratin (K) layer on the mucosal surface and invasion of the wall by nodules (arrows) of neoplastic squamous cells that have extended through the peritoneal surface of the stomach. H&E; 40x PLATE 

### 12 An alveolar/bronchiolar adenoma of the lung in a male B6C3F 1 mouse exposed to 20,000 ppm l-amino-2,4-dibromoanthraquinone in feed for 2 years forms a non-encapsulated subpleural nodule. H&E; 60*81



# DISCUSSION AND CONCLUS IONS

Anthraquinones represent the largest group of natu

rall y occurring quinones. Both natural and synth eti c anthraquinones hav e been and continue to be wi del y used as color ants in foo d, drugs, cosmetics, hair dyes

, and textiles. Dantron (1,8 -di hydroxyanthraq uinone )
and emod in (1,3,8 -tr ihydroxy- 6-meth ylanthraquinone

)

are also u sed therape

utically as catharti

cs and purga

tives. Chry sophanol (1,8- dihydr oxy

3-methyl anthraquinone) occurs in cascara sagrada,

senna, and v arious s

pecie s of R umex

and Rheum (rhubar b).

Anthraquinone and five substitu te d a nthraquinone s

were selec ted fo r toxicol

ogic characterization from

a lar ge group of am

ino-, alk yl-, nitro -, or halog encontaining

anthraquino nes. The basis for selec tion
centered mainl

y on four criteria: 1) lack of availa ble
da ta on carcinog enici ty, 2) magnitude of product io n and use patterns, 3) awarenes s of potential and actua l
human exposure, 4) and represen ta tion of as broad

a spectrum of stru ctural d iver sity within this class as possibl e

. 1-Ami no-2,4- dibromoanthraquino ne wa s selected from a group of 36 enviro nmen ta lly signif

i

cant ary l br omides. Since

all other substituted anthraquinone chem

icals alre ady ev aluated for long

term effects induced carci noge nic res ponses in labora

tory anima ls ( NCI, 1 978a, 1978b, 1978c; IARC, 1982

, 1987; NTP, 1986 a ), 1-am ino-2,4

- dibromoanthraquino

ne was predicted to be carc ino

genic in labo rator y animals as well (Fung et al., 1993).

1-Am ino-2,4- dibr omoanthraq

uinone was studied fo r long- te rm toxicity and carcino genes is us ing a "start

stop" experimental design. One of the first c hemical s
to be studied by the N ational Toxicology Program (NTP) with a start-sto p protoc ol , 1-am

ino2,4-dibrom

oanthraquinone was predicted to be ca rci

nogenic, so the exper

i mental design was selected in a n
attem pt to g ain som

e insig ht into the progression
and/or regression of che mical-ind uced lesions as wel l

as t o p erhaps gain knowled ge about potential mecha

nisms of action.

Because 1-amin

o-2,4- dibromoanthraquino

ne cause d significant carcinogeni c respon ses in male and femal e rats and mice and in several organs and

tissues, the
di scussio n of lesions that follows has been grouped b y
organ.

Li ver: 1-Am

ino-2,4- dibromoanthraquino ne diff er s from other chem

icals studied by t he NTP because i t induced great er than 90% inci dences of mu ltiple benig n and mali gnant hepatocellular neop las ms in rat s with frequent metastases ( almost 50%) of the ma lig

nant liver neoplas

ms. Althou

gh oth er chem

icals including 3,3 N -dim eth ylbenzidine dihyd

rochlo ride (NTP, 1991) and furan (N TP, 1993a) have caused significant

increases in benign and malignant liver

neop las ms tha

t approach a 100% incidence, metastase s occurred in only one o r

two instances in a gro

up of
50 ra t s. Only in the 18-mont h study of methyl
carbam ate (NTP, 1988) has a simila rly hig h incidenc e of malig nant, metas

tatic live r neop las ms occurred i n male rats.

The liver lesions prese nt aft er 13 weeks in the 25,00 0 and 50,000 ppm gr oups of rats inc luded prolifer ativ e bile duct le sions (c

holang iofibros is) an d foci of hep atocellular alte ration. Based on m orphological
features of this pr olife rative bile duct lesion and
results of transpl antation and stop- exposu re studies

, cholang iofibro sis has been considered a "prem aligna

nt" l esio n which is autonom ous and

progre ssive and no t qualit ativel y diff erent f ro m chola ngio car cin oma (Maronpot et a l., 1991). Whil e
t here is some disagreement on the biological behav io r of chol angiofibrosis, this has gener ally been consid

ered to be a preneoplast ic lesion (Ba nn asch and Massner, 1976; Ohshim a et al ., 1984)

. Cholan giofib rosis has bee n descr ibed in toxicity
studies of methap yrile ne (Ohshima et al. , 1984 ),
aflatoxi n (Wi lson et al., 1985), and furan (N TP, 1993a) in rats.

Exposu re conc entrati ons of 1-amin o

2,4-dibrom oanthraquinone administered in th e 2-year study were below those which produced cholangiof

ibrosis at 13 weeks; howev er, many o f
the benign and malignant liver neop las ms in the 2-year study in rats were composed of a mixed growt h pattern of both hepatocytes and bile duct
formation. The incidences of foci82 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### of hepatocellular alteration were increased with
1-amin o-2,4- dibromoanthraquino ne exposu re at 13 weeks and all scheduled intervals e xamined during the 2-y

ear study. A detailed ana lysis of foc i
of hepato cellular

alterati on in th e liv e r of rats fro m this stu d y has been reported by Harada et al. (1989)

. In addition to overa ll exposure-related increased
i nci dences of eosinop hilic and clear cell foci of alteration, the re w ere increases in size, numb er, an d
volum e fraction of atypical eosinophi lic, basoph ilic

, a nd clear cell foc i in rats that correlated with concen

tration and d u r ation o f 1-amin o

2,4-dibrom

oanthraquinone exposure.

Althou gh foci of hepatocel lular al teration in rats ar e believed to be precurs ors of liver neoplasm s, t heir biological nature and potential for progression to

neop las ms is uncerta in (Popp and Goldsw orthy, 1989; Squire, 19 89)

. Some of this uncerta inty result s fro m a conside rable variation in phenotypes o f hepatocellular foci and the different bioma rkers us e d
in their cl assifica

tio n. In many

studies, the conv er

sion rate of foci to neop las ms has been co nsidered t o be extrem ely low, and, in some i nstance s, incre ase s in the incidences of basoph ilic foci have not been

asso ciated with liver neop las ms (MacDon ald et al.

, 1988; Harada et al., 1989; Squire, 1989). Clear an d acidoph ilic cell foci have been suggested to be important in the developmen t of some chem icalinduced liver neop las ms (Bannasch et al.

, 1989; Bannasch and Zerban, 1992). The atypical
eosi nophili c foci that occurred in rats admin istere d
1-amin o-2,4- dibromoanthraquino ne were rarely
observed in control s or in groups of rats recei vi n g
ot her hepatocarcinogens (Harada et al.

, 1989 )

. Adenomas in the livers of rats treated with 1-amin o

2,4-dibrom

oanthraquinone often contained cells morph ologic ally identical to those in the atypical eosinophi l ic foci, suggesting that some of these foc i

may have been precurs ors for the hepatic neop las ms

.

Li ver effects in male mice administered 1-amin o2,4-dibrom

oanthraq uinone for 13 weeks consisted o f pigmentation and hype

rtrophy that persisted th

rough

out the 2-year stud

y. Althoug

h hypertrop hy did no t

occur in femal e mice during thi s early period, pi gmentation in the liver was present by 15 months.
Foci of hepatocellular alteration were n ot present i n the 13-week s tudy, and after 2 years, the incidence s

were only slig

htly increased in m ice. A fter 2 y ears , there were in creased incidences of liver neop las ms i n all groups of m i ce exposed to 1-amin o2,4-dibrom

oanthraqui none in fee d. This respons e

was more prominent in females that a ls o had a greater number of hepatocellular carcinomas and

more multi

ple liver neop las ms than male mic e. Unlike the highly metas tatic liver neop las ms observed in rats, only a few neop las ms in mice had
dete ctable metas tatic foci. The incidences o f hepatoblastomas were al so increased in t he expose d

groups of ma

le and f emale mice . These distinct

iv e liver neoplasms rar

ely occu r in control animals bu t h av e been induced in mice administered acetylaminofluore ne (Non oyama et al.

, 1988) or N -nitrosodiethy lamine (Ward et al

., 1983).

Large In testine: Adenomatous p o lyps (adenomas )
and carcin omas of the lar

ge intestine (distal colon
and rec tum) in rats were gener ally observed after
15 m onths of exposu re to 1-am ino

2,4-dibrom

oanthraquinone

, altho ugh one adenom a

tous polyp (adenoma) was observed as early as 9 month s in the 20,00 0 ppm group of ma le rats i n th e

" stop-exposure" study. Furthe r, these lesions wer

e often grossl

y visible. Even when exposu re wa s stopped after 9 months, th e perc entage of chemical

indu ced rectal n eopl asms was equal to or greater tha n that observed with continuous exposu re fo r 15 months. In many rats, these neop las ms were multi p l e and malig nant, based upon local invasion and/or

metas tases. Neoplasms of the large intestine have

no t been observed for other previo usly tested anthraquinones. On e other chem ical stu died by th e NTP

, bromodichlorom

ethane ( NTP, 1987a), resulte d
in simila rly high i ncidences of benign and maligna n t

neop las ms of the large intestine.

Kidney: Accumulation of pigment i n the kidney was observed in both male and female rats by 13 weeks
and throug hout th e 2- year s t udy. 1-Amino2,4-dibrom

oanthraquinon e (or me tabolite) pigmen t in the kidn ey o f mic e was not eviden t until after
the 15- month

evalu ation; t here were no increased
incidences of other nonneoplast ic lesions or neoplasm

s of the ki dney i n mice . In the kidney of rats, several changes in addition to pig ment were presen

t at 13 weeks. In m

ale rat s, inc reased accumulation of hyaline droplets was observed in the cytoplasm o f
th e ren al tubule epi the liu m, yet no chem ical- relate d
exacerbation of renal tubule epithel ial regener atio n

was observed at 13 weeks. There wa s a s lig ht enlargement (karyomegal y) of some nuclei in the rena l
tubule epithelium of male and female rat s. AtDiscussion and Conclusions 83



### the 15-m onth evaluation, this slight nuclear enlar ge

ment was still evide nt, and the seve rity of

ne phrop athy (tubule epithelial regeneration; transi

tional cell hyper

plasia of the rena l p elvis) was increased in expose d male and femal e rats. Accu mu

lation of h yaline droplet s was not pres ent in expose d
male rats a fter the 9-m

onth evaluation. The mor

pho

logical appearance of hyaline droplet s and their

presence only in males is suggesti ve of accum ulatio n of " 2 : 

### -g lobulin in the renal tubule epi the liu m, although the identi ty of the protein droplet s was not determ in ed. Their absence in the kidney tubule cell s of exposed male rats after the 9- month eva l uation i s consistent with the norma

lly de creased p roduction o f

" 2 : 

### -g lobulin by the liver beginning at 5 months of

age (Baetck e et al ., 19 91). Chem icals that cause a hyaline droplet nephropathy syndr ome are often empir ically associated with increases in the incidenc es of benign and malignant renal tubule

ne oplasms, linear foci of mineralization of the ren a l medulla, and enhanc ed nephropathy in male rats afte r 2 years (Baetck

e et al. , 19 91); howev

er, other alte r

native mech anistic explanations exist that do not sho w a dom

inant ro le for " 2 : 

### -globulin. The key to this view centers on se veral chemical s that incite th e "hyaline droplet syndrome, " yet do not indu ce tubul e cell neopl asms of the kidney (Barrett and Huff, 1991 ; Hu ff, 199 2, 1993; Melnick, 1992). Another stron g neoplas tic response in the kidney of femal e rats shows that mechani sms other than th ose associa

te d
with hyaline drople t nephr opath y were invo lved i n the renal tubule neoplasm res ponse i n rats a dmini s

tered 1-amin

o-2,4- dibromoanthraqu

inone. Inc rease d
incidences of benign and malignant n eopl asms of th e kidney occurred in male and femal e r ats in the NT P study of bromodichlorom ethane (NTP, 1987a).

Urinary Bladder: Chemical- related increased
incidences of proli ferat ive lesi ons (hy perp lasia an d neoplasia) of the t

ransitio nal cell

e pit helium

of th e urinary bladder occurred i n male and femal e rats wit h

a greater num

ber of neoplas ms observe d in femal e
rats (45/146, 31%) than in

male rats (16/146 , 1 1%)

. In the stop- exposu

re groups, transit

i onal cell hyper

plasia was present in four f emale rats at 9 months, and, with the absence of continued chem ic al expo

sure, hyp erplasia did not dev elop in male rats a t th e 15-m onth evaluation. A transitional cell car cin om a

occ urred in one femal e rat from the 15-m onth e xpo

sure group. With continuous e xposure to 1- amino

2,4-d ibr omo anth raquinone, benign and m alignan t

neo plas ms of the urinary bladde r d eveloped by 15 months in femal e rats and in bot h
male and femal e rats after 2 years of exposure. In

rats following c hronic admin istra tion of 1,4,5,8-tetra aminoanthraquinone

, a spectrum of nonneoplas tic lesions and neop las ms of the urinar y bladder was observed with similar morph ologic
features including squamous meta pl asia, squamou s cell carcinoma, and pr oliferation of fat (fatty metaplasia) in the wall of

the urina ry bladder (NTP

, 1986a). In that study, calculi were present in the
urin ary bladder of most rat s, ye t there w as a sig nifi

cant increase in the incidence of smoo th muscl e
neop las ms of the wall of the urinary bladder

. The
h ypoth esis of ce ll proliferation and developmen t o f
urin ary bladder neop las ms has been described (Greenf ield et al.

, 1984; Cohen et al.

, 1991). The
mech anism for formation of neop las ms of the urinar y bladder is uncertain. Increased cell proliferation
evidenced by transitional cell hyper plasia in the urinary bladder did no t occur before 9 months in rats

. Poten tial local irritant effects and associated neo

plasm formation in

the urinary bladd

er attributed t o calculus forma

ti on (Okum

ara et al.

, 1992) were no t identified i n this study. Most mice in that study ha d
calculi of the u rin ary bladder

, yet did not have any evidence of carcino genic activi ty. No scientif ic

consensus exists t hat endorses the notion that calc ul i

or stones c ause c ancer; there may be some cocarcino genic or promotion activi ty, yet even this doe s
not occur consist ently (Huff, 1992, 1993).

Forestomach: In both rats and mice, there were several nonneo

plastic prolifer

a tive and inflammator

y lesions in the forestomach at the end of the 2-year studies. These forestomach lesions were not obs erved in either

species in the 1 3-w eek st

udies o r i n
rats at the 9- a

nd 1 5-m onth evaluations. In t he stop

exposu re evaluation, rats exposed to 20,000 ppm develope d nonneo

plastic l esions of the forestomac h by 15 months. H owev er, in the 9-m onth stopexposur e group, forestomach lesions we re obser ve d
in a few m ales by the 15-m onth eva luation , but th e incidences wer e higher tha n those observed in mal e
rats with the continuous 15-m onth exposure. In femal e rats, forestomach lesions were present at 15 mo nths with continuous exposu re but were no t observed at 9 months or after 6 mont hs o f

nonexp osure

. Chemical-

related le sions consisted o f hyperplasia, h

yperk erato sis and associated inf

lamma

tion, and focal e rosion or ulceration of th e squamou s

mucosa. The infl amm

ato ry and ulcer ative lesions

were gener ally more severe and more common in84 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### rats, but significant increases in the incidences of benig n an d malignant forestomach neop las ms wer e limited to mice. These data provi de further evide nc e that inflammation or ulceration does not always
result in neoplas ia (Berenblum, 1944; Huff, 199 2, 1993; Melnick et al

., 1 993a, 1993b)

. The abse nce o f
forestomach neop las ms i n rats may ha ve been relate d

to l ower exposu

re concentrations. Many of t h e malignant forestomach neop las ms of mice
metastasized or invaded adjacent organs.

Increa ses in the incidences o f forestomach neoplasm s have not been observed in mice or rats following long- term admi nistrati o n of fou

r other structur

all y related anth

raquinones. Howev

er, admin istra tion o f
1-amin o-2- methylanthraqu

inone (NCI, 1978b) to rat s for 62 weeks followed by a 6-m onth nonexp osure period was a ssociated wi th an increased incidence i n hyper plasia of the forestomac

h. Adm inistra tion o f
2-meth yl-1- nitroanthraquinon e (NCI, 1978c) to ra t s for 78 weeks followed by a 6-m onth nonexp osure period was a lso asso cia ted with an increased incidence of proli ferat ive lesions of the forestomach.
Marked increases in t

he incidence s of forestomac h

neop las ms have bee n reported for some chem ical s that caused a sustained proli ferat ive response i n th e
squa mous mucosa that was evident within the f irst 2

to 13 weeks of c h emical administration (NTP, 1987b)

. Howev er, a n u mber of other chem icals causing forestomach neoplasm s in r odents have no t been associated with an early, sustained increase i n the incidence of

hyperplasia (NTP, 1 9 90a, 1990b, 1990c).

Lung: The incidences of alveolar/bronchiolar
aden oma and mult

iple alveo lar/bro nchiolar adenom

a (males only) of the lung were sign ificantl y increase d
in mice in the 10,000 and 20,0 00 ppm groups. Althou gh there wa s no evidence for an increase in th e incidence of hyperplasia or for a prog ression of th e lung neop las ms to malignan cy, the incidence of
aden oma in all f

our e xposure groups exceed ed th e NTP h istorical con trol ran ges. Adm inistrat ion of a
structur ally related anthraquinone, C.I. Disperse
Blue 1 (1,4,5,8-tetraa

minoanthraquinone) (NTP, 1986a), resulted in a marginal inc rea se in the
incidence of alveolar/bronch iolar ad enom a in m ale
mice with no associated increase in the i ncide nce o f alveolar/bronchiolar h yperplasia. Ot

her chemical s tested by th e N TP have also caused increased
incidences of lung neop las ms without increased
incidences of a lveolar/bronchiolar hyper plasia (NTP

, 1994), but more comm only an i ncrease in the incidence of alveo lar/bron

chiola r hyper plasia or inf

lam

mation is also presen

t with i ncre ased incide nces o f lung neoplasms (NTP, 1986b, 1989, 1990a, 1992)

. Althou gh the incidence of alve o lar/bronchiolar

car cin oma was not increased in the lungs of mice
admin istered 1-am

ino- 2,4- dib romoanthraquinone, a number of chem icals have induced both alveo lar/
bronchiolar a denomas and carcinomas (NTP, 1990b

, 1990c, 1993b; Huff, 1994).



# C ONCLUSIONS
Under the conditions of these 2-year feed studies,
there was clear ev id

ence of carcino genic acti vity *
of 1 -am ino- 2,4- dibrom

oanthra quin one in male an d
femal e F344/N rats ba sed on increased inc idences o f neopl asms in the l iv er, large intestine, kidney, and urinary bladder

. There was clear evidence o f

carcino genic activity of 1-ami n o

- 2,4-dibrom

oanthraquinone in male and femal e B6C3F 1 

### m ice based on increased incidences of

neop las ms in the liver, forestomach, and lung.

Exposu re of male a nd femal e rats to 1-amin o2,4-dibrom

oanthraquinone for 2 ye ars was associa te d
with ba sophilic focus (males only), clear cell focus

, eosinop hilic focus, and pigmentation in the liver;
renal tubule hyper

pla sia, ren al tubule pigmentation

, and t ransitional cell hyper

plasia in the ki dney; transi

tional cell hyperplasia, squamous metaplasia, and
stromal metap lasia (females only) in the urinary
bladder ; s quamous hyperplasia, hyperkeratosis, ulceration, and inflammation of the forestomach

mucosa; and s emi nal vesicl e atr ophy. Expo sure o f
male and fe ma le mice to 1-amin o

2,4-dibrom

oanthraquinone for 2 years was ass ociate d
with centrilobular hepat o cellular hypertrophy (male s only), basoph ilic focus, clear cell focus (females only), eosinop hilic focus, and pigmentation i n t he liver; pigm

entati on in the kidney; a nd hy perplas ia

, basal cell hyperplasia, hyper kerato sis

, a

nd inf lamma

tion of the forestomach mucosa.

__________

* Explan ation of Levels of Eviden ce of Carc inogeni c Activi ty is on p age 13. A summary of t he Techn ical Rep orts Revi ew Subcom mittee

comme

nts and the

public di scussio

n on this Techn

ical Re port app ears on page

15.85



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# APPENDIX A
SUMMARY OF LESIONS IN MALE RATS
IN THE 2-YEAR FEED STUDY OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 T ABLE 

### A3 Statistical Analysis of Pr imary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 T ABLE 

### A4a Historical Incidence of Hepatocellular Neoplasms in Untreated Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 T ABLE 

### A4b Historical Incidence of Large Intestine Neoplasms in Untreated Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 T ABLE 

### A4c Historical Incidence of Renal Tubule Neoplasms in Untreated Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 T ABLE 

### A4d Historical Incidence of Urinary Bladder Neoplasms in Untreated Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 T ABLE 

### A4e Historical Incidence of Forestomach Squamous Cell Neoplasms in Untreated Male F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 T ABLE 

### A5 Summary of the Incidence of No nneoplastic Lesions in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13894 1-Amino-2,4-dibromoanthraquinone, NTP TR 383Lesions in Male Rats 95



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone a

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Disposition Summary Animals initially in study 70 50 70 70 9-Month interim evaluation 10 10 10 10 15-Month interim evaluation 10 10 Early deaths

Moribund 19 15 34 33 Natural deaths 5 1 5 7 Survivors

Died last week of study 3 Terminal sacrifice 23 24 21 10

Animals examined microscopically 70 50 69 b 70



### 9-Month Interim Evaluation Alimentary System Intestine large, colon (10) (10) (10) (10) Liver (10) (10) (10) (10)

Hepatocellular adenoma 1 (10%)

Endocrine System Adrenal medulla (10) (10) (10) (10) Ganglioneuroma 1 (10%) Thyroid gland (10) (10) (10) (10)

Nervous System Brain (10) (10) (10) (10)

Cerebrum, meningioma benign 1 (10%)



### Systems Examined With No Neoplasms Observed Cardiovascular System General Body System Genital System Hematopoietic System Integumentary System Musculoskeletal System Respiratory System Special Senses System Urinary System96 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

T ABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation Alimentary System Intestine large, rectum (9) (10) Polyp adenomatous 6 (60%)
Liver (10) (10) Hepatocellular carcinoma 4 (40%) Hepatocellular carcinoma, multiple 3 (30%) Hepatocellular adenoma 2 (20%) Hepatocellular adenoma, multiple 2 (20%)
Pancreas (10)
Adenoma 1 (10%) Stomach, forestomach (10) (10)

Endocrine System Pituitary gland (8) Pars distalis, adenoma 1 (13%) Thyroid gland (10) (1)
Adenoma 1 (10%) C-cell, adenoma 1 (10%)

Genital System Preputial gland (9)
Testes (10) (8)
Adenoma 1 (10%) Interstitial cell, adenoma 2 (20%) 5 (63%)

Respiratory System Lung (10) (3)
Alveolar/bronchiolar adenoma 1 (10%) 1 (33%)

Urinary System Kidney (10) (10) Renal tubule, adenoma 2 (20%) Urinary bladder (10) (10)

Systemic Lesions Multiple organs c (10) (10)
Leukemia mononuclear 2 (20%)

Mesothelioma malignant 1 (10%)Lesions in Male Rats 97



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) 

### Systems Examined With No Neoplasms Observed Cardiovascular System General Body System Hematopoietic System Integumentary System Musculoskeletal System Nervous System Special Senses System



### 2-Year Study Alimentary System Intestine large, colon (47) (40) (59) (49)

Adenocarcinoma 1 (2%) 4 (8%) Polyp adenomatous 1 (3%) Polyp adenomatous, multiple 1 (2%) 3 (6%) Intestine large, rectum (46) (40) (58) (49)

Adenocarcinoma 1 (3%) 10 (17%) 12 (24%) Ad e no c a r c i noma, multiple 3 ( 6 % ) Polyp adenomatous 12 (30%) 17 (29%) 10 (20%) Po l y p a denom a tous, mu l tip l e 1 (3%) 3 4 (59%) 3 0 (61%) Intestine large, cecum (48) (40) (59) (50) Intestine small, duodenum (48) (40) (59) (50) Intestine small, jejunum (48) (38) (57) (48) Intestine sm all

, i l eum (48) (40) (57) (49) Liver (50) (40) (59) (50)

Cholangiocarcinoma 1 ( 2 % ) Cholangioma 2 (3%)
H e p a toce ll u la r c a rcinoma 1 (2%) 11 (28%) 12 (20%) 9 (18%)
H e pato c e llular c a r c inoma, multiple 1 ( 3 %) 4 3 ( 73 %) 3 7 ( 74 %)
H e p a toce ll u la r a denoma 1 (2%) 10 (25%) 17 (29%) 10 (20%) Hepatocellular adenoma, multiple 10 (25%) 23 (39%) 24 (48%) Hepatocholangiocarcinoma 5 (8%) 2 (4%) Hepatocholangiocarcinoma, multiple 1 (2%) Hepatocholangioma 1 (2%) 1 (2%) Myxoma 1 (2%) Mesentery (3) (2) (4) (4) Pancreas (50) (40) (59) (50)

Adenocarcinoma, metastatic, intestine large, rectum 1 (2%) Adenoma 2 (4%) Salivary glands (50) (40) (58) (49) Stomach, forestomach (49) (39) (59) (49)

Squamous cell carcinoma 1 (2%) Squamous cell papilloma 2 (5%) 1 (2%) Stomach, glandular (50) (40) (59) (50)

Cardiovascular System Heart (50) (40) (59) (49)98 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Endocrine System Adrenal cortex (50) (40) (58) (50)
H e pato c e llular c a r c inoma, m e ta s tati c , liv e r 1 (2%)

Adren a l medu ll a (50) (40) (58) (50) Pheochromocytoma malignant 1 (2%) 2 (5%) 1 (2%) Pheochromocytoma benign 11 (22%) 1 2 (30%) 1 1 (19%) 5 (10%) Bilateral, pheochromocytoma benign 1 (2%) 3 (8%) 2 (3%) 2 (4%) Islets, pancreatic (50) (40) (58) (50)

Adenoma 2 (4%) 2 (5%) 1 (2%) Parathyroid gland (43) (35) (55) (44)

Adenoma 1 ( 2 %) Pituitary gland (48) (40) (56) (49)

P a rs dist al is, a denoma 20 (42%) 12 (30%) 9 (16%) 10 (20%) Pa r s di s tali s , ad e noma, multiple 1 ( 2 %) 2 ( 5 %) 1 ( 2 %) Pa r s di s tali s , c a r c inoma 1 ( 3 %) Pars intermedia, adenoma 1 (3%) Thyroid gland (49) (40) (59) (50)

Adenoma 1 (2%) C-cell, adenoma 9 (18%) 5 (13%) 3 (5%) 3 (6%) Follicular cell, adenoma 2 (3%) 1 (2%) Follicular cell, carcinoma 2 (4%) 3 (8%) 1 (2%) 3 (6%)

General Body System

None

Genital System Coagulating gland (2) (2) Epididymis (50) (40) (59) (50) Preputial gland (49) (39) (58) (47)

Adenocarcinoma 1 (2%) 1 (2%) Adenoma 3 (6%) 2 (5%) 1 (2%) 1 (2%) Prostate (50) (40) (59) (49) Seminal vesicle (49) (40) (59) (50) Testes (50) (40) (59) (50) Bilateral, interstitial cell, adenoma 40 (80%) 34 (85%) 49 (83%) 38 (76%) Interstitial cell, adenoma 3 (6%) 3 (8%) 6 (10%) 5 (10%)

Hematopoietic System Bone marrow (50) (40) (59) (50) Lymph node (17) (5) (12) (20)

Lumbar, adenocarcinoma, metastatic, intestine large, rectum 1 (8%) Lymph node, mandibular (50) (40) (54) (48) Lymph node, mesenteric (48) (40) (57) (49)

Adenocarcinoma, metastatic, intestine large, colon 1 (2%)Lesions in Male Rats 99



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Spleen (50) (40) (58) (50)

Fibroma 1 (2%) Sarcoma 1 (3%) Thymus (37) (32) (41) (34)

Thymoma benign 1 (2%)

Integumentary System Mammary gland (27) (22) (29) (24)

Adenocarcinoma 1 (3%) Fibroadenoma 1 (5%) Fibroadenoma, multiple 1 (4%) Skin (50) (38) (58) (50)

Basal cell adenoma 1 (2%) 1 (2%) Basal cell carcinoma 2 (4%) Keratoacanthoma 1 (2%) 1 (3%) 1 (2%) Squamous cell papilloma 1 (2%) 3 (5%) 1 (2%) Trichoepithelioma 1 (2%) Pinna, squamous cell papilloma 2 (5%) 2 (3%) 1 (2%) Subcutaneous tissue, fibroma 2 (4%) 3 (5%) 1 (2%) Subcutaneous tissue, fibrosarcoma 1 (2%) 1 (2%) Subcutaneous tissue, lipoma 1 (2%) Subcutaneous tissue, sarcoma 1 (2%) 3 (8%) 1 (2%)

Musculoskeletal System Skeletal muscle (2) (1) (1)

Nervous System Brain (50) (40) (59) (50)

Carcinoma, metastatic, pituitary gland 1 (3%) Oligodendroglioma benign 1 (2%) Meninges, granular cell tumor benign 1 (2%) Peripher a l nerve (2)

Squamous cell carcinoma, metastatic, uncertain primary site 1 (50%) Spinal cord (5) (1) (1) (4)

Respiratory System Lung (50) (40) (59) (49)

Adenocarcinoma, metastatic, kidney 1 (2%) Adenocarcinoma, metastatic, intestine large, colon 1 (2%) Alveolar/bronchiolar adenoma 2 (5%) 2 (3%) 3 (6%) Alveolar/bronchiolar carcinoma 1 (3%) 2 (3%) Hepatocellular carcinoma, metastatic, liver 1 (3%) 18 (31%) 19 (39%) Sarcoma, metastatic, kidney 1 (2%) Sarcoma, metastatic, skin 1 (2%) Mediastinum, alveolar/bronchiolar carcinoma 1 (2%)100 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Respiratory System (continued) Nose (48) (40) (59) (50) Trachea (50) (40) (59) (50)

Special Senses System Ear (3) (7) (2) (1)

Fibrosarcoma 1 (33%) Eye (6) (3) (1) (6) Zymbal's gland (1) (1) (1) (2) Carcinoma 1 (100%) 1 (100%) 1 (50%) Squamous cell carcinoma 1 (100%) 1 (50%)

Urinary System Kidney (50) (40) (59) (50)

Hepatocellular carcinoma, metastatic, liver 1 (3%) Pelvis, transitional epithelium, papilloma 1 (2%) Renal tubule, adenoma 2 (4%) 6 (15%) 7 (12%) 9 (18%) Renal tubule, adenoma, multiple 4 (10%) 4 (7%) 5 (10%) Renal tubule, carcinoma 2 (3%) 1 (2%) Urinary bladder (50) (38) (58) (50)

Transitional epithelium, carcinoma 1 (2%) 4 (8%) Transitional epithelium, papilloma 1 (3%) 2 (3%) 8 (16%)

Systemic Lesions Multiple organs (50) (40) (59) (50) Leukemia mononuclear 25 (50%) 5 (13%) 3 (5%) 1 (2%) Mesothelioma malignant 1 (2%) 1 (3%) 1 (2%) 1 (2%)

Neoplasm Summary Total animals with primary neoplasms d

9-Month interim evaluation 1 2 15-Month interim evaluation 7 10 2-Year study 49 40 59 48 Total primary neoplasms 9-Month interim evaluation 1 2 15-Month interim evaluation 8 28 2-Year study 140 160 297 258 Total animals with benign neoplasms 9-Month interim evaluation 1 2 15-Month interim evaluation 7 10 2-Year study 49 40 58 47 Total benign neoplasms 9-Month interim evaluation 1 2 15-Month interim evaluation 8 18 2-Year study 103 129 209 176Lesions in Male Rats 101



### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Neoplasm Summary (continued) Total animals with malignant neoplasms 15-Month interim evaluation 8 2-Year study 29 24 57 47 Total malignant neoplasms 15-Month interim evaluation 10 2-Year study 37 31 88 82 Total animals with metastatic neoplasms 2-Year study 2 3 20 22 Total metastatic neoplasms 2-Year study 2 3 23 22 Total animals with malignant neoplasms

of uncertain primary site 2-Year study 1

a Number of animals examined microscopically at site and number of animals with neoplasm b One animal not examined microscopically

c Number of animals with any tissue examined microscopically d Primary neoplasms: all neoplasms except metastatic neoplasms102 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm

4445555555666666666677777 Number of Days on Study 2231456778345677779902223
1754962347164614492671171

1100101110000000100000101 Carcass ID Number 1278134321835361081927153
5255435445424154532334331

Alimentary System Esophagus + + + + + + + + + + M + + + + + + + + + + + + + + Intestine large, colon A + + + + + + + + + A + M + + + + + + + + + + + + Intestine large, rectum A + + + + + + + + + A + + + + + + + + + + + M + + Intestine large, cecum A + + + + + + + + + A + + + + + + + + + + + + + + Intestine small, duodenum A + + + + + + + + + A + + + + + + + + + + + + + + Intestine small, jejunum A + + + + + + + + + A + + + + + + + + + + + + + + Intestine small, ileum A + + + + + + + + + A + + + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma X Hepatocellular adenoma Mesentery + + + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant X Pheochromocytoma benign X X X X X Bilateral, pheochromocytoma benign Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma Parathyroid gland + + + M + + + + + + + + + M + M + + + + + M + + M Pituitary gland + + M + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X Pars distalis, adenoma, multiple X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X X X X Follicular cell, carcinoma X

General Body System

None

Genital System Coagulating gland + Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X Adenoma X

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examinedLesions in Male Rats 103



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
1134444444444444444455555

1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 1 Total Carcass ID Number 3 4 4 2 2 4 5 5 6 6 7 8 9 0 0 0 1 2 2 4 1 6 7 8 4 Tissues/
2 2 3 1 2 2 1 2 1 3 2 1 1 1 3 4 2 1 3 1 1 4 3 2 4 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine large, rectum + + + + + + + + + + + + M + + + + + + + + + + + + 46 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatocellular carcinoma 1 Hepatocellular adenoma X 1 Mesentery 3 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + M + + + + + + + + + + + + + + + + + 49 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant 1 Pheochromocytoma benign X X X X X X 11 Bilateral, pheochromocytoma benign X 1 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X X 2 Parathyroid gland + + + + + + + + + + + + + M + + + + + + + + + M + 43 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + I + 48

Pars distalis, adenoma X X X X X X X X X X 20 Pars distalis, adenoma, multiple 1 Thyroid gland + + + + + + + + + + + + + A + + + + + + + + + + + 49

C-cell, adenoma X X X X 9 Follicular cell, carcinoma X 2

General Body System

None

Genital System Coagulating gland + 2 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 Preputial gland + + + + + + + + + + + + + + M + + + + + + + + + + 49

Adenocarcinoma 1 Adenoma X X 3104 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

4445555555666666666677777 Number of Days on Study 2231456778345677779902223
1754962347164614492671171

1100101110000000100000101 Carcass ID Number 1278134321835361081927153
5255435445424154532334331

Genital System (continued) Prostate + + + + + + + + + + + + + + + + + + + + + + + + + Seminal vesicle + + + M + + + + + + + + + + + + + + + + + + + + + Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X Interstitial cell, adenoma X X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + + + + + + + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + M + + + + + + + + + + + M + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + M + + M + + + + + M + + M + + + M M + + M + +

Integumentary System Mammary gland M + M + + + M M + M + + M + + M M + + + M + M M M

Fibroadenoma, multiple X Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell carcinoma Keratoacanthoma X Squamous cell papilloma Trichoepithelioma Subcutaneous tissue, fibroma X Subcutaneous tissue, fibrosarcoma X Subcutaneous tissue, lipoma X Subcutaneous tissue, sarcoma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Oligodendroglioma benign X Spinal cord + + + + +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant,

metastatic, adrenal medulla X Sarcoma, metastatic, skin X Nose + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Male Rats 105



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
1134444444444444444455555

1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 1 Total Carcass ID Number 3 4 4 2 2 4 5 5 6 6 7 8 9 0 0 0 1 2 2 4 1 6 7 8 4 Tissues/
2 2 3 1 2 2 1 2 1 3 2 1 1 1 3 4 2 1 3 1 1 4 3 2 4 Tumors

Genital System (continued) Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 50 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 49 Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X 40 Interstitial cell, adenoma X 3

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node + + + + + + 17 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 48 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus M M M + M + + + + M + + + + + + + + M + + + + + + 37

Integumentary System Mammary gland M M + + + M M + + + + MMMM+M++MM + + + + 27

Fibroadenoma, multiple 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell carcinoma X X 2 Keratoacanthoma 1 Squamous cell papilloma X 1 Trichoepithelioma X 1 Subcutaneous tissue, fibroma X 2 Subcutaneous tissue, fibrosarcoma 1 Subcutaneous tissue, lipoma 1 Subcutaneous tissue, sarcoma 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50 Skeletal muscle + 2

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Oligodendroglioma benign 1 Spinal cord 5

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma malignant,

metastatic, adrenal medulla 1 Sarcoma, metastatic, skin 1 Nose + + M + + + + + + + + + + + + + + + + + + + + + + 48 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50106 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

4445555555666666666677777 Number of Days on Study 2231456778345677779902223
1754962347164614492671171

1100101110000000100000101 Carcass ID Number 1278134321835361081927153
5255435445424154532334331

Special Senses System

Ear + + +

Fibrosarcoma X Eye + + + + + Zymbal's gland

Carcinoma

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Renal tubule, adenoma Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X X X X X X X X X X X X X X Mesothelioma malignantLesions in Male Rats 107



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
1134444444444444444455555

1 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 1 Total Carcass ID Number 3 4 4 2 2 4 5 5 6 6 7 8 9 0 0 0 1 2 2 4 1 6 7 8 4 Tissues/
2 2 3 1 2 2 1 2 1 3 2 1 1 1 3 4 2 1 3 1 1 4 3 2 4 Tumors

Special Senses System

Ear 3

Fibrosarcoma 1 Eye I + 6 Zymbal's gland + 1

Carcinoma X 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Renal tubule, adenoma X X 2 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X X X X X 25 Mesothelioma malignant X 1108 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm

6666666666666777777777777 Number of Days on Study 0124555677799012333333333
4890039834534080000111111

1112212212212211222111111 Carcass ID Number 5682260450174067333558889
4542144333452234345121231

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp adenomatous Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X Polyp adenomatous X X X X X X X Polyp adenomatous, multiple Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + + + + + + + + + M + + M + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma X X X X X X X Hepatocellular carcinoma, multiple Hepatocellular adenoma X X X X X Hepatocellular adenoma, multiple X X X X X X Mesentery + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + M + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma X X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant X X Pheochromocytoma benign X X X X X X X X Bilateral, pheochromocytoma benign X X Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X X Parathyroid gland + + + + + + + + + + + + + + + + + M + M + + + + + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X Pars distalis, adenoma, multiple X Pars distalis, carcinoma X Pars intermedia, adenoma Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X X Follicular cell, carcinoma X X X

General Body System

NoneLesions in Male Rats 109



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
111111444444444

1 1 1 2 2 2 1 1 1 1 1 2 2 2 2 Total Carcass ID Number 9 9 9 0 3 3 6 6 7 7 7 1 1 1 4 Tissues/
2 3 4 1 1 2 1 2 1 2 3 1 2 3 1 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + 40 Intestine large, colon + + + + + + + + + + + + + + + 40

Polyp adenomatous X 1 Intestine large, rectum + + + + + + + + + + + + + + + 40

Adenocarcinoma 1 Polyp adenomatous X X X X X 12 Polyp adenomatous, multiple X 1 Intestine large, cecum + + + + + + + + + + + + + + + 40 Intestine small, duodenum + + + + + + + + + + + + + + + 40 Intestine small, jejunum + + + + + + + + + + + + + + + 38 Intestine small, ileum + + + + + + + + + + + + + + + 40 Liver + + + + + + + + + + + + + + + 40

Hepatocellular carcinoma X X X X 11 Hepatocellular carcinoma, multiple X 1 Hepatocellular adenoma X X X X X 10 Hepatocellular adenoma, multiple X X X X 10 Mesentery + 2 Pancreas + + + + + + + + + + + + + + + 40 Salivary glands + + + + + + + + + + + + + + + 40 Stomach, forestomach + + + + + + + + + + + + + + + 39

Squamous cell papilloma 2 Stomach, glandular + + + + + + + + + + + + + + + 40

Cardiovascular System

Heart + + + + + + + + + + + + + + + 40

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + 40 Adrenal medulla + + + + + + + + + + + + + + + 40

Pheochromocytoma malignant 2 Pheochromocytoma benign X X X X 12 Bilateral, pheochromocytoma benign X 3 Islets, pancreatic + + + + + + + + + + + + + + + 40

Adenoma 2 Parathyroid gland M + + + + + + + M + M + + + + 35 Pituitary gland + + + + + + + + + + + + + + + 40

Pars distalis, adenoma X X X 12 Pars distalis, adenoma, multiple X 2 Pars distalis, carcinoma 1 Pars intermedia, adenoma X 1 Thyroid gland + + + + + + + + + + + + + + + 40

C-cell, adenoma X X 5 Follicular cell, carcinoma 3

General Body System

None110 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm (continued)

6666666666666777777777777 Number of Days on Study 0124555677799012333333333
4890039834534080000111111

1112212212212211222111111 Carcass ID Number 5682260450174067333558889
4542144333452234345121231

Genital System Coagulating gland + + Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + + + + + + + + + + + + + + + + + + M + + + + +

Adenoma X X Prostate + + + + + + + + + + + + + + + + + + + + + + + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X Interstitial cell, adenoma X X X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma Thymus M M M M +++M+++M+++M+++++++M+

Integumentary System Mammary gland + + + M + M + M + M M + + + M M + MMM+M+++

Fibroadenoma Skin + + + + + + + + + + + + + + + + + + + + + I + + +

Keratoacanthoma X Pinna, squamous cell papilloma X Subcutaneous tissue, sarcoma X X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma, metastatic, pituitary

gland X Spinal cord +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma X Hepatocellular carcinoma, metastatic,

liver Nose + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Male Rats 111



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
111111444444444

1 1 1 2 2 2 1 1 1 1 1 2 2 2 2 Total Carcass ID Number 9 9 9 0 3 3 6 6 7 7 7 1 1 1 4 Tissues/
2 3 4 1 1 2 1 2 1 2 3 1 2 3 1 Tumors

Genital System Coagulating gland 2 Epididymis + + + + + + + + + + + + + + + 40 Preputial gland + + + + + + + + + + + + + + + 39

Adenoma 2 Prostate + + + + + + + + + + + + + + + 40 Seminal vesicle + + + + + + + + + + + + + + + 40 Testes + + + + + + + + + + + + + + + 40

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X 34 Interstitial cell, adenoma 3

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + 40 Lymph node 5 Lymph node, mandibular + + + + + + + + + + + + + + + 40 Lymph node, mesenteric + + + + + + + + + + + + + + + 40 Spleen + + + + + + + + + + + + + + + 40

Sarcoma X 1 Thymus + + + + + + + + + + + + + + + 32

Integumentary System Mammary gland + M + + + + M + M + M M M M + 22

Fibroadenoma X 1 Skin + + + + + + + + + I + + + + + 38

Keratoacanthoma 1 Pinna, squamous cell papilloma X 2 Subcutaneous tissue, sarcoma X 3

Musculoskeletal System

Bone + + + + + + + + + + + + + + + 40 Skeletal muscle 1

Nervous System

Brain + + + + + + + + + + + + + + + 40

Carcinoma, metastatic, pituitary

gland 1 Spinal cord 1

Respiratory System Lung + + + + + + + + + + + + + + + 40

Alveolar/bronchiolar adenoma X X 2 Alveolar/bronchiolar carcinoma 1 Hepatocellular carcinoma, metastatic,

liver X 1 Nose + + + + + + + + + + + + + + + 40 Trachea + + + + + + + + + + + + + + + 40112 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm (continued)

6666666666666777777777777 Number of Days on Study 0124555677799012333333333
4890039834534080000111111

1112212212212211222111111 Carcass ID Number 5682260450174067333558889
4542144333452234345121231

Special Senses System

Ear + + + + + + + Eye I + Zymbal's gland +

Carcinoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liver X Renal tubule, adenoma X X X Renal tubule, adenoma, multiple X X X X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + M +

Transitional epithelium, papilloma X

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X Mesothelioma malignant XLesions in Male Rats 113



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
111111444444444

1 1 1 2 2 2 1 1 1 1 1 2 2 2 2 Total Carcass ID Number 9 9 9 0 3 3 6 6 7 7 7 1 1 1 4 Tissues/
2 3 4 1 1 2 1 2 1 2 3 1 2 3 1 Tumors

Special Senses System

Ear 7 Eye + + 3 Zymbal's gland 1

Carcinoma 1

Urinary System Kidney + + + + + + + + + + + + + + + 40

Hepatocellular carcinoma, metastatic,

liver 1 Renal tubule, adenoma X X X 6 Renal tubule, adenoma, multiple 4 Urinary bladder + + + + M + + + + + + + + + + 38

Transitional epithelium, papilloma 1

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + 40

Leukemia mononuclear X X 5 Mesothelioma malignant 1114 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 5,000 ppm

445555556666666666666666666667 Number of Days on Study 672267880002223335555567778890
581136245893996670023914445730

332332333322333332332322333323 Carcass ID Number 526507328799154438285457135870
533445425543432335145144322333

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + M + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma Polyp adenomatous, multiple Intestine large, rectum + + + + + M + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X X X X X Polyp adenomatous X X X X X X X X X X X Polyp adenomatous, multiple X X X X X X X X X X X Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + A + + + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Cholangioma X Hepatocellular carcinoma X X X X X X Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X X X X X Hepatocellular adenoma X X X X X X X X X Hepatocellular adenoma, multiple X X X X X X X X Hepatocholangiocarcinoma X Hepatocholangiocarcinoma, multiple X Hepatocholangioma X Myxoma Mesentery + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + M + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Tooth + +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma malignant X Pheochromocytoma benign X X X X X X Bilateral, pheochromocytoma benign Islets, pancreatic + A + + + + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + + + + + + + + M + + + + + + + + + + + + + + + M + + + +

Adenoma Pituitary gland + + + + + + + + + + + + M + + + + + + + + + + + + M + + + +

Pars distalis, adenoma X X Pars distalis, adenoma, multiple X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma Follicular cell, adenoma X Follicular cell, carcinoma XL

esion s in M ale R ats 115

T ABLE A2

Individu al Anim al Tum or Pa thology of M ale Rat s in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 5,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 0 0 0 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3
0 2 9 2 4 5 0 8 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0

3 3 3 2 2 3 2 3 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carc ass ID N um ber 7 1 6 6 8 6 5 6 5 5 6 7 7 8 8 8 9 9 0 0 1 3 5 6 6 7 7 8 8 Tissues/
3 2 5 2 4 4 3 3 1 2 1 1 2 1 2 3 1 2 1 2 1 1 1 1 2 1 2 1 2 Tum ors

Alimen tary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58
Intest ine lar ge, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Adeno car ci noma X 1
Po lyp ade no ma tou s, m ultip le X 1
Intest ine lar ge, rect um + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Adeno car ci noma X X X X X 10 Polyp adenomatous X X X X X X 17
Po lyp ade no ma tou s, m ultip le X X X X X X X X X X X X X X X X X X X X X X X 34
Intest ine lar ge, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59
Intest ine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59
Intest ine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + M + + + + 57
Intest ine small, ileum + + + M + + + + + + + + + + + + + + + + + + + + + + + + + 57 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Chol ang io ma X 2 Hepa to cel lu la r c ar ci noma X X X X X X 12
He pat oce llula r ca rcin om a, m ultip le X X X X X X X X X X X X X X X X X X X X X X 43 Hepa to cel lu la r a den oma X X X X X X X X 17
He pat oce llula r ad eno ma , m ultip le X X X X X X X X X X X X X X X 23 Hepa to cho la ngi oca rc in oma X X X X 5
He pat och ola ng ioc arc ino ma , m ultip le 1 Hepa to cho la ngi oma 1 Myxoma X 1
Me sentery + + + 4
Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Salivary gl ands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Stomach , forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Stomach , glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59
To oth 2

Card iovascular System

Hear t + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

En docrine S ystem
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + M + + + + 58 Ad ren al m edu lla + + + + + + + + + + + + + + + + + + + + + + + + M + + + + 58 Pheochromocytoma malignant 1 Pheochromocytoma benign X X X X X 11 Bilater al, pheochromocytoma benign X X 2 Isle ts, p anc rea tic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58 Parathyroid gl and + + + + M + + + + + + + + + + + + M + + + + + + + + + + + 55 Adeno ma X 1 Pituit ary gland + + + + + M + + + + + + + + + + + + + + + + + + + + + + + 56
Par s di st al is , a den oma X X X X X X X 9
Pa rs d ista lis, a den om a, m ultip le 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59
C-c el l, ade noma X X X 3 Fol li cul ar cel l, ade noma X 2 Fol li cul ar cel l, car ci noma 1116 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 5,000 ppm (continued)

445555556666666666666666666667 Number of Days on Study 672267880002223335555567778890
581136245893996670023914445730

332332333322333332332322333323 Carcass ID Number 526507328799154438285457135870
533445425543432335145144322333

General Body System

None

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X Adenoma X Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X X Interstitial cell, adenoma X X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + +

Lumbar, adenocarcinoma, metastatic,

intestine large, rectum X Lymph node, mandibular + + + + + + M M + + + + + + + + + + + + + + + + + + M + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + M

Adenocarcinoma, metastatic,

intestine large, colon Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Fibroma Thymus + M + + M + M M M M ++++++++ MMM++M++M+++

Thymoma benign

Integumentary System Mammary gland M M + + M + + + + + + + + M + M + MMMM ++++ MMM++

Adenocarcinoma X Skin + I + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma Squamous cell papilloma X Pinna, squamous cell papilloma Subcutaneous tissue, fibroma Subcutaneous tissue, fibrosarcoma X Subcutaneous tissue, sarcoma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle +Lesions in Male Rats 117



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 5,000 ppm (continued)

77777777777777777777777777777 Number of Days on Study 00011122222222222222233333333
02924508999999999999900000000

3 3 3 2 2 3 2 3 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carcass ID Number 7 1 6 6 8 6 5 6 5 5 6 7 7 8 8 8 9 9 0 0 1 3 5 6 6 7 7 8 8 Tissues/
3 2 5 2 4 4 3 3 1 2 1 1 2 1 2 3 1 2 1 2 1 1 1 1 2 1 2 1 2 Tumors

General Body System

None

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Preputial gland + M + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Adenocarcinoma 1 Adenoma 1 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Testes + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X X X X 49 Interstitial cell, adenoma X X X X 6

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Lymph node + + + + + + + 12

Lumbar, adenocarcinoma, metastatic,

intestine large, rectum 1 Lymph node, mandibular + + M + + + + + + + + + + + + + + + + + + M + + + + + + + 54 Lymph node, mesenteric M + + + + + + + + + + + + + + + + + + + + + + + + + + + + 57

Adenocarcinoma, metastatic,

intestine large, colon X 1 Spleen + + + + + + + M + + + + + + + + + + + + + + + + + + + + + 58

Fibroma X 1 Thymus M + M M + + M M + + M + + + + + + + + + + + + + + + + + M 41

Thymoma benign X 1

Integumentary System Mammary gland M M M M M M M + M M M M M M + M M M + M + + + + + M + + + 29

Adenocarcinoma 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58

Basal cell adenoma X 1 Squamous cell papilloma X X 3 Pinna, squamous cell papilloma X X 2 Subcutaneous tissue, fibroma X X X 3 Subcutaneous tissue, fibrosarcoma 1 Subcutaneous tissue, sarcoma 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Skeletal muscle 1118 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 5,000 ppm (continued)

445555556666666666666666666667 Number of Days on Study 672267880002223335555567778890
581136245893996670023914445730

332332333322333332332322333323 Carcass ID Number 526507328799154438285457135870
533445425543432335145144322333

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Meninges, granular cell tumor benign Peripheral nerve +

Squamous cell carcinoma, metastatic,

uncertain primary site Spinal cord +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma, metastatic, kidney X Adenocarcinoma, metastatic,

intestine large, colon Alveolar/bronchiolar adenoma X Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic,

liver X X X X X X X X X X X X X Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System

Ear + Eye + Zymbal's gland +

Squamous cell carcinoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Pelvis, transitional epithelium,

papilloma X Renal tubule, adenoma X X X Renal tubule, adenoma, multiple X X Renal tubule, carcinoma X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + M +

Transitional epithelium, carcinoma Transitional epithelium, papilloma

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X Mesothelioma malignantL

esion s in M ale R ats 119

T ABLE A2

Individu al Anim al Tum or Pa thology of M ale Rat s in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 5,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 0 0 0 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3
0 2 9 2 4 5 0 8 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0

3 3 3 2 2 3 2 3 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carc ass ID N um ber 7 1 6 6 8 6 5 6 5 5 6 7 7 8 8 8 9 9 0 0 1 3 5 6 6 7 7 8 8 Tissues/
3 2 5 2 4 4 3 3 1 2 1 1 2 1 2 3 1 2 1 2 1 1 1 1 2 1 2 1 2 Tum ors

Nerv ous System

Br ain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Meninges, granular ce ll t umor benign X 1 Peripheral nerve + 2 Squam ous cell carcinoma, metastatic,

un cer tain prim ary site X 1 Spina l cord 1

Resp iratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Adeno carcinoma, metastatic, kidney 1 Adeno carcinoma, metastatic,
intest ine lar ge, colon X 1 Alve ol ar /b ron chi ol ar

ade noma X 2 Alve ol ar /b ron chi ol ar

car ci noma X X 2 Hepatocellular carcinoma, m etastatic,
liver X X X X X 18
Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Special Senses S

ystem

Ear + 2 Eye 1 Zymbal's gland 1 Squa mous c el l c ar ci noma 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Pel vi s, tr ans it io nal epi th el iu m,
pap il lo ma 1
Rena l t ubul e, ade noma X X X X 7
Re nal tub ule , ad eno ma , m ultip le X X 4
Rena l t ubul e, car ci noma X 2 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 58
Tra nsi ti ona l e pi th el iu m, c ar ci noma X 1
Tra nsi ti ona l e pi th el iu m, pa pi ll oma X X 2

Syst emic Les io ns Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Leukem ia monon uclear X X 3 Mesothelioma malignant X 1120 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm

2334444455555555555666666 Number of Days on Study 8563379924557788999012223
1245663772233718016502451

4454443444534444443544444 Carcass ID Number 4904939758098526359124193
5544445553242432332523431

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X X Polyp adenomatous, multiple X Intestine large, rectum + + + + + + + + + M + + + + + + + + + + + + + + +

Adenocarcinoma X X X X Adenocarcinoma, multiple X X Polyp adenomatous X X X X X X X X Polyp adenomatous, multiple X X X X X X X Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + A + + + + A + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + A + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Cholangiocarcinoma Hepatocellular carcinoma X X X X X X X X Hepatocellular carcinoma, multiple X X X X X X X X X X X X X Hepatocellular adenoma X X X X Hepatocellular adenoma, multiple X X X X X X X Hepatocholangiocarcinoma X Hepatocholangioma X Mesentery + + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma, metastatic,

intestine large, rectum X Pharynx + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + M + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma Squamous cell papilloma Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tongue

Cardiovascular System

Heart + + + + + + + + + + + + M + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X Bilateral, pheochromocytoma benign X Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

AdenomaLesions in Male Rats 121



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666666777777777777777777 Number of Days on Study 3477778000112222222222222
1614499239451449999999999

5 5 5 5 5 4 4 5 4 3 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 Total Carcass ID Number 2 0 2 1 2 0 7 1 9 9 5 7 2 7 8 0 1 1 4 4 6 9 1 1 2 Tissues/
4 1 3 4 2 4 4 3 2 1 2 3 1 1 1 1 2 3 1 2 1 1 1 2 1 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + M + + + + + + 49

Adenocarcinoma X X 4 Polyp adenomatous, multiple X X 3 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenocarcinoma X X X X X X X X 12 Adenocarcinoma, multiple X 3 Polyp adenomatous X X 10 Polyp adenomatous, multiple X X X X X X X X X X X X X X X X X X X X X X X 30 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Cholangiocarcinoma X 1 Hepatocellular carcinoma X 9 Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X X X X X X X X 37 Hepatocellular adenoma X X X X X X 10 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X 24 Hepatocholangiocarcinoma X 2 Hepatocholangioma 1 Mesentery + + 4 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenocarcinoma, metastatic,

intestine large, rectum 1 Pharynx 1 Salivary glands + + M + + + + + + + + + + + + + + + + + + + + + + 49 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49

Squamous cell carcinoma X 1 Squamous cell papilloma X 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Tongue + 1

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 49

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatocellular carcinoma, metastatic,

liver X 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign X X X X 5 Bilateral, pheochromocytoma benign X 2 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1122 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

2334444455555555555666666 Number of Days on Study 8563379924557788999012223
1245663772233718016502451

4454443444534444443544444 Carcass ID Number 4904939758098526359124193
5544445553242432332523431

Endocrine System (continued) Parathyroid gland + + + + + + + + + + M + + M + + + + M + + + + + + Pituitary gland + + + M + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X C-cell, adenoma Follicular cell, adenoma Follicular cell, carcinoma X

General Body System

None

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + Preputial gland + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X Prostate + + + + + M + + + + + + + + + + + + + + + + + + + Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X Interstitial cell, adenoma

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + + + Lymph node, mandibular + + + + + + M + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + M + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus M + + M + + + M + + + + M + + M + M + + + M + M +

Integumentary System Mammary gland M + M M M M +++++M++++M++++++MM Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Basal cell adenoma Keratoacanthoma X Squamous cell papilloma Pinna, squamous cell papilloma Subcutaneous tissue, fibroma

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Spinal cord + + +Lesions in Male Rats 123



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666666777777777777777777 Number of Days on Study 3477778000112222222222222
1614499239451449999999999

5 5 5 5 5 4 4 5 4 3 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 Total Carcass ID Number 2 0 2 1 2 0 7 1 9 9 5 7 2 7 8 0 1 1 4 4 6 9 1 1 2 Tissues/
4 1 3 4 2 4 4 3 2 1 2 3 1 1 1 1 2 3 1 2 1 1 1 2 1 Tumors

Endocrine System (continued) Parathyroid gland + + + + + M + + + + M + + + + M + + + + + + + + + 44 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

Pars distalis, adenoma X X X X X 10 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma 1 C-cell, adenoma X X X 3 Follicular cell, adenoma X 1 Follicular cell, carcinoma X X 3

General Body System

None

Genital System Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50 Preputial gland M + + + + + M + + + + + + + + + + + M + + + + + + 47

Adenoma 1 Prostate + + + + + + + + + + + + + + + + + + + + + + + + + 49 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 50 Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Bilateral, interstitial cell, adenoma X X X X X X X X X X X X X X X X X X X X X 38 Interstitial cell, adenoma X X X X X 5

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node + + + + + + + + + + + + + 20 Lymph node, mandibular + + + + + + + + + + + + + + + + + + M + + + + + + 48 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 49 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus M M + + M + M + M + + + M + M M + + + + + + + + + 34

Integumentary System Mammary gland M + M + M M M + + M M M + M M M M M M M M + + + M 24 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basal cell adenoma X 1 Keratoacanthoma 1 Squamous cell papilloma X 1 Pinna, squamous cell papilloma X 1 Subcutaneous tissue, fibroma X 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Spinal cord + 4124 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

2334444455555555555666666 Number of Days on Study 8563379924557788999012223
1245663772233718016502451

4454443444534444443544444 Carcass ID Number 4904939758098526359124193
5544445553242432332523431

Respiratory System Lung + + + + + + + + + + + + M + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X Hepatocellular carcinoma, metastatic,

liver X X X X X X X X X Sarcoma, metastatic, kidney X Mediastinum, alveolar/bronchiolar

carcinoma X Nose + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System

Ear Eye M Zymbal's gland +

Carcinoma X Squamous cell carcinoma

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Renal tubule, adenoma X Renal tubule, adenoma, multiple X Renal tubule, carcinoma X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Transitional epithelium, carcinoma Transitional epithelium, papilloma X X X

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X Mesothelioma malignant XLesions in Male Rats 125



### T ABLE 

### A2 Individual Animal Tumor Pathology of Male Rats in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666666777777777777777777 Number of Days on Study 3477778000112222222222222
1614499239451449999999999

5 5 5 5 5 4 4 5 4 3 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 Total Carcass ID Number 2 0 2 1 2 0 7 1 9 9 5 7 2 7 8 0 1 1 4 4 6 9 1 1 2 Tissues/
4 1 3 4 2 4 4 3 2 1 2 3 1 1 1 1 2 3 1 2 1 1 1 2 1 Tumors

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 49

Alveolar/bronchiolar adenoma X X 3 Hepatocellular carcinoma, metastatic,

liver X X X X X X X X X X 19 Sarcoma, metastatic, kidney 1 Mediastinum, alveolar/bronchiolar

carcinoma 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System

Ear + 1 Eye + + + + + + 6 Zymbal's gland + 2

Carcinoma 1 Squamous cell carcinoma X 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Renal tubule, adenoma X X X X X X X X 9 Renal tubule, adenoma, multiple X X X X 5 Renal tubule, carcinoma 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Transitional epithelium, carcinoma X X X X 4 Transitional epithelium, papilloma X X X X X 8

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear 1 Mesothelioma malignant 1126 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Adrenal Medulla: Benign Pheochromocytoma
Overall rate a 12/50 (24%) 15/40 (38%) 13/58 (22%) 7/50 (14%) Adjusted rate b 36.6% 45.3% 38.7% 39.0% Terminal rate c 7/26 (27%) 7/24 (29%) 4/20 (20%) 2/10 (20%) First incidence (days) 574 629 521 590 Life table test d P=0.496 P=0.257 P=0.407 P=0.475 Logistic regression test d P=0.130N P=0.160 P=0.536N P=0.357N Cochran-Armitage test d P=0.041N Fisher exact test d P=0.124 P=0.512N P=0.154N

Adrenal Medulla: Malignant Pheochromocytoma Overall rate 1/50 (2%) 2/40 (5%) 1/58 (2%) 0/50 (0%) Adjusted rate 3.1% 6.9% 3.2% 0.0% Terminal rate 0/26 (0%) 1/24 (4%) 0/20 (0%) 0/10 (0%) First incidence (days) 692 653 693

) e
Life table test P=0.359N P=0.470 P=0.757 P=0.615N Logistic regression test P=0.250N P=0.429 P=0.738N P=0.557N Cochran-Armitage test P=0.207N Fisher exact test P=0.416 P=0.714N P=0.500N

Adrenal Medulla: Benign or Malignant Pheochromocytoma Overall rate 13/50 (26%) 17/40 (43%) 14/58 (24%) 7/50 (14%) Adjusted rate 38.6% 50.0% 40.7% 39.0% Terminal rate 7/26 (27%) 8/24 (33%) 4/20 (20%) 2/10 (20%) First incidence (days) 574 629 521 590 Life table test P=0.504N P=0.200 P=0.409 P=0.547 Logistic regression test P=0.075N P=0.231 P=0.525N P=0.277N Cochran-Armitage test P=0.020N Fisher exact test P=0.077 P=0.499N P=0.105N

Kidney (Renal Tubule): Adenoma Overall rate 2/50 (4%) 10/40 (25%) 11/59 (19%) 14/50 (28%) Adjusted rate 7.7% 33.6% 34.6% 68.3% Terminal rate 2/26 (8%) 6/24 (25%) 4/21 (19%) 5/10 (50%) First incidence (days) 729 (T) 618 636 588 Life table test P<0.001 P=0.012 P=0.007 P<0.001 Logistic regression test P<0.001 P=0.007 P=0.014 P<0.001 Cochran-Armitage test P=0.008 Fisher exact test P=0.004 P=0.017 P<0.001

Kidney (Renal Tubule): Adenoma or Carcinoma Overall rate 2/50 (4%) 10/40 (25%) 13/59 (22%) 15/50 (30%) Adjusted rate 7.7% 33.6% 39.4% 69.1% Terminal rate 2/26 (8%) 6/24 (25%) 4/21 (19%) 5/10 (50%) First incidence (days) 729 (T) 618 636 497 Life table test P<0.001 P=0.012 P=0.002 P<0.001 Logistic regression test P<0.001 P=0.007 P=0.005 P<0.001 Cochran-Armitage test P=0.004 Fisher exact test P=0.004 P=0.006 P<0.001Lesions in Male Rats 127



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Large Intestine (Colon): Adenomatous Polyp Overall rate 0/50 (0%) 1/40 (3%) 1/59 (2%) 3/50 (6%) Adjusted rate 0.0% 4.2% 4.3% 19.9% Terminal rate 0/26 (0%) 1/24 (4%) 0/21 (0%) 1/10 (10%) First incidence (days)

) 729 (T) 720 590 Life table test P=0.009 P=0.484 P=0.454 P=0.037 Logistic regression test P= 0.027 P=0.484 P=0.494 P=0.081 Cochran-Armitage test P=0.065 Fisher exact test P=0.444 P=0.541 P=0.121

Large Intestine (Colon): Carcinoma Overall rate 0/50 (0%) 0/40 (0%) 1/59 (2%) 4/50 (8%) Adjusted rate 0.0% 0.0% 4.8% 20.4% Terminal rate 0/26 (0%) 0/24 (0%) 1/21 (5%) 0/10 (0%) First incidence (days)

) ) 729 (T) 590 Life table test P<0.001

) P=0.457 P=0.018 Logistic regression test P=0.003

) P=0.457 P=0.046 Cochran-Armitage test P=0.007 Fisher exact test

) P=0.541 P=0.059

Large Intestine (Rectum): Adenomatous Polyp Overall rate 0/50 (0%) 13/40 (33%) 51/59 (86%) 40/50 (80%) Adjusted rate 0.0% 45.8% 100.0% 100.0% Terminal rate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%) First incidence (days)

) 659 478 352 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Large Intestine (Rectum): Carcinoma Overall rate 0/50 (0%) 1/40 (3%) 10/59 (17%) 15/50 (30%) Adjusted rate 0.0% 3.8% 32.4% 63.0% Terminal rate 0/26 (0%) 0/24 (0%) 5/21 (24%) 4/10 (40%) First incidence (days)

) 718 608 493 Life table test P<0.001 P=0.478 P=0.001 P<0.001 Logistic regression test P<0.001 P=0.480 P=0.003 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.444 P=0.001 P<0.001

Large Intestine (All Sites): Adenomatous Polyp Overall rate 0/50 (0%) 13/40 (33%) 51/59 (86%) 40/50 (80%) Adjusted rate 0.0% 45.8% 100.0% 100.0% Terminal rate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%) First incidence (days)

) 659 478 352 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001128 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Large Intestine (All Sites): Carcinoma Overall rate 0/50 (0%) 1/40 (3%) 11/59 (19%) 17/50 (34%) Adjusted rate 0.0% 3.8% 36.6% 67.1% Terminal rate 0/26 (0%) 0/24 (0%) 6/21 (29%) 4/10 (40%) First incidence (days)

) 718 608 493 Life table test P<0.001 P=0.478 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.480 P=0.002 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.444 P<0.001 P<0.001

Liver: Hepatocellular Adenoma Overall rate 1/50 (2%) 20/40 (50%) 40/59 (68%) 34/50 (68%) Adjusted rate 3.8% 71.3% 92.3% 97.0% Terminal rate 1/26 (4%) 16/24 (67%) 18/21 (86%) 9/10 (90%) First incidence (days) 729 (T) 675 521 435 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Liver: Hepatocellular Carcinoma Overall rate 1/50 (2%) 12/40 (30%) 55/59 (93%) 46/50 (92%) Adjusted rate 2.7% 43.5% 100.0% 100.0% Terminal rate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%) First incidence (days) 666 650 465 436 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 2/50 (4%) 25/40 (63%) 57/59 (97%) 47/50 (94%) Adjusted rate 6.4% 83.1% 100.0% 100.0% Terminal rate 1/26 (4%) 19/24 (79%) 21/21 (100%) 10/10 (100%) First incidence (days) 666 650 465 435 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Liver: Hepatocholangiocarcinoma Overall rate 0/50 (0%) 0/40 (0%) 6/59 (10%) 2/50 (4%) Adjusted rate 0.0% 0.0% 19.1% 12.1% Terminal rate 0/26 (0%) 0/24 (0%) 2/21 (10%) 1/10 (10%) First incidence (days)

) ) 563 527 Life table test P=0.025

) P=0.019 P=0.133 Logistic regression test P=0.110

) P=0.029 P=0.250 Cochran-Armitage test P=0.117 Fisher exact test

) P=0.022 P=0.247Lesions in Male Rats 129



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Lung: Alveolar/bronchiolar Adenoma Overall rate 0/50 (0%) 2/40 (5%) 2/59 (3%) 3/49 (6%) Adjusted rate 0.0% 8.3% 7.2% 15.5% Terminal rate 0/26 (0%) 2/24 (8%) 1/21 (5%) 1/10 (10%) First incidence (days)

) 729 (T) 653 527 Life table test P=0.024 P=0.220 P=0.219 P=0.052 Logistic regression test P= 0.103 P=0.220 P=0.269 P=0.124 Cochran-Armitage test P=0.130 Fisher exact test P=0.195 P=0.291 P=0.117

Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 0/50 (0%) 3/40 (8%) 4/59 (7%) 4/49 (8%) Adjusted rate 0.0% 12.0% 16.5% 17.3% Terminal rate 0/26 (0%) 2/24 (8%) 3/21 (14%) 1/10 (10%) First incidence (days)

) 720 653 435 Life table test P=0.010 P=0.105 P=0.046 P=0.028 Logistic regression test P= 0.089 P=0.099 P=0.068 P=0.094 Cochran-Armitage test P=0.100 Fisher exact test P=0.084 P=0.082 P=0.056

Pancreatic Islets: Adenoma Overall rate 2/50 (4%) 2/40 (5%) 0/58 (0%) 1/50 (2%) Adjusted rate 7.7% 7.4% 0.0% 10.0% Terminal rate 2/26 (8%) 1/24 (4%) 0/21 (0%) 1/10 (10%) First incidence (days) 729 (T) 675

) 729 (T) Life table test P=0.519N P=0.664 P=0.286N P=0.671 Logistic regression test P= 0.415N P=0.660 P=0.286N P=0.671 Cochran-Armitage test P=0.255N Fisher exact test P=0.603 P=0.212N P=0.500N

Pituitary Gland (Pars Distalis): Adenoma Overall rate 21/48 (44%) 14/40 (35%) 10/56 (18%) 10/49 (20%) Adjusted rate 56.3% 40.8% 33.0% 51.1% Terminal rate 10/25 (40%) 6/24 (25%) 4/21 (19%) 4/10 (40%) First incidence (days) 549 604 608 577 Life table test P=0.298N P=0.190N P=0.041N P=0.430N Logistic regression test P=0.006N P=0.155N P=0.005N P=0.035N Cochran-Armitage test P=0.004N Fisher exact test P=0.269N P=0.004N P=0.012N

Pituitary Gland (Pars Dista lis): Adenoma or Carcinoma Overall rate 21/48 (44%) 15/40 (38%) 10/56 (18%) 10/49 (20%) Adjusted rate 56.3% 42.3% 33.0% 51.1% Terminal rate 10/25 (40%) 6/24 (25%) 4/21 (19%) 4/10 (40%) First incidence (days) 549 604 608 577 Life table test P=0.273N P=0.248N P=0.041N P=0.430N Logistic regression test P=0.004N P=0.222N P=0.005N P=0.035N Cochran-Armitage test P=0.003N Fisher exact test P=0.354N P=0.004N P=0.012N130 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Preputial Gland: Adenoma Overall rate 3/49 (6%) 2/39 (5%) 1/58 (2%) 1/47 (2%) Adjusted rate 11.0% 7.2% 2.4% 2.5% Terminal rate 2/25 (8%) 1/23 (4%) 0/21 (0%) 0/9 (0%) First incidence (days) 696 659 650 552 Life table test P=0.368N P=0.543N P=0.332N P=0.557N Logistic regression test P=0.198N P=0.554N P=0.264N P=0.384N Cochran-Armitage test P=0.179N Fisher exact test P=0.608N P=0.248N P=0.324N

Preputial Gland: Adenoma or Carcinoma Overall rate 3/49 (6%) 2/39 (5%) 2/58 (3%) 1/47 (2%) Adjusted rate 11.0% 7.2% 5.6% 2.5% Terminal rate 2/25 (8%) 1/23 (4%) 0/21 (0%) 0/9 (0%) First incidence (days) 696 659 650 552 Life table test P=0.432N P=0.543N P=0.525N P=0.557N Logistic regression test P=0.247N P=0.554N P=0.448N P=0.384N Cochran-Armitage test P=0.212N Fisher exact test P=0.608N P=0.420N P=0.324N

Skin: Squamous Cell Papilloma Overall rate 1/50 (2%) 2/40 (5%) 5/59 (8%) 2/50 (4%) Adjusted rate 3.8% 7.7% 18.9% 10.5% Terminal rate 1/26 (4%) 1/24 (4%) 2/21 (10%) 0/10 (0%) First incidence (days) 729 (T) 700 693 674 Life table test P=0.117 P=0.469 P=0.079 P=0.278 Logistic regression test P= 0.351 P=0.466 P=0.100 P=0.366 Cochran-Armitage test P=0.413 Fisher exact test P=0.416 P=0.146 P=0.500

Skin: Trichoepithelioma, Basal Cell Adenoma, or Basal Cell Carcinoma Overall rate 3/50 (6%) 0/40 (0%) 1/59 (2%) 1/50 (2%) Adjusted rate 11.5% 0.0% 4.3% 5.0% Terminal rate 3/26 (12%) 0/24 (0%) 0/21 (0%) 0/10 (0%) First incidence (days) 729 (T)

) 720 679 Life table test P=0.564N P=0.134N P=0.386N P=0.627N Logistic regression test P=0.462N P=0.134N P=0.351N P=0.501N Cochran-Armitage test P=0.294N Fisher exact test P=0.167N P=0.249N P=0.309N

Skin: Squamous Cell Papilloma, Keratoac anthoma, Trichoepithelioma, Basal Ce ll Adenoma, or Basal Cell Carcinoma Overall rate 5/50 (10%) 3/40 (8%) 6/59 (10%) 4/50 (8%) Adjusted rate 18.0% 10.1% 22.5% 17.0% Terminal rate 4/26 (15%) 1/24 (4%) 2/21 (10%) 0/10 (0%) First incidence (days) 692 629 693 527 Life table test P=0.205 P=0.403N P=0.394 P=0.377 Logistic regression test P= 0.448 P=0.421N P=0.494 P=0.629 Cochran-Armitage test P=0.483N Fisher exact test P=0.488N P=0.616 P=0.500NLesions in Male Rats 131



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Skin (Subcutaneous Tissue): Fibroma Overall rate 2/50 (4%) 0/40 (0%) 3/59 (5%) 1/50 (2%) Adjusted rate 6.3% 0.0% 12.9% 3.8% Terminal rate 1/26 (4%) 0/24 (0%) 1/21 (5%) 0/10 (0%) First incidence (days) 646

) 715 631 Life table test P=0.389 P=0.258N P=0.437 P=0.707N Logistic regression test P=0.568 P=0.283N P=0.529 P=0.543N Cochran-Armitage test P=0.549N Fisher exact test P=0.306N P=0.579 P=0.500N

Skin (Subcutaneous Tissue): Sarcoma Overall rate 1/50 (2%) 3/40 (8%) 1/59 (2%) 0/50 (0%) Adjusted rate 2.3% 11.1% 3.0% 0.0% Terminal rate 0/26 (0%) 1/24 (4%) 0/21 (0%) 0/10 (0%) First incidence (days) 562 675 685

) Life table test P=0.283N P=0.272 P=0.732N P=0.529N Logistic regression test P=0.156N P=0.187 P=0.741N P=0.416N Cochran-Armitage test P=0.152N Fisher exact test P=0.229 P=0.709N P=0.500N

Skin (Subcutaneous Tissue): Fibrosarcoma or Sarcoma Overall rate 2/50 (4%) 3/40 (8%) 2/59 (3%) 0/50 (0%) Adjusted rate 4.7% 11.1% 4.7% 0.0% Terminal rate 0/26 (0%) 1/24 (4%) 0/21 (0%) 0/10 (0%) First incidence (days) 562 675 465

) Life table test P=0.207N P=0.456 P=0.638N P=0.304N Logistic regression test P=0.062N P=0.321 P=0.703 P=0.175N Cochran-Armitage test P=0.103N Fisher exact test P=0.394 P=0.626N P=0.247N

Skin (Subcutaneous Tissue): Fibroma, Fibrosarcoma, or Sarcoma Overall rate 4/50 (8%) 3/40 (8%) 5/59 (8%) 1/50 (2%) Adjusted rate 10.7% 11.1% 17.0% 3.8% Terminal rate 1/26 (4%) 1/24 (4%) 1/21 (5%) 0/10 (0%) First incidence (days) 562 675 465 631 Life table test P=0.412N P=0.550N P=0.493 P=0.351N Logistic regression test P=0.136N P=0.637 P=0.601 P=0.160N Cochran-Armitage test P=0.148N Fisher exact test P=0.624N P=0.605 P=0.181N

Stomach (Forestomach): Squamous Cell Papilloma Overall rate 0/50 (0%) 2/40 (5%) 0/59 (0%) 1/50 (2%) Adjusted rate 0.0% 7.7% 0.0% 5.6% Terminal rate 0/26 (0%) 1/24 (4%) 0/21 (0%) 0/10 (0%) First incidence (days)

) 700

) 702 Life table test P=0.427 P=0.220

) P=0.398 Logistic regression test P=0.487 P=0.215

) P=0.426 Cochran-Armitage test P=0.604 Fisher exact test P=0.195

) P=0.500132 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Stomach (Forestomach): Squamous Ce ll Papilloma or Squamous Cell Carcinoma Overall rate 0/50 (0%) 2/40 (5%) 0/59 (0%) 2/50 (4%) Adjusted rate 0.0% 7.7% 0.0% 15.0% Terminal rate 0/26 (0%) 1/24 (4%) 0/21 (0%) 1/10 (10%) First incidence (days)

) 700

) 702 Life table test P=0.117 P=0.220

) P=0.099 Logistic regression test P=0.157 P=0.215

) P=0.130 Cochran-Armitage test P=0.299 Fisher exact test P=0.195

) P=0.247

Testes: Adenoma Overall rate 43/50 (86%) 37/40 (93%) 55/59 (93%) 42/50 (84%) Adjusted rate 97.7% 100.0% 100.0% 100.0% Terminal rate 25/26 (96%) 24/24 (100%) 21/21 (100%) 10/10 (100%) First incidence (days) 421 604 521 476 Life table test P<0.001 P=0.404N P=0.022 P<0.001 Logistic regression test P= 0.068 P=0.471N P=0.235 P=0.150 Cochran-Armitage test P=0.341N Fisher exact test P=0.265 P=0.177 P=0.500N

Thyroid Gland (C-cell): Adenoma Overall rate 9/49 (18%) 5/40 (13%) 3/59 (5%) 3/50 (6%) Adjusted rate 26.8% 20.8% 11.6% 23.5% Terminal rate 4/25 (16%) 5/24 (21%) 1/21 (5%) 2/10 (20%) First incidence (days) 427 729 (T) 702 671 Life table test P=0.231N P=0.243N P=0.088N P=0.348N Logistic regression test P=0.052N P=0.332N P=0.030N P=0.085N Cochran-Armitage test P=0.026N Fisher exact test P=0.324N P=0.030N P=0.056N

Thyroid Gland (Follicular Cell): Carcinoma Overall rate 2/49 (4%) 3/40 (8%) 1/59 (2%) 3/50 (6%) Adjusted rate 8.0% 12.5% 2.1% 18.7% Terminal rate 2/25 (8%) 3/24 (13%) 0/21 (0%) 1/10 (10%) First incidence (days) 729 (T) 729 (T) 623 577 Life table test P=0.201 P=0.481 P=0.519N P=0.205 Logistic regression test P= 0.396 P=0.481 P=0.445N P=0.369 Cochran-Armitage test P=0.546 Fisher exact test P=0.404 P=0.430N P=0.510

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma Overall rate 2/49 (4%) 3/40 (8%) 3/59 (5%) 4/50 (8%) Adjusted rate 8.0% 12.5% 8.8% 27.7% Terminal rate 2/25 (8%) 3/24 (13%) 1/21 (5%) 2/10 (20%) First incidence (days) 729 (T) 729 (T) 623 577 Life table test P=0.051 P=0.481 P=0.482 P=0.079 Logistic regression test P= 0.178 P=0.481 P=0.576 P=0.192 Cochran-Armitage test P=0.332 Fisher exact test P=0.404 P=0.588 P=0.349Lesions in Male Rats 133



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Urinary Bladder: Papilloma Overall rate 0/50 (0%) 1/38 (3%) 2/58 (3%) 8/50 (16%) Adjusted rate 0.0% 3.7% 9.5% 40.3% Terminal rate 0/26 (0%) 0/22 (0%) 2/21 (10%) 2/10 (20%) First incidence (days)

) 700 729 (T) 493 Life table test P<0.001 P=0.479 P=0.192 P<0.001 Logistic regression test P<0.001 P=0.459 P=0.192 P=0.004 Cochran-Armitage test P<0.001 Fisher exact test P=0.432 P=0.286 P=0.003

Urinary Bladder: Carcinoma Overall rate 0/50 (0%) 0/38 (0%) 1/58 (2%) 4/50 (8%) Adjusted rate 0.0% 0.0% 4.3% 24.5% Terminal rate 0/26 (0%) 0/22 (0%) 0/21 (0%) 1/10 (10%) First incidence (days)

) ) 720 674 Life table test P<0.001

) P=0.454 P=0.012 Logistic regression test P=0.001

) P=0.491 P=0.022 Cochran-Armitage test P=0.007 Fisher exact test

) P=0.537 P=0.059

Urinary Bladder: Papilloma or Carcinoma Overall rate 0/50 (0%) 1/38 (3%) 3/58 (5%) 12/50 (24%) Adjusted rate 0.0% 3.7% 13.5% 56.2% Terminal rate 0/26 (0%) 0/22 (0%) 2/21 (10%) 3/10 (30%) First incidence (days)

) 700 720 493 Life table test P<0.001 P=0.479 P=0.087 P<0.001 Logistic regression test P<0.001 P=0.459 P=0.096 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.432 P=0.151 P<0.001

All Organs: Mononuclear Cell Leukemia Overall rate 25/50 (50%) 5/40 (13%) 3/59 (5%) 1/50 (2%) Adjusted rate 59.0% 18.8% 11.7% 2.9% Terminal rate 9/26 (35%) 4/24 (17%) 2/21 (10%) 0/10 (0%) First incidence (days) 514 604 650 590 Life table test P<0.001N P<0.001N P<0.001N P<0.001N Logistic regression test P<0.001N P<0.001N P<0.001N P<0.001N Cochran-Armitage test P<0.001N Fisher exact test P<0.001N P<0.001N P<0.001N

All Organs: Benign Neoplasms Overall rate 49/50 (98%) 40/40 (100%) 58/59 (98%) 47/50 (94%) Adjusted rate 100.0% 100.0% 100.0% 100.0% Terminal rate 26/26 (100%) 24/24 (100%) 21/21 (100%) 10/10 (100%) First incidence (days) 421 604 478 352 Life table test P<0.001 P=0.263N P=0.074 P<0.001 Logistic regression test P=0.543

) f P=0.486N P=0.727 Cochran-Armitage test P=0.102N Fisher exact test P=0.556 P=0.709 P=0.309N134 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A3 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

All Organs: Malignant Neoplasms Overall rate 29/50 (58%) 24/40 (60%) 57/59 (97%) 47/50 (94%) Adjusted rate 67.0% 71.7% 100.0% 100.0% Terminal rate 12/26 (46%) 15/24 (63%) 21/21 (100%) 10/10 (100%) First incidence (days) 514 604 465 435 Life table test P<0.001 P=0.398N P<0.001 P<0.001 Logistic regression test P<0.001 P=0.583 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.510 P<0.001 P<0.001

All Organs: Benign or Malignant Neoplasms Overall rate 49/50 (98%) 40/40 (100%) 59/59 (100%) 48/50 (96%) Adjusted rate 100.0% 100.0% 100.0% 100.0% Terminal rate 26/26 (100%) 24/24 (100%) 21/21 (100%) 10/10 (100%) First incidence (days) 421 604 465 352 Life table test P<0.001 P=0.263N P=0.061 P<0.001 Logistic regression test P=0.296

) P=0.943 P=0.501 Cochran-Armitage test P=0.252N Fisher exact test P=0.556 P=0.459 P=0.500N

(T)Terminal sacrifice

a Number of lesion-bearing animals/number of animals examined. De nominator is number of animals examined microscopically for adr enal gland, kidney, liver, lung, pancreatic islets, pituitary gland, preputial gland, stomach, testes, thyroid gland, and urinary bladder; for other tissues, de nominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Observed incidence at terminal kill d Beneath the control incidence are the P valu es associated with the trend test. Bene ath the exposed group incidence are the P v alues corresponding to pairwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare di rectly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed.Lesions in Male Rats 135



### T ABLE 

### A4a Historical Incidence of Hepatocellula r Neoplasms in Untreated Male F344/N Rats a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 1/50 1/50 2/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 1/50 0/50 1/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 2/49 1/49 3/49 Quercetin 3/50 0/50 3/50 Turmeric Oleoresin 0/50 1/50 1/50

Overall Historical Incidence

Total 35/1,350 (2.6%) 14/1,350 (1.0%) 45/1,350 (3.3%) Standard deviation 3.2% 1.8% 3.6% Range 0%-10% 0%-6% 0%-10%

a Data as of 31 March 1993



### T ABLE 

### A4b Historical Incidence of Large Intestin e Neoplasms in Untreated Male F344/N Rats a

Incidence in Controls Study Adenomatous Polyp Car cinoma Adenomatous Polyp

(Adenoma) (Adenoma) or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/50 0/50 0/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/50 0/50 0/50 Turmeric Oleoresin 0/50 1/50 1/50

Overall Historical Incidence

Total 0/1,353 (0.0%) 1/1,353 (0.1%) 1/1,353 (0.1%) Standard deviation 0.4% 0.4% Range 0%-2% 0%-2%

a Data as of 31 March 1993; the data include incidences for the colon and rectum.136 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A4c Historical Incidence of Renal Tubule Neoplasms in Untreated Male F344/N Rats a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 2/50 0/50 2/50 Acetaminophen 3/50 0/50 3/50 HC Yellow 4 1/50 0/50 1/50 Methylphenidate Hydrochloride 0/49 0/49 0/49 Pentaerythritol Tetranitrate 0/49 0/49 0/49 Quercetin 0/50 0/50 0/50 Turmeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 9/1,350 (0.7%) 6/1,350 (0.4%) 15/1,350 (1.1%) Standard deviation 1.5% 1.0% 1.7% Range 0%-6% 0%-4% 0%-6%

a Data as of 31 March 1993



### T ABLE 

### A4d Historical Incidence of Urinary Bladder Neoplasms in Untreated Male F344/N Rats a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/49 0/49 0/49 HC Yellow 4 0/48 0/48 0/48 Methylphenidate Hydrochloride 0/43 0/43 0/43 Pentaerythritol Tetranitrate 0/46 0/46 0/46 Quercetin 0/50 0/50 0/50 Turmeric Oleoresin 0/49 0/49 0/49

Overall Historical Incidence

Total 3/1,329 (0.2%) 0/1,329 (0.0%) 3/1,329 (0.2%) Standard deviation 0.6% 0.6% Range 0%-2% 0%-2%

a Data as of 31 March 1993Lesions in Male Rats 137



### T ABLE 

### A4e Historical Incidence of Fore stomach Squamous Cell Neoplasms in Untreated Male F344/N Rats a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/50 1/50 1/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/50 0/50 0/50 Tumeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 1/1,353 (0.1%) 3/1,353 (0.2%) 4/1,353 (0.3%) Standard deviation 0.4% 0.6% 0.7% Range 0%-2% 0%-2% 0%-2%

a Data as of 31 March 1993138 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Disposition Summary Animals initially in study 70 50 70 70 9-Month interim evaluation 10 10 10 10 15-Month interim evaluation 10 10 Early deaths

Moribund 19 15 34 33 Natural deaths 5 1 5 7 Survivors

Died last week of study 3 Terminal sacrifice 23 24 21 10

Animals examined microscopically 70 50 69 b 70



### 9-Month Interim Evaluation Alimentary System Intestine large, colon (10) (10) (10) (10)

Parasite metazoan 2 (20%) 3 (30%) 3 (30%) 2 (20%) Intestine large, rectum (10) (10) (10) (10)

Parasite metazoan 2 (20%) Intestine large, cecum (10) (10) (10) (10)

Parasite metazoan 1 (10%) Lymphoid tissue, hemorrhage 1 (10%) Liver (10) (10) (10) (10)

Basophilic focus 1 (10%) 1 (10%) Clear cell focus 4 (40%) Developmental malformation 1 (10%) Eosinophilic focus 1 (10%) Fatty change 3 (30%) 7 (70%) 6 (60%) Inflammation, chronic 1 (10%) Inflammation, chronic active 1 (10%) 1 (10%) Necrosis, coagulative 1 (10%) 2 (20%) 2 (20%) Pigmentation 1 (10%) 6 (60%) Bile duct, hyperplasia 1 (10%) 1 (10%) 5 (50%) Periportal, inflammation 1 (10%) Periportal, inflammation, chronic active 9 (90%) 10 (100%) 10 (100%) 8 (80%) Mesentery (1) (1) Hemorrhage 1 (100%) Inflammation, chronic, granulomatous 1 (100%) Inflammation, chronic active 1 (100%) Necrosis 1 (100%) Necrosis, coagulative 1 (100%) Pancreas (10) (10) (10) (10)

Atrophy 2 (20%) 1 (10%) 1 (10%) Infiltration cellular, mononuclear cell 3 (30%) 5 (50%) 2 (20%) 3 (30%) Infiltration cellular, mixed cell 1 (10%) Inflammation, chronic 1 (10%) Inflammation, chronic active 1 (10%)

a Number of animals examined microscopically at site and number of animals with lesion b One animal not examined microscopicallyLesions in Male Rats 139



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) Alimentary System (continued) Pancreas (continued) (10) (10) (10) (10)

Necrosis, coagulative 2 (20%) Pigmentation 1 (10%) Acinus, atrophy 1 (10%)

Cardiovascular System Heart (10) (10) (10) (10)

Cardiomyopathy 9 (90%) 8 (80%) 8 (80%) 6 (60%) Infiltration cellula r, mononuclear cell 1 (10%)

Endocrine System Adrenal cortex (10) (10) (10) (10) Adrenal medulla (10) (10) (10) (10) Hyperplasia 1 (10%) Pituitary gland (9) (10) (9) (9)

Cyst 1 (11%) Pars distalis, cyst 1 (11%) 1 (11%) 2 (22%) Pars distalis, hyperplasia 3 (33%) 4 (40%) 1 (11%) 5 (56%) Pars nervosa, cyst 1 (11%) Thyroid gland (10) (10) (10) (10) Infiltration cellular, mononuclear cell 1 (10%) Ultimobranchial cyst 1 (10%) 1 (10%) 1 (10%)

Genital System Preputial gland (9) (9) (8) (9)

Inflammation, acute 1 (13%) Inflammation, chronic 2 (22%) 1 (13%) 2 (22%) Inflammation, chronic acti ve 7 (78%) 6 (67%) 2 (25%) 2 (22%) Prostate (10) (10) (9) (10)

Inflammation, acute 1 (10%) 2 (20%) Inflammation, chronic active 4 (40%) 1 (11%) 1 (10%) Testes (10) (10) (10) (10)

Abscess, chronic 1 (10%) Atrophy 1 (10%) 1 (10%) Infarct 1 (10%) 1 (10%) Infarct, chronic 1 (10%) Mineralization 1 (10%) Interstitial cell, hyperpla sia 1 (10%) 9 (90%) 5 (50%) 5 (50%)

Hematopoietic System Lymph node (2) (1) (6) (2)

Lumbar, pigmentation 1 (17%) Mediastinal, hemorrhage 1 (50%) 4 (67%) 2 (100%) Mediastinal, pigmentation 1 (50%) 2 (100%) Pancreatic, hemorrhage 1 (17%)140 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) Hematopoietic System (continued) Lymph node (continued) (2) (1) (6) (2)

Pancreatic, infiltration cellu lar, histiocyte 1 (17%) 1 (50%) Pancreatic, inflammation, chronic active 1 (17%) Pancreatic, pigmentation 1 (50%) Renal, hemorrhage 1 (50%) 1 (100%) Renal, pigmentation 1 (50%) 1 (100%) Lymph node, mandibular (10) (10) (10) (10)

Hemorrhage 9 (90%) 5 (50%) 7 (70%) 7 (70%) Infiltration cellular, histiocyte 1 (10%) Lymph node, mesenteric (10) (10) (10) (10)

Hemorrhage 1 (10%) Infiltration cellular, histio cyte 10 (100%) 10 (100%) 10 (100%) 10 (100%) Pigmentation 1 (10%) 6 (60%) Spleen (10) (10) (10) (10)

Depletion lymphoid 1 (10%) Pigmentation 2 (20%) Thymus (9) (9) (10) (10) Depletion lymphoid 3 (33%) 4 (44%) 5 (50%) 7 (70%) Hemorrhage 1 (11%) 1 (10%) 5 (50%)

Integumentary System Mammary gland (8) (7) (5) (5) Hyperplasia 7 (88%) 7 (100%) 5 (100%) 5 (100%)

Respiratory System Lung (10) (10) (10) (10)

Hemorrhage 1 (10%) Infiltration cellular, histio cyte 1 (10%) 1 (10%) 2 (20%) 1 (10%) Alveolar epithelium, hyperplasia 1 (10%) Artery, mineralization 6 ( 60%) 3 (30%) 5 (50%) 4 (40%) Nose (10) (10) (10) (10)

Special Senses System Ear (1)

Inflammation, chronic active 1 (100%) Ulcer 1 (100%)

Urinary System Kidney (10) (10) (10) (10)

Granuloma 1 (10%) Infiltration cellular, mononu clear cell 9 (90%) 9 (90%) 9 (90%) Inflammation, chronic 4 (40%) Renal tubule, degeneration, hyaline 10 (100%) 10 (100%) 10 (100%) Renal tubule, pigmentation 10 (100%) 10 (100%) 10 (100%) Renal tubule, regeneration 10 (100%) 10 (100%) 10 (100%) 9 (90%)Lesions in Male Rats 141



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) Urinary System (continued) Urinary bladder (10) (10) (9) (10)

Calculus, microscopic observation only 2 (20%) 1 (11%) Serosa, mineralization 1 (10%)



### Systems Examined With No Lesions Observed General Body System Musculoskeletal System Nervous System



### 15-Month Interim Evaluation Alimentary System Intestine large, colon (10) (2)

Parasite metazoan 4 (40%) 2 (100%) Intestine large, rectum (9) (10)

Parasite metazoan 1 (11%) Intestine large, cecum (10)

Parasite metazoan 3 (30%) Intestine small, ileum (10) Liver (10) (10)

Basophilic focus 1 (10%) 5 (50%) Clear cell focus 7 (70%) Cytomegaly 1 (10%) Degeneration 2 (20%) 1 (10%) Eosinophilic focus 1 (10%) Fatty change 6 (60%) 9 (90%) Hematopoietic cell proliferation 2 (20%) Inflammation, chronic, gr anulomatous 2 (20%) 1 (10%) Inflammation, chronic active 6 (60%) Mixed cell focus 1 (10%) Necrosis, coagulative 6 (60%) 2 (20%) Pigmentation 10 (100%) Bile duct, hyperplasia 10 (100%) 10 (100%) Periportal, inflammation, chronic 10 (100%) 10 (100%) Pancreas (10)

Atrophy 4 (40%) Inflammation, chronic 8 (80%) Salivary glands (10) Stomach, forestomach (10) (10)

Arteriole, mineralization 1 (10%) Muscularis, mineralization 1 (10%) 1 (10%) Stomach, glandular (10) (10)

Inflammation, chronic 1 (10%) Muscularis, mineralization 1 (10%)142 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) Cardiovascular System Heart (10)

Cardiomyopathy 10 (100%)

Endocrine System Adrenal cortex (10) Hyperplasia 1 (10%) Bilateral, vacuolization cytoplasmic 1 (10%) Adrenal medulla (9) Bilateral, hyperplasia 1 (11%) Islets, pancreatic (4)

Hemorrhage 1 (25%) Pituitary gland (8)

Pars distalis, hyperplasia 8 (100%) Thyroid gland (10) (1) C-cell, hyperplasia 1 (10%) 1 (100%)

Genital System Epididymis (10) Preputial gland (9)

Inflammation, chronic 5 (56%) Inflammation, chronic active 4 (44%) Prostate (10)

Inflammation, acute 1 (10%) Inflammation, chronic active 6 (60%) Testes (10) (8) Interstitial cell, hyperplasia 10 (100%) 8 (100%) Seminiferous tubule, atrophy 2 (20%) 3 (38%)

Hematopoietic System Lymph node (3)

Mediastinal, pigmentation 1 (33%) Pancreatic, infiltration cellular, histiocyte 3 (100%) Pancreatic, pigmentation 3 (100%) Lymph node, mandibular (10) (1)

Hemorrhage 3 (30%) 1 (100%) Lymph node, mesenteric (10) (1)

Depletion lymphoid 1 (10%) Hemorrhage 1 (10%) Infiltration cellular, histiocyte 10 (100%) 1 (100%) Pigmentation 9 (90%) 1 (100%) Thymus (10) (9) Cyst 1 (10%) Depletion lymphoid 3 (33%)Lesions in Male Rats 143



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) Integumentary System Mammary gland (4) Hyperplasia 2 (50%) Skin (10)

Respiratory System Lung (10) (3)

Hemorrhage 1 (33%) Infiltration cellular, histiocyte 2 (20%) 2 (67%) Alveolar epithelium, hyperplasia 3 (30%) Alveolus, mineralization 1 (10%) Artery, mineralization 8 (80%) 2 (67%) Nose (10) Glands, inflammation, acute 3 (30%) Glands, inflammation, chronic active 1 (10%) Nasolacrimal duct, inflammation, chronic

active 1 (10%)

Urinary System Kidney (10) (10)

Nephropathy 10 (100%) 10 (100%) Renal tubule, hyperplasia 2 (20%) Renal tubule, pigmentation 10 (100%) Transitional epithelium, hyperplasia 4 (40%) Urinary bladder (10) (10)

Metaplasia, squamous 1 (10%) Transitional epithelium, hyperplasia 3 (30%)



### Systems Examined With No Lesions Observed General Body System Musculoskeletal System Nervous System Special Senses System



### 2-Year Study Alimentary System Intestine large, colon (47) (40) (59) (49)

Autolysis 1 (2%) 1 (3%) 4 (7%) 5 (10%)

Hyperplasia, lymphoid 1 (2%) Parasite metazoan 13 (28%) 14 (35%) 9 (15%) 3 (6%) Polyarteritis 1 (3%) 1 (2%) Ulcer 1 (2%)144 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Intestine large, rectum (46) (40) (58) (49)

Atypia cellular 2 (3%) Autolysis 1 (2%) 5 (10%) Inflammation, chronic active 1 (2%) Parasite metazoan 6 (13%) 6 (15%) 9 (16%) 2 (4%) Artery, neovascularization 2 (3%) Artery, thrombosis 1 (2%) Epithelium, hyperplasia 1 (2%) 1 (2%) Intestine large, cecum (48) (40) (59) (50)

Atypia cellular 1 (2%) Autolysis 2 (4%) 1 (3%) 3 (5%) 5 (10%) Hyperplasia, lymphoid 1 (2%) Inflammation, chronic active 1 (3%) 1 (2%) Parasite metazoan 4 (8%) 8 (20%) 10 (17%) 3 (6%) Ulcer, acute 1 (3%) Intestine small, duodenum (48) (40) (59) (50)

Autolysis 2 (4%) 1 (3%) 3 (5%) 5 (10%) Ectopic tissue 1 (2%) Inflammation, chronic active 1 (2%) Intestine small, jejunum (48) (38) (57) (48)

Autolysis 2 (4%) 1 (3%) 5 (9%) 6 (13%) Intestine small, ileum (48) (40) (57) (49)

Autolysis 3 (6%) 1 (3%) 4 (7%) 9 (18%) Hyperplasia, lymphoid 1 (2%) 2 (4%) Liver (50) (40) (59) (50)

Angiectasis 3 (6%) 3 (8%) Autolysis 2 (4%) 2 (4%) Basophilic focus 9 (18%) 12 (30%) 24 (41%) 22 (44%) Clear cell focus 3 (6%) 26 (65%) 39 (66%) 27 (54%) Cyst 1 (2%) 2 (4%) Degeneration 10 (20%) 3 (8%) 12 (20%) 3 (6%) Eosinophilic focus 1 (2%) 13 (33%) 14 (24%) 6 (12%) Fatty change 7 (14%) 4 (10%) 7 (12%) 2 (4%) Fibrosis 1 (2%) Hematopoietic cell proliferation 4 (7%) Hemorrhage 1 (2%) 1 (2%) Hepatodiaphragmatic nodule 3 (6%) 2 (5%) Hyperplasia 1 (2%) Infarct 1 (3%) 1 (2%) Inflammation, chronic active 3 (6%) 1 (3%) 4 (7%) 6 (12%) Mixed cell focus 2 (4%) 17 (43%) 8 (14%) 1 (2%) Necrosis, coagulative 3 (6%) 6 (15%) 8 (14%) 6 (12%) Pigmentation 3 (6%) 19 (48%) 48 (81%) 39 (78%) Regeneration 4 (8%) Bile duct, cyst 1 (3%) Bile duct, hyperplasia 45 (90%) 9 (23%) 54 (92%) 45 (90%) Periportal, inflammation, chronic active 2 (4%) 2 (5%) 51 (86%) 36 (72%)Lesions in Male Rats 145



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Mesentery (3) (2) (4) (4) Fibrosis 2 (100%) 2 (50%) 2 (50%) Hemorrhage 1 (25%) 1 (25%) Inflammation, chronic acti ve 1 (33%) 2 (100%) 3 (75%) 3 (75%) Necrosis, coagulative 1 (50%) 2 (50%) 1 (25%) Polyarteritis 1 (50%) Pancreas (50) (40) (59) (50)

Atrophy 11 (22%) 26 (65%) 29 (49%) 20 (40%) Autolysis 2 (3%) 2 (4%) Cytoplasmic alteration 1 (2%) Ectopic tissue 1 (2%) Inflammation, chronic active 10 (20%) 28 (70%) 45 (76%) 26 (52%) Vacuolization cytoplasmic 17 (34%) 2 (5%) 6 (10%) 15 (30%) Acinus, hyperplasia 1 (2%) 1 (2%) Artery, fibrosis 1 (2%) 1 (2%) Artery, polyarteritis 4 (8%) 8 (20%) 4 (7%) 3 (6%) Duct, hyperplasia 1 (2%) 1 (2%) Pharynx (1)

Palate, hyperkeratosis 1 (100%) Salivary glands (50) (40) (58) (49)

Duct, submandibular gland, inflammation,
acute 1 (2%) Parotid gland, inflammation, chronic 1 (2%) Submandibular gland, inflammation, chronic 2 (3%) Submandibular gland, metaplasia, squamous 1 (2%) Stomach, forestomach (49) (39) (59) (49)

Erosion 1 (2%) 1 (2%) Fibrosis 1 (2%) 1 (2%) Hyperkeratosis 5 (10%) 18 (46%) 21 (36%) 20 (41%) Hyperplasia, basal cell 11 (22%) 18 (46%) 23 (39%) 23 (47%) Hyperplasia, squamous 3 (6%) 19 (49%) 25 (42%) 26 (53%) Infarct 1 (2%) Inflammation, chronic active 3 (6%) 12 (31%) 11 (19%) 11 (22%) Ulcer 3 (6%) 10 (26%) 15 (25%) 16 (33%) Stomach, glandular (50) (40) (59) (50)

Autolysis 2 (3%) 2 (4%) Inflammation, chronic active 3 (6%) 2 (5%) 2 (3%) 3 (6%) Mucosa, hyperplasia 1 (2%) Mucosa, necrosis, coagulative 1 (2%)

Cardiovascular System Heart (50) (40) (59) (49)

Autolysis 1 (3%) Cardiomyopathy 47 (94%) 38 (95%) 57 (97%) 48 (98%) Metaplasia, osseous 1 (2%) Polyarteritis 1 (3%) Thrombosis 2 (4%) 1 (2%)146 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Endocrine System Adrenal cortex (50) (40) (58) (50)

Angiectasis 2 (4%) 5 (13%) 13 (22%) 15 (30%) Autolysis 2 (4%) Hematopoietic cell proliferation 2 (4%) 1 (2%) Hyperplasia 4 (8%) 1 (3%) 2 (3%) 1 (2%) Necrosis, coagulative 1 (2%) Vacuolization cytoplasmic 23 (46%) 18 (45%) 19 (33%) 11 (22%) Adrenal medulla (50) (40) (58) (50)

Autolysis 2 (4%) Fibrosis 1 (2%) Hematopoietic cell proliferation 1 (2%) Hyperplasia 17 (34%) 25 (63%) 24 (41%) 21 (42%) Necrosis, coagulative 2 (4%) Islets, pancreatic (50) (40) (58) (50)

Autolysis 1 (2%) 1 (2%) Hyperplasia 1 (2%) 2 (4%) Parathyroid gland (43) (35) (55) (44)

Hyperplasia 4 (9%) 12 (34%) 1 (2%) Pituitary gland (48) (40) (56) (49)

Autolysis 1 (2%) Pars distalis, cyst 6 (13%) 4 (10%) 7 (13%) 7 (14%) Pars distalis, hyperplasia 12 (25%) 11 (28%) 23 (41%) 27 (55%) Pars intermedia, cyst 2 (4%) 1 (3%) 2 (4%) Thyroid gland (49) (40) (59) (50)

Autolysis 2 (3%) 2 (4%) Inflammation, chronic active 2 (4%) 1 (3%) 1 (2%) Ultimobranchial cyst 2 (5%) 3 (5%) 1 (2%) C-cell, hyperplasia 10 (20%) 8 (20%) 5 (8%) 3 (6%) Follicle, cyst 2 (4%) 1 (3%) 7 (12%) 3 (6%) Follicular cell, hyperplasia 3 (6%) 1 (3%) 2 (3%)

General Body System

None

Genital System Coagulating gland (2) (2)

Inflammation, chronic active 1 (50%) 2 (100%) Epididymis (50) (40) (59) (50)

Aspermia 1 (2%) Autolysis 2 (3%) Inflammation, chronic active 3 (6%) 2 (5%) 10 (17%) 3 (6%) Vacuolization cytoplasmic 1 (2%) 1 (2%) Preputial gland (49) (39) (58) (47)

Abscess 6 (12%) 1 (3%) 1 (2%) Cyst 2 (4%) 1 (2%) 1 (2%) Hyperplasia 2 (3%) Inflammation, chronic active 46 (94%) 38 (97%) 57 (98%) 43 (91%)Lesions in Male Rats 147



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Genital System (continued) Prostate (50) (40) (59) (49)

Abscess 3 (8%) 1 (2%) 1 (2%) Fibrosis 1 (2%) Inflammation, chronic active 11 (22%) 18 (45%) 24 (41%) 23 (47%) Metaplasia, squamous 1 (2%) 11 (28%) 6 (10%) 1 (2%) Polyarteritis 1 (3%) Epithelium, hyperplasia 8 (16%) 4 (10%) 2 (3%) 1 (2%) Seminal vesicle (49) (40) (59) (50)

Atrophy 1 (2%) 30 (75%) 35 (59%) 23 (46%) Autolysis 1 (2%) Inflammation, chronic active 3 (8%) 2 (3%) 1 (2%) Metaplasia, squamous 1 (3%) Testes (50) (40) (59) (50) Infarct 1 (3%) Inflammation, chronic 1 (2%) Polyarteritis 1 (2%) Interstitial cell, hyperplasia 16 (32%) 3 (8%) 27 (46%) 19 (38%) Seminiferous tubule, atrophy 41 (82%) 37 (93%) 55 (93%) 36 (72%) Seminiferous tubule, mineralization 8 (16%) 2 (3%) 6 (12%)

Hematopoietic System Bone marrow (50) (40) (59) (50)

Sternal, autolysis 1 (2%) Sternal, hypoplasia 1 (2%) Sternal, myelofibrosis 1 (2%) Lymph node (17) (5) (12) (20)

Deep cervical, hyperplasia 1 (5%) Lumbar, hyperplasia 1 (20%) 1 (5%) Mediastinal, hemorrhage 1 (6%) Mediastinal, infiltration cellular,

histiocyte 1 (20%) 2 (10%) Mediastinal, sinus, ectasia 1 (6%) Pancreatic, hyperplasia 1 (20%) 1 (8%) 2 (10%) Pancreatic, infiltration cellular, histiocyte 2 (40%) 9 (75%) 19 (95%) Renal, infiltration cellu lar, histiocyte 1 (6%) 1 (8%) Lymph node, mandibular (50) (40) (54) (48)

Hyperplasia 11 (22%) 7 (18%) 7 (13%) 4 (8%) Infiltration cellular, hi stiocyte 2 (4%) 1 (2%) 8 (17%) Inflammation, chronic active 1 (2%) Lymph node, mesenteric (48) (40) (57) (49)

Autolysis 1 (2%) Hyperplasia 1 (2%) 2 (4%) Infiltration cellular, histio cyte 44 (92%) 40 (100%) 56 (98%) 49 (100%) Pigmentation 1 (2%)148 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Spleen (50) (40) (58) (50)

Autolysis 1 (2%) 2 (4%) Depletion lymphoid 12 (24%) 2 (5%) 2 (4%) Fibrosis 6 (12%) 3 (8%) 2 (3%) 5 (10%) Hyperplasia, RE cell 1 (2%) Infiltration cellular, histiocyte 1 (2%) Necrosis, coagulative 2 (4%) Thymus (37) (32) (41) (34) Cyst 2 (5%)

Integumentary System Mammary gland (27) (22) (29) (24) Galactocele 3 (11%) 1 (5%) 1 (3%) Hyperplasia 16 (59%) 18 (82%) 17 (59%) 12 (50%) Skin (50) (38) (58) (50)

Abscess 2 (5%) 1 (2%) Cyst epithelial inclusion 1 (2%) 3 (5%) Fibrosis 1 (2%) Inflammation, chronic active 1 (2%) 2 (3%) 2 (4%)

Musculoskeletal System Bone (50) (40) (59) (50)

Cranium, abscess 1 (2%) Joint, tarsal, hyperostosis 1 (2%) Joint, tarsal, inflammati on, chronic active 1 (2%) Rib, hyperostosis 1 (3%) Skeletal muscle (2) (1) (1)

Fibrosis 1 (100%) Polyarteritis 1 (100%)

Nervous System Brain (50) (40) (59) (50)

Infarct 3 (6%) Spinal cord (5) (1) (1) (4)

Infarct 1 (20%) 1 (100%)

Respiratory System Lung (50) (40) (59) (49)

Crystals 1 (2%) Fibrosis 3 (5%) Foreign body 1 (2%) Infiltration cellular, histio cyte 21 (42%) 22 (55%) 32 (54%) 23 (47%) Inflammation, chronic active 1 (3%) 5 (8%) 3 (6%) Metaplasia, osseous 1 (2%) Respiratory epithelium, hyperplasia 2 (4%) 1 (2%) 2 (4%)Lesions in Male Rats 149



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Respiratory System (continued) Lung (continued) (50) (40) (59) (49)

Necrosis, coagulative 1 (2%) Alveolar epithelium, hyperplasia 4 (8%) 1 (3%) 7 (12%) 1 (2%) Artery, mineralization 22 (44%) 14 (35%) 36 (61%) 28 (57%) Artery, thrombosis 1 (2%) Bronchiole, epithelium, hyperplasia 1 (2%) Mediastinum, polyarteritis 2 (5%) Nose (48) (40) (59) (50)

Autolysis 1 (2%) Foreign body 2 (5%) 7 (12%) 1 (2%) Hemorrhage 3 (6%) Inflammation, chronic active 26 (54%) 25 (63%) 27 (46%) 18 (36%) Metaplasia, squamous 10 (21%) 7 (18%) 7 (12%) 6 (12%) Ulcer 2 (4%) Glands, hyperplasia 9 (19%) 3 (6%) Respiratory epithelium, hyperplasia 2 (4%) 1 (2%) 2 (4%) Trachea (50) (40) (59) (50)

Autolysis 1 (2%) Inflammation, chronic active 1 (3%) 3 (5%) 2 (4%)

Special Senses System Ear (3) (7) (2) (1)

Acanthosis 1 (33%) 1 (14%) 1 (50%) Hyperkeratosis 1 (33%) Hyperplasia, basal cell 1 (33%) 1 (14%) 1 (50%) Inflammation, chronic active 1 (33%) Submucosa, abscess 1 (100%) Eye (6) (3) (1) (6)

Cataract 1 (100%) Synechia 1 (17%) Anterior, synechia 1 (17%) Cornea, inflammation, chronic active 1 (17%) Lens, cataract 3 (50%) Retina, degeneration 1 (17%) 1 (100%) 3 (50%)

Urinary System Kidney (50) (40) (59) (50)

Autolysis 2 (4%) 1 (2%) 4 (8%) Cyst 4 (8%) 3 (8%) 1 (2%) Cyst, multiple 1 (2%) Fibrosis, focal 1 (3%) Hydronephrosis 1 (2%) Nephropathy 50 (100%) 40 (100%) 59 (100%) 49 (98%) Collecting tubule, mineralization 1 (2%) Renal tubule, degeneration, hyaline 1 (2%) 1 (2%) Renal tubule, hyperplasia 9 (18%) 30 (75%) 25 (42%) 19 (38%) Renal tubule, hyperplasia, oncocytic 1 (3%) 1 (2%)150 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### A5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Urinary System (continued) Kidney (continued) (50) (40) (59) (50)

Renal tubule, inflammation, chronic active 14 (28%) 10 (25%) 22 (37%) 14 (28%) Renal tubule, mineralization 1 (2%) 2 (5%) 13 (22%) 4 (8%) Renal tubule, pigmentation 5 (10%) 40 (100%) 58 (98%) 49 (98%) Transitional epithelium, hyperp lasia 30 (60%) 40 (100%) 51 (86%) 35 (70%) Urinary bladder (50) (38) (58) (50)

Autolysis 1 (2%) 1 (2%) Edema 1 (3%) Fibrosis 1 (3%) Hemorrhage 2 (4%) Hyperplasia, lymphoid 4 (8%) Inflammation, chronic 1 (2%) Inflammation, chronic active 1 (2%) 3 (8%) 5 (10%) Polyarteritis 1 (3%) Transitional epithelium, hyperplasia 1 (2%) 5 (13%) 17 (29%) 30 (60%) Transitional epithelium, metaplasia, squamous 3 (6%)151



# APPENDIX B
SUMMARY OF LESIONS IN FEMALE RATS
IN THE 2-YEAR FEED STUDY OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 T ABLE 

### B4a Historical Incidence of Hepatocellular Neoplasms in Untreated Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 T ABLE 

### B4b Historical Incidence of Large Intestine Neoplasms in Untreated Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 T ABLE 

### B4c Historical Incidence of Renal Tubule Neoplasms in Untreated Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 T ABLE 

### B4d Historical Incidence of Urinary Bladder Neoplasms in Untreated Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 T ABLE 

### B4e Historical Incidence of Forestomach Squamous Cell Neoplasms in Untreated Female F344/N Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 T ABLE 

### B5 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192152 1-Amino-2,4-dibromoanthraquinone, NTP TR 383Lesions in Female Rats 153



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Disposition Summary Animals initially in study 70 50 70 70 9-Month interim evaluation 10 10 10 10 15-Month interim evaluation 10 10 Early deaths

Moribund 8 5 15 29 Natural deaths 4 3 7 8 Survivors

Died last week of study 1 Terminal sacrifice 37 32 38 12 Missexed 1

Animals examined microscopically 70 50 70 69



### 9-Month Interim Evaluation Genital System Uterus (10) (10) (10) (10)

Polyp stromal 1 (10%)



### Systems Examined With No Neoplasms Observed Alimentary System Cardiovascular System Endocrine System General Body System Hematopoietic System Integumentary System Musculoskeletal System Nervous System Respiratory System Special Senses System Urinary System



### 15-Month Interim Evaluation Alimentary System Intestine large, rectum (10) (10)

Polyp adenomatous 2 (20%) Liver (10) (10)

Hepatocellular carcinoma 3 (30%) Hepatocellular carcinoma, multiple 3 (30%) Hepatocellular adenoma 3 (30%) Hepatocellular adenoma, multiple 5 (50%)

Endocrine System Adrenal medulla (10) (1) Pheochromocytoma benign 1 (100%) Pituitary gland (10)

Pars distalis, adenoma 2 (20%)154 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) Genital System Uterus (10) (3)

Polyp stromal 1 (10%) 1 (33%)

Integumentary System Mammary gland (7) Fibroadenoma 1 (14%) Skin (10) (1)

Subcutaneous tissue, fibroma 1 (100%)

Urinary System Kidney (10) (10) Urinary bladder (10) (10)

Carcinoma 2 (20%) Papilloma 1(10%) Squamous cell carcinoma 2 (20%)



### Systems Examined With No Neoplasms Observed Cardiovascular System General Body System Hematopoietic System Musculoskeletal System Nervous System Respiratory System Special Senses System



### 2-Year Study Alimentary System Esophagus (50) (40) (57) (48) Intestine large, colon (49) (40) (59) (47)

Adenocarcinoma 2 (3%) 1 (2%) Adenocarcinoma, multiple 1 (3%) Polyp adenomatous 1 (2%) 1 (2%) Polyp adenomatous, multiple 1 (3%) 1 (2%) 1 (2%) Intestine large, rectum (49) (40) (60) (47)

Adenocarcinoma 1 (3%) 18 (30%) 6 (13%) Adenocarcinoma, multiple 1 (2%) 1 (2%) Polyp adenomatous 10 (25%) 8 (13%) 12 (26%) Polyp adenomatous, multiple 17 (43%) 45 (75%) 31 (66%) Intestine large, cecum (50) (40) (60) (47)

Polyp adenomatous 1 (2%) Intestine small, jejunum (48) (40) (59) (46) Intestine small, ileum (49) (39) (59) (44)

Leiomyoma 1 (2%)Lesions in Female Rats 155



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Liver (50) (40) (60) (48)

Cholangioma 1 (2%) Hepatocellular carcinoma 5 (13%) 6 (10%) 4 (8%) Hepatocellular carcinoma, multiple 7 (18%) 51 (85%) 41 (85%) Hepatocellular adenoma 10 (25%) 8 (13%) 7 (15%) Hepatocellular adenoma, multiple 18 (45%) 39 (65%) 22 (46%) Hepatocholangiocarcinoma 10 (17%) 8 (17%) Hepatocholangiocarcinoma, multiple 1 (2%) 5 (10%) Hepatocholangioma 1 (2%) Hepatocholangioma, multiple 1 (2%) Mesentery (4) (6) (1) (4) Hepatocellular carcinoma, metastatic, liver 3 (75%) Pancreas (50) (40) (60) (47)

Adenoma 2 (3%) Hepatocellular carcinoma, metastatic, liver 1 (2%) Pharynx (1)

Palate, squamous cell papilloma, multiple 1 (100%) Stomach, forestomach (49) (40) (60) (47)

Squamous cell carcinoma 1 (3%) 1 (2%) 1 (2%) Squamous cell papilloma 1 (2%) Stomach, glandular (50) (40) (60) (48)

Hepatocellular carcinoma, metastatic, liver 1 (2%) Tongue (1) Squamous cell papilloma 1 (100%) Tooth (1) (1) (1) Gingiva, squamous cell carcinoma 1 (100%)

Cardiovascular System Heart (50) (40) (60) (49)

Hepatocellular carcinoma, metastatic, liver 1 (2%) 2 (4%)

Endocrine System Adrenal cortex (47) (40) (59) (47)

Adenoma 2 (4%) 1 (3%) Carcinoma, metastatic, kidney 1 (2%) Adrenal medulla (47) (40) (59) (47) Pheochromocytoma complex 1 (2%) Pheochromocytoma benign 2 (4%) 3 (8%) 1 (2%) 1 (2%) Bilateral, pheochromocytoma benign 1 (2%) Islets, pancreatic (50) (40) (60) (47)

Adenoma 1 (3%) Hepatocellular carcinoma, metastatic, liver 1 (2%) Parathyroid gland (43) (37) (57) (38)

Adenoma 1 (2%) Pituitary gland (50) (39) (60) (47)

Pars distalis, adenoma 27 (54%) 16 (41%) 25 (42%) 13 (28%) Pars distalis, adenoma, multiple 5 (10%) 3 (8%) 7 (12%) Pars intermedia, adenoma 1 (3%)156 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE B1 Summary of t he Inci dence of Neoplasms in Femal e Rats i n the 2- Year Feed Study

of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 2,000 ppm 5,000 ppm 10,00 0 ppm



### 2-Year Study

(conti nued) Endocrine System

(conti nued) Thyr oid gl and (50) (40) (60) (49) C-cell, ade

noma 5 (10%) 5 (13%) 5 (8%) C-cell, adenoma, multip le 1 (2%) C-cell, car

cinoma 1 (2%) 1 (2%)
Foll icular cell , adenoma 1 (3%) 1 (2%) 2 (4%)

General Body System

Tissue NOS (2) (1) (1) Basosqu amous t umor m aligna nt 1 (100%) Neoplasm NOS 1 (100%)
Sarcoma 2 (100%)

Genital System

Clitor al gl and (45) (36) (60) (45)
Adenoma 5 (11%) 3 (8%) 3 (5%) 2 (4%) Ovary (50) (40) (60) (47) Granulosa cell tumor benign 1 (2%) 1 (3%) Hepatoc ellular ca rcinom a, meta stati c, liver 1 (2%) 1 (2%)
Uter us (50) (40) (60) (47)
Adenocarcinom

a 1 (2%) Hemangioma 1 (2%) Polyp s tromal 7 (14%) 12 (30%) 7 (12%) 5 (11%) Polyp stromal, multiple 1 (2%) 3 (8%)
Sarcoma strom

al 1 (2%)

Hematop oietic System

Bone marrow (50) (40) (60) (49) Ly mph node (3) (5) (25) (14) Mediastinal, hepatocel

lular carcinoma,

metast atic, liver 1 (7%) Lymph no de, mandibular (50) (39) (56) (45) Ly mph node , mese

nteric (50) (40) (59) (46) Spleen (50) (40) (60) (48) Hepatoc ellular ca rcinom a, meta stati c, liver 1 (2%) Lipoma 1 (3%) Thym us (42) (31) (51) (38) Hepatoc ellular ca rcinom a, meta stati c, liver 1 (3%)

Integumen

tary System Mammary

gland (49) (34) (50) (41)
Adenocarcinom

a 2 (4%)
Fibroadenoma 17 (35%) 8 (24%) 9 (18%) 5 (12%) Fibroadenoma, multip le 4 (8%) 2 (6%)
Fibroma 1 (2%)Lesions in Female Rats 157



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Integumentary System (continued) Skin (50) (39) (60) (49)

Squamous cell papilloma 1 (2%) 1 (2%) 1 (2%) Pinna, granular cell tumor benign 1 (2%) Pinna, squamous cell papilloma 2 (3%) 2 (4%) Subcutaneous tissue, fibroma 2 (4%) 2 (5%) Subcutaneous tissue, sarcoma 1 (2%)

Musculoskeletal System Bone (50) (40) (60) (49)

Basosquamous tumor malignant, metastatic,

tissue NOS 1 (3%) Squamous cell carcinoma, metastatic,

uncertain primary site 1 (2%) Skeletal muscle (1)

Rhabdomyosarcoma 1 (100%)

Nervous System Brain (50) (40) (60) (49)

Oligodendroglioma benign 1 (2%)

Respiratory System Lung (50) (40) (60) (49)

Alveolar/bronchiolar adenoma 1 (2%) 2 (5%) 1 (2%) Alveolar/bronchiolar adenoma, multiple 1 (3%) Alveolar/bronchiolar carcinoma 1 (2%) Hepatocellular carcinoma, metastatic, liver 1 (3%) 22 (37%) 24 (49%) Neoplasm NOS, metastatic, uncertain primary

site 1 (2%) Sarcoma, metastatic, thymus 1 (2%) Squamous cell carcinoma 1 (3%) Nose (50) (40) (60) (49)

Special Senses System

Ear (8) (3) Zymbal's gland (1) (2)

Adenoma 1 (50%) Carcinoma 1 (50%) Squamous cell carcinoma 1 (100%)158 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Urinary System Kidney (50) (40) (60) (48)

Squamous cell carcinoma, metastatic,

urinary bladder 1 (2%) Pelvis, transitional epithelium, carcinoma 1 (3%) Pelvis, transitional epithelium, papilloma 1 (2%) Renal tubule, adenoma 3 (8%) 11 (18%) 11 (23%) Renal tubule, adenoma, multiple 5 (8%) 5 (10%) Renal tubule, carcinoma 2 (4%) Urinary bladder (50) (40) (60) (46)

Transitional epithelium, carcinoma 8 (13%) 16 (35%) Transitional epithelium, papilloma 2 (5%) 6 (10%) 9 (20%) Transitional epithelium, papilloma, multiple 1 (2%) Transitional epithelium, squamous cell

carcinoma 1 (2%) Transitional epithelium, squamous cell

papilloma 1 (2%) 1 (2%) Transitional epithelium, squamous cell

papilloma, multiple 1 (2%)

Systemic Lesions Multiple organs b (50) (40) (60) (49) Leukemia mononuclear 9 (18%) 1 (3%) 5 (8%) 1 (2%) Lymphoma malignant histiocytic 1 (2%)

Neoplasm Summary Total animals with primary neoplasms c
9-Month interim evaluation 1 15-Month interim evaluation 2 9 2-Year study 46 40 60 48 Total primary neoplasms 9-Month interim evaluation 1 15-Month interim evaluation 4 24 2-Year study 100 148 306 228 Total animals with benign neoplasms 9-Month interim evaluation 1 15-Month interim evaluation 2 8 2-Year study 43 40 60 46 Total benign neoplasms 9-Month interim evaluation 1 15-Month interim evaluation 4 14 2-Year study 83 128 197 139 Total animals with malignant neoplasms 15-Month interim evaluation 9 2-Year study 16 15 60 46 Total malignant neoplasms 15-Month interim evaluation 10 2-Year study 17 20 108 89Lesions in Female Rats 159



### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Neoplasm Summary (continued) Total animals with metastatic neoplasms 2-Year study 1 2 22 27 Total metastatic neoplasms 2-Year study 1 2 26 38 Total animals with malignant neoplasms of uncertain primary site 2-Year study 2 Total animals with uncertain neoplasms -

benign or malignant 2-Year study 1 Total uncertain neoplasms 2-Year study 1

a Number of animals examined microscopically at site and number of animals with neoplasm b Number of animals with any tissue examined microscopically

c Primary neoplasms: all neoplasms except metastatic neoplasms160 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm

2666666677777777777777777 Number of Days on Study 6002455700113333333333333
8160268548691444444444444

6667777777777666667777777 Carcass ID Number 8687323016843667773445556
5343433442452121231121231

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + M + + + + + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + M + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + A + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + + Mesentery + + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + M + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tooth +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + M + M + + + M + + + + + + + + + + + M + M + +

Adenoma Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X Pars distalis, adenoma, multiple Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X X C-cell, carcinoma X

General Body System
Tissue NOS + +

Sarcoma X X

Genital System Clitoral gland + M M + + + + + M + + + + + + + + + + + + + + + +

Adenoma X X X X Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Granulosa cell tumor benign Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X Hemangioma X Polyp stromal X X Polyp stromal, multiple

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examinedLesions in Female Rats 161



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
5555555555566666666666666

6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total Carcass ID Number 8 8 8 9 9 4 4 5 7 9 9 0 0 0 1 1 1 2 2 7 8 8 8 9 9 Tissues/
1 2 3 1 2 3 4 4 2 3 4 1 2 3 1 2 3 1 2 1 1 2 3 1 2 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine large, rectum + + + + + + + + + + + + + + + + + M + + + + + + + 49 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, jejunum + + M + + + + + + + + + + + + + + + + + + + + + + 48 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50 Mesentery + + 4 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 49 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Tooth 1

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System
Adrenal cortex + + + + + + + M + M + M + + + + + + + + + + + + + 47

Adenoma X 2 Adrenal medulla + + + + + + + M + M + M + + + + + + + + + + + + + 47

Pheochromocytoma benign X X 2 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 Parathyroid gland + + + + + + + M + + + + + + + + + + + + M + + + + 43

Adenoma X 1 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pars distalis, adenoma X X X X X X X X X X X X X X 27 Pars distalis, adenoma, multiple X X X X X 5 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

C-cell, adenoma X X 5 C-cell, carcinoma 1

General Body System
Tissue NOS 2

Sarcoma 2

Genital System Clitoral gland + + + + + + + + + + + M + + + + + + + + M + + + + 45

Adenoma X 5 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 50

Granulosa cell tumor benign X 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenocarcinoma 1 Hemangioma 1 Polyp stromal X X X X X 7 Polyp stromal, multiple X 1162 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

2666666677777777777777777 Number of Days on Study 6002455700113333333333333
8160268548691444444444444

6667777777777666667777777 Carcass ID Number 8687323016843667773445556
5343433442452121231121231

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + M + + + M + + + + I M + + + + + + + M

Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + M + + + +

Adenocarcinoma X Fibroadenoma X X X X X Fibroadenoma, multiple X X X Fibroma Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma Subcutaneous tissue, fibroma Subcutaneous tissue, sarcoma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X Sarcoma, metastatic, thymus X Nose + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System Eye + + + + + + + + + + Zymbal's gland +

Squamous cell carcinoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X X XLesions in Female Rats 163



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
5555555555566666666666666

6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Total Carcass ID Number 8 8 8 9 9 4 4 5 7 9 9 0 0 0 1 1 1 2 2 7 8 8 8 9 9 Tissues/
1 2 3 1 2 3 4 4 2 3 4 1 2 3 1 2 3 1 2 1 1 2 3 1 2 Tumors

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node + + 3 Lymph node, mandibular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 50 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + M + + + + + + + + M + + + + M + + + + + + 42

Integumentary System Mammary gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenocarcinoma X 2 Fibroadenoma X X X X X X X X X X X X 17 Fibroadenoma, multiple X 4 Fibroma X 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell papilloma X 1 Subcutaneous tissue, fibroma X X 2 Subcutaneous tissue, sarcoma 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma 1 Sarcoma, metastatic, thymus 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + 50 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses System Eye + + + 13 Zymbal's gland 1

Squamous cell carcinoma 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Leukemia mononuclear X X X X 9164 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm

6666666677777777777777777 Number of Days on Study 0115667833333333333333333
0169140900333333333333334

8888888888888888888888888 Carcass ID Number 0103476433677778888899990
4435354234312341234512341

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma, multiple Polyp adenomatous, multiple X Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma Polyp adenomatous X X X X X X Polyp adenomatous, multiple X X X X X X X X X X Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + M + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma X Hepatocellular carcinoma, multiple X X X X Hepatocellular adenoma X X X X X X X Hepatocellular adenoma, multiple X X X X X X X X X X X X X Mesentery + + + + + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Pharynx +

Palate, squamous cell papilloma,

multiple X Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tooth +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X Parathyroid gland + + + + + + + + + + + + + + M + + + M + + + + + + Pituitary gland + M + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X Pars distalis, adenoma, multiple X X Pars intermedia, adenoma X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X Follicular cell, adenoma XLesions in Female Rats 165



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
444444444444444

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 Total Carcass ID Number 0 1 1 1 2 2 2 3 3 4 5 5 5 6 6 Tissues/
2 1 2 3 1 2 3 1 2 1 1 2 3 1 2 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + 40 Intestine large, colon + + + + + + + + + + + + + + + 40

Adenocarcinoma, multiple X 1 Polyp adenomatous, multiple 1 Intestine large, rectum + + + + + + + + + + + + + + + 40

Adenocarcinoma X 1 Polyp adenomatous X X X X 10 Polyp adenomatous, multiple X X X X X X X 17 Intestine large, cecum + + + + + + + + + + + + + + + 40 Intestine small, duodenum + + + + + + + + + + + + + + + 40 Intestine small, jejunum + + + + + + + + + + + + + + + 40 Intestine small, ileum + + + + + + + + + + + + + + + 39 Liver + + + + + + + + + + + + + + + 40

Hepatocellular carcinoma X X X X 5 Hepatocellular carcinoma, multiple X X X 7 Hepatocellular adenoma X X X 10 Hepatocellular adenoma, multiple X X X X X 18 Mesentery + 6 Pancreas + + + + + + + + + + + + + + + 40 Pharynx 1

Palate, squamous cell papilloma,

multiple 1 Salivary glands + + + + + + + + + + + + + + + 40 Stomach, forestomach + + + + + + + + + + + + + + + 40

Squamous cell carcinoma X 1 Stomach, glandular + + + + + + + + + + + + + + + 40 Tooth 1

Cardiovascular System

Heart + + + + + + + + + + + + + + + 40

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + 40

Adenoma X 1 Adrenal medulla + + + + + + + + + + + + + + + 40

Pheochromocytoma benign X X 3 Islets, pancreatic + + + + + + + + + + + + + + + 40

Adenoma 1 Parathyroid gland + + + + + + + M + + + + + + + 37 Pituitary gland + + + + + + + + + + + + + + + 39

Pars distalis, adenoma X X X X X X 16 Pars distalis, adenoma, multiple X 3 Pars intermedia, adenoma 1 Thyroid gland + + + + + + + + + + + + + + + 40

C-cell, adenoma X X X X 5 Follicular cell, adenoma 1166 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm (continued)

6666666677777777777777777 Number of Days on Study 0115667833333333333333333
0169140900333333333333334

8888888888888888888888888 Carcass ID Number 0103476433677778888899990
4435354234312341234512341

General Body System
Tissue NOS

Basosquamous tumor malignant

Genital System Clitoral gland + + + + + M M + + + + + + + + + + + + + + + + + +

Adenoma X X Ovary + + + + + + + + + + + + + + + + + + + + + + + + +

Granulosa cell tumor benign X Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X X X X X X X X Polyp stromal, multiple X X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + Lymph node, mandibular M + + + + + + + + + + + + + + + + + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Lipoma Thymus M + + + M + M + + + M + + + + + + + + + + + + + +

Integumentary System Mammary gland + + + + + + + + + + + M M + + + + + M + + + + M +

Fibroadenoma X X X X X Fibroadenoma, multiple X X Skin + + + + + + + + + + + + M + + + + + + + + + + + +

Subcutaneous tissue, fibroma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Basosquamous tumor malignant,

metastatic, tissue NOS Skeletal muscle

Rhabdomyosarcoma

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X Alveolar/bronchiolar adenoma,

multiple X Hepatocellular carcinoma, metastatic,

liver X Squamous cell carcinoma Nose + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Female Rats 167



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
444444444444444

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 Total Carcass ID Number 0 1 1 1 2 2 2 3 3 4 5 5 5 6 6 Tissues/
2 1 2 3 1 2 3 1 2 1 1 2 3 1 2 Tumors

General Body System
Tissue NOS + 1

Basosquamous tumor malignant X 1

Genital System Clitoral gland + + + + + + + + + + M + M + + 36

Adenoma X 3 Ovary + + + + + + + + + + + + + + + 40

Granulosa cell tumor benign 1 Uterus + + + + + + + + + + + + + + + 40

Polyp stromal X X X 12 Polyp stromal, multiple X 3

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + 40 Lymph node + + + 5 Lymph node, mandibular + + + + + + + + + + + + + + + 39 Lymph node, mesenteric + + + + + + + + + + + + + + + 40 Spleen + + + + + + + + + + + + + + + 40

Lipoma X 1 Thymus + M + + + M + + + M M + M + + 31

Integumentary System Mammary gland + + M + + + + + + + + M + + + 34

Fibroadenoma X X X 8 Fibroadenoma, multiple 2 Skin + + + + + + + + + + + + + + + 39

Subcutaneous tissue, fibroma X 2

Musculoskeletal System

Bone + + + + + + + + + + + + + + + 40

Basosquamous tumor malignant,

metastatic, tissue NOS X 1 Skeletal muscle + 1

Rhabdomyosarcoma X 1

Nervous System

Brain + + + + + + + + + + + + + + + 40

Respiratory System Lung + + + + + + + + + + + + + + + 40

Alveolar/bronchiolar adenoma X 2 Alveolar/bronchiolar adenoma,

multiple 1 Hepatocellular carcinoma, metastatic,

liver 1 Squamous cell carcinoma X 1 Nose + + + + + + + + + + + + + + + 40 Trachea + + + + + + + + + + + + + + + 40168 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm (continued)

6666666677777777777777777 Number of Days on Study 0115667833333333333333333
0169140900333333333333334

8888888888888888888888888 Carcass ID Number 0103476433677778888899990
4435354234312341234512341

Special Senses System Eye + + + + + +

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Pelvis, transitional epithelium,

carcinoma Renal tubule, adenoma X X X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Transitional epithelium, papilloma X

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear XLesions in Female Rats 169



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 2,000 ppm (continued)

777777777777777 Number of Days on Study 333333333333333
444444444444444

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 Total Carcass ID Number 0 1 1 1 2 2 2 3 3 4 5 5 5 6 6 Tissues/
2 1 2 3 1 2 3 1 2 1 1 2 3 1 2 Tumors

Special Senses System Eye + 7

Urinary System Kidney + + + + + + + + + + + + + + + 40

Pelvis, transitional epithelium,

carcinoma X 1 Renal tubule, adenoma 3 Urinary bladder + + + + + + + + + + + + + + + 40

Transitional epithelium, papilloma X 2

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + 40

Leukemia mononuclear 1170 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 5,000 ppm

5566666666666666677777777777777 Number of Days on Study 7800034444666799901112222222222
5216922799007015684695999999999

1010100101010001011001000000000 Carcass ID Number 0909099090909990900990999999999
0726098342830671610532000011112 5355454444335424333443123412341

Alimentary System Esophagus + + + + + + + M + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + M + + + + +

Adenocarcinoma X Polyp adenomatous Polyp adenomatous, multiple X Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X X X X X X X Adenocarcinoma, multiple Polyp adenomatous X X X X X X Polyp adenomatous, multiple X X X X X X X X X X X X X X X X X X Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp adenomatous Intestine small, duodenum + + + + + + + + + + + + M + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + M + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + M + + + + + + + + +

Leiomyoma Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma X X X X X Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X X X X X X X Hepatocellular adenoma X X X X X Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X Hepatocholangiocarcinoma X X X X X Hepatocholangiocarcinoma, multiple X Hepatocholangioma X Hepatocholangioma, multiple Mesentery + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liver X

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma complex X Pheochromocytoma benign Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Female Rats 171



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 5,000 ppm (continued)

77777777777777777777777777777 Number of Days on Study 22222223333333333333333333333
99999990000000000000000000000

00011110000000000000000001111 Carcass ID Number 9 9 9 0 0 0 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 Total 2 2 2 0 0 3 3 3 3 3 4 4 4 5 5 5 6 6 7 8 8 9 9 9 9 1 1 2 2 Tissues/ 2 3 4 1 2 1 2 1 2 3 1 2 3 1 2 3 1 2 1 1 2 1 2 3 4 1 2 1 2 Tumors

Alimentary System Esophagus + + + + + + + + + + + M + + + + + + + + + + M + + + + + + 57 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Adenocarcinoma X 2 Polyp adenomatous X 1 Polyp adenomatous, multiple 1 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenocarcinoma X X X X X X X X X X X 18 Adenocarcinoma, multiple X 1 Polyp adenomatous X X 8 Polyp adenomatous, multiple X X X X X X X X X X X X X X X X X X X X X X X X X X X 45 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp adenomatous X 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59

Leiomyoma X 1 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma X 6 Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X X X X X X X X X X X X 51 Hepatocellular adenoma X X X 8 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X X 39 Hepatocholangiocarcinoma X X X X X 10 Hepatocholangiocarcinoma, multiple 1 Hepatocholangioma 1 Hepatocholangioma, multiple X 1 Mesentery 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 2 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell carcinoma 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma, metastatic,

liver 1

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + M + + + + + + + + + + + + + + 59 Adrenal medulla + + + + + + + + + + + + + + M + + + + + + + + + + + + + + 59

Pheochromocytoma complex 1 Pheochromocytoma benign X 1 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60172 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 5,000 ppm (continued)

5566666666666666677777777777777 Number of Days on Study 7800034444666799901112222222222
5216922799007015684695999999999

1010100101010001011001000000000 Carcass ID Number 0909099090909990900990999999999
0726098342830671610532000011112 5355454444335424333443123412341

Endocrine System (continued) Parathyroid gland + + + + + + + + + + + + + + + + + + + + + M + + + + + + M + + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X X X X X X X X X X Pars distalis, adenoma, multiple X X X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma X X X C-cell, carcinoma X Follicular cell, adenoma X

General Body System
Tissue NOS

Neoplasm NOS

Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X X Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X X X Sarcoma stromal

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + + + + + Lymph node, mandibular + + + + + M + + + + + + + + + + M + + + + + M + + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + M + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liver X Thymus + M M + I M + M + M + + + M + + + + + + M + + + + + + + + + +

Integumentary System Mammary gland + + + M + + + + + + + + + + + M + + + + + + + + + + + + + M +

Fibroadenoma X X X X X Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma X Pinna, granular cell tumor benign Pinna, squamous cell papilloma

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Female Rats 173



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone: 5,000 ppm (continued)

77777777777777777777777777777 Number of Days on Study 22222223333333333333333333333
99999990000000000000000000000

00011110000000000000000001111 Carcass ID Number 9 9 9 0 0 0 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 Total
2 2 2 0 0 3 3 3 3 3 4 4 4 5 5 5 6 6 7 8 8 9 9 9 9 1 1 2 2 Tissues/
2 3 4 1 2 1 2 1 2 3 1 2 3 1 2 3 1 2 1 1 2 1 2 3 4 1 2 1 2 Tumors

Endocrine System (continued) Parathyroid gland + + + + + + + + + + + + M + + + + + + + + + + + + + + + + 57 Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Pars distalis, adenoma X X X X X X X X X X 25 Pars distalis, adenoma, multiple X X X X 7 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

C-cell, adenoma X X 5 C-cell, carcinoma 1 Follicular cell, adenoma 1

General Body System

Tissue NOS + 1

Neoplasm NOS X 1

Genital System Clitoral gland + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Adenoma X 3 Ovary + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma, metastatic,

liver X 1
Uterus + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Polyp stromal X X X 7
Sarcoma stromal X 1

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Lymph node + + + + + + + + + + + + + + + + 25 Lymph node, mandibular + + + + M + + + + + + + + + + + + + + + + + + + + + + + + 56 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 59 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Hepatocellular carcinoma, metastatic,

liver 1 Thymus + + M + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Integumentary System Mammary gland M + + + + M + M + M + + + M M + + + + + + M + + + + + + + 50

Fibroadenoma X X X X 9
Skin + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Squamous cell papilloma 1 Pinna, granular cell tumor benign X 1 Pinna, squamous cell papilloma X X2

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60174 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 5,000 ppm (continued)

5566666666666666677777777777777 Number of Days on Study 7800034444666799901112222222222
5216922799007015684695999999999

1010100101010001011001000000000 Carcass ID Number 0909099090909990900990999999999
0726098342830671610532000011112 5355454444335424333443123412341

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X Hepatocellular carcinoma, metastatic,

liver X X X X X X X X X Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System

Ear + + + Eye + Zymbal's gland +

Adenoma Carcinoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

urinary bladder X Renal tubule, adenoma X X X X X X Renal tubule, adenoma, multiple X X Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Transitional epithelium, carcinoma X X Transitional epithelium, papilloma X X X X Transitional epithelium, papilloma,

multiple Transitional epithelium, squamous

cell carcinoma X Transitional epithelium, squamous

cell papilloma

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X X X XL

esion s in F em ale R ats 175

T ABLE B2

Individu al Anim al Tum or Pa thology of Fem ale Rat s in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 5,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 Carc ass ID N um ber 9 9 9 0 0 0 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 0 0 0 0 Total
2 2 2 0 0 3 3 3 3 3 4 4 4 5 5 5 6 6 7 8 8 9 9 9 9 1 1 2 2 Tissues/
2 3 4 1 2 1 2 1 2 3 1 2 3 1 2 3 1 2 1 1 2 1 2 3 4 1 2 1 2 Tum ors

Resp iratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Alve ol ar /b ron chi ol ar

ade noma 1 Hepatocellular carcinoma, m etastatic,
liver X X X X X X X X X X X X X 22
Nose + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60

Special Senses S

ystem

Ear + + + + + 8 Eye + + + + + + 7 Zymbal's gland + 2 Adeno ma X 1
Car ci noma 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Squam ous cell carcinoma, metastatic, urinary bladder 1
Rena l t ubul e, ade noma X X X X X 11
Re nal tub ule , ad eno ma , m ultip le X X X 5 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60
Tra nsi ti ona l e pi th el iu m, c ar ci noma X X X X X X 8
Tra nsi ti ona l e pi th el iu m, pa pi ll oma X X 6 Transitional epithelium, papilloma,

mu ltipl e X 1 Transiti onal epit helium, squamous
cel l c ar ci noma 1 Transiti onal epit helium, squamous
cel l pa pi ll oma X 1

Syst emic Les io ns Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 60 Leukem ia monon uclear X 5176 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm

344555555556666666666666 Number of Days on Study 616145667890111233333566
780239696080046556678822

111111111111111111111111 Carcass ID Number 101101001011010010010101
774593475813805909948266 353545444434344331222133

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + M + + + + + + +

Adenocarcinoma Polyp adenomatous Polyp adenomatous, multiple Intestine large, rectum + + + + + + + + + + + + + + + + M + + + + + + +

Adenocarcinoma Adenocarcinoma, multiple X Polyp adenomatous X X X X X X X X X X Polyp adenomatous, multiple X X X X X X X X X Intestine large, cecum + + + + + + + + + A + + + + + + M + + + + + + + Intestine small, duodenum + + + + M + + + + + + + + + + + M + + + + + + A Intestine small, jejunum + + + + + + + + + A + + + + + + M + + + + + + A Intestine small, ileum + + + + + + + + + A + + + M + + M + + + + + + A Liver + + + + + + + + + + + + + + + + M + + + + + + +

Cholangioma Hepatocellular carcinoma X X X Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X Hepatocellular adenoma X X X X Hepatocellular adenoma, multiple X X X X X X X X X X X X X Hepatocholangiocarcinoma X X X Hepatocholangiocarcinoma, multiple X X Mesentery +

Hepatocellular carcinoma, metastatic,

liver Pancreas + + + + + + + + + + + + + + + + M + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Salivary glands + + + M + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + M + + + + + + +

Squamous cell carcinoma X Squamous cell papilloma Stomach, glandular + + + + + + + + + + + + + + + + M + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Tongue +

Squamous cell papilloma X Tooth

Gingiva, squamous cell carcinoma

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + +

Hepatocellular carcinoma, metastatic,

liverLesions in Female Rats 177



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666677777777777777777777 Number of Days on Study 6778900011122222222222222
4345522545902999999999999

1111111111111111111111111 Carcass ID Number 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 Total 6 5 7 6 4 5 8 3 1 3 4 6 7 4 4 5 5 7 0 0 1 3 5 5 6 Tissues/ 2 3 3 2 3 3 1 3 2 2 1 1 2 1 2 1 2 1 1 2 1 1 1 2 1 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + M + + + + + + 48 Intestine large, colon + + + + + + + + + + M + + + + + + + + + + + + + + 47

Adenocarcinoma X 1 Polyp adenomatous X 1 Polyp adenomatous, multiple X 1 Intestine large, rectum + + + + + M + + + + + + + + + + + + + + + + + + + 47

Adenocarcinoma X X X X X X 6 Adenocarcinoma, multiple 1 Polyp adenomatous X X 12 Polyp adenomatous, multiple X X X X X X X X X X X X X X X X X X X X X X 31 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 46 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 46 Intestine small, ileum + + + + + M + + + + + + + + + + + + + + + + + + + 44 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 48

Cholangioma X 1 Hepatocellular carcinoma X 4 Hepatocellular carcinoma, multiple X X X X X X X X X X X X X X X X X X X X X X X X 41 Hepatocellular adenoma X X X 7 Hepatocellular adenoma, multiple X X X X X X X X X 22 Hepatocholangiocarcinoma X X X X X 8 Hepatocholangiocarcinoma, multiple X X X 5 Mesentery + + + 4

Hepatocellular carcinoma, metastatic,

liver X X X 3 Pancreas + + + + + + + M + + + + + + + + + + + + + + + + + 47

Hepatocellular carcinoma, metastatic,

liver X 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 48 Stomach, forestomach + + + + + + + + + + + + + + + + + M + + + + + + + 47

Squamous cell carcinoma 1 Squamous cell papilloma X 1 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 48

Hepatocellular carcinoma, metastatic,

liver X 1 Tongue 1

Squamous cell papilloma 1 Tooth + 1

Gingiva, squamous cell carcinoma X 1

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 49

Hepatocellular carcinoma, metastatic,

liver X X 2178 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

344555555556666666666666 Number of Days on Study 616145667890111233333566
780239696080046556678822

111111111111111111111111 Carcass ID Number 101101001011010010010101
774593475813805909948266 353545444434344331222133

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + M + + + + + + +

Carcinoma, metastatic, kidney Adrenal medulla + + + + + + + + + + + + + + + + M + + + + + + +

Pheochromocytoma benign Bilateral, pheochromocytoma benign X Islets, pancreatic + + + + + + + + + + + + + + + + M + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Parathyroid gland + + M + + M + M + M + + M + + + M + + + + + M + Pituitary gland + + + + + + + + + + + + + + + + + + + + + + + +

Pars distalis, adenoma X X X X X X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + +

C-cell, adenoma, multiple Follicular cell, adenoma

General Body System

None

Genital System Clitoral gland + + M + + + + + + + + + + + + + M + + M + + + +

Adenoma X Ovary + + + + + + + + + + + + + + + + M + + + + + + +

Hepatocellular carcinoma, metastatic,

liver Uterus + + + + + + + + + + + + + + + + M + + + + + + +

Polyp stromal X X X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + +

Mediastinal, hepatocellular

carcinoma, metastatic, liver Lymph node, mandibular + + + + + + + + + + + + + + + M M + + + + + + + Lymph node, mesenteric + + + + + + + + + + + + + + + + M + + + + + + + Spleen + + + + + + + + + + + + + + + + M + + + + + + + Thymus + + + M + M + + + + + M + + + + M + + M + + + +

Hepatocellular carcinoma, metastatic,

liver

Integumentary System Mammary gland + + + + + + + + + + + + + + + M M + + + + + + +

Fibroadenoma X X X Skin + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma X Pinna, squamous cell papillomaLesions in Female Rats 179



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666677777777777777777777 Number of Days on Study 6778900011122222222222222
4345522545902999999999999

1111111111111111111111111 Carcass ID Number 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 Total 6 5 7 6 4 5 8 3 1 3 4 6 7 4 4 5 5 7 0 0 1 3 5 5 6 Tissues/ 2 3 3 2 3 3 1 3 2 2 1 1 2 1 2 1 2 1 1 2 1 1 1 2 1 Tumors

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + M + + + 47

Carcinoma, metastatic, kidney X 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + M + + + 47

Pheochromocytoma benign X 1 Bilateral, pheochromocytoma benign 1 Islets, pancreatic + + + + + + + M + + + + + + + + + + + + + + + + + 47

Hepatocellular carcinoma, metastatic,

liver X 1 Parathyroid gland + M + + + + + + + + + + M + + + + + + + M M + + + 38 Pituitary gland + + + + + + + + + + + M + + + + M + + + + + + + + 47

Pars distalis, adenoma X X X X X X X 13 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

C-cell, adenoma, multiple X 1 Follicular cell, adenoma X X 2

General Body System

None

Genital System Clitoral gland + + + + + M + + + + + + + + + + + + + + + + + + + 45

Adenoma X 2 Ovary + + + + + M + + + + + + + + + + + + + + + + + + + 47

Hepatocellular carcinoma, metastatic,

liver X 1 Uterus + + + + + M + + + + + + + + + + + + + + + + + + + 47

Polyp stromal X X 5

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + + + + + + 14

Mediastinal, hepatocellular

carcinoma, metastatic, liver X 1 Lymph node, mandibular + + M + + + + + + + + + M + + + + + + + + + + + + 45 Lymph node, mesenteric + + + + + + + + + + M + + + M + + + + + + + + + + 46 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 48 Thymus + + + + M M + + M + + + M + + M + + + + + + + + M 38

Hepatocellular carcinoma, metastatic,

liver X 1

Integumentary System Mammary gland + + + + M + + M + + + + + + M + M + + + M + + + M 41

Fibroadenoma X X 5 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 49

Squamous cell papilloma 1 Pinna, squamous cell papilloma X X 2180 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

344555555556666666666666 Number of Days on Study 616145667890111233333566
780239696080046556678822

111111111111111111111111 Carcass ID Number 101101001011010010010101
774593475813805909948266 353545444434344331222133

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

uncertain primary site X

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + +

Oligodendroglioma benign X Spinal cord +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar carcinoma Hepatocellular carcinoma, metastatic,

liver X X X X X X Neoplasm NOS, metastatic, uncertain

primary site X Nose + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses System

Ear Eye +

Urinary System Kidney + + + + + + + + + + + + + + + + M + + + + + + +

Pelvis, transitional epithelium,

papilloma Renal tubule, adenoma X X X X Renal tubule, adenoma, multiple Renal tubule, carcinoma Urinary bladder + + + + + + + + + + + + + + + + M + + + + + + A

Transitional epithelium, carcinoma X X X X X X X X X Transitional epithelium, papilloma X X Transitional epithelium, squamous

cell papilloma Transitional epithelium, squamous

cell papilloma, multiple

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + +

Leukemia mononuclear X Lymphoma malignant histiocyticLesions in Female Rats 181



### T ABLE 

### B2 Individual Animal Tumor Pathology of Female Rats in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

6666677777777777777777777 Number of Days on Study 6778900011122222222222222
4345522545902999999999999

1111111111111111111111111 Carcass ID Number 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 Total 6 5 7 6 4 5 8 3 1 3 4 6 7 4 4 5 5 7 0 0 1 3 5 5 6 Tissues/ 2 3 3 2 3 3 1 3 2 2 1 1 2 1 2 1 2 1 1 2 1 1 1 2 1 Tumors

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 49

Squamous cell carcinoma, metastatic,

uncertain primary site 1

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 49

Oligodendroglioma benign 1 Spinal cord 1

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 49

Alveolar/bronchiolar carcinoma X 1 Hepatocellular carcinoma, metastatic,

liver X X X X X X X X X X X X X X X X X X 24 Neoplasm NOS, metastatic, uncertain

primary site 1 Nose + + + + + + + + + + + + + + + + + + + + + + + + + 49 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 49

Special Senses System

Ear + + + 3 Eye + + + + + + 7

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 48

Pelvis, transitional epithelium,

papilloma X 1 Renal tubule, adenoma X X X X X X X 11 Renal tubule, adenoma, multiple X X X X X 5 Renal tubule, carcinoma X X 2 Urinary bladder + + + + + M + + + + + + + + + + + + + + + + + + + 46

Transitional epithelium, carcinoma X X X X X X X 16 Transitional epithelium, papilloma X X X X X X X 9 Transitional epithelium, squamous

cell papilloma X 1 Transitional epithelium, squamous

cell papilloma, multiple X 1

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 49

Leukemia mononuclear 1 Lymphoma malignant histiocytic X 1182 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Adrenal Medulla: Benign Pheochromocytoma
Overall rate a 2/47 (4%) 3/40 (8%) 1/59 (2%) 2/47 (4%) Adjusted rate b 5.7% 9.4% 2.7% 7.7% Terminal rate c 2/35 (6%) 3/32 (9%) 1/37 (3%) 0/11 (0%) First incidence (days) 729 (T) 729 (T) 729 (T) 636 Life table test d P=0.396 P=0.459 P=0.481N P=0.379 Logistic regression test d P=0.602N P=0.459 P=0.481N P=0.624 Cochran-Armitage test d P=0.441N Fisher exact test d P=0.423 P=0.415N P=0.692N

Adrenal Medulla: Benign or Complex Pheochromocytoma Overall rate 2/47 (4%) 3/40 (8%) 2/59 (3%) 2/47 (4%) Adjusted rate 5.7% 9.4% 4.6% 7.7% Terminal rate 2/35 (6%) 3/32 (9%) 1/37 (3%) 0/11 (0%) First incidence (days) 729 (T) 729 (T) 660 636 Life table test P=0.354 P=0.459 P=0.665N P=0.379 Logistic regression test P= 0.583N P=0.459 P=0.617N P=0.624 Cochran-Armitage test P=0.467N Fisher exact test P=0.423 P=0.601N P=0.692N

Clitoral Gland: Adenoma Overall rate 5/45 (11%) 3/36 (8%) 3/60 (5%) 2/45 (4%) Adjusted rate 13.3% 10.0% 7.4% 10.8% Terminal rate 4/36 (11%) 3/30 (10%) 2/38 (5%) 1/12 (8%) First incidence (days) 708 729 (T) 696 610 Life table test P=0.524N P=0.465N P=0.330N P=0.664 Logistic regression test P=0.265N P=0.488N P=0.263N P=0.365N Cochran-Armitage test P=0.137N Fisher exact test P=0.488N P=0.212N P=0.217N

Kidney (Renal Tubule): Adenoma Overall rate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%) Adjusted rate 0.0% 8.0% 36.0% 69.7% Terminal rate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%) First incidence (days)

) e 600 601 625 Life table test P<0.001 P=0.093 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.049 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.084 P<0.001 P<0.001

Kidney (Renal Tubule): Adenoma or Carcinoma Overall rate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%) Adjusted rate 0.0% 8.0% 36.0% 69.7% Terminal rate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%) First incidence (days)

) 600 601 625 Life table test P<0.001 P=0.093 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.049 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.084 P<0.001 P<0.001Lesions in Female Rats 183



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Large Intestine (Rectum): Adenomatous Polyp Overall rate 0/50 (0%) 27/40 (68%) 53/60 (88%) 43/49 (88%) Adjusted rate 0.0% 75.0% 100.0% 100.0% Terminal rate 0/38 (0%) 23/32 (72%) 38/38 (100%) 12/12 (100%) First incidence (days)

) 616 582 512 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Large Intestine (Rectum): Carcinoma Overall rate 0/50 (0%) 1/40 (3%) 19/60 (32%) 7/49 (14%) Adjusted rate 0.0% 3.1% 41.7% 41.9% Terminal rate 0/38 (0%) 1/32 (3%) 13/38 (34%) 4/12 (33%) First incidence (days)

) 729 (T) 606 625 Life table test P<0.001 P=0.466 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.466 P<0.001 P=0.001 Cochran-Armitage test P=0.005 Fisher exact test P=0.444 P<0.001 P=0.006

Large Intestine (All Sites): Adenomatous Polyp Overall rate 0/50 (0%) 28/40 (70%) 53/60 (88%) 43/49 (88%) Adjusted rate 0.0% 77.7% 100.0% 100.0% Terminal rate 0/38 (0%) 24/32 (75%) 38/38 (100%) 12/12 (100%) First incidence (days)

) 616 582 512 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Large Intestine (All Sites): Carcinoma Overall rate 0/50 (0%) 2/40 (5%) 21/60 (35%) 8/49 (16%) Adjusted rate 0.0% 6.3% 45.1% 46.3% Terminal rate 0/38 (0%) 2/32 (6%) 14/38 (37%) 4/12 (33%) First incidence (days)

) 729 (T) 606 625 Life table test P<0.001 P=0.201 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.201 P<0.001 P<0.001 Cochran-Armitage test P=0.004 Fisher exact test P=0.195 P<0.001 P=0.003

Liver: Hepatocellular Adenoma Overall rate 0/50 (0%) 28/40 (70%) 47/60 (78%) 29/48 (60%) Adjusted rate 0.0% 75.5% 83.7% 83.6% Terminal rate 0/38 (0%) 23/32 (72%) 29/38 (76%) 8/12 (67%) First incidence (days)

) 600 575 418 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001184 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Liver: Hepatocellular Carcinoma Overall rate 0/50 (0%) 12/40 (30%) 57/60 (95%) 45/48 (94%) Adjusted rate 0.0% 37.5% 98.3% 100.0% Terminal rate 0/38 (0%) 12/32 (38%) 37/38 (97%) 12/12 (100%) First incidence (days)

) 729 (T) 575 460 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 0/50 (0%) 33/40 (83%) 59/60 (98%) 47/48 (98%) Adjusted rate 0.0% 89.1% 100.0% 100.0% Terminal rate 0/38 (0%) 28/32 (88%) 38/38 (100%) 12/12 (100%) First incidence (days)

) 600 575 418 Life table test P<0.001 P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001 P<0.001

Liver: Hepatocholangiocarcinoma Overall rate 0/50 (0%) 0/40 (0%) 11/60 (18%) 13/48 (27%) Adjusted rate 0.0% 0.0% 27.1% 56.9% Terminal rate 0/38 (0%) 0/32 (0%) 9/38 (24%) 5/12 (42%) First incidence (days)

) ) 670 600 Life table test P<0.001

) P<0.001 P<0.001 Logistic regression test P<0.001

) P=0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test

) P<0.001 P<0.001

Lung: Alveolar/bronchiolar Adenoma Overall rate 1/50 (2%) 3/40 (8%) 1/60 (2%) 0/49 (0%) Adjusted rate 2.5% 9.4% 1.9% 0.0% Terminal rate 0/38 (0%) 3/32 (9%) 0/38 (0%) 0/12 (0%) First incidence (days) 716 729 (T) 647

) Life table test P=0.338N P=0.244 P=0.739N P=0.695N Logistic regression test P=0.196N P=0.229 P=0.732N P=0.616N Cochran-Armitage test P=0.154N Fisher exact test P=0.229 P=0.705N P=0.505N

Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 1/50 (2%) 3/40 (8%) 1/60 (2%) 1/49 (2%) Adjusted rate 2.5% 9.4% 1.9% 5.0% Terminal rate 0/38 (0%) 3/32 (9%) 0/38 (0%) 0/12 (0%) First incidence (days) 716 729 (T) 647 702 Life table test P=0.572 P=0.244 P=0.739N P=0.559 Logistic regression test P= 0.472N P=0.229 P=0.732N P=0.682 Cochran-Armitage test P=0.380N Fisher exact test P=0.229 P=0.705N P=0.747Lesions in Female Rats 185



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Mammary Gland: Fibroadenoma Overall rate 21/50 (42%) 10/40 (25%) 9/60 (15%) 5/49 (10%) Adjusted rate 52.2% 29.1% 20.7% 22.3% Terminal rate 19/38 (50%) 8/32 (25%) 6/38 (16%) 1/12 (8%) First incidence (days) 606 659 582 610 Life table test P=0.058N P=0.055N P=0.008N P=0.210N Logistic regression test P=0.001N P=0.065N P=0.002N P=0.008N Cochran-Armitage test P<0.001N Fisher exact test P=0.071N P=0.002N P<0.001N

Mammary Gland: Fibroma, Fibroadenoma, or Carcinoma Overall rate 23/50 (46%) 10/40 (25%) 9/60 (15%) 5/49 (10%) Adjusted rate 57.2% 29.1% 20.7% 22.3% Terminal rate 21/38 (55%) 8/32 (25%) 6/38 (16%) 1/12 (8%) First incidence (days) 606 659 582 610 Life table test P=0.030N P=0.024N P=0.003N P=0.147N Logistic regression test P<0.001N P=0.029N P<0.001N P=0.004N Cochran-Armitage test P<0.001N Fisher exact test P=0.033N P<0.001N P<0.001N

Pituitary Gland (Pars Distalis): Adenoma Overall rate 32/50 (64%) 19/39 (49%) 32/60 (53%) 13/47 (28%) Adjusted rate 74.3% 55.6% 62.3% 55.7% Terminal rate 27/38 (71%) 17/32 (53%) 19/38 (50%) 4/11 (36%) First incidence (days) 642 616 575 512 Life table test P=0.285 P=0.060N P=0.510N P=0.431 Logistic regression test P=0.015N P=0.081N P=0.208N P=0.013N Cochran-Armitage test P<0.001N Fisher exact test P=0.109N P=0.175N P<0.001N

Skin: Squamous Cell Papilloma Overall rate 1/50 (2%) 0/40 (0%) 3/60 (5%) 3/49 (6%) Adjusted rate 2.6% 0.0% 7.9% 19.4% Terminal rate 1/38 (3%) 0/32 (0%) 3/38 (8%) 2/12 (17%) First incidence (days) 729 (T)

) 729 (T) 637 Life table test P=0.007 P=0.534N P=0.305 P=0.056 Logistic regression test P= 0.033 P=0.534N P=0.305 P=0.171 Cochran-Armitage test P=0.109 Fisher exact test P=0.556N P=0.381 P=0.301

Skin (Subcutaneous Tissue): Fibroma Overall rate 2/50 (4%) 2/40 (5%) 0/60 (0%) 0/49 (0%) Adjusted rate 5.3% 6.0% 0.0% 0.0% Terminal rate 2/38 (5%) 1/32 (3%) 0/38 (0%) 0/12 (0%) First incidence (days) 729 (T) 670

) ) Life table test P=0.148N P=0.625 P=0.238N P=0.513N Logistic regression test P=0.089N P=0.616 P=0.238N P=0.513N Cochran-Armitage test P=0.068N Fisher exact test P=0.603 P=0.204N P=0.253N186 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Skin (Subcutaneous Tissue): Fibroma or Sarcoma Overall rate 3/50 (6%) 2/40 (5%) 0/60 (0%) 0/49 (0%) Adjusted rate 7.5% 6.0% 0.0% 0.0% Terminal rate 2/38 (5%) 1/32 (3%) 0/38 (0%) 0/12 (0%) First incidence (days) 704 670

) ) Life table test P=0.080N P=0.591N P=0.122N P=0.355N Logistic regression test P=0.039N P=0.599N P=0.100N P=0.247N Cochran-Armitage test P=0.031N Fisher exact test P=0.606N P=0.091N P=0.125N

Thyroid Gland (C-cell): Adenoma Overall rate 5/50 (10%) 5/40 (13%) 5/60 (8%) 1/49 (2%) Adjusted rate 13.2% 15.6% 12.7% 6.7% Terminal rate 5/38 (13%) 5/32 (16%) 4/38 (11%) 0/12 (0%) First incidence (days) 729 (T) 729 (T) 714 719 Life table test P=0.405N P=0.519 P=0.627N P=0.509N Logistic regression test P=0.303N P=0.519 P=0.604N P=0.421N Cochran-Armitage test P=0.058N Fisher exact test P=0.481 P=0.509N P=0.107N

Thyroid Gland (C-cell): Adenoma or Carcinoma Overall rate 5/50 (10%) 5/40 (13%) 6/60 (10%) 1/49 (2%) Adjusted rate 13.2% 15.6% 15.2% 6.7% Terminal rate 5/38 (13%) 5/32 (16%) 5/38 (13%) 0/12 (0%) First incidence (days) 729 (T) 729 (T) 714 719 Life table test P=0.469N P=0.519 P=0.503 P=0.509N Logistic regression test P=0.360N P=0.519 P=0.530 P=0.421N Cochran-Armitage test P=0.069N Fisher exact test P=0.481 P=0.622N P=0.107N

Urinary Bladder: Papilloma Overall rate 0/50 (0%) 2/40 (5%) 7/60 (12%) 9/46 (20%) Adjusted rate 0.0% 6.3% 17.6% 39.5% Terminal rate 0/38 (0%) 2/32 (6%) 6/38 (16%) 1/12 (8%) First incidence (days)

) 729 (T) 691 637 Life table test P<0.001 P=0.201 P=0.010 P<0.001 Logistic regression test P<0.001 P=0.201 P=0.012 P=0.003 Cochran-Armitage test P<0.001 Fisher exact test P=0.195 P=0.012 P<0.001

Urinary Bladder: Carcinoma Overall rate 0/50 (0%) 0/40 (0%) 8/60 (13%) 16/46 (35%) Adjusted rate 0.0% 0.0% 19.5% 55.8% Terminal rate 0/38 (0%) 0/32 (0%) 6/38 (16%) 4/12 (33%) First incidence (days)

) ) 670 367 Life table test P<0.001

) P=0.006 P<0.001 Logistic regression test P<0.001

) P=0.008 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test

) P=0.006 P<0.001Lesions in Female Rats 187



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Urinary Bladder: Papilloma, Squamous Cell Pa pilloma, Carcinoma, or Sq uamous Cell Carcinoma Overall rate 0/50 (0%) 2/40 (5%) 17/60 (28%) 26/46 (57%) Adjusted rate 0.0% 6.3% 40.9% 78.1% Terminal rate 0/38 (0%) 2/32 (6%) 14/38 (37%) 6/12 (50%) First incidence (days)

) 729 (T) 670 367 Life table test P<0.001 P=0.201 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.201 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.195 P<0.001 P<0.001

Uterus: Stromal Polyp Overall rate 8/50 (16%) 15/40 (38%) 7/60 (12%) 5/49 (10%) Adjusted rate 20.2% 41.4% 16.6% 17.4% Terminal rate 7/38 (18%) 11/32 (34%) 5/38 (13%) 0/12 (0%) First incidence (days) 656 600 601 512 Life table test P=0.498N P=0.030 P=0.482N P=0.393 Logistic regression test P=0.041N P=0.019 P=0.371N P=0.295N Cochran-Armitage test P=0.038N Fisher exact test P=0.019 P=0.350N P=0.290N

Uterus: Stromal Polyp or Stromal Sarcoma Overall rate 8/50 (16%) 15/40 (38%) 8/60 (13%) 5/49 (10%) Adjusted rate 20.2% 41.4% 19.2% 17.4% Terminal rate 7/38 (18%) 11/32 (34%) 6/38 (16%) 0/12 (0%) First incidence (days) 656 600 601 512 Life table test P=0.540N P=0.030 P=0.589N P=0.393 Logistic regression test P=0.050N P=0.019 P=0.477N P=0.295N Cochran-Armitage test P=0.042N Fisher exact test P=0.019 P=0.449N P=0.290N

All Organs: Mononuclear Cell Leukemia Overall rate 9/50 (18%) 1/40 (3%) 5/60 (8%) 1/49 (2%) Adjusted rate 21.5% 3.0% 10.0% 3.7% Terminal rate 6/38 (16%) 0/32 (0%) 1/38 (3%) 0/12 (0%) First incidence (days) 620 689 601 662 Life table test P=0.112N P=0.026N P=0.177N P=0.162N Logistic regression test P=0.011N P=0.023N P=0.111N P=0.024N Cochran-Armitage test P=0.016N Fisher exact test P=0.019N P=0.110N P=0.009N

All Organs: Benign Neoplasms Overall rate 43/50 (86%) 40/40 (100%) 60/60 (100%) 46/49 (94%) Adjusted rate 91.5% 100.0% 100.0% 100.0% Terminal rate 34/38 (89%) 32/32 (100%) 38/38 (100%) 12/12 (100%) First incidence (days) 606 600 575 418 Life table test P<0.001 P=0.223 P=0.007 P<0.001 Logistic regression test P=0.009 P=0.025 P=0.004 P=0.015 Cochran-Armitage test P=0.164 Fisher exact test P=0.013 P=0.003 P=0.167188 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B3 Statistical Analysis of Prim ary Neoplasms in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

All Organs: Malignant Neoplasms Overall rate 16/50 (32%) 15/40 (38%) 60/60 (100%) 46/49 (94%) Adjusted rate 33.9% 45.5% 100.0% 100.0% Terminal rate 8/38 (21%) 14/32 (44%) 38/38 (100%) 12/12 (100%) First incidence (days) 268 689 575 367 Life table test P<0.001 P=0.424 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.341 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.373 P<0.001 P<0.001

All Organs: Benign or Malignant Neoplasms Overall rate 46/50 (92%) 40/40 (100%) 60/60 (100%) 49/49 (100%) Adjusted rate 92.0% 100.0% 100.0% 100.0% Terminal rate 34/38 (89%) 32/32 (100%) 38/38 (100%) 12/12 (100%) First incidence (days) 268 600 575 367 Life table test P<0.001 P=0.435 P=0.035 P<0.001 Logistic regression test P= 0.049 P=0.086 P=0.063 P=0.289 Cochran-Armitage test P=0.021 Fisher exact test P=0.090 P=0.040 P=0.061

(T)Terminal sacrifice

a Number of lesion-bearing animals/number of animals examined. De nominator is number of animals examined microscopically for adr enal gland, clitoral gland, kidney, liver, lung, pituitary gland, thyroid gland, urinary bladder, and uterus; for other tissues, denominator is number of animals necropsi ed. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Observed incidence at terminal kill d Beneath the control incidence are the P valu es associated with the trend test. Bene ath the exposed group incidence are the P v alues corresponding to pairwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare di rectly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal groupLesions in Female Rats 189



### T ABLE 

### B4a Historical Incidence of Hepatocellular Neoplasms in Untreated Female F344/N Rats a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/50 1/50 1/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/50 0/50 0/50 Turmeric Oleoresin 1/50 0/50 1/50

Overall Historical Incidence

Total 8/1,351 (0.6%) 1/1,351 (0.1%) 9/1,351 (0.7%) Standard deviation 1.5% 0.4% 1.5% Range 0%-6% 0%-2% 0%-6%

a Data as of 31 March 1993



### T ABLE 

### B4b Historical Incidence of Large Intestine Neoplasms in Untreated Female F344/N Rats a

Incidence in Controls Study Adenomatous Polyp Car cinoma Adenomatous Polyp (Adenoma) (Adenoma) or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/50 0/50 0/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/50 0/50 0/50 Turmeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 0/1,351 (0.0%) 0/1,351 (0.0%) 0/1,351 (0.0%)

a Data as of 31 March 1993; the data include incidences for the colon and rectum.190 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B4c Historical Incidence of Renal Tubule Neoplasms in Untreated Female F344/N Rats a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/49 0/49 0/49 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/49 0/49 0/49 Turmeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 1/1,348 (0.1%) 0/1,348 (0.0%) 1/1,348 (0.1%) Standard deviation 0.4% 0.4% Range 0%-2% 0%-2%

a Data as of 31 March 1993



### T ABLE 

### B4d Historical Incidence of Ur inary Bladder Neoplasms in Untreated Female F344/N Rats a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/44 0/44 0/44 HC Yellow 4 0/48 0/48 0/48 Methylphenidate Hydrochloride 0/47 0/47 0/47 Pentaerythritol Tetranitrate 0/49 0/49 0/49 Quercetin 1/50 0/50 1/50 Turmeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 3/1,334 (0.2%) 0/1,334 (0.0%) 3/1,334 (0.2%) Standard deviation 0.6% 0.6% Range 0%-2% 0%-2%

a Data as of 31 March 1993Lesions in Female Rats 191



### T ABLE 

### B4e Historical Incidence of Forest omach Squamous Cell Neoplasms in Untreated Female F344/N Rats a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 0/50 0/50 0/50 Methylphenidate Hydrochloride 0/50 0/50 0/50 Pentaerythritol Tetranitrate 0/50 0/50 0/50 Quercetin 0/50 0/50 0/50 Tumeric Oleoresin 0/50 1/50 1/50

Overall Historical Incidence

Total 0/1,351 (0%) 2/1,351 (0.2%) 2/1,351 (0.2%) Standard deviation 0.5% 0.5% Range 0%-2% 0%-2%

a Data as of 31 March 1993192 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm

Disposition Summary Animals initially in study 70 50 70 70 9-Month interim evaluation 10 10 10 10 15-Month interim evaluation 10 10 Early deaths

Moribund 8 5 15 29 Natural deaths 4 3 7 8 Survivors

Died last week of study 1 Terminal sacrifice 37 32 38 12 Missexed 1

Animals examined microscopically 70 50 70 69



### 9-Month Interim Evaluation Alimentary System Intestine large, colon (10) (10) (10) (10)

Parasite metazoan 1 (10%) 4 (40%) 2 (20%) 1 (10%) Lymphoid tissue, hemorrhage 1 (10%) Intestine large, rectum (10) (10) (10) (10)

Parasite metazoan 2 (20%) 1 (10%) Intestine large, cecum (10) (10) (10) (10)

Parasite metazoan 1 (10%) Liver (10) (10) (10) (10)

Basophilic focus 1 (10%) 1 (10%) Clear cell focus 1 (10%) Developmental malformation 1 (10%) Fatty change 1 (10%) 1 (10%) Inflammation, chronic active 2 (20%) 2 (20%) Necrosis, coagulative 4 (40%) Pigmentation 2 (20%) 6 (60%) 6 (60%) Bile duct, hyperplasia 1 (10%) 5 (50%) 9 (90%) 3 (30%) Periportal inflammation, chronic active 6 (60%) 10 (100%) 10 (100%) 8 (80%) Pancreas (10) (10) (10) (10)

Atrophy 1 (10%) 2 (20%) Ectopic tissue 1 (10%) Infiltration cellular, mononuclear cell 3 (30%) 7 (70%) 1 (10%) 1 (10%) Infiltration cellular, mixed cell 1 (10%) Salivary glands (10) (10) (10) (10)

Infiltration cellula r, mononuclear cell 2 (20%) Infiltration cellular, lymphocyte 1 (10%) Stomach, forestomach (10) (10) (10) (10) Stomach, glandular (10) (10) (10) (10)

Muscularis, mineralization 1 (10%)

Cardiovascular System Heart (10) (10) (10) (10)

Cardiomyopathy 7 (70%) 8 (80%) 6 (60%) 3 (30%)

a Number of animals examined microscopically at site and number of animals with lesionLesions in Female Rats 193



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) Endocrine System Adrenal cortex (10) (10) (10) (10)

Angiectasis 3 (30%) 1 (10%) Hyperplasia 1 (10%) Capsule, fibrosis 1 (10%) Capsule, inflammation, chronic 1 (10%) Zona reticularis, hyperplasia 1 (10%) Pituitary gland (10) (10) (10) (10)

Pars distalis, angiectasis 3 (30%) Pars distalis, cyst 2 (20%) 1 (10%) 4 (40%) 1 (10%) Pars distalis, hemorrhage 1 (10%) Pars distalis, hyperplasia 1 (10%) 1 (10%) Pars distalis, pars intermedia, cyst 2 (20%) Pars intermedia, cyst 1 (10%) 2 (20%) Thyroid gland (10) (10) (10) (10) Infiltration cellular, mononuclear cell 1 (10%) Ultimobranchial cyst 1 (10%) C-cell, hyperplasia 1 (10%) Follicular cell, hyperplasia 1 (10%)

General Body System
Tissue NOS (1) Hemorrhage 1 (100%)

Genital System Clitoral gland (10) (10) (8) (10)

Infiltration cellular, mononuclear cell 1 (10%) Inflammation, chronic 3 (30%) Inflammation, chronic active 2 (20%) 3 (30%) 4 (50%) Ovary (10) (10) (10) (10)

Congestion 1 (10%) Cyst 1 (10%) Pigmentation 1 (10%) Periovarian tissue, cyst 4 (40%) 3 (30%) Uterus (10) (10) (10) (10)

Decidual reaction 1 (10%) Hydrometra 1 (10%) 2 (20%) 1 (10%) 5 (50%)

Hematopoietic System Lymph node (3) (6) (3) (7)

Pigmentation 1 (14%) Lumbar, hemorrhage 1 (14%) Lumbar, infiltration cellular, histiocyte 1 (14%) Lumbar, pigmentation 1 (14%) Mediastinal, hemorrhage 3 (100%) 1 (17%) 3 (100%) 3 (43%) Mediastinal, infiltration cellular,

histiocyte 1 (17%) 1 (14%) Mediastinal, pigmentation 1 (17%) 2 (67%) 2 (29%)194 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) Hematopoietic System (continued) Lymph node (continued) (3) (6) (3) (7)

Pancreatic, depletion lymphoid 1 (17%) Pancreatic, hemorrhage 2 (33%) 1 (33%) 3 (43%) Pancreatic, infiltration cellular, histiocyte 3 (50%) Pancreatic, pigmentation 3 (50%) 1 (33%) 1 (14%) Renal, hemorrhage 1 (17%) 1 (14%) Renal, infiltration cellular, histiocyte 1 (14%) Renal, pigmentation 1 (17%) 1 (14%) Lymph node, mandibular (9) (10) (10) (8)

Hemorrhage 7 (78%) 3 (30%) 5 (50%) 4 (50%) Infiltration cellular, histiocyte 1 (10%) Pigmentation 1 (10%) Lymph node, mesenteric (10) (10) (10) (10)

Hemorrhage 4 (40%) 4 (40%) 2 (20%) Infiltration cellular, histio cyte 10 (100%) 10 (100%) 10 (100%) 10 (100%) Pigmentation 7 (70%) 8 (80%) Spleen (10) (10) (10) (10)

Congestion 1 (10%) Depletion lymphoid 2 (20%) Thymus (10) (10) (10) (8) Congestion 1 (10%) Depletion lymphoid 1 (10%) Hemorrhage 1 (10%) 1 (10%)

Respiratory System Lung (10) (10) (10) (10)

Hemorrhage 1 (10%) Infiltration cellular, histio cyte 1 (10%) 2 (20%) 3 (30%) 2 (20%) Alveolar epithelium, hyperplasia 1 (10%) Artery, mineralization 4 ( 40%) 3 (30%) 3 (30%) 2 (20%)

Urinary System Kidney (10) (10) (10) (10)

Hydronephrosis 1 (10%) Infiltration cellular 1 (10%) Infiltration cellular, mononu clear cell 9 (90%) 7 (70%) 3 (30%) Inflammation, chronic 4 (40%) Mineralization 3 (30%) 1 (10%) Pigmentation 1 (10%) Renal tubule, pigmentation 10 (100%) 10 (100%) 10 (100%) Renal tubule, regeneration 4 (40%) 5 (50%) 7 (70%) 4 (40%) Transitional epithelium, mineralization 1 (10%) 1 (10%) Urinary bladder (10) (10) (10) (10)

Infiltration cellula r, mononuclear cell 1 (10%) Transitional epithelium, hyperplasia 2 (20%) Transitional epithelium, infiltration cellular, mononuclear cell 1 (10%)Lesions in Female Rats 195



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 9-Month Interim Evaluation (continued) 

### Systems Examined With No Lesions Observed Integumentary System Musculoskeletal System Nervous Sytem Special Senses System



### 15-Month Interim Evaluation Alimentary System Intestine large, colon (10)

Parasite metazoan 2 (20%) Intestine large, rectum (10) (10)

Parasite metazoan 1 (10%) Intestine large, cecum (10)

Parasite metazoan 2 (20%) Liver (10) (10)

Abscess 1 (10%) Basophilic focus 8 (80%) 9 (90%) Clear cell focus 5 (50%) Eosinophilic focus 2 (20%) Fatty change 8 (80%) Hepatodiaphragmatic nodule 2 (20%) 1 (10%) Inflammation, chronic, gr anulomatous 6 (60%) 6 (60%) Inflammation, chronic active 1 (10%) Necrosis, coagulative 1 (10%) 4 (40%) Pigmentation 1 (10%) 10 (100%) Bile duct, hyperplasia 7 (70%) 10 (100%) Periportal, inflammation, chronic 10 (100%) 10 (100%) Mesentery (1) Inflammation, chronic 1 (100%) Pigmentation 1 (100%) Pancreas (10) (1)

Atrophy 1 (10%) Ectopic tissue 1 (10%) Inflammation, chronic 9 (90%) 1 (100%) Pharynx (1)

Hemorrhage 1 (100%) Salivary glands (10) Stomach, forestomach (10) (10)

Hyperplasia, basal cell 1 (10%) Inflammation, chronic 1 (10%) Stomach, glandular (10) (10)

Muscularis, mineralization 1 (10%)

Cardiovascular System Heart (10)

Cardiomyopathy 10 (100%)196 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) Endocrine System Adrenal cortex (10) (1)

Angiectasis 5 (50%) Vacuolization cytoplasmic 1 (100%) Adrenal medulla (10) (1) Pituitary gland (10)

Pars distalis, cyst 8 (80%) Pars distalis, hyperplasia 1 (10%) Pars intermedia, cyst 3 (30%) Thyroid gland (10) C-cell, hyperplasia 4 (40%)

Genital System Clitoral gland (10) (3)

Inflammation, chronic 7 (70%) 3 (100%) Inflammation, chronic active 1 (10%) Ovary (10) (4)

Cyst 2 (50%) Periovarian tissue, cyst 4 (100%) Uterus (10) (3)

Hydrometra 2 (20%) 3 (100%) Endometrium, hyperplasia 1 (10%)

Hematopoietic System Lymph node (1) (3)

Mediastinal, hemorrhage 1 (100%) Mediastinal, pigmentation 1 (100%) Pancreatic, infiltration cellular, histiocyte 3 (100%) Pancreatic, pigmentation 3 (100%) Lymph node, mandibular (7)

Hemorrhage 1 (14%) Lymph node, mesenteric (10) (4)

Hemorrhage 1 (25%) Infiltration cellular, histiocyte 10 (100%) 4 (100%) Pigmentation 10 (100%) 4 (100%) Thymus (10) (9) Cyst 1 (10%) 1 (11%) Depletion lymphoid 4 (44%) Pigmentation 1 (11%)

Integumentary System Mammary gland (7) Hyperplasia 6 (86%)

Musculoskeletal System Bone (10)

Osteopetrosis 1 (10%)Lesions in Female Rats 197



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 15-Month Interim Evaluation (continued) Respiratory System Lung (10) (5)

Hemorrhage 1 (10%) Infiltration cellular, histiocyte 6 (60%) 5 (100%) Inflammation, acute 1 (20%) Alveolus, mineralization 1 (10%) Artery, mineralization 4 (40%) 2 (40%) Nose (10) Submucosa, inflammation, chronic 1 (10%)

Urinary System Kidney (10) (10)

Nephropathy 10 (100%) 10 (100%) Renal tubule, hyperplasia 3 (30%) Renal tubule, mineralization 1 (10%) Renal tubule, pigmentation 10 (100%) Transitional epithelium, hyperplasia 3 (30%) 4 (40%) Transitional epithelium, mineralization 2 (20%) 1 (10%) Urinary bladder (10) (10)

Inflammation, chronic 1 (10%) Transitional epithelium, hyperplasia 9 (90%)



### Systems Examined With No Lesions Observed General Body System Nervous System Special Senses System



### 2-Year Study Alimentary System Esophagus (50) (40) (57) (48)

Autolysis 1 (2%) Intestine large, colon (49) (40) (59) (47)

Autolysis 3 (6%) 5 (8%) 3 (6%) Parasite metazoan 14 (29%) 9 (23%) 7 (12%) 6 (13%) Intestine large, rectum (49) (40) (60) (47)

Atypia cellular 1 (3%) 6 (10%) Autolysis 4 (8%) 6 (10%) 1 (2%) Diverticulum 1 (2%) Fibrosis 1 (3%) Inflammation, acute, necrotizing 3 (6%) Parasite metazoan 10 (20%) 4 (10%) 8 (13%) 8 (17%) Ulcer 1 (2%) Epithelium, hyperplasia 3 (5%)198 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Intestine large, cecum (50) (40) (60) (47)

Autolysis 4 (8%) 1 (3%) 6 (10%) 4 (9%) Hemorrhage 1 (3%) Mineralization 1 (2%) Parasite metazoan 4 (8%) 6 (15%) 5 (8%) 2 (4%) Intestine small, duodenum (50) (40) (59) (46)

Autolysis 2 (4%) 5 (8%) 1 (2%) Intestine small, jejunum (48) (40) (59) (46)

Autolysis 3 (6%) 1 (3%) 8 (14%) 3 (7%) Intestine small, ileum (49) (39) (59) (44)

Autolysis 3 (6%) 1 (3%) 8 (14%) 4 (9%) Parasite metazoan 1 (3%) Ulcer 1 (2%) Liver (50) (40) (60) (48)

Angiectasis 1 (2%) 1 (3%) Autolysis 4 (7%) Basophilic focus 39 (78%) 15 (38%) 22 (37%) 16 (33%) Clear cell focus 3 (6%) 28 (70%) 39 (65%) 17 (35%) Degeneration 3 (5%) Eosinophilic focus 7 (14%) 23 (58%) 12 (20%) 1 (2%) Fatty change 8 (16%) 3 (5%) 5 (10%) Hematopoietic cell proliferation 10 (17%) 2 (4%) Hemorrhage 1 (2%) Hepatodiaphragmatic nodule 8 (16%) 4 (10%) 1 (2%) Hepatodiaphragmatic nodule, multiple 1 (2%) Hyperplasia, focal 1 (3%) Infarct 1 (2%) Inflammation, chronic active 21 (42%) 16 (40%) 23 (38%) 5 (10%) Mixed cell focus 1 (2%) 11 (28%) 5 (8%) 4 (8%) Necrosis, coagulative 3 (6%) 4 (10%) 11 (18%) 9 (19%) Pigmentation 1 (2%) 19 (48%) 51 (85%) 45 (94%) Thrombosis 1 (2%) 1 (2%) Bile duct, hyperplasia 33 (66%) 5 (13%) 56 (93%) 42 (88%) Centrilobular, hemorrhage 1 (2%) Centrilobular, necrosis, coagulative 1 (2%) Periportal, inflammation, chronic active 25 (50%) 2 (5%) 54 (90%) 39 (81%) Mesentery (4) (6) (1) (4) Fibrosis 2 (50%) 5 (83%) 1 (100%) 1 (25%) Inflammation, chronic acti ve 3 (75%) 3 (50%) 1 (100%) 1 (25%) Necrosis, coagulative 2 (50%) 5 (83%) 1 (100%) Polyarteritis 1 (17%) 1 (25%) Pancreas (50) (40) (60) (47)

Atrophy 20 (40%) 17 (43%) 16 (27%) 15 (32%) Autolysis 1 (2%) 1 (2%) 1 (2%) Cytoplasmic alteration 1 (3%) 2 (3%) 1 (2%) Ectopic liver 2 (3%) Ectopic tissue 1 (2%) Inflammation, chronic active 29 (58%) 24 (60%) 30 (50%) 22 (47%) Polyarteritis 2 (5%) Vacuolization cytoplasmic 5 (10%) 12 (20%) 3 (6%) Acinus, hyperplasia 1 (2%) 1 (2%) Duct, hyperplasia 1 (2%)Lesions in Female Rats 199



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Salivary glands (50) (40) (60) (48)

Parotid gland, inflammation , chronic active 2 (3%) 1 (2%) Sublingual gland, inflammation, chronic

active 1 (2%) Submandibular gland, inflammation, chronic

active 1 (2%) 1 (2%) Stomach, forestomach (49) (40) (60) (47)

Autolysis 1 (2%) Cyst epithelial inclusion 1 (2%) Erosion 2 (3%) 3 (6%) Hyperkeratosis 2 (4%) 7 (18%) 23 (38%) 28 (60%) Hyperplasia, basal cell 2 (4%) 7 (18%) 35 (58%) 28 (60%) Hyperplasia, squamous 2 (4%) 7 (18%) 26 (43%) 33 (70%) Inflammation, chronic active 1 (3%) 13 (22%) 10 (21%) Ulcer 1 (2%) 2 (5%) 7 (12%) 17 (36%) Stomach, glandular (50) (40) (60) (48)

Autolysis 2 (4%) 3 (5%) 1 (2%) Fibrosis 1 (2%) Inflammation, chronic active 3 (6%) 1 (3%) 6 (10%) 6 (13%) Necrosis, coagulative 1 (2%) 1 (2%) Ulcer 1 (2%) Tongue (1) Foreign body 1 (100%) Inflammation, chronic 1 (100%) Tooth (1) (1) (1) Cyst 1 (100%)

Cardiovascular System Heart (50) (40) (60) (49)

Cardiomyopathy 46 (92%) 38 (95%) 54 (90%) 40 (82%)

Endocrine System Adrenal cortex (47) (40) (59) (47)

Angiectasis 25 (53%) 29 (73%) 40 (68%) 26 (55%) Autolysis 1 (2%) 3 (6%) Hyperplasia 3 (6%) 4 (10%) 3 (5%) 1 (2%) Hypertrophy 1 (2%) Necrosis, coagulative 1 (2%) Thrombosis, multiple 1 (2%) Vacuolization cytoplasmic 17 (36%) 16 (40%) 35 (59%) 19 (40%) Capsule, hyperplasia 1 (2%) Adrenal medulla (47) (40) (59) (47)

Autolysis 1 (2%) 1 (2%) Hyperplasia 11 (23%) 5 (13%) 11 (19%) 14 (30%) Islets, pancreatic (50) (40) (60) (47)

Autolysis 1 (2%) Parathyroid gland (43) (37) (57) (38)

Hyperplasia 3 (8%) 1 (2%)200 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Endocrine System (continued) Pituitary gland (50) (39) (60) (47)

Thrombosis 1 (2%) Pars distalis, angiectasis 1 (2%) Pars distalis, autolysis 2 (3%) 1 (2%) Pars distalis, cyst 14 (28%) 20 (51%) 19 (32%) 17 (36%) Pars distalis, cyst, multiple 3 (6%) Pars distalis, hyperplasia 12 (24%) 9 (23%) 15 (25%) 8 (17%) Pars distalis, necrosis, coagulative 1 (2%) Pars intermedia, cyst 1 (2%) Rathke's cleft, cyst 1 (2%) Thyroid gland (50) (40) (60) (49)

Autolysis 2 (4%) 1 (2%) 4 (8%) Inflammation, chronic 1 (3%) Inflammation, chronic active 1 (2%) Ultimobranchial cyst 1 (2%) C-cell, hyperplasia 7 (14%) 2 (5%) 3 (5%) 1 (2%) Follicle, cyst 1 (2%) Follicular cell, hyperplasia 1 (2%)

General Body System

None

Genital System Clitoral gland (45) (36) (60) (45)

Abscess 2 (4%) 3 (5%) 1 (2%) Cyst 1 (2%) 1 (3%) Hyperplasia 1 (3%) Inflammation, chronic active 34 (76%) 18 (50%) 47 (78%) 31 (69%) Duct, metaplasia, squamous 1 (2%) Ovary (50) (40) (60) (47)

Cyst 11 (22%) 2 (5%) 5 (8%) 4 (9%) Uterus (50) (40) (60) (47)

Abscess 6 (15%) 8 (13%) Autolysis 1 (2%) Cyst 1 (2%) 5 (13%) 3 (6%) Fibrosis 3 (6%) 1 (2%) 1 (2%) Hydrometra 5 (10%) 4 (10%) 8 (13%) 4 (9%) Inflammation, acute 1 (2%) Inflammation, chronic active 1 (2%) Endometrium, hyperplasia 4 (8%) 9 (23%) 2 (3%) 1 (2%)

Hematopoietic System Bone marrow (50) (40) (60) (49)

Autolysis 1 (2%) Myelofibrosis 1 (3%)Lesions in Female Rats 201



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Lymph node (3) (5) (25) (14)

Iliac, infiltration cellular, histiocyte 1 (7%) Lumbar, infiltration cellular, histiocyte 1 (7%) Mediastinal, hyperplasia 1 (33%) Mediastinal, infiltration cellular,

histiocyte 1 (33%) 2 (8%) 2 (14%) Pancreatic, hyperplasia 2 (67%) 5 (20%) 1 (7%) Pancreatic, infiltration cellular, histiocyte 3 (100%) 5 (100%) 21 (84%) 8 (57%) Renal, infiltration ce llular, histiocyte 1 (20%) Lymph node, mandibular (50) (39) (56) (45)

Autolysis 1 (2%) Depletion lymphoid 1 (2%) Hyperplasia 1 (3%) 2 (4%) 1 (2%) Infiltration cellular, hist iocyte 1 (2%) 1 (3%) 1 (2%) 9 (20%) Inflammation, chronic active 1 (2%) Lymph node, mesenteric (50) (40) (59) (46)

Autolysis 1 (2%) 2 (4%) Depletion lymphoid 2 (4%) 1 (3%) 2 (3%) 8 (17%) Infiltration cellular, histio cyte 49 (98%) 39 (98%) 57 (97%) 45 (98%) Spleen (50) (40) (60) (48)

Autolysis 1 (2%) Depletion lymphoid 8 (16%) 1 (3%) 3 (5%) 5 (10%) Fibrosis 1 (3%) 1 (2%) 1 (2%) Hematopoietic cell proliferation 1 (2%) Hyperplasia 1 (2%) Infiltration cellular, histiocyte 1 (2%) Thymus (42) (31) (51) (38) Cyst 1 (3%) Hemorrhage 1 (3%)

Integumentary System Mammary gland (49) (34) (50) (41) Galactocele 1 (2%) Hyperplasia 41 (84%) 29 (85%) 41 (82%) 26 (63%) Inflammation, chronic active 1 (2%) Skin (50) (39) (60) (49)

Abscess 1 (2%) 1 (2%) Cyst epithelial inclusion 2 (3%) Foreign body 1 (2%) Inflammation, chronic active 2 (4%) 6 (10%) 2 (4%) Foot, acanthosis 1 (2%) Foot, hyperkeratosis 1 (2%) Foot, inflammation, chronic active 1 (3%) 1 (2%)

Musculoskeletal System Bone (50) (40) (60) (49)

Cranium, osteopetrosis 1 (2%) Femur, osteopetrosis 1 (2%) Sternum, osteopetrosis 1 (2%) 3 (6%)202 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Nervous System Brain (50) (40) (60) (49)

Hydrocephalus 1 (2%) 2 (5%) Infarct 1 (2%)

Respiratory System Lung (50) (40) (60) (49)

Autolysis 1 (2%) Fibrosis 2 (3%) Infiltration cellular, histio cyte 31 (62%) 31 (78%) 50 (83%) 36 (73%) Inflammation, chronic active 4 (8%) 3 (5%) 2 (4%) Polyarteritis 1 (2%) Alveolar epithelium, hyperplasia 1 (2%) Artery, mineralization 23 (46%) 6 (15%) 20 (33%) 14 (29%) Bronchiole, epithelium, hyperplasia 1 (2%) Mediastinum, polyarteritis 2 (5%) Nose (50) (40) (60) (49)

Autolysis 1 (2%) Foreign body 1 (2%) 4 (7%) Inflammation, chronic active 9 (18%) 8 (20%) 13 (22%) 12 (24%) Metaplasia, squamous 1 (2%) 4 (10%) 4 (7%) 2 (4%) Arteriole, thrombosis 1 (2%) Trachea (50) (40) (60) (49)

Autolysis 1 (2%)

Special Senses System

Ear (8) (3)

External ear, inflammation, acute 2 (25%) Internal ear, inflammation, acute 1 (13%) Eye (13) (7) (7) (7)

Phthisis bulbi 1 (14%) 1 (14%) Synechia 1 (8%) 1 (14%) Cornea, inflammation, acute 1 (14%) Lens, cataract 1 (8%) 1 (14%) Retina, degeneration 1 (8%) 3 (43%) 1 (14%) Zymbal's gland (1) (2)

Autolysis 1 (100%)Lesions in Female Rats 203



### T ABLE 

### B5 Summary of the Incidence of Nonneoplastic Lesion s in Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm



### 2-Year Study (continued) Urinary System Kidney (50) (40) (60) (48)

Abscess 1 (2%) Autolysis 2 (4%) 3 (5%) 3 (6%) Cyst 1 (3%) 2 (3%) Fibrosis 1 (2%) Hydronephrosis 2 (4%) 2 (3%) Infarct 1 (3%) Inflammation, chronic active 1 (2%) Nephropathy 50 (100%) 39 (98%) 60 (100%) 46 (96%) Polyarteritis 1 (3%) Artery, thrombosis 1 (2%) Papilla, necrosis, coagulative 2 (4%) Pelvis, inflammation, chronic active 1 (2%) 1 (3%) 4 (7%) 6 (13%) Pelvis, metaplasia, squamous 3 (5%) 2 (4%) Renal tubule, hyperplasia 1 (2%) 12 (30%) 23 (38%) 27 (56%) Renal tubule, hyperplasia, oncocytic 1 (2%) Renal tubule, inflammation, acute 1 (2%) 7 (18%) 3 (5%) Renal tubule, inflammation, chronic active 2 (3%) 7 (15%) Renal tubule, pigmentation 40 (100%) 60 (100%) 48 (100%) Transitional epithelium, hyperplasia 10 (20%) 16 (40%) 44 (73%) 21 (44%) Transitional epithelium, mineralization 4 (10%) Urinary bladder (50) (40) (60) (46)

Autolysis 1 (2%) 2 (3%) Calculus, gross observation 2 (5%) 1 (2%) Calculus, microscopic observation only 1 (2%) Fibrosis 1 (2%) Hemorrhage 2 (4%) Hyperplasia, lymphoid 15 (30%) 2 (5%) 3 (5%) Inflammation, chronic active 1 (2%) 1 (3%) 3 (5%) 1 (2%) Necrosis 1 (2%) Fat, proliferation 4 (7%) 2 (4%) Transitional epithelium, hyperplasia 1 (2%) 2 (5%) 41 (68%) 41 (89%) Transitional epithelium, metaplasia, squamous 1 (3%) 4 (7%) 8 (17%)204 1-Amino-2,4-dibromoanthraquinone, NTP TR 383205



# APPENDIX C
SUMMARY OF LESIONS IN MALE MICE
IN THE 2-YEAR FEED STUDY OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 T ABLE 

### C3 Statistical Analysis of Pr imary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 T ABLE 

### C4a Historical Incidence of Hepatocellular Neoplasms in Untreated Male B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 T ABLE 

### C4b Historical Incidence of Forestomach Squamous Cell Neoplasms in Untreated Male B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 T ABLE 

### C4c Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Male B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 T ABLE 

### C5 Summary of the Incidence of No nneoplastic Lesions in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239206 1-Amino-2,4-dibromoanthraquinone, NTP TR 383Lesions in Male Mice 207



### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm

Disposition Summary Animals initially in study 60 60 60 15-Month interim evaluation 10 9 10 Early deaths Accidental death 1 Moribund 7 23 21 Natural deaths 3 5 6 Survivors Terminal sacrifice 40 22 23

Animals examined microscopically 60 60 60



### 15-Month Interim Evaluation Alimentary System Liver (10) (9) (10) Hepatocellular carcinoma 1 (11%) Hepatocellular adenoma 2 (22%) 4 (40%) Stomach, forestomach (9) (9) (10) Squamous cell carcinoma 1 (10%) Squamous cell papilloma 5 (50%)

Genital System Preputial gland (5) (1) Squamous cell carcinoma 1 (20%)

Respiratory System Lung (10) (9) (10) Alveolar/bronchiolar adenoma 3 (33%) 5 (50%)

Special Senses System Lacrimal gland (1) Adenoma 1 (100%)

Urinary System Urinary bladder (10) (9) (10) Papilloma 1 (10%)



### Systems Examined With No Neoplasms Observed Cardiovascular System Endocrine System General Body System Hematopoietic system Integumentary System Musculoskeletal System Nervous System208 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study Alimentary System Gallbladder (46) (47) (42) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) 1 (2%) Intestine large, colon (50) (49) (50) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Intestine large, rectum (48) (49) (48) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Intestine large, cecum (49) (51) (50) Adenocarcinoma 1 (2%) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Intestine small, duodenum (50) (50) (46) Mast cell tumor malignant 1 (2%) Polyp adenomatous 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Intestine small, jejunum (50) (47) (48) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Intestine small, ileum (50) (49) (47) Adenocarcinoma 1 (2%) 2 (4%) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Liver (50) (51) (50) Fibrosarcoma, metastatic, stomach,

forestomach 1 (2%) Hemangiosarcoma 2 (4%) 1 (2%) Hemangiosarcoma, multiple 3 (6%) Hepatoblastoma 3 (6%) 5 (10%) Hepatocellular carcinoma 8 (16%) 15 (29%) 12 (24%) Hepatocellular carcinoma, multiple 1 (2%) 3 (6%) 9 (18%) Hepatocellular adenoma 4 (8%) 9 (18%) 8 (16%) Hepatocellular adenoma, multiple 6 (12%) 29 (57%) 31 (62%) Hepatocholangiocarcinoma, multiple 1 (2%) Histiocytic sarcoma 1 (2%) 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic,

tissue NOS 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 4 (8%) 4 (8%)Lesions in Male Mice 209



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0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Mesentery (2) (7) (9) Sarcoma, metastatic, stomach, forestomach 1 (14%) Squamous cell carcinoma, metastatic, stomach,

forestomach 2 (29%) 5 (56%) Pancreas (50) (50) (48) Histiocytic sarcoma 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) 1 (2%) Salivary glands (50) (51) (50) Stomach, forestomach (50) (50) (50) Leiomyosarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma 12 (24%) 13 (26%) Squamous cell papilloma 11 (22%) 11 (22%) Squamous cell papilloma, multiple 2 (4%) 5 (10%) Stomach, glandular (50) (50) (49) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Sarcoma 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 3 (6%) 4 (8%) Tooth (4) (7)

Cardiovascular System Heart (50) (51) (50)

Endocrine System Adrenal cortex (50) (51) (50) Adenoma 2 (4%) 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Adrenal medulla (50) (50) (50) Pheochromocytoma benign 1 (2%) Pituitary gland (43) (45) (47) Pars distalis, adenoma 2 (4%) Thyroid gland (49) (50) (49) C-cell, adenoma 1 (2%)

General Body System Tissue NOS (1) (1) (1) Squamous cell carcinoma 1 (100%)

Genital System Coagulating gland (1) (1) (1) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (100%) 1 (100%)210 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Genital System (continued) Epididymis (49) (51) (50) Fibrosarcoma, metastatic, stomach,

forestomach 1 (2%) Histiocytic sarcoma 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) 1 (2%) Preputial gland (16) (16) (12) Squamous cell carcinoma 1 (6%) Prostate (47) (46) (48) Histiocytic sarcoma 1 (2%) Squamous cell carcinoma, metastatic,

preputial gland 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Seminal vesicle (49) (50) (45) Fibrosarcoma, metastatic, stomach,

forestomach 1 (2%) Histiocytic sarcoma 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Testes (50) (50) (50) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%)

Hematopoietic System Bone marrow (49) (50) (49) Mast cell tumor malignant 1 (2%) Lymph node (8) (8) (11) Lumbar, histiocytic sarcoma 1 (13%) Mediastinal, histiocytic sarcoma 1 (13%) Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach 2 (25%) 1 (9%) Pancreatic, histiocytic sarcoma 1 (13%) Pancreatic, squamous cell carcinoma,

metastatic, stomach, forestomach 1 (9%) Renal, histiocytic sarcoma 1 (13%) Lymph node, mandibular (32) (34) (26) Histiocytic sarcoma 1 (3%) Mast cell tumor malignant 1 (3%) Lymph node, mesenteric (46) (47) (47) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 2 (4%)Lesions in Male Mice 211



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0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Spleen (50) (51) (50) Hemangiosarcoma 1 (2%) 1 (2%) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 2 (4%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (2%) Thymus (37) (35) (33) Mast cell tumor malignant 1 (3%) Squamous cell carcinoma, metastatic, stomach,

forestomach 1 (3%)

Integumentary System Skin (50) (48) (48) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma 1 (2%) Squamous cell papilloma 1 (2%) Subcutaneous tissue, fibroma 2 (4%) 2 (4%) 1 (2%) Subcutaneous tissue, fibroma, multiple 1 (2%) Subcutaneous tissue, fibrosarcoma 3 (6%) 8 (17%) 2 (4%) Subcutaneous tissue, sarcoma 1 (2%)

Musculoskeletal System Skeletal muscle (3) (3) (3) Abdominal, squamous cell carcinoma,

metastatic, stomach, forestomach 2 (67%) Diaphragm, sarcoma, metastatic, stomach,

forestomach 1 (33%) Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach 2 (67%) 1 (33%)

Nervous System Brain (50) (51) (50)

Respiratory System Lung (50) (51) (50) Alveolar/bronchiolar adenoma 7 (14%) 20 (39%) 15 (30%) Alveolar/bronchiolar adenoma, multiple 6 (12%) 9 (18%) Alveolar/bronchiolar ca rcinoma 3 (6%) 3 (6%) 1 (2%) Alveolar/bronchiolar carcinoma, multiple 1 (2%) Hepatocellular carcinoma, metastatic 1 (2%) 2 (4%) Hepatocellular carcinoma, metastatic, liver 2 (4%) Histiocytic sarcoma 1 (2%) 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 3 (6%) Mediastinum, hemangiosarcoma, metastatic,

spleen 1 (2%)212 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Special Senses System Ear (1) (1) (1) Fibrosarcoma 1 (100%) Harderian gland (2) (2) (1) Adenoma 2 (100%) 2 (100%) 1 (100%)

Urinary System Kidney (50) (51) (50) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 1 (2%) Squamous cell carcinoma, metastatic, stomach,

forestomach 2 (4%) Urinary bladder (49) (50) (49) Histiocytic sarcoma 1 (2%) Mast cell tumor malignant 1 (2%)

Systemic Lesions Multiple organs b (50) (51) (50) Histiocytic sarcoma 1 (2%) 1 (2%) Lymphoma malignant histiocytic 1 (2%) 1 (2%) Lymphoma malignant lymphocytic 1 (2%) 1 (2%) Lymphoma malignant mixed 7 (14%) 3 (6%) 5 (10%) Lymphoma malignant undifferentiated cell 2 (4%) 2 (4%) 1 (2%) Mesothelioma malignant 1 (2%)

Neoplasm Summary Total animals with primary neoplasms c
15-Month interim evaluation 1 4 8 2-Year study 37 49 47 Total primary neoplasms 15-Month interim evaluation 1 6 12 2-Year study 57 162 139 Total animals with benign neoplasms 15-Month interim evaluation 4 7 2-Year study 20 43 43 Total benign neoplasms 15-Month interim evaluation 5 11 2-Year study 25 85 83Lesions in Male Mice 213



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### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of 1- Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Neoplasm Summary (continued) Total animals with malignant neoplasms 15-Month interim evaluation 1 1 1 2-Year study 25 39 39 Total malignant neoplasms 15-Month interim evaluation 1 1 1 2-Year study 32 77 56 Total animals with metastatic neoplasms 2-Year study 4 7 9 Total metastatic neoplasms 2-Year study 4 37 36

a Number of animals examined microscopically at site and number of animals with neoplasm b Number of animals with any tissue examined microscopically

c Primary neoplasms: all neoplasms except metastatic neoplasms214 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm

2344567777777777777777777 Number of Days on Study 9948220022333333333333333
2350115533001111111111111

0000000000000000000000000 Carcass ID Number 2343314636222445555566677
5554353524134141234512323
Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + Gallbladder + + A + + + + + + M + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + M + M + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + + + M + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp adenomatous X Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangiosarcoma, multiple Hepatocellular carcinoma X X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X Hepatocellular adenoma, multiple X Mesentery + + Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tooth
Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +
Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma X Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + Parathyroid gland + + + + MMMMM++MM +++++++M++MM Pituitary gland + + I + M + + + + MMM+M +++++++++++ Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +
General Body System
Tissue NOS
Genital System Coagulating gland + Epididymis + + I + + + + + + + + + + + + + + + + + + + + + + Preputial gland M + + + + + + + + + +

Squamous cell carcinoma Prostate + + + + + M + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

preputial gland Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + Testes + + + + + + + + + + + + + + + + + + + + + + + + +

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examinedLesions in Male Mice 215



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### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
1111111122222222222222222

0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 Total Carcass ID Number 7 7 9 0 0 1 2 2 1 1 1 3 8 8 8 8 9 9 9 0 1 1 1 2 2 Tissues/
4 5 5 3 4 4 4 5 1 2 4 1 1 2 3 5 1 3 4 1 1 2 3 2 3 Tumors
Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50 Gallbladder + + + + + M + + + + + + + + + + + + + + + + + M + 46 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp adenomatous 1 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hemangiosarcoma, multiple X X X 3 Hepatocellular carcinoma X X X X 8 Hepatocellular carcinoma, multiple 1 Hepatocellular adenoma X X 4 Hepatocellular adenoma, multiple X X X X X 6 Mesentery 2 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 50 Tooth + + + + 4
Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50
Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 2 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50 Parathyroid gland + + M + M M + + M + M M + + + + M M + M M + + + + 30 Pituitary gland + + + + + + + + + + + + + + + + M + + + + + + + + 43 Thyroid gland + + + + + + + + + + M + + + + + + + + + + + + + + 49
General Body System

Tissue NOS + 1
Genital System Coagulating gland 1 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 49 Preputial gland + + + + + + 16

Squamous cell carcinoma X 1 Prostate + + + + + M + + + + + + + + + + + + + + + + + + M 47

Squamous cell carcinoma, metastatic,

preputial gland X 1 Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + + + 49 Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50216 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

2344567777777777777777777 Number of Days on Study 9948220022333333333333333
2350115533001111111111111

0000000000000000000000000 Carcass ID Number 2343314636222445555566677
5554353524134141234512323
Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + Lymph node, mandibular M + + + M + + M M + M + + + + MMM+MM+M++ Lymph node, mesenteric + + M + + + + + + + + + + + + M + + + + + + + + + Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus M + + + M M + M + M M M M M + M ++++++++I
Integumentary System Mammary gland M M M M + M M M M M M M M M M M M M M M M M M M M Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma Subcutaneous tissue, fibroma X X Subcutaneous tissue, fibrosarcoma X X Subcutaneous tissue, sarcoma X
Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + + +
Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +
Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X X X Alveolar/bronchiolar carcinoma X X X Hepatocellular carcinoma, metastatic Hepatocellular carcinoma, metastatic,

liver X Nose + + + + + + + M + + + + + + + + + + + M + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +
Special Senses System

Ear

Fibrosarcoma Eye + Harderian gland +

Adenoma X
Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + M +
Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Lymphoma malignant histiocytic X Lymphoma malignant lymphocytic Lymphoma malignant mixed X X X X Lymphoma malignant undifferentiated

cell type X XLesions in Male Mice 217



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### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
1111111122222222222222222

0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 Total Carcass ID Number 7 7 9 0 0 1 2 2 1 1 1 3 8 8 8 8 9 9 9 0 1 1 1 2 2 Tissues/
4 5 5 3 4 4 4 5 1 2 4 1 1 2 3 5 1 3 4 1 1 2 3 2 3 Tumors
Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + + + 8 Lymph node, mandibular M + M M + + + + M + + + + + M M + + + + M + + + + 32 Lymph node, mesenteric + + + + + + + + M + + + + + + M + + + + + + + + + 46 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + + + + + I + + + + + + + + + + + + + + + + M + 37
Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell papilloma X 1 Subcutaneous tissue, fibroma 2 Subcutaneous tissue, fibrosarcoma X 3 Subcutaneous tissue, sarcoma 1
Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 49 Skeletal muscle 3
Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50
Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X X X X 7 Alveolar/bronchiolar carcinoma 3 Hepatocellular carcinoma, metastatic X 1 Hepatocellular carcinoma, metastatic,

liver X 2 Nose + + + + + + M + + + + + + + + + + + + + M + + M + 43 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50
Special Senses System

Ear + 1

Fibrosarcoma X 1 Eye + 2 Harderian gland + 2

Adenoma X 2
Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 49
Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymphoma malignant histiocytic 1 Lymphoma malignant lymphocytic X 1 Lymphoma malignant mixed X X X 7 Lymphoma malignant undifferentiated

cell type 2218 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



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### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm

1444455555556666666666666 Number of Days on Study 5155600577881111334445778
2417415028231238332784339

1212121212212111112111111 Carcass ID Number 8360903162270648594985334
5551555445454254545333344
Alimentary System Esophagus + + + + M + + M + + + + + + + + + + M + + + + + Gallbladder + + + + + + M + + M + + + + + + + + + + M + + + +

Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + M

Mast cell tumor malignant X Intestine large, rectum + + + + M + + + + M + + + + + + + + + + + + + + + Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + +

Mast cell tumor malignant X Intestine small, duodenum + + + + + + + + + + + + + + A + + + + + + + + + +

Mast cell tumor malignant X Intestine small, jejunum A + + + + + + + + + + + + M A + + + + + + + + + M

Mast cell tumor malignant X Intestine small, ileum + + + + + + + + + + + + + + A M + + + + + + + + +

Adenocarcinoma Histiocytic sarcoma X Mast cell tumor malignant X Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangiosarcoma X Hepatoblastoma X X X Hepatocellular carcinoma X X X X X Hepatocellular carcinoma, multiple X Hepatocellular adenoma X X X X X X Hepatocellular adenoma, multiple X X X X X X X X X X Histiocytic sarcoma X Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

tissue NOS X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X X Mesentery + + + + + +

Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X X Pancreas + + + + + + + + + + M + + + + + + + + + + + + + +

Histiocytic sarcoma X Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Male Mice 219



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77777777777777777777777777

Number of Days on Study 00112233333333333333333333
69579900000000000000000000

2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 Total Carcass ID Number 1 0 3 2 3 3 4 4 5 5 6 7 7 7 8 9 9 0 2 2 3 3 4 4 4 4 Tissues/
3 3 4 3 1 2 1 3 1 2 1 1 2 4 2 1 2 2 1 2 1 3 1 2 3 4 Tumors
Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + + 47 Gallbladder + + + + + + + + + + + + + + + + M + + + + + + + + + 47

Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 49

Mast cell tumor malignant 1 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Mast cell tumor malignant 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + + 50

Mast cell tumor malignant 1 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + + 47

Mast cell tumor malignant 1 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenocarcinoma X 1 Histiocytic sarcoma 1 Mast cell tumor malignant 1 Liver + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Hemangiosarcoma X 2 Hepatoblastoma 3 Hepatocellular carcinoma X X X X X X X X X X 15 Hepatocellular carcinoma, multiple X X 3 Hepatocellular adenoma X X X 9 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X 29 Histiocytic sarcoma 1 Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

tissue NOS 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 4 Mesentery + 7

Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 2 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1 Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + + 51220 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 10,000 ppm (continued)

1444455555556666666666666 Number of Days on Study 5155600577881111334445778
2417415028231238332784339

1212121212212111112111111 Carcass ID Number 8360903162270648594985334
5551555445454254545333344
Alimentary System (continued) Stomach, forestomach + + + + + + + + + + + + M + + + + + + + + + + + +

Leiomyosarcoma X Mast cell tumor malignant X Squamous cell carcinoma X X X X X X X Squamous cell papilloma X X Squamous cell papilloma, multiple Stomach, glandular + + + + + + + + + + + + I + + + + + + + + + + + +

Histiocytic sarcoma X Mast cell tumor malignant X Sarcoma X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X
Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +
Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma Sarcoma, metastatic, stomach,

forestomach X Adrenal medulla + + + + + M + + + + + + + + + + + + + + + + + + + Islets, pancreatic + + + + + + + + + + M + + + + + + + + + + + + + + Parathyroid gland M + M + + M + + M + + + + M + + M M + M M + + + Pituitary gland + + + + + + + M + + + + + + + + + + + + + + + M

Pars distalis, adenoma X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + +
General Body System
Tissue NOS +

Squamous cell carcinoma X
Genital System Coagulating gland +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Penis Preputial gland + + + + + + + + + + Prostate + + + + + + + + + + + + MMM+++++++++

Histiocytic sarcoma X Seminal vesicle + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma, metastatic, stomach,

forestomach XLesions in M

ale M ice 221

T ABLE C2

Individu al Anim al Tum or Pa thology of M ale M ice in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 10,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 0 0 1 1 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
6 9 5 7 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 Total Carc ass ID N um ber 1 0 3 2 3 3 4 4 5 5 6 7 7 7 8 9 9 0 2 2 3 3 4 4 4 4 Tissues/
3 3 4 3 1 2 1 3 1 2 1 1 2 4 2 1 2 2 1 2 1 3 1 2 3 4 Tum ors

Alimen tary System

(continued)
Stomach , forestomach + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lei omyos ar coma 1 Mast cell t umor malignant 1 Squa mous c el l c ar ci noma X X X X X 12 Squa mous c el l pa pi ll oma X X X X X X X X X 11
S q ua m o u s c e ll p a pill o m a , m ulti p le X X 2

Stomach , glandular + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Hist io cyt ic sar coma 1 Mast cell t umor malignant 1
Sar coma 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 3

Card iovascular System

Hear t + + + + + + + + + + + + + + + + + + + + + + + + + + 51

En docrine S ystem
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + + 51 Adeno ma X 1 Sarcoma, metastat ic, st omach,
forestomach 1 Ad ren al m edu lla + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Isle ts, p anc rea tic + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Parathyroid gl and + + + + + + + M + + + + + + M + M + + + + + + M + + 37 Pituit ary gland + + + + + + + + + + + + + M M + + + + + M + + + + + 45
Par s di st al is , a den oma X 2 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + + 50

G eneral B ody S ystem
Ti ss ue NOS 1 Squa mous c el l c ar ci noma 1

G enital System Coagulating gla nd 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 1 Ep idid ym is + + + + + + + + + + + + + + + + + + + + + + + + + + 51 Hist io cyt ic sar coma 1 Sarcoma, metastat ic, st omach,
forestomach 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 1
Pe nis + 1 Preputial gland + M + + + + + 16
Pro stat e + + + I + + + + + + + + + + + + + + + + + + + + + + 46 Hist io cyt ic sar coma 1
Se mi nal ves icle + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Hist io cyt ic sar coma 1
Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50 Sarcoma, metastat ic, st omach,
forestomach 1222 1-Am ino-2, 4-dibromoanthraqu

inone, NTP T

R 383

T ABLE C2

Individu al Anim al Tum or Pa thology of M ale M ice in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 10,000 ppm

(continued)

1 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 Num ber of Days on St udy 5 1 5 5 6 0 0 5 7 7 8 8 1 1 1 1 3 3 4 4 4 5 7 7 8
2 4 1 7 4 1 5 0 2 8 2 3 1 2 3 8 3 3 2 7 8 4 3 3 9

1 2 1 2 1 2 1 2 1 2 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 Carc ass ID N um ber 8 3 6 0 9 0 3 1 6 2 2 7 0 6 4 8 5 9 4 9 8 5 3 3 4
5 5 5 1 5 5 5 4 4 5 4 5 4 2 5 4 5 4 5 3 3 3 3 4 4

H em atopoietic System

Bone marrow M + + + + + + + + + + + + + + + + + + + + + + + + Mast cell t umor malignant Lymph node + + + + + + Lumba r, his ti ocy ti c sa rc oma X Medi ast in al , hi st io cyt ic sar coma X Med iastinal, squamous cell carcinoma, metastatic, stomach, forestomach X X
Panc re at ic , hi st io cyt ic sar coma X
Rena l, his ti ocy ti c sa rc oma X Lymp h node, m andibular + + + + M + M + + + + + M + + M M M + M M M + + M Hist io cyt ic sar coma X Mast cell t umor malignant X Ly mp h n od e, m ese nte ric M + + + + + + + + + + + + + + + + + + + + + + + M Hist io cyt ic sar coma X Mast cell t umor malignant X Sarcoma, metastat ic, st omach,
forestomach X Squam ous cell carcinoma, metastatic, stomach, forestom

ach X X Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Heman gi osa rc oma Hist io cyt ic sar coma X Mast cell t umor malignant X Squam ous cell carcinoma, metastatic, stomach, forestom

ach X Thymus + M + M + M M M + + + M M + + M + + + + M + M Mast cell t umor malignant Squam ous cell carcinoma, metastatic, stomach, forestom

ach X

Integum entary S ystem

Mam mary gland M M M M M M M M M M M M M M M M M M M M M M M M Sk in M M M + + + + + + + + + + + + + + + + + + + + + + Mast cell t umor malignant X Squa mous c el l c ar ci noma X Subc ut ane ous t is sue , f

ib roma Subcutaneo

us tissue, fibroma,

mu ltipl e Subc ut ane ous t is sue , f

ib ros ar coma X X X X X

M usculoskeletal System

Bone M + + + + + + + + + + + + + + + + + + + + + + + + Sk eleta l m usc le + + + Diaphragm

, sarcoma, metastatic, stomach, forestom

ach X Diaphragm

, squamou s cell carcinoma, metastatic, stomach, forestomach X XLesions in M

ale M ice 223

T ABLE C2

Individu al Anim al Tum or Pa thology of M ale M ice in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 10,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 0 0 1 1 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
6 9 5 7 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 Total Carc ass ID N um ber 1 0 3 2 3 3 4 4 5 5 6 7 7 7 8 9 9 0 2 2 3 3 4 4 4 4 Tissues/
3 3 4 3 1 2 1 3 1 2 1 1 2 4 2 1 2 2 1 2 1 3 1 2 3 4 Tum ors

H em atopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Mast cell t umor malignant X 1 Lymph node + + 8 Lumba r, his ti ocy ti c sa rc oma 1 Medi ast in al , hi st io cyt ic sar coma 1 Med iastinal, squamous cell carcinoma, metastatic, stomach, forestomach 2
Panc re at ic , hi st io cyt ic sar coma 1
Rena l, his ti ocy ti c sa rc oma 1 Lymp h node, m andibular M + M + M M + + + + M I + + + M + + + + + + + + + + 34 Hist io cyt ic sar coma 1 Mast cell t umor malignant 1 Ly mp h n od e, m ese nte ric + + + + + + + + + + + + + + + + + + + + + + M + + 47 Hist io cyt ic sar coma 1 Mast cell t umor malignant 1 Sarcoma, metastat ic, st omach,
forestomach 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 2 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + + 51
Heman gi osa rc oma X 1 Hist io cyt ic sar coma 1 Mast cell t umor malignant X 2 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 1 Thymus M + M + + + + + + + + + + M + + + + + + + + + + + M 35 Mast cell t umor malignant X 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 1

Integum entary S ystem

Mam mary gland M M M M M M M M M M M M M M M M M M M + M M M M M M 1 Sk in + + + + + + + + + + + + + + + + + + + + + + + + + + 48 Mast cell t umor malignant 1 Squa mous c el l c ar ci noma 1 Subc ut ane ous t is sue , f

ib roma X X 2 Subcutaneo

us tissue, fibroma,

mu ltipl e X 1 Subc ut ane ous t is sue , f

ib ros ar coma X X X 8

M usculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Sk eleta l m usc le 3 Diaphragm

, sarcoma, metastatic, stomach, forestom

ach 1 Diaphragm

, squamou s cell carcinoma, metastatic, stomach, forestomach 2224 1-Am ino-2, 4-dibromoanthraqu

inone, NTP T

R 383

T ABLE C2

Individu al Anim al Tum or Pa thology of M ale M ice in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 10,000 ppm

(continued)

1 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 Num ber of Days on St udy 5 1 5 5 6 0 0 5 7 7 8 8 1 1 1 1 3 3 4 4 4 5 7 7 8
2 4 1 7 4 1 5 0 2 8 2 3 1 2 3 8 3 3 2 7 8 4 3 3 9

1 2 1 2 1 2 1 2 1 2 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 Carc ass ID N um ber 8 3 6 0 9 0 3 1 6 2 2 7 0 6 4 8 5 9 4 9 8 5 3 3 4
5 5 5 1 5 5 5 4 4 5 4 5 4 2 5 4 5 4 5 3 3 3 3 4 4

Nerv ous System

Br ain + + + + + + + + + + + + + + + + + + + + + + + + +

Resp iratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + Alve ol ar /b ron chi ol ar

ade noma X X X X X X X X X X Alveolar/bronchiolar adeno ma,

mu ltipl e X X Alve ol ar /b ron chi ol ar

car ci noma Alveolar/bronchiolar carcinom a,

mu ltipl e X Hist io cyt ic sar coma X Sarcoma, metastat ic, st omach,
forestomach X Squam ous cell carcinoma, metastatic, stomach, forestom

ach X X X
Nose M + + + + + + + + + + + + + + + + + + + + + M + + Trachea + + + + + + + + + + + + + + + + + + + + + + + +

Special Senses S

ystem

Ear Eye Harderian gland + Adeno ma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Hist io cyt ic sar coma X Mast cell t umor malignant X Squam ous cell carcinoma, metastatic, stomach, forestom

ach X X Urinary bladder + + + + I + + + + + + + + + + + + + + + + + + + + Hist io cyt ic sar coma X Mast cell t umor malignant X

Syst emic Les io ns Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + Hist io cyt ic sar coma X Ly mp ho ma ma lign ant his tioc ytic Lymp homa m alignant mixed X Lymp homa m alignant undifferentiated
cell t ype X Mesothelioma malignant XLesions in M

ale M ice 225

T ABLE C2

Individu al Anim al Tum or Pa thology of M ale M ice in the 2-Ye ar Fe ed Stu dy of 1-Am

ino-2,4-d ibrom oanth raqu inone: 10,000 ppm

(continued)

7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Num ber of Days on St udy 0 0 1 1 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
6 9 5 7 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 Total Carc ass ID N um ber 1 0 3 2 3 3 4 4 5 5 6 7 7 7 8 9 9 0 2 2 3 3 4 4 4 4 Tissues/
3 3 4 3 1 2 1 3 1 2 1 1 2 4 2 1 2 2 1 2 1 3 1 2 3 4 Tum ors

Nerv ous System

Br ain + + + + + + + + + + + + + + + + + + + + + + + + + + 51

Resp iratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + + 51 Alve ol ar /b ron chi ol ar

ade noma X X X X X X X X X X 20 Alveolar/bronchiolar adeno ma,

mu ltipl e X X X X 6 Alve ol ar /b ron chi ol ar

car ci noma X X X 3 Alveolar/bronchiolar carcinom a,

mu ltipl e 1 Hist io cyt ic sar coma 1 Sarcoma, metastat ic, st omach,
forestomach 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 3
Nose + + + + + + + + + + + + + + + + + + + + + + + + + + 49 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + + 50

Special Senses S

ystem

Ear + 1 Eye + 1 Harderian gland + 2 Adeno ma X 2

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + 51 Hist io cyt ic sar coma 1 Mast cell t umor malignant 1 Squam ous cell carcinoma, metastatic, stomach, forestom

ach 2 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + + 50 Hist io cyt ic sar coma 1 Mast cell t umor malignant 1

Syst emic Les io ns Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + + 51 Hist io cyt ic sar coma 1 Ly mp ho ma ma lign ant his tioc ytic X 1 Lymp homa m alignant mixed X X 3 Lymp homa m alignant undifferentiated
cell t ype X 2 Mesothelioma malignant 1226 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm

1122455555666666666666677 Number of Days on Study 0344811239011123446688902
9689427353638813582599692

3233233332223323223323333 Carcass ID Number 5635642126973694862385026
5454351552554544514341534
Alimentary System Esophagus + + + + + M + + + + + + + + + + + + + + + + + + + Gallbladder A + M + + M + + + + + + + M + + + + + + M + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + M +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + +

Adenocarcinoma X Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine small, duodenum M A + + + + + + + + + + + + + + + + + A + + M + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine small, jejunum A A + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Intestine small, ileum M A + + + + + M + + + + + + + + + + + + + + + + +

Adenocarcinoma X Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Fibrosarcoma, metastatic, stomach,

forestomach X Hemangiosarcoma X Hepatoblastoma X X X Hepatocellular carcinoma X X X X X X X X Hepatocellular carcinoma, multiple X X Hepatocellular adenoma X X X X X Hepatocellular adenoma, multiple X X X X X X X X X X X Hepatocholangiocarcinoma, multiple Histiocytic sarcoma X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X Mesentery + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X X X Pancreas + + + + + + + + + + + M + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma X X X X X X X X Squamous cell papilloma X X X Squamous cell papilloma, multiple X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X X XLesions in Male Mice 227



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222222222222222222
4699999999999999999999999

3 3 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carcass ID Number 4 0 5 5 5 5 7 7 7 7 8 8 9 9 0 0 1 1 1 1 2 3 4 6 6 Tissues/
3 4 2 3 4 5 1 2 3 4 1 3 1 2 1 3 1 2 3 4 2 1 1 1 2 Tumors
Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 49 Gallbladder + + + + + + + + + + + + M + + + M + + + + + + + 42

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine large, rectum M + + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenocarcinoma 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 46

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 47

Adenocarcinoma X 2 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Fibrosarcoma, metastatic, stomach,

forestomach 1 Hemangiosarcoma 1 Hepatoblastoma X X 5 Hepatocellular carcinoma X X X X 12 Hepatocellular carcinoma, multiple X X X X X X X 9 Hepatocellular adenoma X X X 8 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X 31 Hepatocholangiocarcinoma, multiple X 1 Histiocytic sarcoma 1 Squamous cell carcinoma, metastatic,

stomach, forestomach X 4 Mesentery + + + 9

Squamous cell carcinoma, metastatic,

stomach, forestomach X X 5 Pancreas M + + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma X X X X X 13 Squamous cell papilloma X X X X X X X X 11 Squamous cell papilloma, multiple X X X X 5 Stomach, glandular M + + + + + + + + + + + + + + + + + + + + + + + + 49

Squamous cell carcinoma, metastatic,

stomach, forestomach X 4228 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm (continued)

1122455555666666666666677 Number of Days on Study 0344811239011123446688902
9689427353638813582599692

3233233332223323223323333 Carcass ID Number 5635642126973694862385026
5454351552554544514341534
Alimentary System (continued) Tongue + Tooth + + +
Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +
Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + +

Pheochromocytoma benign X Islets, pancreatic + + + + + + + + + + + M + + + + + + + + + + + + + Parathyroid gland + + + + M + M + + + + M + + + + + + + M M + + + M Pituitary gland + + + + + + + + + + + + + + + + + + M + + + + M + Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + M

C-cell, adenoma
General Body System
Tissue NOS
Genital System Coagulating gland +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Epididymis + + + + + + + + + + + + + + + + + + + + + + + + +

Fibrosarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Penis Preputial gland + + + + + + Prostate + + + + + + + + + M + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Seminal vesicle + + + + + + + + + + + + + + + + + + + + + +

Fibrosarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Testes + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X
Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + +

Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach X Pancreatic, squamous cell carcinoma,

metastatic, stomach, forestomach Lymph node, mandibular M M + M + + M M M M M ++++M+MM+MM+++ Lymph node, mesenteric M + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Male Mice 229



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222222222222222222
4699999999999999999999999

3 3 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carcass ID Number 4 0 5 5 5 5 7 7 7 7 8 8 9 9 0 0 1 1 1 1 2 3 4 6 6 Tissues/
3 4 2 3 4 5 1 2 3 4 1 3 1 2 1 3 1 2 3 4 2 1 1 1 2 Tumors
Alimentary System (continued) Tongue 1 Tooth + + + + 7
Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50
Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 50

Pheochromocytoma benign 1 Islets, pancreatic M + + + + + + + + + + + + + + + + + + + + + + + + 48 Parathyroid gland M M + + + + + + M + + + M + + + + M + + M + + + + 38 Pituitary gland + + + + + + + + + + + + M + + + + + + + + + + + + 47 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 49

C-cell, adenoma X 1
General Body System
Tissue NOS + 1
Genital System Coagulating gland 1

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Epididymis + + + + + + + + + + + + + + + + + + + + + + + + + 50

Fibrosarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Penis + + 2 Preputial gland + + + + M + + 12 Prostate + M + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Seminal vesicle M M + + + + + + + + + + + + + + + + + + + + + + + 45

Fibrosarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Testes + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma, metastatic,

stomach, forestomach 1
Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + + + 11

Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach 1 Pancreatic, squamous cell carcinoma,

metastatic, stomach, forestomach X 1 Lymph node, mandibular + + + + + + M M + M + M M + + + M I M M M + + + M 26 Lymph node, mesenteric M + + + + + + + + + + + + + + + + + + + + + + + M 47230 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm (continued)

1122455555666666666666677 Number of Days on Study 0344811239011123446688902
9689427353638813582599692

3233233332223323223323333 Carcass ID Number 5635642126973694862385026
5454351552554544514341534
Hematopoietic System (continued) Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangiosarcoma X Thymus + M + + M + M + M + + + + + + MMMM++MM+M
Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M Skin + + + + + + + + + + + + + + + + + + + + + + M + +

Subcutaneous tissue, fibroma X Subcutaneous tissue, fibrosarcoma X X
Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + + +

Abdominal, squamous cell carcinoma,

metastatic, stomach, forestomach X X Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach X
Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +
Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X X X X X X X X Alveolar/bronchiolar adenoma,

multiple X X X Alveolar/bronchiolar carcinoma X Hepatocellular carcinoma, metastatic X Histiocytic sarcoma X Mediastinum, hemangiosarcoma,

metastatic, spleen X Nose + + M + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +
Special Senses System

Ear Harderian gland

Adenoma
Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + M + + + + + + + + + + + + + + + + + + + + + + +
Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Histiocytic sarcoma X Lymphoma malignant lymphocytic X Lymphoma malignant mixed X Lymphoma malignant undifferentiated

cell type XLesions in Male Mice 231



### T ABLE 

### C2 Individual Animal Tumor Pathology of Male Mice in the 2-Year Feed Study of 1-Amin o-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222222222222222222
4699999999999999999999999

3 3 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 Total Carcass ID Number 4 0 5 5 5 5 7 7 7 7 8 8 9 9 0 0 1 1 1 1 2 3 4 6 6 Tissues/
3 4 2 3 4 5 1 2 3 4 1 3 1 2 1 3 1 2 3 4 2 1 1 1 2 Tumors
Hematopoietic System (continued) Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hemangiosarcoma 1 Thymus M + + + M + + + + + + + + I + + + M M + M + + + + 33
Integumentary System Mammary gland M M M M M M M M M M M M M M M M M M M M M M M M M Skin + + + + + + + + + + + + + + + + + + + + + + M + + 48

Subcutaneous tissue, fibroma 1 Subcutaneous tissue, fibrosarcoma 2
Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + 49 Skeletal muscle 3

Abdominal, squamous cell carcinoma,

metastatic, stomach, forestomach 2 Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach 1
Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50
Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X X X X X X X 15 Alveolar/bronchiolar adenoma,

multiple X X X X X X 9 Alveolar/bronchiolar carcinoma 1 Hepatocellular carcinoma, metastatic X 2 Histiocytic sarcoma 1 Mediastinum, hemangiosarcoma,

metastatic, spleen 1 Nose + + + + + + + M + + + + + + + + + + + + + + + + + 48 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50
Special Senses System

Ear + 1 Harderian gland + 1

Adenoma X 1
Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 49
Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Histiocytic sarcoma 1 Lymphoma malignant lymphocytic 1 Lymphoma malignant mixed X X X X 5 Lymphoma malignant undifferentiated

cell type 1232 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,000 ppm 20,000 ppm

Liver: Hemangiosarcoma
Overall rate a 3/50 (6%) 2/51 (4%) 1/50 (2%) Adjusted rate b 7.5% 6.5% 2.2% Terminal rate c 3/40 (8%) 1/22 (5%) 0/23 (0%) First incidence (days) 729 (T) 414 484 Life table test d P=0.366N P=0.658 P=0.461N

Logistic regression test d P=0.189N P=0.481N P=0.301N

Cochran-Armitage test d P=0.221N Fisher exact test d P=0.491N P=0.309N

Liver: Hepatocellular Adenoma Overall rate 10/50 (20%) 38/51 (75%) 39/50 (78%) Adjusted rate 24.3% 94.7% 95.0% Terminal rate 9/40 (23%) 20/22 (91%) 21/23 (91%) First incidence (days) 723 451 484 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Liver: Hepatocellular Carcinoma Overall rate 9/50 (18%) 18/51 (35%) 21/50 (42%) Adjusted rate 21.1% 58.1% 58.4% Terminal rate 7/40 (18%) 10/22 (45%) 9/23 (39%) First incidence (days) 445 505 535 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P=0.002 P=0.017 P=0.003

Cochran-Armitage test P=0.007 Fisher exact test P=0.040 P=0.008

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 18/50 (36%) 43/51 (84%) 42/50 (84%) Adjusted rate 41.7% 97.7% 97.7% Terminal rate 15/40 (38%) 21/22 (95%) 22/23 (96%) First incidence (days) 445 451 484 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Liver: Hepatoblastoma Overall rate 0/50 (0%) 3/51 (6%) 5/50 (10%) Adjusted rate 0.0% 8.2% 16.8% Terminal rate 0/40 (0%) 0/22 (0%) 2/23 (9%) First incidence (days)

) e 572 633 Life table test P=0.011 P=0.090 P=0.011

Logistic regression test P=0.021 P=0.151 P=0.026

Cochran-Armitage test P=0.022 Fisher exact test P=0.125 P=0.028Lesions in Male Mice 233



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Liver: Hepatocellular Ca rcinoma or Hepatoblastoma Overall rate 9/50 (18%) 20/51 (39%) 24/50 (48%) Adjusted rate 21.1% 60.2% 65.3% Terminal rate 7/40 (18%) 10/22 (45%) 11/23 (48%) First incidence (days) 445 505 535 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P=0.007 P<0.001

Cochran-Armitage test P=0.001 Fisher exact test P=0.016 P=0.001

Liver: Hepatocellular Adenoma, Hepa tocellular Carcinoma, or Hepatoblastoma Overall rate 18/50 (36%) 43/51 (84%) 42/50 (84%) Adjusted rate 41.7% 97.7% 97.7% Terminal rate 15/40 (38%) 21/22 (95%) 22/23 (96%) First incidence (days) 445 451 484 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Lung: Alveolar/bronchiolar Adenoma Overall rate 7/50 (14%) 26/51 (51%) 24/50 (48%) Adjusted rate 16.8% 71.0% 66.5% Terminal rate 6/40 (15%) 12/22 (55%) 12/23 (52%) First incidence (days) 445 578 248 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Lung: Alveolar/bronchiolar Carcinoma Overall rate 3/50 (6%) 4/51 (8%) 1/50 (2%) Adjusted rate 6.9% 15.9% 3.0% Terminal rate 2/40 (5%) 2/22 (9%) 0/23 (0%) First incidence (days) 393 673 648 Life table test P=0.454N P=0.259 P=0.442N

Logistic regression test P=0.259N P=0.512 P=0.251N

Cochran-Armitage test P=0.252N Fisher exact test P=0.511 P=0.309N

Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 10/50 (20%) 28/51 (55%) 25/50 (50%) Adjusted rate 23.3% 75.0% 67.5% Terminal rate 8/40 (20%) 13/22 (59%) 12/23 (52%) First incidence (days) 393 578 248 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P=0.002

Cochran-Armitage test P=0.002 Fisher exact test P<0.001 P=0.002234 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Skin (Subcutaneous Tissue): Fibroma Overall rate 2/50 (4%) 3/51 (6%) 1/50 (2%) Adjusted rate 5.0% 11.9% 3.8% Terminal rate 2/40 (5%) 1/22 (5%) 0/23 (0%) First incidence (days) 729 (T) 706 722 Life table test P=0.578 P=0.262 P=0.679N

Logistic regression test P=0.547N P=0.330 P=0.650N

Cochran-Armitage test P=0.399N Fisher exact test P=0.509 P=0.500N

Skin (Subcutaneous Tissue): Fibrosarcoma Overall rate 3/50 (6%) 8/51 (16%) 2/50 (4%) Adjusted rate 6.9% 22.8% 5.1% Terminal rate 1/40 (3%) 2/22 (9%) 0/23 (0%) First incidence (days) 621 501 606 Life table test P=0.526 P=0.037 P=0.638N

Logistic regression test P=0.381N P=0.136 P=0.488N

Cochran-Armitage test P=0.429N Fisher exact test P=0.106 P=0.500N

Skin (Subcutaneous Tissue): Fibrosarcoma or Sarcoma Overall rate 4/50 (8%) 8/51 (16%) 2/50 (4%) Adjusted rate 9.3% 22.8% 5.1% Terminal rate 2/40 (5%) 2/22 (9%) 0/23 (0%) First incidence (days) 621 501 606 Life table test P=0.501N P=0.066 P=0.508N

Logistic regression test P=0.268N P=0.222 P=0.339N

Cochran-Armitage test P=0.303N Fisher exact test P=0.188 P=0.339N

Skin (Subcutaneous Tissue): Fibroma, Fibrosarcoma, or Sarcoma Overall rate 6/50 (12%) 10/51 (20%) 3/50 (6%) Adjusted rate 14.1% 29.5% 8.7% Terminal rate 4/40 (10%) 3/22 (14%) 0/23 (0%) First incidence (days) 621 501 606 Life table test P=0.494N P=0.053 P=0.476N

Logistic regression test P=0.233N P=0.220 P=0.280N

Cochran-Armitage test P=0.225N Fisher exact test P=0.220 P=0.243N

Stomach (Forestomach): Squamous Cell Papilloma Overall rate 0/50 (0%) 13/51 (25%) 16/50 (32%) Adjusted rate 0.0% 51.0% 55.6% Terminal rate 0/40 (0%) 10/22 (45%) 11/23 (48%) First incidence (days)

) 613 606 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001Lesions in Male Mice 235



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Stomach (Forestomach): Squamous Cell Carcinoma Overall rate 0/50 (0%) 12/51 (24%) 13/50 (26%) Adjusted rate 0.0% 36.5% 37.7% Terminal rate 0/40 (0%) 4/22 (18%) 4/23 (17%) First incidence (days)

) 505 523 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Stomach (Forestomach): Squamous Ce ll Papilloma or Squamous Cell Carcinoma Overall rate 0/50 (0%) 19/51 (37%) 27/50 (54%)

Adjusted rate 0.0% 61.2% 73.9% Terminal rate 0/40 (0%) 11/22 (50%) 14/23 (61%) First incidence (days)

) 505 523 Life table test P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

All Organs: Hemangiosarcoma Overall rate 3/50 (6%) 2/51 (4%) 2/50 (4%) Adjusted rate 7.5% 6.5% 4.4% Terminal rate 3/40 (8%) 1/22 (5%) 0/23 (0%) First incidence (days) 729 (T) 414 484 Life table test P=0.560N P=0.658 P=0.650N

Logistic regression test P=0.340N P=0.481N P=0.455N

Cochran-Armitage test P=0.406N Fisher exact test P=0.491N P=0.500N

All Organs: Malignant Lymphoma (Histiocytic, Lymphocyt ic, Mixed, or Undifferentiated Cell Type) Overall rate 10/50 (20%) 6/51 (12%) 7/50 (14%) Adjusted rate 21.9% 20.8% 23.3% Terminal rate 5/40 (13%) 2/22 (9%) 4/23 (17%) First incidence (days) 480 612 512 Life table test P=0.517 P=0.565N P=0.567 Logistic regression test P=0.282N P=0.218N P=0.306N

Cochran-Armitage test P=0.243N Fisher exact test P=0.195N P=0.298N

All Organs: Benign Neoplasms Overall rate 20/50 (40%) 43/51 (84%) 44/50 (88%) Adjusted rate 47.4% 100.0% 100.0% Terminal rate 18/40 (45%) 22/22 (100%) 23/23 (100%) First incidence (days) 445 451 248 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001236 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C3 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

All Organs: Malignant Neoplasms Overall rate 26/50 (52%) 39/51 (76%) 39/50 (78%) Adjusted rate 53.1% 82.9% 88.3% Terminal rate 17/40 (43%) 14/22 (64%) 18/23 (78%) First incidence (days) 393 414 484 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P=0.002 P=0.012 P=0.003

Cochran-Armitage test P=0.003 Fisher exact test P=0.009 P=0.006

All Organs: Benign or Malignant Neoplasms Overall rate 37/50 (74%) 49/51 (96%) 48/50 (96%) Adjusted rate 75.5% 100.0% 100.0% Terminal rate 28/40 (70%) 22/22 (100%) 23/23 (100%) First incidence (days) 393 414 248 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P=0.002 P=0.002

(T)Terminal sacrifice

a Number of lesion-bearing animals/number of animals examined. De nominator is number of animals examined microscopically for liv er, lung, and stomach; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Observed incidence at terminal kill d Beneath the control incidence are the P valu es associated with the trend test. Bene ath the exposed group incidence are the P v alues corresponding to pairwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare di rectly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal groupLesions in Male Mice 237



### T ABLE 

### C4a Historical Incidence of Hepatocellula r Neoplasms in Untreated Male B6C3F 1 

### Mice a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 10/50 9/50 18/50 Acetaminophen 11/50 7/50 16/50 HC Yellow 4 8/49 5/49 13/49 Methylphenidate Hydrochloride 18/50 10/50 24/50 Pentaerythritol Tetranitrate 9/48 3/48 11/48 Turmeric Oleoresin 25/50 12/50 30/50

Overall Historical Incidence

Total 347/1,466 (23.7%) 241/1,466 (16.4%) 531/1,466 (36.2%) Standard deviation 13.6% 7.0% 14.1% Range 4%-60% 3%-29% 10%-68%

a Data as of 31 March 1993



### T ABLE 

### C4b Historical Incidence of Fore stomach Squamous Cell Neoplasms in Untreated Male B6C3F 1 

### Mice a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 1/50 0/50 1/50 HC Yellow 4 3/50 0/50 3/50 Methylphenidate Hydrochloride 1/50 0/50 1/50 Pentaerythritol Tetranitrate 0/49 0/49 0/49 Turmeric Oleoresin 2/50 0/50 2/50

Overall Historical Incidence

Total 20/1,474 (1.4%) 2/1,474 (0.1%) 22/1,474 (1.5%) Standard deviation 2.0% 0.5% 2.0% Range 0%-6% 0%-2% 0%-6%

a Data as of 31 March 1993238 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C4c Historical Incidence of Al veolar/bronchiolar Neoplasms in Untreated Male B6C3F 1 

### Mice a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 7/50 3/50 10/50 Acetaminophen 8/50 0/50 8/50 HC Yellow 4 7/50 2/50 8/50 Methylphenidate Hydrochloride 14/50 4/50 16/50 Pentaerythritol Tetranitrate 11/47 1/47 11/47 Turmeric Oleoresin 11/50 4/50 14/50

Overall Historical Incidence

Total 201/1,469 (13.7%) 73/1,469 (5.0%) 265/1,469 (18.0%) Standard deviation 6.2% 4.0% 7.6% Range 4%-28% 0%-14% 4%-32%

a Data as of 31 March 1993Lesions in Male Mice 239



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm

Disposition Summary Animals initially in study 60 60 60 15-Month interim evaluation 10 9 10 Early deaths Accidental death 1 Moribund 7 23 21 Natural deaths 3 5 6 Survivors Terminal sacrifice 40 22 23

Animals examined microscopically 60 60 60



### 15-Month Interim Evaluation Alimentary System Gallbladder (10) (9) (9) Inflammation, chronic 1 (11%) Inflammation, chronic active 1 (10%) Intestine large, cecum (10) (9) (10) Ulcer, acute 1 (10%) Liver (10) (9) (10) Basophilic focus 1 (10%) Fatty change 7 (70%) 3 (33%) Inflammation, acute 1 (10%) 9 (100%) 3 (30%) Inflammation, chronic active 4 (40%) 5 (50%) Necrosis, coagulative 4 (40%) 8 (89%) 8 (80%) Pigmentation 9 (100%) 10 (100%) Centrilobular, cytoplasmic alteration 9 (100%) 8 (80%) Mesentery (1) Inflammation, chronic 1 (100%) Necrosis, coagulative 1 (100%) Pancreas (10) (9) (10) Atrophy 1 (10%) Cytoplasmic alteration 1 (10%) Inflammation, chronic 2 (20%) Artery, inflammation, chronic active 1 (10%) Salivary glands (10) (8) (10) Submandibular gland, inflammation, chronic 8 (80%) 4 (40%) Stomach, forestomach (9) (9) (10) Acanthosis 2 (22%) 3 (30%) Hyperkeratosis 1 (11%) 2 (22%) 3 (30%) Hyperplasia, basal cell 1 (11%) 1 (10%) Inflammation, acute 1 (10%) Inflammation, chronic active 2 (20%) Stomach, glandular (9) (9) (10) Inflammation, chronic 1 (11%) 5 (56%) 2 (20%) Inflammation, chronic active 1 (11%) 1 (10%) Muscularis, mineralization 1 (11%)

a Number of animals examined microscopically at site and number of animals with lesion240 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 15-Month Interim Evaluation (continued) Cardiovascular System Heart (10) (9) (10) Cardiomyopathy 3 (30%) 1 (10%)

Endocrine System Islets, pancreatic (1) (1) Hyperplasia 1 (100%) Inflammation, chronic 1 (100%) Pituitary gland (9) (9) (9) Pars distalis, hyperplasia 2 (22%) Thyroid gland (10) (9) (10) Follicle, cyst 1 (10%)

Genital System Epididymis (10) (9) (10) Inflammation, chronic active 1 (10%) Penis (1) Inflammation, acute 1 (100%) Preputial gland (5) (1) Abscess 1 (100%) Inflammation, chronic 2 (40%) Inflammation, chronic active 2 (40%) Duct, dilatation 1 (20%) Prostate (10) (9) (9) Inflammation, chronic 5 (50%) 2 (22%) Inflammation, chronic active 1 (10%) Artery, inflammation, chronic active 1 (10%) Testes (10) (9) (10) Atrophy 2 (20%) Seminiferous tubule, atrophy 6 (60%) 2 (20%)

Hematopoietic System Lymph node (2) Inguinal, hyperplasia, lymphoid 1 (50%) Lumbar, hyperplasia 1 (50%) Lymph node, mesenteric (8) (9) (8) Hemorrhage 3 (38%) Infiltration cellular, histiocyte 1 (13%) Pigmentation 1 (13%) Spleen (10) (9) (8) Hematopoietic cell proliferation 1 (10%) Thymus (7) (9) (8) Cyst 3 (38%)Lesions in Male Mice 241



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 15-Month Interim Evaluation (continued) Integumentary System Skin (10) (9) (10) Inflammation, acute 1 (10%) Inflammation, chronic active 1 (10%) Subcutaneous tissue, inflammation, acute 2 (20%) Subcutaneous tissue, infl ammation, chronic active 1 (10%)

Musculoskeletal System Bone (2) (2) (10)

Joint, tarsal, hyperostosis 2 (100%) 2 (100%)

Nervous System Brain (10) (9) (10) Thalamus, mineralization 9 (90%) 6 (60%)

Respiratory System Lung (10) (9) (10) Congestion 1 (10%) Hemorrhage 1 (10%) 1 (10%) Inflammation, chronic active 1 (11%) Peribronchiolar, inflammation, chronic 1 (10%) Perivascular, inflammation, chronic 1 (10%) Nose (10) (9) (10) Crystals 1 (10%) Inflammation, acute 2 (20%) Glands, inflammation, acute 2 (20%) Respiratory epithelium, necrosis 1 (10%)

Urinary System Kidney (10) (1) (10) Inflammation, chronic 10 (100%) 1 (100%) 8 (80%) Renal tubule, regeneration 4 (40%) Urinary bladder (10) (9) (10) Inflammation, chronic 3 (30%) 7 (78%) 5 (50%) Inflammation, chronic active 1 (10%)



### Systems Examined With No Lesions Observed General Body System Special Senses System242 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study Alimentary System Gallbladder (46) (47) (42) Autolysis 1 (2%) 2 (4%) Inflammation, chronic active 11 (24%) 12 (26%) 8 (19%) Epithelium, pigmentation 1 (2%) Intestine large, colon (50) (49) (50) Autolysis 1 (2%) 2 (4%) Inflammation, chronic active 1 (2%) 1 (2%) 2 (4%) Peyer's patch, hyperplasia 1 (2%) Intestine large, rectum (48) (49) (48) Autolysis 2 (4%) 2 (4%) Intestine large, cecum (49) (51) (50) Autolysis 2 (4%) 2 (4%) Hemorrhage 1 (2%) Hyperplasia, lymphoid 1 (2%) Epithelium, hyperplasia 1 (2%) Peyer's patch, hyperplasia 1 (2%) 1 (2%) Serosa, inflammation, chronic active 1 (2%) Intestine small, duodenum (50) (50) (46) Autolysis 2 (4%) 1 (2%) Mesothelium, hyperplasia 1 (2%) Peyer's patch, inflammation, chronic,

granulomatous 1 (2%) Serosa, fibrosis 1 (2%) Intestine small, jejunum (50) (47) (48) Autolysis 2 (4%) 1 (2%) Hyperplasia, lymphoid 1 (2%) Inflammation, acute 1 (2%) Epithelium, pigmentation 1 (2%) Peyer's patch, inflammation, chronic,

granulomatous 1 (2%) Intestine small, ileum (50) (49) (47) Autolysis 2 (4%) 1 (2%) Hyperplasia, lymphoid 1 (2%) Inflammation, acute, necrotizing 1 (2%) Inflammation, chronic active 1 (2%) Peyer's patch, hyperplasia 1 (2%) 2 (4%) 1 (2%) Peyer's patch, hyperplasia, lymphoid 1 (2%) Peyer's patch, inflammation, chronic,

granulomatous 1 (2%) Liver (50) (51) (50) Angiectasis 1 (2%) Basophilic focus 4 (8%) 3 (6%) Basophilic focus, focal 1 (2%) Clear cell focus 4 (8%) 4 (8%) 2 (4%) Cytoplasmic alteration 1 (2%) Eosinophilic focus 6 (12%) 1 (2%) Fatty change 4 (8%) 3 (6%) 7 (14%) Fibrosis 1 (2%) Hematopoietic cell proliferation 1 (2%) 5 (10%) 10 (20%) Infarct 1 (2%)Lesions in Male Mice 243



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Liver (continued) (50) (51) (50) Inflammation, chronic active 11 (22%) 10 (20%) 22 (44%) Karyomegaly 1 (2%) Mineralization 1 (2%) 1 (2%) Necrosis, coagulative 5 (10%) 12 (24%) 20 (40%) Nuclear alteration 1 (2%) Pigmentation 1 (2%) 50 (98%) 47 (94%) Thrombosis 1 (2%) Bile duct, hyperplasia 1 (2%) 2 (4%) 1 (2%) Hepatocyte, centrilobular, hypertrophy 17 (33%) 13 (26%) Mesentery (2) (7) (9) Abscess 1 (11%) Fibrosis 1 (11%) Inflammation, chronic active 4 (57%) 5 (56%) Mineralization 1 (11%) Necrosis, coagulative 2 (22%) Thrombosis 1 (14%) Pancreas (50) (50) (48) Atrophy 2 (4%) Autolysis 1 (2%) 1 (2%) Cytoplasmic alteration 4 (8%) 1 (2%) Ectopic liver 1 (2%) Inflammation, chronic active 19 (38%) 12 (24%) 8 (17%) Vacuolization cytoplasmic 20 (40%) 18 (36%) 4 (8%) Acinus, atrophy 1 (2%) Salivary glands (50) (51) (50) Inflammation, chronic 2 (4%) Duct, parotid gland, mineralization 1 (2%) Parotid gland, inflammation, chronic 2 (4%) Sublingual gland, inflammation, chronic 1 (2%) Sublingual gland, submandibular gland,

inflammation, chronic 1 (2%) Submandibular gland, atrophy 1 (2%) Submandibular gland, inflammation, chronic 37 (74%) 29 (57%) 36 (72%) Stomach, forestomach (50) (50) (50) Abscess 1 (2%) Acanthosis 1 (2%) 9 (18%) 4 (8%) Autolysis 1 (2%) Cyst 1 (2%) Diverticulum 1 (2%) Edema 1 (2%) Erosion 1 (2%) Hyperkeratosis 1 (2%) 7 (14%) 6 (12%) Hyperplasia, basal cell 2 (4%) Inflammation, chronic active 2 (4%) 6 (12%) 12 (24%) Inflammation, chronic active, necrotizing 1 (2%) Ulcer 2 (4%)244 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Stomach, glandular (50) (50) (49) Autolysis 1 (2%) Edema 1 (2%) Infiltration cellular, mast cell 1 (2%) Inflammation, chronic active 14 (28%) 24 (48%) 22 (45%) Mineralization 4 (8%) 3 (6%) Tooth (4) (7) Dysplasia 4 (100%) 6 (86%)

Cardiovascular System Heart (50) (51) (50) Inflammation, chronic 7 (14%) 17 (33%) 5 (10%) Mineralization 1 (2%) Necrosis, coagulative 1 (2%) Coronary artery, thrombosis 1 (2%)

Endocrine System Adrenal cortex (50) (51) (50) Hematopoietic cell proliferation 1 (2%) Hyperplasia 3 (6%) 2 (4%) Inflammation, chronic active 3 (6%) Pigmentation 2 (4%) Vacuolization cytoplasmic 1 (2%) Capsule, inflammation, chronic active 1 (2%) Adrenal medulla (50) (50) (50) Hyperplasia 1 (2%) 1 (2%) Inflammation, acute 1 (2%) Pigmentation 1 (2%) Islets, pancreatic (50) (50) (48) Hyperplasia 8 (16%) 1 (2%) 1 (2%) Pituitary gland (43) (45) (47) Pars distalis, cyst 5 (12%) 4 (9%) 1 (2%) Pars distalis, hyperplasia 13 (30%) 9 (20%) 4 (9%) Thyroid gland (49) (50) (49) Inflammation, chronic 1 (2%) 1 (2%) 1 (2%) Ultimobranchial cyst 1 (2%) 1 (2%) Follicular cell, hyperplasia 2 (4%) 3 (6%) 3 (6%)

General Body System

NoneLesions in Male Mice 245



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Genital System Coagulating gland (1) (1) (1) Inflammation, chronic active 1 (100%) Epididymis (49) (51) (50) Inflammation, chronic active 29 (59%) 21 (41%) 26 (52%) Epithelium, hyperplasia 1 (2%) Serosa, hyperplasia 1 (2%) Penis (1) (2) Hemorrhage 1 (100%) Preputial gland (16) (16) (12) Abscess 1 (6%) 3 (19%) 1 (8%) Inflammation, chronic active 12 (75%) 7 (44%) 11 (92%) Duct, dilatation 11 (69%) 10 (63%) 2 (17%) Prostate (47) (46) (48) Inflammation 1 (2%) Inflammation, chronic active 31 (66%) 29 (63%) 20 (42%) Epithelium, hyperplasia 1 (2%) Seminal vesicle (49) (50) (45) Atrophy 1 (2%) 1 (2%) Cyst 1 (2%) Fibrosis 3 (6%) Inflammation, chronic active 4 (8%) 14 (28%) 13 (29%) Artery, thrombosis 1 (2%) Testes (50) (50) (50) Inflammation, chronic active 2 (4%) Interstitial cell, hyperplasia 1 (2%) Seminiferous tubule, atrophy 1 (2%) 2 (4%) Seminiferous tubule, mine ralization 2 (4%) 1 (2%) 3 (6%)

Hematopoietic System Bone marrow (49) (50) (49) Myeloid cell, sternal, hyperplasia 1 (2%) 2 (4%) 1 (2%) Sternal, infiltration ce llular, mast cell 1 (2%) Sternal, inflammation, granulomatous 1 (2%) Lymph node (8) (8) (11) Hyperplasia, lymphoid 1 (9%) Lumbar, hyperplasia, lymphoid 1 (13%) 1 (13%) Lumbar, hyperplasia, plasma cell 2 (18%) Lumbar, inflammation, chronic active 1 (13%) Mediastinal, infiltration cellular, histiocyte 1 (13%) Mediastinal, inflammation, chronic active 1 (9%) Mediastinal, pigmentation 1 (13%) Pancreatic, angiectasis 1 (9%) Pancreatic, hyperplasia, lymphoid 2 (25%) Pancreatic, infiltration cellular, histiocyte 1 (13%) Pancreatic, pigmentation 1 (13%) Renal, angiectasis 1 (13%) Renal, hyperplasia, lymphoid 1 (13%) Renal, inflammation, chronic active 1 (13%)246 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Lymph node, mandibular (32) (34) (26) Depletion lymphoid 3 (9%) 1 (3%) 1 (4%) Hyperplasia, lymphoid 3 (9%) 1 (3%) 1 (4%) Hyperplasia, plasma cell 1 (3%) Infiltration cellular, histiocyte 3 (9%) 8 (31%) Inflammation, chronic active 1 (3%) 1 (3%) 1 (4%) Necrosis, coagulative 1 (4%) Pigmentation 5 (16%) 4 (12%) 9 (35%) Lymph node, mesenteric (46) (47) (47) Angiectasis 24 (52%) 11 (23%) 21 (45%) Congestion 1 (2%) Depletion lymphoid 1 (2%) 1 (2%) Hematopoietic cell proliferation 2 (4%) Hemorrhage 1 (2%) 1 (2%) Hyperplasia 1 (2%) Hyperplasia, lymphoid 6 (13%) 9 (19%) 4 (9%) Hyperplasia, plasma cell 1 (2%) Infiltration cellular, histiocyte 24 (52%) 22 (47%) 21 (45%) Inflammation, chronic active 7 (15%) 4 (9%) Pigmentation 23 (50%) 22 (47%) 19 (40%) Spleen (50) (51) (50) Angiectasis 1 (2%) Congestion 1 (2%) Depletion lymphoid 1 (2%) 3 (6%) 3 (6%) Fibrosis 1 (2%) Hematopoietic cell proliferation 5 (10%) 14 (27%) 12 (24%) Hyperplasia, lymphoid 2 (4%) 1 (2%) Inflammation, chronic active 1 (2%) 1 (2%) 2 (4%) Necrosis, coagulative 1 (2%) Thymus (37) (35) (33) Cyst 2 (5%) 6 (17%) 9 (27%) Cyst, multiple 2 (5%) Depletion lymphoid 2 (5%) 8 (23%) 6 (18%) Hyperplasia, lymphoid 1 (3%) Infiltration cellular, histiocyte 1 (3%) Necrosis, coagulative 1 (3%) 1 (3%) Pigmentation 1 (3%) 1 (3%)

Integumentary System Skin (50) (48) (48) Inflammation, chronic active 4 (8%) 9 (19%) 12 (25%) Subcutaneous tissue, abscess 1 (2%) Subcutaneous tissue, cyst 1 (2%) Subcutaneous tissue, fibrosis 2 (4%) 1 (2%)Lesions in Male Mice 247



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Musculoskeletal System Bone (49) (50) (49) Cartilage, tarsal, hyperplasia 1 (2%) Joint, tarsal, hyperostosis 10 (20%) 11 (22%) 12 (24%) Skeletal muscle (3) (3) (3) Fibrosis 1 (33%) Intercostal, inflammation, chronic active 1 (33%)

Nervous System Brain (50) (51) (50) Cyst epithelial inclusion 1 (2%) Cerebellum, necrosis 1 (2%) Cerebrum, necrosis 1 (2%) Thalamus, mineralization 17 (34%) 20 (39%) 13 (26%)

Respiratory System Lung (50) (51) (50) Bronchiectasis 1 (2%) Congestion 1 (2%) Crystals 1 (2%) Foreign body 1 (2%) Granuloma 1 (2%) Hemorrhage 8 (16%) 9 (18%) 9 (18%) Infiltration cellular, hi stiocyte 2 (4%) 2 (4%) 6 (12%) Inflammation, acute 2 (4%) Inflammation, chronic active 1 (2%) 2 (4%) 1 (2%) Mineralization 1 (2%) Pigmentation 1 (2%) Alveolar epithelium, hyperplasia 1 (2%) 4 (8%) Bronchiole, hyperplasia 1 (2%) Bronchiole, metaplasia, squamous 1 (2%) Nose (43) (49) (48) Inflammation, acute 11 (26%) 11 (22%) 6 (13%) Glands, crystals 2 (5%) Nasolacrimal duct, inflammation, acute 2 (5%) Trachea (50) (50) (50) Inflammation, acute 2 (4%) 3 (6%)

Special Senses System Eye (2) (1) Anterior, synechia 1 (100%) Cornea, fibrosis 1 (100%) Cornea, inflammation, chronic active 1 (50%) Harderian gland (2) (2) (1) Inflammation, chronic active 1 (50%)248 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### C5 Summary of the Incidence of Nonneoplastic Lesi ons in Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Urinary System Kidney (50) (51) (50) Autolysis 1 (2%) Congestion 1 (2%) Cyst 1 (2%) 1 (2%) Glomerulosclerosis 3 (6%) Infiltration cellular, mononuclear cell 1 (2%) Inflammation, acute, necrotizing 1 (2%) Inflammation, chronic active 47 (94%) 42 (82%) 47 (94%) Metaplasia, osseous 1 (2%) Mineralization 1 (2%) Necrosis, coagulative 1 (2%) Artery, necrosis, fibrinoid 1 (2%) Pelvis, transitional epithelium, hyperplasia 1 (2%) Proximal convoluted renal tubule, hyperplasia 1 (2%) Proximal convoluted renal tubule, regeneration 2 (4%) 11 (22%) 1 (2%) Proximal convoluted renal tubule, vacuolization cytoplasmic 1 (2%) Renal tubule, mineralization 1 (2%) 4 (8%) 1 (2%) Renal tubule, pigmentation 42 (82%) 43 (86%) Renal tubule, regeneration 4 (8%) 1 (2%) Urinary bladder (49) (50) (49) Autolysis 2 (4%) 3 (6%) Calculus, microscopic observation only 5 (10%) 2 (4%) Inflammation, chronic active 36 (73%) 31 (62%) 34 (69%) Transitional epithelium, hyperplasia 2 (4%)249



# APPENDIX D
SUMMARY OF LESIONS IN FEMALE MICE
IN THE 2-YEAR FEED STUDY OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 T ABLE 

### D3 Statistical Analysis of Prim ary Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 T ABLE 

### D4a Historical Incidence of Hepatocellular Neoplasms in Untreated Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 T ABLE 

### D4b Historical Incidence of Forestomach Squamous Cell Neoplasms in Untreated Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 T ABLE 

### D4c Historical Incidence of Alveolar/bronchiolar Neoplasms in Untreated Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 T ABLE 

### D4d Historical Incidence of Uterine Neoplasms in Untreated Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . 279 T ABLE 

### D4e Historical Incidence of Pituitar y Gland Pars Distalis Neoplasms in Untreated Female B6C3F 1 

### Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 T ABLE 

### D5 Summary of the Incidence of Nonne oplastic Lesions in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281250 1-Amino-2,4-dibromoanthraquinone, NTP TR 383Lesions in Female Mice 251



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm

Disposition Summary Animals initially in study 60 60 60 15-Month interim evaluation 10 10 10 Early deaths Moribund 5 11 11 Natural deaths 6 5 6 Survivors Died last week of study 1 1 Terminal sacrifice 38 33 33

Animals examined microscopically 60 60 60



### 15-Month Interim Evaluation Alimentary System Liver (10) (10) (10) Hepatocellular carcinoma 1 (10%) Hepatocellular adenoma 1 (10%) 3 (30%) Hepatocellular adenoma, multiple 1 (10%) 4 (40%) Stomach, forestomach (10) (10) (10) Squamous cell papilloma 4 (40%) 2 (20%)

Respiratory System Lung (10) (10) (10) Alveolar/bronchiolar adenoma 3 (30%) 2 (20%)



### Systems Examined With No Neoplasms Observed Cardiovascular System Endocrine System General Body System Genital System Hematopoietic System Integumentary System Musculoskeletal System Nervous System Special Senses System Urinary System



### 2-Year Study Alimentary System Esophagus (50) (45) (47) Basosquamous tumor malignant, metastatic,

uterus 1 (2%) Gallbladder (49) (47) (44) Basosquamous tumor malignant, metastatic, uterus 1 (2%)252 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Intestine large, colon (50) (50) (48) Intestine small, duodenum (48) (50) (46) Intestine small, jejunum (47) (50) (46) Intestine small, ileum (48) (50) (46) Liver (50) (50) (50) Fibrosarcoma, metastatic, skin 1 (2%) Hemangioma 1 (2%) Hepatoblastoma 2 (4%) Hepatocellular carcinoma 10 (20%) 14 (28%) Hepatocellular carcinoma, multiple 13 (26%) 13 (26%) Hepatocellular adenoma 6 (12%) 5 (10%) 4 (8%) Hepatocellular adenoma, multiple 40 (80%) 45 (90%) Squamous cell carcinoma, metastatic, stomach, forestomach 3 (6%) 4 (8%) Mesentery (6) (9) (8) Basosquamous tumor malignant, metastatic, uterus 1 (17%) Fibrosarcoma, metastatic, skin 1 (11%) Sarcoma, metastatic, stomach, forestomach 1 (13%) Squamous cell carcinoma, metastatic, stomach, forestomach 2 (22%) 3 (38%) Pancreas (50) (50) (49) Basosquamous tumor malignant, metastatic, uterus 1 (2%) Fibrosarcoma, metastatic, skin 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) 2 (4%) Salivary glands (49) (50) (47) Stomach, forestomach (48) (50) (50) Sarcoma 1 (2%) Squamous cell carcinoma 12 (24%) 11 (22%) Squamous cell papilloma 2 (4%) 12 (24%) 13 (26%) Squamous cell papilloma, multiple 4 (8%) 14 (28%) Stomach, glandular (49) (48) (48) Sarcoma 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 3 (6%) 3 (6%)

Cardiovascular System Heart (50) (50) (49)

Endocrine System Adrenal cortex (50) (49) (48) Carcinoma 1 (2%) Squamous cell carcinoma, metastatic 1 (2%) Adrenal medulla (49) (49) (48) Islets, pancreatic (49) (50) (48) Adenoma 2 (4%) 1 (2%)Lesions in Female Mice 253



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Endocrine System (continued) Pituitary gland (43) (45) (43) Pars distalis, adenoma 1 (2%) 8 (18%) 4 (9%) Pars distalis, adenoma, multiple 1 (2%) Pars intermedia, adenoma 1 (2%) Pars nervosa, adenoma 1 (2%) Thyroid gland (50) (50) (48) Follicular cell, adenoma 3 (6%) 1 (2%) 2 (4%)

General Body System
Tissue NOS (1)

Genital System Ovary (49) (49) (47) Basosquamous tumor malignant, metastatic, uterus 1 (2%) Granulosa cell tumor benign 1 (2%) Hemangioma 1 (2%) Luteoma 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) 3 (6%) Uterus (49) (50) (49) Leiomyoma 2 (4%) Polyp stromal 2 (4%) Sarcoma stromal 3 (6%) Cervix, basosquamous tumor malignant 1 (2%) Vagina (1) Squamous cell carcinoma 1 (100%)

Hematopoietic System Bone marrow (50) (49) (49) Lymph node (7) (12) (12) Bronchial, sarcoma, metastatic, stomach, forestomach 1 (8%) Mediastinal, squamous cell carcinoma, metastatic, stomach, forestomach 1 (8%) 1 (8%) Renal, basosquamous tumor malignant, metastatic, uterus 1 (14%) Lymph node, mandibular (32) (45) (31) Lymph node, mesenteric (49) (46) (44) Basosquamous tumor malignant, metastatic, uterus 1 (2%) Sarcoma, metastatic, stomach, forestomach 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) 1 (2%)254 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Spleen (50) (50) (50) Hemangioma 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) Thymus (40) (38) (34) Osteosarcoma, metastatic, bone 1 (3%)

Integumentary System Mammary gland (25) (25) (20) Fibroadenoma 1 (4%) Skin (50) (50) (50) Squamous cell papilloma 1 (2%) Subcutaneous tissue, fibrosarcoma 2 (4%) 1 (2%) Subcutaneous tissue, hemangioma 1 (2%)

Musculoskeletal System Bone (50) (48) (49) Rib, osteosarcoma 1 (2%) Skeletal muscle (4) (2) (2) Abdominal, fibrosarcoma, metastatic, skin 1 (50%) Diaphragm, basosquamous tumor malignant, metastatic, uterus 1 (25%) Diaphragm, squamous cell carcinoma, metastatic, stomach, forestomach 1 (50%) 1 (50%)

Nervous System Brain (49) (50) (50)

Respiratory System Lung (50) (50) (49) Alveolar/bronchiolar adenoma 4 (8%) 15 (30%) 12 (24%) Alveolar/bronchiolar adenoma, multiple 2 (4%) 1 (2%) Alveolar/bronchiolar carcinoma 2 (4%) Basosquamous tumor malignant, metastatic, uterus 1 (2%) Hemangioma 1 (2%) Hepatocellular carcinoma, metastatic, liver 1 (2%) Osteosarcoma, metastatic, bone 1 (2%) Squamous cell carcinoma, metastatic, stomach, forestomach 1 (2%) Nose (48) (45) (44)Lesions in Female Mice 255



### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Special Senses System Harderian gland (1) (3) (2) Adenoma 1 (100%) 2 (67%) 2 (100%) Carcinoma 1 (33%)

Urinary System Kidney (50) (50) (50) Osteosarcoma, metastatic, bone 1 (2%) Urinary bladder (49) (50) (50)

Systemic Lesions Multiple organs b (50) (50) (50) Lymphoma malignant histiocytic 1 (2%) Lymphoma malignant lymphocytic 1 (2%) Lymphoma malignant mixed 9 (18%) 13 (26%) 13 (26%) Lymphoma malignant undifferentiated cell 2 (4%)

Neoplasm Summary Total animals with primary neoplasms c
15-Month interim evaluation 5 9 2-Year study 32 50 50 Total primary neoplasms 15-Month interim evaluation 9 12 2-Year study 45 149 157 Total animals with benign neoplasms 15-Month interim evaluation 5 8 2-Year study 24 46 50 Total benign neoplasms 15-Month interim evaluation 9 11 2-Year study 29 94 99 Total animals with malignant neoplasms 15-Month interim evaluation 1 2-Year study 14 40 37 Total malignant neoplasms 15-Month interim evaluation 1 2-Year study 16 55 58 Total animals with metastatic neoplasms 2-Year study 2 4 6 Total metastatic neoplasms 2-Year study 12 18 24

a Number of animals examined microscopically at site and number of animals with neoplasm b Number of animals with any tissue examined microscopically

c Primary neoplasms: all neoplasms except metastatic neoplasms256 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm

0456666777777777777777777 Number of Days on Study 7791258000033333333333333
6820835116823333333333333

4333443444433333444444444 Carcass ID Number 4989667088678899000111222
5554545454342312123145123

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + +

Basosquamous tumor malignant,

metastatic, uterus X Gallbladder + + + + + + + + + M + + + + + + + + + + + + + + +

Basosquamous tumor malignant,

metastatic, uterus X Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, duodenum A + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum A + + A + + + + + + A + + + + + + + + + + + + + + Intestine small, ileum A + + A + + + + + + + + + + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangioma Hepatocellular adenoma X X X Mesentery + + + + +

Basosquamous tumor malignant,

metastatic, uterus X Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Basosquamous tumor malignant,

metastatic, uterus X Salivary glands + + M + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + M + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + Tooth +

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + +

Carcinoma Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + Islets, pancreatic + + + + + + + + + + + + + + + + + M + + + + + + +

Adenoma X Parathyroid gland M + M + + + + + + + + + + + + MMM+M++M++ Pituitary gland M + + + + + M + + + + + + M + + + + + + + + + + +

Pars distalis, adenoma X Pars intermedia, adenoma X Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

Follicular cell, adenoma

General Body System

None

+: Tissue examined microscopically M: Missing tissue X: Lesion present A: Autolysis precludes examination I: Insufficient tissue Blank: Not examinedLesions in Female Mice 257



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
3333333333333333444444444

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 4 4 4 4 4 4 4 Total Carcass ID Number 2 2 3 3 3 3 3 4 4 4 5 5 6 7 7 7 7 8 4 5 5 6 8 8 8 Tissues/
4 5 1 2 3 4 5 1 2 3 1 3 1 1 3 4 3 4 4 4 5 2 1 2 3 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basosquamous tumor malignant,

metastatic, uterus 1 Gallbladder + + + + + + + + + + + + + + + + + + + + + + + + + 49

Basosquamous tumor malignant,

metastatic, uterus 1 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + M + 48 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 48 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hemangioma X 1 Hepatocellular adenoma X X X 6 Mesentery + 6

Basosquamous tumor malignant,

metastatic, uterus 1 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Basosquamous tumor malignant,

metastatic, uterus 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 49 Stomach, forestomach + + + + + + + + + + + + M + + + + + + + + + + + + 48

Squamous cell papilloma X X 2 Stomach, glandular + + + + + + + + + + + + M + + + + + + + + + + + + 49 Tooth 1

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 50

Carcinoma X 1 Adrenal medulla + + + + + + + M + + + + + + + + + + + + + + + + + 49 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 49

Adenoma X 2 Parathyroid gland + M M M + + + + M + M + + M M + M + + M + M + + + 33 Pituitary gland + + M + M + + + + + + + + + + + + M M + + + + + + 43

Pars distalis, adenoma 1 Pars intermedia, adenoma 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Follicular cell, adenoma X X X 3

General Body System

None258 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

0456666777777777777777777 Number of Days on Study 7791258000033333333333333
6820835116823333333333333

4333443444433333444444444 Carcass ID Number 4989667088678899000111222
5554545454342312123145123

Genital System Ovary + + + + + + + + + + + + + + + + + + + + + + + + M

Basosquamous tumor malignant,

metastatic, uterus X Granulosa cell tumor benign Hemangioma Uterus M + + + + + + + + + + + + + + + + + + + + + + + +

Leiomyoma Cervix, basosquamous tumor malignant X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + +

Renal, basosquamous tumor malignant,

metastatic, uterus X Lymph node, mandibular M + M M + + + + + + M + + M + + + + M M + + M + M Lymph node, mesenteric M + + + + + + + + + + + + + + + + + + + + + + + +

Basosquamous tumor malignant,

metastatic, uterus X Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Hemangioma Thymus M + + M M + I + M M + + + + + + + + + + + + + + +

Osteosarcoma, metastatic, bone X

Integumentary System Mammary gland M + M + + + + M + MMM++M++++M++M++

Fibroadenoma X Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell papilloma X Subcutaneous tissue, fibrosarcoma X X Subcutaneous tissue, hemangioma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + +

Rib, osteosarcoma X Skeletal muscle + + + +

Diaphragm, basosquamous tumor

malignant, metastatic, uterus X

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X Basosquamous tumor malignant,

metastatic, uterus X Osteosarcoma, metastatic, bone X Nose + + + + + + + + + + + + + + M + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Female Mice 259



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
3333333333333333444444444

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 4 4 4 4 4 4 4 Total Carcass ID Number 2 2 3 3 3 3 3 4 4 4 5 5 6 7 7 7 7 8 4 5 5 6 8 8 8 Tissues/
4 5 1 2 3 4 5 1 2 3 1 3 1 1 3 4 3 4 4 4 5 2 1 2 3 Tumors

Genital System Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 49

Basosquamous tumor malignant,

metastatic, uterus 1 Granulosa cell tumor benign X 1 Hemangioma X 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 49

Leiomyoma X X 2 Cervix, basosquamous tumor malignant 1

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 50 Lymph node + 7

Renal, basosquamous tumor malignant,

metastatic, uterus 1 Lymph node, mandibular + + M M + + + + I + + M + M + M M + + + + M M + + 32 Lymph node, mesenteric + + + + + + + + + + + + + + + + + + + + + + + + + 49

Basosquamous tumor malignant,

metastatic, uterus 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hemangioma X 1 Thymus + + + + + + + M + + + + M + M + + + + + + + + + M 40

Osteosarcoma, metastatic, bone 1

Integumentary System Mammary gland M M + + M M M M + M + + M + M M + M M M + M M M + 25

Fibroadenoma 1 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell papilloma 1 Subcutaneous tissue, fibrosarcoma 2 Subcutaneous tissue, hemangioma 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 50

Rib, osteosarcoma 1 Skeletal muscle 4

Diaphragm, basosquamous tumor

malignant, metastatic, uterus 1

Nervous System

Brain + + + + + + + + + + M + + + + + + + + + + + + + + 49

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X X X 4 Basosquamous tumor malignant,

metastatic, uterus 1 Osteosarcoma, metastatic, bone 1 Nose + M + + + + + + + + + + + + + + + + + + + + + + + 48 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 50260 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

0456666777777777777777777 Number of Days on Study 7791258000033333333333333
6820835116823333333333333

4333443444433333444444444 Carcass ID Number 4989667088678899000111222
5554545454342312123145123

Special Senses System

Ear + Eye Harderian gland

Adenoma

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + +

Osteosarcoma, metastatic, bone X Urinary bladder + + + + + + A + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Lymphoma malignant histiocytic Lymphoma malignant lymphocytic Lymphoma malignant mixed X X X X X XLesions in Female Mice 261



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 0 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
3333333333333333444444444

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 4 4 4 4 4 4 4 Total Carcass ID Number 2 2 3 3 3 3 3 4 4 4 5 5 6 7 7 7 7 8 4 5 5 6 8 8 8 Tissues/
4 5 1 2 3 4 5 1 2 3 1 3 1 1 3 4 3 4 4 4 5 2 1 2 3 Tumors

Special Senses System

Ear 1 Eye + 1 Harderian gland + 1

Adenoma X 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50

Osteosarcoma, metastatic, bone 1 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 49

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymphoma malignant histiocytic X 1 Lymphoma malignant lymphocytic X 1 Lymphoma malignant mixed X X X 9262 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm

2345556666777777777777777 Number of Days on Study 0740585577011112333333333
2223572719633367000000000

5455655455555555444555555 Carcass ID Number 6976040977613935999000011
5554545443355545123123412

Alimentary System Esophagus + + + + M + + M + + + + M + + + + + + + + + + + + Gallbladder + + + M + + + + + + + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine large, cecum + + + + + + M + + + + + + + + + + + + + + + + + + Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Fibrosarcoma, metastatic, skin X Hepatocellular carcinoma X X X X Hepatocellular carcinoma, multiple X X X X X X Hepatocellular adenoma X X X X Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X Squamous cell carcinoma, metastatic,

stomach, forestomach X X Mesentery + + + + + +

Fibrosarcoma, metastatic, skin X Squamous cell carcinoma, metastatic,

stomach, forestomach X X Pancreas + + + + + + + + + + + + + + + + + + + + + + + + +

Fibrosarcoma, metastatic, skin X Squamous cell carcinoma, metastatic,

stomach, forestomach Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma X X X X X X Squamous cell papilloma X X X X X Squamous cell papilloma, multiple X Stomach, glandular + + + + + + + + + + + + + + + + + + M + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X X

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + M + + + + + + + + + + + Adrenal medulla + + + + + + + + + + + + + M + + + + + + + + + + + Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + +

Adenoma Parathyroid gland M + + + + + + + + + + M M + + + + + + + + M + + Pituitary gland + + + + + + + + + + I + + + + + + + I M + + + + +

Pars distalis, adenoma X X X Pars distalis, adenoma, multiple Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + +

Follicular cell, adenomaLesions in Female Mice 263



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
0000000000000002333333333

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 Total Carcass ID Number 1 2 2 2 2 3 3 4 4 4 5 5 6 6 7 8 7 8 8 8 9 9 9 0 0 Tissues/
3 1 2 3 4 1 2 1 2 3 3 4 1 2 2 5 1 1 3 4 1 2 3 3 4 Tumors

Alimentary System Esophagus + + + + + + + + + + + + M + + + + + + M + + + + + 45 Gallbladder + M + + + + + + + + + + + + M + + + + + + + + + + 47 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + + 49 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + + 50 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Fibrosarcoma, metastatic, skin 1 Hepatocellular carcinoma X X X X X X 10 Hepatocellular carcinoma, multiple X X X X X X X 13 Hepatocellular adenoma X 5 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X X X X 40 Squamous cell carcinoma, metastatic,

stomach, forestomach X 3 Mesentery + + + 9

Fibrosarcoma, metastatic, skin 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 2 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 50

Fibrosarcoma, metastatic, skin 1 Squamous cell carcinoma, metastatic,

stomach, forestomach X 1 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + + 50 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma X X X X X X 12 Squamous cell papilloma X X X X X X X 12 Squamous cell papilloma, multiple X X X 4 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic,

stomach, forestomach X 3

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 50

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + + 49 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + + 49 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 50

Adenoma X 1 Parathyroid gland + + M + + + + M + M + + M + + M + + + M + + + + + 39 Pituitary gland + + + I + + + + I + + + + + + + + + + + + + + + + 45

Pars distalis, adenoma X X X X X 8 Pars distalis, adenoma, multiple X 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 50

Follicular cell, adenoma X 1264 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

2345556666777777777777777 Number of Days on Study 0740585577011112333333333
2223572719633367000000000

5455655455555555444555555 Carcass ID Number 6976040977613935999000011
5554545443355545123123412

General Body System
Tissue NOS +

Genital System Clitoral gland M Ovary + + + + + + + + + + + + + + + + + + + + + + + +

Luteoma X Squamous cell carcinoma, metastatic,

stomach, forestomach X Uterus + + + + + + + + + + + + + + + + + + + + + + + + +

Polyp stromal X X Sarcoma stromal X

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + +

Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach X Lymph node, mandibular + + + + + + + M + + + + + + + + + + + + + + + + M Lymph node, mesenteric + + + M + + M + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Spleen + + + + + + + + + + + + + + + + + + + + + + + + + Thymus + + + + + + M M + M + M + M + M + + + + + + + + +

Integumentary System Mammary gland M + + M + + M + M M M + + M + + + + + M + MMM+ Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Subcutaneous tissue, fibrosarcoma X

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + +

Abdominal, fibrosarcoma, metastatic,

skin X Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach X

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + +

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X X X X X X X X Alveolar/bronchiolar adenoma,

multiple Squamous cell carcinoma, metastatic,

stomach, forestomach X Nose + + + + + + + + + + + + + + + + + + + + + + + + Trachea + + + + + + + + + + + + + + + + + + + + + + + + +Lesions in Female Mice 265



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
0000000000000002333333333

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 Total Carcass ID Number 1 2 2 2 2 3 3 4 4 4 5 5 6 6 7 8 7 8 8 8 9 9 9 0 0 Tissues/
3 1 2 3 4 1 2 1 2 3 3 4 1 2 2 5 1 1 3 4 1 2 3 3 4 Tumors

General Body System
Tissue NOS 1

Genital System Clitoral gland Ovary + + + + + + + + + + + + + + + + + + + + + + + + + 49

Luteoma 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 50

Polyp stromal 2 Sarcoma stromal X X 3

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + + + + 12

Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach 1 Lymph node, mandibular + + I + + + + + + + + M + + + M + + + + + + + + + 45 Lymph node, mesenteric M + + + + + + + + + + + + + + + M + + + + + + + + 46

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50 Thymus + + M M + + + + M + + + M + + M + + + + M + + + + 38

Integumentary System Mammary gland M M M M M M + M M + + M + + M +++M++ M M M + 25 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Subcutaneous tissue, fibrosarcoma 1

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + 48 Skeletal muscle 2

Abdominal, fibrosarcoma, metastatic,

skin 1 Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach 1

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 50

Alveolar/bronchiolar adenoma X X X X X X X 15 Alveolar/bronchiolar adenoma,

multiple X X 2 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Nose + + + + M + + M + + + + + M + + + + + + + + + + 45 Trachea + + + + + + M + + + + + + + + + + + + + + + + + + 49266 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

2345556666777777777777777 Number of Days on Study 0740585577011112333333333
2223572719633367000000000

5455655455555555444555555 Carcass ID Number 6976040977613935999000011
5554545443355545123123412

Special Senses System Harderian gland + +

Adenoma X Carcinoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Lymphoma malignant mixed X X X X X Lymphoma malignant undifferentiated

cell type X XLesions in Female Mice 267



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 10,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 3333333333333333333333333
0000000000000002333333333

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 Total Carcass ID Number 1 2 2 2 2 3 3 4 4 4 5 5 6 6 7 8 7 8 8 8 9 9 9 0 0 Tissues/
3 1 2 3 4 1 2 1 2 3 3 4 1 2 2 5 1 1 3 4 1 2 3 3 4 Tumors

Special Senses System Harderian gland + 3

Adenoma X 2 Carcinoma 1

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymphoma malignant mixed X X X X X X X X 13 Lymphoma malignant undifferentiated

cell type 2268 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm

5555556666666777777777777 Number of Days on Study 0356692236689112222222222
1832863312407383499999999

6766776677766666766666666 Carcass ID Number 3179113320169113211122223
4545433555244542412312341

Alimentary System Esophagus + + M + + + + + + M + + + + + + + + + + + + + + + Gallbladder + A M + + + + + + M + + + + + + + + + + + + + + + Intestine large, colon + + + + + + + + + + + + + + A + + + + + + + + + + Intestine large, rectum + + + + + + + + + + + M + + A + + + + + + + + + + Intestine large, cecum + + + + + + + + + + + M + + A + + + + + + + + + + Intestine small, duodenum + A A + + + + + + + + + + + A + + + + + + + + + + Intestine small, jejunum + A A + + + + + + + + + + + A + + + + + + + + + + Intestine small, ileum A + A + + + + + + + + + + + A + + + + + + + + + + Liver + + + + + + + + + + + + + + + + + + + + + + + + +

Hepatoblastoma X X Hepatocellular carcinoma X X X X X X X X X X X Hepatocellular carcinoma, multiple X X X X X X Hepatocellular adenoma X X X Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X X Mesentery + + + + + + + +

Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X Pancreas + + + + + M + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X X Salivary glands + + M + + + + + + M + + + + + + + + + + + + + + + Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma X Squamous cell carcinoma X X X X X X X X X X Squamous cell papilloma X X X X Squamous cell papilloma, multiple X X X X X X Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + +

Sarcoma X Squamous cell carcinoma, metastatic,

stomach, forestomach X X X

Cardiovascular System

Heart + + M + + + + + + + + + + + + + + + + + + + + + +

Endocrine System
Adrenal cortex + + + + + + + + + + + + + M + + + + + + + + + + +

Squamous cell carcinoma, metastatic X Adrenal medulla + + + + + + + + + + + + + M + + + + + + + + + + + Islets, pancreatic + + + + + M + + + + + + + + A + + + + + + + + + + Parathyroid gland M M M + + + + M + M + + + M M + + + + + + + M M + Pituitary gland + + + + + + + + + M + M + M + + + M + + + + + + +

Pars distalis, adenoma X X Pars nervosa, adenoma X Thyroid gland + + M + + + + + + M + + + + + + + + + + + + + + +

Follicular cell, adenomaLesions in Female Mice 269



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222333333333333333
9999999999000000000000000

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 Total Carcass ID Number 4 4 4 4 5 8 8 8 8 8 5 5 5 6 6 6 7 7 7 9 9 0 0 1 2 Tissues/
1 2 4 5 5 1 2 3 4 5 2 3 4 1 2 3 1 2 3 1 3 3 4 1 3 Tumors

Alimentary System Esophagus + + + + + + + + + + + + + + + + M + + + + + + + + 47 Gallbladder + + + + + + M + + + + M + + + + + + + + + + M + + 44 Intestine large, colon + + + + + + + + + + + + + + + + + + + + + + + + 48 Intestine large, rectum + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine large, cecum + + + + + + + + + + + + + + + + + + + + + + + + 47 Intestine small, duodenum + + + + + + + + + + + + + + + + + + + + + + + + 46 Intestine small, jejunum + + + + + + + + + + + + + + + + + + + + + + + + 46 Intestine small, ileum + + + + + + + + + + + + + + + + + + + + + + + + 46 Liver + + + + + + + + + + + + + + + + + + + + + + + + + 50

Hepatoblastoma 2 Hepatocellular carcinoma X X X 14 Hepatocellular carcinoma, multiple X X X X X X X 13 Hepatocellular adenoma X 4 Hepatocellular adenoma, multiple X X X X X X X X X X X X X X X X X X X X X X X X 45 Squamous cell carcinoma, metastatic,

stomach, forestomach 4 Mesentery 8

Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 3 Pancreas + + + + + + + + + + + + + + + + + + + + + + + + + 49

Squamous cell carcinoma, metastatic,

stomach, forestomach 2 Salivary glands + + + + + + + + + + + + + + + + + + + + + + + + M 47 Stomach, forestomach + + + + + + + + + + + + + + + + + + + + + + + + + 50

Sarcoma 1 Squamous cell carcinoma X 11 Squamous cell papilloma X X X X X X X X X 13 Squamous cell papilloma, multiple X X X X X X X X 14 Stomach, glandular + + + + + + + + + + + + + + + + + + + + + + + + 48

Sarcoma 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 3

Cardiovascular System

Heart + + + + + + + + + + + + + + + + + + + + + + + + + 49

Endocrine System
Adrenal cortex + + + + + + + + + + + + + + + + + + + + + + + + 48

Squamous cell carcinoma, metastatic 1 Adrenal medulla + + + + + + + + + + + + + + + + + + + + + + + + 48 Islets, pancreatic + + + + + + + + + + + + + + + + + + + + + + + + + 48 Parathyroid gland + M + M + + M + + + + + M + M + M + + M + + + M + 33 Pituitary gland + + + M + + + + + + M + M + + + + + + + + + + + + 43

Pars distalis, adenoma X X 4 Pars nervosa, adenoma 1 Thyroid gland + + + + + + + + + + + + + + + + + + + + + + + + + 48

Follicular cell, adenoma X X 2270 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm (continued)

5555556666666777777777777 Number of Days on Study 0356692236689112222222222
1832863312407383499999999

6766776677766666766666666 Carcass ID Number 3179113320169113211122223
4545433555244542412312341

General Body System

None

Genital System Clitoral gland + Ovary + + + + + + + + + + + + + M M + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X X X Uterus + + + + + + + + + + + + + + A + + + + + + + + + + Vagina +

Squamous cell carcinoma X

Hematopoietic System

Bone marrow + + M + + + + + + + + + + + + + + + + + + + + + + Lymph node + + + + + + + +

Bronchial, sarcoma, metastatic,

stomach, forestomach X Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach X Lymph node, mandibular + + M + + M + M + M M + + + + + + + + + + + + + I Lymph node, mesenteric + + M + M + + + + M + + + + M + + + + + + M + + +

Sarcoma, metastatic, stomach,

forestomach X Squamous cell carcinoma, metastatic,

stomach, forestomach X Spleen + + + + + + + + + + + + + + + + + + + + + + + + +

Squamous cell carcinoma, metastatic,

stomach, forestomach X Thymus M + M + M M + + + M M M M M M + M M + + M ++++

Integumentary System Mammary gland + + M M + M + + M + M M + M M M M M + + M + M M M Skin + + + + + + + + + + + + + + + + + + + + + + + + +

Musculoskeletal System

Bone + + M + + + + + + + + + + + + + + + + + + + + + + Skeletal muscle + +

Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach X

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + Spinal cord +Lesions in Female Mice 271



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222333333333333333
9999999999000000000000000

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 Total Carcass ID Number 4 4 4 4 5 8 8 8 8 8 5 5 5 6 6 6 7 7 7 9 9 0 0 1 2 Tissues/
1 2 4 5 5 1 2 3 4 5 2 3 4 1 2 3 1 2 3 1 3 3 4 1 3 Tumors

General Body System

None

Genital System Clitoral gland 1 Ovary + M + + + + + + + + + + + + + + + + + + + + + + + 47

Squamous cell carcinoma, metastatic,

stomach, forestomach 3 Uterus + + + + + + + + + + + + + + + + + + + + + + + + + 49 Vagina 1

Squamous cell carcinoma 1

Hematopoietic System

Bone marrow + + + + + + + + + + + + + + + + + + + + + + + + + 49 Lymph node + + + + 12

Bronchial, sarcoma, metastatic,

stomach, forestomach 1 Mediastinal, squamous cell carcinoma,

metastatic, stomach, forestomach 1 Lymph node, mandibular M + M + M M I + + M M M + + + M M + + + + I M + M 31 Lymph node, mesenteric + + + + + + + + + + M + + + + + + + + + + + + + + 44

Sarcoma, metastatic, stomach,

forestomach 1 Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Spleen + + + + + + + + + + + + + + + + + + + + + + + + + 50

Squamous cell carcinoma, metastatic,

stomach, forestomach 1 Thymus + + + + + + + + + + + + + + + + + + + M M M + + + 34

Integumentary System Mammary gland + M M M M + M M + + M + M + + M M M + M M M + + M 20 Skin + + + + + + + + + + + + + + + + + + + + + + + + + 50

Musculoskeletal System

Bone + + + + + + + + + + + + + + + + + + + + + + + + + 49 Skeletal muscle 2

Diaphragm, squamous cell carcinoma,

metastatic, stomach, forestomach 1

Nervous System

Brain + + + + + + + + + + + + + + + + + + + + + + + + + 50 Spinal cord 1272 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm (continued)

5555556666666777777777777 Number of Days on Study 0356692236689112222222222
1832863312407383499999999

6766776677766666766666666 Carcass ID Number 3179113320169113211122223
4545433555244542412312341

Respiratory System Lung + + M + + + + + + + + + + + + + + + + + + + + + +

Alveolar/bronchiolar adenoma X X X X X X Alveolar/bronchiolar adenoma,

multiple X Alveolar/bronchiolar carcinoma X Hemangioma Hepatocellular carcinoma, metastatic,

liver Nose + + + + + + + + + + + + + + M + + + + + + + + M + Trachea + + M + + + + + + M + + + + + + + I + + + + + + +

Special Senses System Eye + Harderian gland +

Adenoma X

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + +

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + +

Lymphoma malignant mixed X X X X X XLesions in Female Mice 273



### T ABLE 

### D2 Individual Animal Tumor Pathology of Female Mice in th e 2-Year Feed Study of 1-Am ino-2,4-dibromoanthraquinone: 20,000 ppm (continued)

7777777777777777777777777 Number of Days on Study 2222222222333333333333333
9999999999000000000000000

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 Total Carcass ID Number 4 4 4 4 5 8 8 8 8 8 5 5 5 6 6 6 7 7 7 9 9 0 0 1 2 Tissues/
1 2 4 5 5 1 2 3 4 5 2 3 4 1 2 3 1 2 3 1 3 3 4 1 3 Tumors

Respiratory System Lung + + + + + + + + + + + + + + + + + + + + + + + + + 49

Alveolar/bronchiolar adenoma X X X X X X 12 Alveolar/bronchiolar adenoma,

multiple 1 Alveolar/bronchiolar carcinoma X 2 Hemangioma X 1 Hepatocellular carcinoma, metastatic,

liver X 1 Nose + + + + + + + M + + + M M + + + M + + + + + + + + 44 Trachea + + + + + + + + + + + + + + + + + + + + + + + + + 47

Special Senses System Eye 1 Harderian gland + 2

Adenoma X 2

Urinary System Kidney + + + + + + + + + + + + + + + + + + + + + + + + + 50 Urinary bladder + + + + + + + + + + + + + + + + + + + + + + + + + 50

Systemic Lesions Multiple organs + + + + + + + + + + + + + + + + + + + + + + + + + 50

Lymphoma malignant mixed X X X X X X X 13274 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D3 Statistical Analysis of Prim ary Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,000 ppm 20,000 ppm

Harderian Gland: Adenoma or Carcinoma
Overall rate a 1/50 (2%) 3/50 (6%) 2/50 (4%) Adjusted rate b 2.6% 7.8% 6.1% Terminal rate c 1/39 (3%) 2/34 (6%) 2/33 (6%) First incidence (days) 729 (T) 372 729 (T) Life table test d P=0.348 P=0.271 P=0.442 Logistic regression test d P=0.393 P=0.314 P=0.442 Cochran-Armitage test d P=0.399 Fisher exact test d P=0.309 P=0.500

Liver: Hepatocellular Adenoma Overall rate 6/50 (12%) 45/50 (90%) 49/50 (98%) Adjusted rate 15.4% 95.7% 100.0% Terminal rate 6/39 (15%) 32/34 (94%) 33/33 (100%) First incidence (days) 729 (T) 442 501 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Liver: Hepatocellular Carcinoma Overall rate 0/50 (0%) 23/50 (46%) 27/50 (54%) Adjusted rate 0.0% 57.2% 60.8% Terminal rate 0/39 (0%) 17/34 (50%) 16/33 (48%) First incidence (days)

) e 503 538 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 6/50 (12%) 46/50 (92%) 50/50 (100%) Adjusted rate 15.4% 97.9% 100.0% Terminal rate 6/39 (15%) 33/34 (97%) 33/33 (100%) First incidence (days) 729 (T) 442 501 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Lung: Alveolar/bronchiolar Adenoma Overall rate 4/50 (8%) 17/50 (34%) 13/49 (27%) Adjusted rate 9.8% 45.6% 33.5% Terminal rate 3/39 (8%) 14/34 (41%) 9/33 (27%) First incidence (days) 685 587 538 Life table test P=0.010 P<0.001 P=0.009

Logistic regression test P=0.017 P=0.001 P=0.015

Cochran-Armitage test P=0.018 Fisher exact test P=0.001 P=0.014Lesions in Female Mice 275



### T ABLE 

### D3 Statistical Analysis of Prim ary Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate 4/50 (8%) 17/50 (34%) 15/49 (31%) Adjusted rate 9.8% 45.6% 37.9% Terminal rate 3/39 (8%) 14/34 (41%) 10/33 (30%) First incidence (days) 685 587 538 Life table test P=0.003 P<0.001 P=0.003

Logistic regression test P=0.006 P=0.001 P=0.005

Cochran-Armitage test P=0.006 Fisher exact test P=0.001 P=0.004

Pituitary Gland (Pars Distalis): Adenoma Overall rate 1/43 (2%) 9/45 (20%) 4/43 (9%) Adjusted rate 2.2% 27.2% 12.2% Terminal rate 0/34 (0%) 7/30 (23%) 3/29 (10%) First incidence (days) 628 671 553 Life table test P=0.145 P=0.007 P=0.151 Logistic regression test P=0.181 P=0.009 P=0.216 Cochran-Armitage test P=0.191 Fisher exact test P=0.009 P=0.180

Stomach (Forestomach): Squamous Cell Papilloma Overall rate 2/50 (4%) 16/50 (32%) 27/50 (54%) Adjusted rate 5.1% 41.7% 72.4% Terminal rate 2/39 (5%) 12/34 (35%) 23/33 (70%) First incidence (days) 729 (T) 671 538 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Stomach (Forestomach): Squamous Cell Carcinoma Overall rate 0/50 (0%) 12/50 (24%) 11/50 (22%) Adjusted rate 0.0% 30.9% 27.3% Terminal rate 0/39 (0%) 8/34 (24%) 5/33 (15%) First incidence (days)

) 587 501 Life table test P=0.001 P<0.001 P<0.001

Logistic regression test P=0.002 P<0.001 P<0.001

Cochran-Armitage test P=0.002 Fisher exact test P<0.001 P<0.001

Stomach (Forestomach): Squamous Ce ll Papilloma or Squamous Cell Carcinoma Overall rate 2/50 (4%) 25/50 (50%) 34/50 (68%) Adjusted rate 5.1% 60.7% 80.5% Terminal rate 2/39 (5%) 18/34 (53%) 25/33 (76%) First incidence (days) 729 587 501 Life table test P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P<0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001276 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D3 Statistical Analysis of Prim ary Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Thyroid Gland (Follicular Cell): Adenoma Overall rate 3/50 (6%) 1/50 (2%) 2/48 (4%) Adjusted rate 7.7% 2.9% 6.1% Terminal rate 3/39 (8%) 1/34 (3%) 2/33 (6%) First incidence (days) 729 (T) 729 (T) 729 (T) Life table test P=0.467N P=0.355N P=0.576N

Logistic regression test P=0.467N P=0.355N P=0.576N

Cochran-Armitage test P=0.415N Fisher exact test P=0.309N P=0.520N

Uterus: Stromal Sarcoma Overall rate 0/50 (0%) 3/50 (6%) 0/50 (0%) Adjusted rate 0.0% 8.1% 0.0% Terminal rate 0/39 (0%) 2/34 (6%) 0/33 (0%) First incidence (days)

) 671

) Life table test P=0.597 P=0.105

)

Logistic regression test P=0.634 P=0.116

)

Cochran-Armitage test P=0.640 Fisher exact test P=0.121

)

Uterus: Stromal Polyp or Stromal Sarcoma Overall rate 0/50 (0%) 5/50 (10%) 0/50 (0%) Adjusted rate 0.0% 12.4% 0.0% Terminal rate 0/39 (0%) 2/34 (6%) 0/33 (0%) First incidence (days)

) 587

) Life table test P=0.562 P=0.029

)

Logistic regression test P=0.608 P=0.034

)

Cochran-Armitage test P=0.610 Fisher exact test P=0.028

)

All Organs: Hemangioma Overall rate 4/50 (8%) 0/50 (0%) 1/50 (2%) Adjusted rate 9.6% 0.0% 3.0% Terminal rate 3/39 (8%) 0/34 (0%) 1/33 (3%) First incidence (days) 478

) 729 (T) Life table test P=0.105N P=0.080N P=0.222N

Logistic regression test P=0.082N P=0.060N P=0.182N

Cochran-Armitage test P=0.082N Fisher exact test P=0.059N P=0.181N

All Organs: Malignant Lymphoma (Histiocytic, Lymphocyt ic, Mixed, or Undifferentiated Cell Type) Overall rate 11/50 (22%) 15/50 (30%) 13/50 (26%) Adjusted rate 25.5% 37.1% 35.7% Terminal rate 7/39 (18%) 10/34 (29%) 10/33 (30%) First incidence (days) 653 202 680 Life table test P=0.237 P=0.181 P=0.268 Logistic regression test P=0.364 P=0.254 P=0.339 Cochran-Armitage test P=0.366 Fisher exact test P=0.247 P=0.408Lesions in Female Mice 277



### T ABLE 

### D3 Statistical Analysis of Prim ary Neoplasms in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

All Organs: Benign Neoplasms Overall rate 24/50 (48%) 46/50 (92%) 50/50 (100%) Adjusted rate 54.1% 95.8% 100.0% Terminal rate 19/39 (49%) 32/34 (94%) 33/33 (100%) First incidence (days) 478 442 501 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

All Organs: Malignant Neoplasms Overall rate 14/50 (28%) 40/50 (80%) 38/50 (76%) Adjusted rate 30.9% 84.9% 80.7% Terminal rate 8/39 (21%) 27/34 (79%) 24/33 (73%) First incidence (days) 478 202 501 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

All Organs: Benign or Malignant Neoplasms Overall rate 32/50 (64%) 50/50 (100%) 50/50 (100%) Adjusted rate 66.7% 100.0% 100.0% Terminal rate 23/39 (59%) 34/34 (100%) 33/33 (100%) First incidence (days) 478 202 501 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P<0.001 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

(T)Terminal sacrifice

a Number of lesion-bearing animals/number of animals examined. De nominator is number of animals examined microscopically for liv er, lung, pituitary gland, stomach, thyroid gland, and uterus; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Observed incidence at terminal kill d Beneath the control incidence are the P valu es associated with the trend test. Bene ath the exposed group incidence are the P v alues corresponding to pairwise comparisons between the controls and that exposed group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests compare di rectly the overall incidence rates. For all tests, a negative trend or a lower incidence in an exposure group is indicated by N

. e Not applicable; no neoplasms in animal group278 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D4a Historical Incidence of Hepatocellular Neoplasms in Untreated Female B6C3F 1 

### Mice a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 6/50 0/50 6/50 Acetaminophen 3/49 0/49 3/49 HC Yellow 4 5/50 1/50 6/50 Methylphenidate Hydrochloride 6/49 5/49 9/49 Pentaerythritol Tetranitrate 5/49 1/49 6/49 Turmeric Oleoresin 7/50 7/50 13/50

Overall Historical Incidence

Total 176/1,462 (12.0%) 89/1,462 (6.1%) 247/1,462 (16.9%) Standard deviation 8.2% 5.4% 10.7% Range 0%-33% 0%-20% 3%-42%

a Data as of 31 March 1993



### T ABLE 

### D4b Historical Incidence of Fore stomach Squamous Cell Neoplasms in Untreated Female B6C3F 1 

### Mice a

Incidence in Controls Study Papilloma Carcinoma Papilloma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 2/50 0/50 2/50 Acetaminophen 0/50 0/50 0/50 HC Yellow 4 3/50 0/50 3/50 Methylphenidate Hydrochloride 1/49 0/49 1/49 Pentaerythritol Tetranitrate 1/50 0/50 1/50 Turmeric Oleoresin 0/50 0/50 0/50

Overall Historical Incidence

Total 31/1,470 (2.1%) 2/1,470 (0.1%) 33/1,470 (2.2%) Standard deviation 2.9% 0.5% 3.1% Range 0%-14% 0%-2% 0%-14%

a Data as of 31 March 1993Lesions in Female Mice 279



### T ABLE 

### D4c Historical Incidence of Al veolar/bronchiolar Neoplasms in Untreated Female B6C3F 1 

### Mice a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 4/50 0/50 4/50 Acetaminophen 1/50 0/50 1/50 HC Yellow 4 3/50 1/50 4/50 Methylphenidate Hydrochloride 1/48 0/48 1/48 Pentaerythritol Tetranitrate 2/50 1/50 3/50 Turmeric Oleoresin 4/50 1/50 5/50

Overall Historical Incidence

Total 82/1,469 (5.6%) 30/1,469 (2.1%) 110/1,469 (7.5%) Standard deviation 4.8% 2.2% 5.0% Range 0%-24% 0%-8% 2%-26%

a Data as of 31 March 1993



### T ABLE 

### D4d Historical Incidence of Uterine Neo plasms in Untreated Female B6C3F 1 

### Mice a

Incidence in Controls Study Stromal Polyp Stromal Sarcoma Stromal Polyp or Stromal Sarcoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 0/50 0/50 0/50 Acetaminophen 1/50 0/50 1/50 HC Yellow 4 2/50 0/50 2/50 Methylphenidate Hydrochloride 2/49 0/49 2/49 Pentaerythritol Tetranitrate 3/50 0/50 3/50 Tumeric Oleoresin 1/50 0/50 1/50

Overall Historical Incidence

Total 44/1,470 (3.0%) 7/1,470 (0.5%) 51/1,470 (3.5%) Standard deviation 3.2% 0.9% 3.1% Range 0%-16% 0%-2% 0%-16%

a Data as of 31 March 1993280 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D4e Historical Incidence of Pituit ary Gland Pars Distalis Neoplasms in Untreated Female B6C3F 1 

### Mice a

Incidence in Controls Study Adenoma Carcinoma Adenoma or Carcinoma

Historical Incidence at EG&G Mason Research Institute

1-Amino-2,4-dibromoanthraquinone 1/43 0/43 1/43 Acetaminophen 14/46 1/46 15/46 HC Yellow 4 5/42 0/42 5/42 Methylphenidate Hydrochloride 7/48 0/48 7/48 Pentaerythritol Tetranitrate 8/45 1/45 9/45 Tumeric Oleoresin 0/46 0/46 0/46

Overall Historical Incidence

Total 212/1,392 (15.2%) 8/1,392 (0.6%) 220/1,392 (15.8%) Standard deviation 9.9% 1.0% 10.3% Range 0%-36% 0%-4% 0%-36%

a Data as of 31 March 1993Lesions in Female Mice 281



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm

Disposition Summary Animals initially in study 60 60 60 15-Month interim evaluation 10 10 10 Early deaths Moribund 5 11 11 Natural deaths 6 5 6 Survivors Died last week of study 1 1 Terminal sacrifice 38 33 33

Animals examined microscopically 60 60 60



### 15-Month Interim Evaluation Alimentary System Gallbladder (10) (10) (10) Inflammation, chronic 1 (10%) 1 (10%) Inflammation, chronic active 1 (10%) Liver (10) (10) (10) Basophilic focus 1 (10%) 1 (10%) Fatty change 1 (10%) 1 (10%) 3 (30%) Inflammation, acute 5 (50%) 4 (40%) Inflammation, chronic active 9 (90%) 4 (40%) 6 (60%) Necrosis, coagulative 7 (70%) 8 (80%) 9 (90%) Pigmentation 10 (100%) 9 (90%) Bile duct, hyperplasia, focal 1 (10%) Pancreas (10) (10) (10) Inflammation, chronic 5 (50%) 1 (10%) Vacuolization cytoplasmic 1 (10%) Salivary glands (10) (10) (10) Inflammation, chronic 5 (50%) Submandibular gland, inflammation, chronic 7 (70%) 4 (40%) Stomach, forestomach (10) (10) (10) Acanthosis 1 (10%) 8 (80%) Hyperkeratosis 2 (20%) 7 (70%) Hyperplasia, basal cell 2 (20%) Inflammation, chronic 2 (20%) Inflammation, chronic active 1 (10%) 3 (30%) Stomach, glandular (10) (10) (10) Inflammation, chronic 2 (20%) 1 (10%) Inflammation, chronic active 1 (10%)

Cardiovascular System Heart (10) (10) (10) Cardiomyopathy 2 (20%)

Endocrine System Pituitary gland (8) (10) (9) Pars distalis, cyst 1 (11%) Pars distalis, hyperplasia 1 (13%) 1 (11%)

a Number of animals examined microscopically at site and number of animals with lesion282 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 15-Month Interim Evaluation (continued) Genital System Ovary (10) (2) (10) Cyst 1 (10%) 1 (50%) 2 (20%) Periovarian tissue, cyst 1 (50%) Periovarian tissue, inflammation, chronic 2 (20%) Uterus (10) (10) (10) Hydrometra 8 (80%) 3 (30%) Endometrium, hyperplasia 6 (60%) 10 (100%)

Hematopoietic System Bone marrow (10) (10) (10) Myelofibrosis 2 (20%) 1 (10%) Lymph node, mandibular (6) (10) (10) Pigmentation 1 (17%) Lymph node, mesenteric (7) (10) (8) Depletion lymphoid 1 (14%) Infiltration cellular, histiocyte 4 (57%) 7 (88%) Pigmentation 3 (43%) 7 (88%) Spleen (10) (10) (9) Depletion lymphoid 1 (11%) Thymus (9) (10) (8) Cyst 6 (67%) 6 (75%)

Nervous System Brain (10) (10) (10) Thalamus, mineralization 3 (30%) 4 (40%)

Respiratory System Nose (10) (10) (9) Glands, inflammation, acute 2 (20%) 1 (11%)

Urinary System Kidney (10) (4) (10) Inflammation, chronic 10 (100%) 4 (100%) 9 (90%) Renal tubule, regeneration 2 (20%) 1 (10%) Urinary bladder (10) (10) (10) Inflammation, chronic 7 (70%) 7 (70%) 8 (80%)



### Systems Examined With No Lesions Observed General Body System Integumentary System Musculoskeletal System Special Senses SystemLesions in Female Mice 283



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study Alimentary System Esophagus (50) (45) (47) Autolysis 1 (2%) Gallbladder (49) (47) (44) Autolysis 3 (6%) 2 (4%) 2 (5%) Inflammation, chronic active 5 (10%) 6 (13%) 8 (18%) Epithelium, hyperplasia 1 (2%) Intestine large, colon (50) (50) (48) Autolysis 3 (6%) 1 (2%) Inflammation, acute 1 (2%) Peyer's patch, hyperplasia 1 (2%) 1 (2%) Intestine large, rectum (50) (50) (47) Autolysis 2 (4%) 1 (2%) Peyer's patch, epithelium, proliferation 1 (2%) Intestine large, cecum (50) (49) (47) Autolysis 4 (8%) 1 (2%) Peyer's patch, hyperplasia 1 (2%) 2 (4%) Intestine small, duodenum (48) (50) (46) Angiectasis 1 (2%) Autolysis 2 (4%) 2 (4%) 2 (4%) Intestine small, jejunum (47) (50) (46) Autolysis 1 (2%) 2 (4%) 2 (4%) Peyer's patch, hyperplasia 2 (4%) Intestine small, ileum (48) (50) (46) Autolysis 2 (4%) 2 (4%) 2 (4%) Inflammation, acute 1 (2%) Peyer's patch, hyperplasia 4 (8%) 3 (6%) 1 (2%) Peyer's patch, inflammation, acute 1 (2%) Liver (50) (50) (50) Autolysis 2 (4%) 1 (2%) Basophilic focus 4 (8%) 5 (10%) Clear cell focus 10 (20%) 9 (18%) Eosinophilic focus 4 (8%) 2 (4%) Fatty change 2 (4%) 3 (6%) 15 (30%) Fibrosis 1 (2%) Hematopoietic cell proliferation 3 (6%) 6 (12%) 6 (12%) Hemorrhage 1 (2%) Inflammation, chronic active 27 (54%) 24 (48%) 23 (46%) Mixed cell focus 2 (4%) Necrosis, coagulative 9 (18%) 18 (36%) 16 (32%) Pigmentation 44 (88%) 49 (98%) Bile duct, hyperplasia 1 (2%) Mesentery (6) (9) (8) Fibrosis 4 (44%) Inflammation, chronic active 3 (50%) 5 (56%) 4 (50%) Mineralization 1 (17%) Necrosis, coagulative 1 (17%) 3 (33%) Pigmentation 1 (17%)284 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Alimentary System (continued) Pancreas (50) (50) (49) Atrophy 1 (2%) 2 (4%) Autolysis 2 (4%) Cytoplasmic alteration 3 (6%) 1 (2%) Fibrosis 1 (2%) Inflammation 1 (2%) Inflammation, chronic active 25 (50%) 24 (48%) 18 (37%) Vacuolization cytoplasmic 19 (38%) 13 (26%) 21 (43%) Duct, dilatation 1 (2%) 1 (2%) 1 (2%) Duct, hyperplasia 1 (2%) Salivary glands (49) (50) (47) Inflammation, chronic 1 (2%) Parotid gland, autolysis 1 (2%) Sublingual gland, infla mmation, chronic 1 (2%) 2 (4%) Submandibular gland, autolysis 1 (2%) Submandibular gland, inflammation, chronic 40 (82%) 32 (64%) 29 (62%) Stomach, forestomach (48) (50) (50) Acanthosis 9 (19%) 15 (30%) 19 (38%) Autolysis 1 (2%) Erosion 1 (2%) 1 (2%) Hyperkeratosis 10 (21%) 14 (28%) 17 (34%) Hyperplasia, basal cell 7 (14%) 3 (6%) Hyperplasia, squamous 2 (4%) Inflammation, chronic active 7 (15%) 10 (20%) 21 (42%) Ulcer 2 (4%) 1 (2%) 2 (4%) Ulcer, multiple 1 (2%) Stomach, glandular (49) (48) (48) Autolysis 2 (4%) 1 (2%) Inflammation, chronic 1 (2%) Inflammation, chronic active 20 (41%) 19 (40%) 21 (44%) Mineralization 1 (2%) 1 (2%) Tooth (1) Abscess 1 (100%) Dysplasia 1 (100%)

Cardiovascular System Heart (50) (50) (49) Autolysis 1 (2%) Inflammation, chronic 9 (18%) 6 (12%) 8 (16%) Mineralization 1 (2%) 1 (2%) Thrombosis 1 (2%)Lesions in Female Mice 285



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Endocrine System Adrenal cortex (50) (49) (48) Angiectasis 2 (4%) Autolysis 1 (2%) Cyst 1 (2%) Degeneration, fatty 1 (2%) Hematopoietic cell proliferation 1 (2%) 3 (6%) 1 (2%) Hyperplasia 1 (2%) 1 (2%) Inflammation, chronic active 2 (4%) 1 (2%) Vacuolization cytoplasmic 1 (2%) Adrenal medulla (49) (49) (48) Autolysis 1 (2%) Hematopoietic cell proliferation 1 (2%) 1 (2%) Hyperplasia 1 (2%) Islets, pancreatic (49) (50) (48) Hyperplasia 3 (6%) 1 (2%) 1 (2%) Parathyroid gland (33) (39) (33) Cyst 1 (3%) Pituitary gland (43) (45) (43) Autolysis 1 (2%) Pars distalis, angiectasis 1 (2%) Pars distalis, cyst 3 (7%) Pars distalis, hyperplasia 7 (16%) 22 (49%) 7 (16%) Pars distalis, pigmentation 1 (2%) Thyroid gland (50) (50) (48) Autolysis 1 (2%) Inflammation, chronic active 1 (2%) 2 (4%) 3 (6%) C-cell, hyperplasia 1 (2%) 1 (2%) 1 (2%) Follicle, cyst 2 (4%) 2 (4%) Follicular cell, hyperpla sia 3 (6%) 19 (38%) 18 (38%)

General Body System

None

Genital System Clitoral gland (1) Duct, dilatation 1 (100%) Ovary (49) (49) (47) Abscess 3 (6%) 2 (4%) Angiectasis 2 (4%) 4 (8%) 4 (9%) Cyst 12 (24%) 11 (22%) 7 (15%) Cyst, multiple 1 (2%) 3 (6%) 1 (2%) Hemorrhage 1 (2%) Inflammation, acute, necrotizing 1 (2%) Mineralization 2 (4%) Pigmentation 1 (2%)286 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Genital System (continued) Ovary (continued) (49) (49) (47) Periovarian tissue, cyst 1 (2%) 1 (2%) Periovarian tissue, cyst, multiple 1 (2%) Periovarian tissue, hemorrhage 1 (2%) Periovarian tissue, inflammation, chronic active 29 (59%) 24 (49%) 28 (60%) Periovarian tissue, mineralization 1 (2%) Periovarian tissue, pigmentation 1 (2%) Uterus (49) (50) (49) Angiectasis 1 (2%) Fibrosis 2 (4%) 1 (2%) Hydrometra 14 (29%) 3 (6%) Inflammation, acute 1 (2%) Inflammation, chronic active 4 (8%) 6 (12%) 7 (14%) Endometrium, hyperplasia 38 (78%) 36 (72%) 35 (71%) Epithelium, metaplasia, squamous 2 (4%) 1 (2%) 1 (2%)

Hematopoietic System Bone marrow (50) (49) (49) Myelofibrosis 5 (10%) Myeloid cell, sternal, hyperplasia 2 (4%) 1 (2%) 2 (4%) Sternal, autolysis 1 (2%) Sternal, myelofibrosis 38 (76%) 32 (65%) 34 (69%) Lymph node (7) (12) (12) Lumbar, hyperplasia, lymphoid 1 (14%) 1 (8%) Lumbar, inflammation, acute 1 (14%) Mediastinal, hyperplasia, lymphoid 1 (8%) Mediastinal, hyperplasia, plasma cell 1 (8%) Mediastinal, inflammati on, chronic active 1 (14%) Pancreatic, hyperplasia, lymphoid 1 (8%) 1 (8%) Pancreatic, infiltration cellular, histiocyte 1 (8%) Pancreatic, pigmentation 1 (8%) Renal, hyperplasia, plasma cell 2 (17%) Renal, sinus, ectasia 1 (8%) Lymph node, mandibular (32) (45) (31) Autolysis 1 (3%) Congestion 1 (3%) Depletion lymphoid 1 (2%) Hyperplasia, lymphoid 3 (9%) 1 (2%) 2 (6%) Hyperplasia, plasma cell 1 (3%) 1 (3%) Inflammation, acute 1 (2%) Pigmentation 2 (6%) 2 (4%) 3 (10%) Sinus, ectasia 2 (4%)Lesions in Female Mice 287



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Hematopoietic System (continued) Lymph node, mesenteric (49) (46) (44) Angiectasis 7 (14%) 4 (9%) 3 (7%) Depletion lymphoid 1 (2%) 1 (2%) Hemorrhage 2 (4%) Hyperplasia, lymphoid 2 (4%) 6 (13%) 10 (23%) Infiltration cellular, histiocyte 32 (65%) 24 (52%) 27 (61%) Inflammation, chronic active 1 (2%) 3 (7%) 2 (5%) Mineralization 1 (2%) Necrosis, coagulative 1 (2%) Pigmentation 31 (63%) 24 (52%) 27 (61%) Sinus, ectasia 1 (2%) Spleen (50) (50) (50) Autolysis 1 (2%) 1 (2%) Depletion lymphoid 4 (8%) 2 (4%) 2 (4%) Fibrosis 1 (2%) Hematopoietic cell proliferation 6 (12%) 13 (26%) 11 (22%) Hyperplasia, lymphoid 15 (30%) 14 (28%) 8 (16%) Inflammation, chronic 1 (2%) Thymus (40) (38) (34) Angiectasis 1 (3%) Cyst 14 (35%) 16 (42%) 22 (65%) Depletion lymphoid 4 (10%) 2 (5%) 6 (18%) Hemorrhage 1 (3%) 1 (3%) Hyperplasia, lymphoid 3 (8%) 1 (3%) 1 (3%)

Integumentary System Mammary gland (25) (25) (20) Hyperplasia 2 (8%) 9 (36%) 3 (15%) Inflammation, chronic 1 (4%) Skin (50) (50) (50) Acanthosis 1 (2%) Autolysis 1 (2%) Inflammation, acute 1 (2%) Epidermis, inflammation, acute 1 (2%) Subcutaneous tissue, cyst epithelial inclusion 1 (2%) Subcutaneous tissue, fibrosis 1 (2%) Subcutaneous tissue, inflammation, chronic active 1 (2%) 2 (4%) 2 (4%) Subcutaneous tissue, mineralization 1 (2%)

Musculoskeletal System

None

Nervous System Brain (49) (50) (50) Perivascular, inflammation, chronic 1 (2%) Thalamus, mineralization 21 (43%) 15 (30%) 21 (42%)288 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### D5 Summary of the Incidence of Nonneoplastic Lesion s in Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm



### 2-Year Study (continued) Respiratory System Lung (50) (50) (49) Autolysis 1 (2%) Congestion 1 (2%) Hemorrhage 4 (8%) 4 (8%) 8 (16%) Infiltration cellular, histiocyte 1 (2%) Inflammation, chronic active 3 (6%) 4 (8%) 6 (12%) Leukocytosis 1 (2%) 1 (2%) Alveolar epithelium, hyperplasia 1 (2%) Pleura, inflammation, acute 1 (2%) Nose (48) (45) (44) Inflammation, acute 21 (44%) 9 (20%) 7 (16%) Nasolacrimal duct, hyperplasia 1 (2%) Nasolacrimal duct, inflammation, acute 1 (2%) Trachea (50) (49) (47) Autolysis 1 (2%)

Special Senses System Eye (1) (1) Cornea, inflammation, chronic active 1 (100%) Cornea, neovascularization 1 (100%) Harderian gland (1) (3) (2) Inflammation, chronic 1 (100%) 2 (100%)

Urinary System Kidney (50) (50) (50) Autolysis 1 (2%) Glomerulosclerosis 2 (4%) 3 (6%) 4 (8%) Hydronephrosis 1 (2%) 1 (2%) Infarct 1 (2%) Inflammation, chronic 43 (86%) 42 (84%) 45 (90%) Metaplasia, osseous 3 (6%) 1 (2%) Papilla, mineralization 1 (2%) Pelvis, transitional epithelium, hyperplasia 1 (2%) Proximal convoluted renal tubule, degeneration, hyaline 1 (2%) Renal tubule, atrophy 1 (2%) 3 (6%) Renal tubule, casts protein 1 (2%) Renal tubule, mineralization 3 (6%) Renal tubule, pigmentation 43 (86%) 43 (86%) Renal tubule, regeneration 1 (2%) 7 (14%) 3 (6%) Transitional epithelium, mineralization 1 (2%) Autolysis 3 (6%) 1 (2%) 2 (4%) Inflammation, chronic active 42 (86%) 43 (86%) 46 (92%) Arteriole, necrosis, fibrinoid 1 (2%) Submucosa, proliferation 1 (2%)289



# APPENDIX E
SUMMARY OF LESIONS IN MALE RATS
IN THE STOP-EXPOSURE EVALUATION OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### E1 Summary of the Incidence of Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . 290 T ABLE 

### E2a Statistical Analysis of Primary Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation . . . . . . . . . . . . . 294 T ABLE 

### E2b Statistical Analysis of Primary Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation . . . . . . . . . . . . . . . . 297 T ABLE 

### E3 Summary of the Incidence of Nonne oplastic Lesions in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . 300290 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### E1 Summary of the Incidence of Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Disposition Summary Animals initially in study 70 10 30 9-Month interim evaluation 10 10 Early deaths Moribund 3 2 Natural death 1 Survivors 7 17

Animals examined microscopically 10 10 20



### 9-Month Interim Evaluation b Alimentary System Intestine large, colon (10) (10) Polyp adenomatous 1 (10%) Liver (10) (10) Hepatocellular carcinoma 2 (20%) Hepatocellular adenoma 2 (20%)

Endocrine System Adrenal medulla (10) (10) Thyroid gland (10) (10) Follicular cell, adenoma 1 (10%)

Nervous System Brain (10) (10) Cerebrum, meningioma benign 1 (10%)

Respiratory System Lung (10) (10) Alveolar/bronchiolar adenoma 1 (10%)

Urinary System Urinary bladder (10) (9) Papilloma 1 (11%)



### Systems Examined With No Neoplasms Observed Cardiovascular System General Body System Genital System Hematopoietic System Integumentary System Musculoskeletal System Special Senses SystemLesion s in Male Stop-Exp osure

Rats 291

T ABLE E1 Summary of t he Inci dence of Neoplasms in Male Rats i n the St op-Exposu re Evaluat ion of 1-Amino-2,4-dibromoanthraquinone (contin ued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 15-Month Evaluation c Alimentary System

Inte stine l arge , rect um (9) (10) (20)
Adenocarcinom

a 2 (10%) Poly p adeno matous 2 (20%) 6 (30%) Polyp adenomatous, multip le 1 (10%) 1 (5%)
Liver (10) (10) (20) Hepatocell

ular carcinoma 4 (40%) 6 (30%) Hepatocellular carcinoma, mu ltiple 3 (30%) 13 (65%) Hepatocell

ular adenoma 7 (70%) 3 (15%) Hepatocholangio carcinoma 1 (10%) 5 (25%)
Pancreas (10) (10)
Adenoma 1 (10%) Stomach, fore stomach (10) (10) (20) Squamous cell

papilloma 1 (5%)

Cardiovascu

lar System

Heart (10) (10) (20) Hepatoc holangi ocarci noma, m etasta tic, li ver 1 (10%)

Endocrine System

Pituitar y gl and (8) (10) (19) Pars distalis, adeno

ma 1 (13%) 1 (5%) Thyr oid gl and (10) (10)
Adenoma 1 (10%) C-cell, ade

noma 1 (10%)

General Body System

None

Genital System Epididy mis (20) Preput ial gl and (9) (19)
Carcinoma 1 (5%)
Testes (10) (10) (20)
Adenoma 1 (10%) Bilateral, inte

rstitial cell , adenoma 1 (10%)
Inters titial cell, adenoma 2 (20%) 3 (30%) 3 (15%)

Hematop oietic System Ly mph node (3) Mediastinal, hepatocel

lular carcinoma,

metast atic, liver 1 (33%)292 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### E1 Summary of the Incidence of Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Integumentary System

None

Musculoskeletal System

None

Nervous System

None

Respiratory System Lung (10) (10) (20) Alveolar/bronchiolar adenoma 1 (10%) Hepatocellular carcinoma, metastatic, liver 5 (50%) 7 (35%) Hepatocholangiocarcinoma, metastatic, liver 1 (10%)

Special Senses System

None

Urinary System Kidney (10) (10) (20) Hepatocellular carcinoma, metastatic, liver 1 (5%) Renal tubule, adenoma 3 (30%) 2 (10%) Urinary bladder (10) (10) (19) Transitional epithelium, papilloma 3 (16%) Squamous cell carcinoma 1 (5%) Transitional epithelium, carcinoma 1 (5%)

Systemic Lesions Multiple organs d (10) (10) (20) Mesothelioma malignant 1 (5%)Lesions in Male Stop-Exposure Rats 293



### T ABLE 

### E1 Summary of the Incidence of Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Neoplasm Summary Total animals with primary neoplasms e 7 10 20 Total primary neoplasms 8 25 50 Total animals with benign neoplasms 7 10 15 Total benign neoplasms 8 17 25 Total animals with malignant neoplasms 8 19 Total malignant neoplasms 8 25 Total animals with metastatic neoplasms 6 7 Total metastatic neoplasms 7 9

a Number of animals examined microscopically at site and number of animals with neopla sm (includes interim and moribund animals) b Controls from the 9-month interim evaluation of the 2-year core study we re used for comparison.

c Controls from the 15-month interim evaluation of the 2-year core study we re used for comparison. d Number of animals with any tissue examined microscopically

e Primary neoplasms: all neoplasms except metastatic neoplasms294 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### E2a Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

Kidney (Renal Tubule): Adenoma
Overall rate a 0/10 (0%) 3/10 (30%) 2/20 (10%) Adjusted rate b 0.0% 60.0% 18.2% Interim evaluation 0/10 (0%) 3/7 (43%) 2/17 (12%) First incidence (days)

) d 458 (I) 457 (I) Life table test c P=0.336 P=0.056 P=0.360 Logistic regression test c P=0.336 P=0.056 P=0.360 Cochran-Armitage test c P=0.385 Fisher exact test c P=0.105 P=0.437

Large Intestine (Rectum): Adenomatous Polyp Overall rate 0/10 (0%) 3/10 (30%) 7/20 (35%) Adjusted rate 0.0% 38.3% 66.3% Interim evaluation 0/10 (0%) 2/7 (29%) 7/17 (41%) First incidence (days)

) 414 456 (I) Life table test P=0.030 P=0.080 P=0.031 Logistic regression test P=0.034 P=0.232 P=0.031 Cochran-Armitage test P=0.037 Fisher exact test P=0.105 P=0.038

Large Intestine (Rectum): Carcinoma Overall rate 0/10 (0%) 0/10 2/20 (10%) Adjusted rate 0.0% 0.0% 29.2% Interim evaluation 0/10 (0%) 0/7 (0%) 1/17 (6%) First incidence (days)

) ) 427 Life table test P=0.229 P=0.369 Logistic regression test P=0.284 P=0.502 Cochran-Armitage test P=0.239 Fisher exact test P=0.437

Liver: Hepatocellular Adenoma Overall rate 0/10 (0%) 7/10 (70%) 8/20 (40%) Adjusted rate 0.0% 86.3% 69.6% Interim evaluation 0/10 (0%) 5/7 (71%) 7/17 (41%) First incidence (days)

) 414 423 Life table test P=0.038 P=0.002 P=0.023 Logistic regression test P=0.049 P=0.006 P=0.026 Cochran-Armitage test P=0.043 Fisher exact test P=0.002 P=0.022

Liver: Hepatocellular Carcinoma Overall rate 0/10 (0%) 7/10 (70%) 19/20 (95%) Adjusted rate 0.0% 77.8% 100.0% Interim evaluation 0/10 (0%) 5/7 (71%) 16/17 (94%) First incidence (days)

) 442 400 Life table test P<0.001 P=0.001 P<0.001

Logistic regression test P<0.001 P=0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P=0.002 P<0.001Lesions in Male Stop-Exposure Rats 295



### T ABLE 

### E2a Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation (continued)

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 0/10 (0%) 9/10 (90%) 20/20 (100%) Adjusted rate 0.0% 90.0% 100.0% Interim evaluation 0/10 (0%) 6/7 (86%) 17/17 (100%) First incidence (days)

) 414 400 Life table test P<0.001 P<0.001 P<0.001 Logistic regression test P<0.001 P=0.001 P<0.001 Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

Pituitary Gland (Pars Distalis): Adenoma Overall rate 1/8 (13%) 0/10 (0%) 1/19 (5%) Adjusted rate 10.0% 0.0% 10.0% Interim evaluation 1/8 (13%) 0/7 (0%) 1/16 (6%) First incidence (days) 456 (I)

) 457 (I) Life table test P=0.498N P=0.527N P=0.601N Logistic regression test P=0.498N P=0.527N P=0.601N Cochran-Armitage test P=0.462N Fisher exact test P=0.444N P=0.513N

Testes: Adenoma Overall rate 3/10 (30%) 4/10 (40%) 3/20 (15%) Adjusted rate 46.7% 65.0% 35.8% Interim evaluation 3/10 (30%) 3/7 (43%) 3/17 (18%) First incidence (days) 456 (I) 447 456 (I) Life table test P=0.328N P=0.300 P=0.397N Logistic regression test P=0.306N P=0.395 P=0.397N Cochran-Armitage test P=0.238N Fisher exact test P=0.500 P=0.306N

Urinary Bladder: Papilloma Overall rate 0/10 (0%) 0/10 (0%) 3/19 (16%) Adjusted rate 0.0% 0.0% 38.6% Interim evaluation 0/10 (0%) 0/7 (0%) 3/17 (18%) First incidence (days)

) ) 457 (I) Life table test P=0.126 P=0.223 Logistic regression P=0.126 P=0.223 Cochran-Armitage test P=0.128 Fisher exact test P=0.265

Urinary Bladder: Carcinoma Overall rate 0/10 (0%) 0/10 (0%) 1/19 (5%) Adjusted rate 0.0% 0.0% 25.0% Interim evaluation 0/10 (0%) 0/7 (0%) 1/17 (6%) First incidence (days)

) ) 458 (I) Life table test P=0.430 P=0.606 Logistic regression P=0.430 P=0.606 Cochran-Armitage test P=0.442 Fisher exact test P=0.655296 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### E2a Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation (continued)

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

All Organs: Benign Neoplasms Overall rate 7/10 (70%) 10/10 (100%) 15/20 (75%) Adjusted rate 73.3% 100.0% 93.4% Interim evaluation 7/10 (70%) 7/7 (100%) 14/17 (82%) First incidence (days) 456 (I) 414 423 Life table test P=0.289 P=0.024 P=0.296 Logistic regression test P=0.420 P=0.178 P=0.388 Cochran-Armitage test P=0.538 Fisher exact test P=0.105 P0.548

All Organs: Malignant Neoplasms Overall rate 0/10 (0%) 8/10 (80%) 19/20 (95%) Adjusted rate 0.0% 80.0% 100.0% Interim evaluation 0/10 (0%) 5/7 (71%) 16/17 (94%) First incidence (days)

) 414 400 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P=0.005 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001

All Organs: Benign or Malignant Neoplasms Overall rate 7/10 (70%) 10/10 (100%) 20/20 (100%) Adjusted rate 73.3% 100.0% 100.0% Interim evaluation 7/10 (70%) 7/7 (100%) 17/17 (100%) First incidence (days) 456 (I) 414 400 Life table test P=0.020 P=0.024 P=0.018 Logistic regression test P=0.015 P=0.178 P=0.042 Cochran-Armitage test P=0.008 Fisher exact test P=0.105 P=0.030

(I) Interim evaluation

a Number of lesion-bearing animals/number of animals examined (inc ludes interim and moribund animals). Denominator is number of animals examined microscopically for kidney, liver, pituitary gland, testes, and urinary bladder; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that expose d group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regressio n test regards these lesions as nonfatal. The Fisher exact test compares directly the overall incidence rates. For all tests, a lower incidence in an exposure group is indicated b y N

. d Not applicable; no neoplasms in animal groupLesions in Male Stop-Exposure Rats 297



### T ABLE 

### E2b Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Kidney (Renal Tubule): Adenoma
Overall rate a 3/10 (30%) 2/20 (10%) Adjusted rate b 60.0% 18.2% Interim evaluation 3/7 (43%) 2/17 (12%) First incidence (days) 458 (I) 457 (I) Life table test c P=0.130N Logistic regression test c P=0.130N Fisher exact test c P=0.191N

Large Intestine (Rectum): Adenomatous Polyp Overall rate 3/10 (30%) 7/20 (35%) Adjusted rate 38.3% 66.3% Interim evaluation 2/7 (29%) 7/17 (41%) First incidence (days) 414 456 (I) Life table test P=0.652N Logistic regression test P=0.561 Fisher exact test P=0.560

Large Intestine (Rectum): Carcinoma Overall rate 0/10 (0%) 2/20 (10%) Adjusted rate 0.0% 29.2% Interim evaluation 0/7 (0%) 1/17 (6%) First incidence (days)

) d 427 Life table test P=0.424 Logistic regression test P=0.400 Fisher exact test P=0.437

Liver: Hepatocellular Adenoma Overall rate 7/10 (70%) 8/20 (40%) Adjusted rate 86.3% 69.6% Interim evaluation 5/7 (71%) 7/17 (41%) First incidence (days) 414 423 Life table test P=0.078N Logistic regression test P=0.122N Fisher exact test P=0.123N

Liver: Hepatocellular Carcinoma Overall rate 7/10 (70%) 19/20 (95%) Adjusted rate 77.8% 100.0% Interim evaluation 5/7 (71%) 16/17 (94%) First incidence (days) 442 400 Life table test P=0.415 Logistic regression test P=0.091 Fisher exact test P=0.095298 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### E2b Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation (continued)

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 9/10 (90%) 20/20 (100%) Adjusted rate 90.0% 100.0% Interim evaluation 6/7 (86%) 17/17 (100%) First incidence (days) 414 400 Life table test P=0.566N Logistic regression test P=0.323 Fisher exact test P=0.333

Pituitary Gland (Pars Distalis): Adenoma Overall rate 0/10 (0%) 1/19 (5%) Adjusted rate 0.0% 10.0% Interim evaluation 0/7 (0%) 1/16 (6%) First incidence (days)

) 457 (I) Life table test P=0.665 Logistic regression test P=0.665 Fisher exact test P=0.655

Testes: Adenoma Overall rate 4/10 (40%) 3/20 (15%) Adjusted rate 65.0% 35.8% Interim evaluation 3/7 (43%) 3/17 (18%) First incidence (days) 447 456 (I) Life table test P=0.095N Logistic regression test P=0.120N Fisher exact test P=0.143N

Urinary Bladder: Papilloma Overall rate 0/10 (0%) 3/19 (16%) Adjusted rate 0.0% 38.6% Interim evaluation 0/7 (0%) 3/17 (18%) First incidence (days)

) 457 (I) Life table test P=0.309 Logistic regression test P=0.309 Fisher exact test P=0.265

Urinary Bladder: Carcinoma Overall rate 0/10 (0%) 1/19 (5%) Adjusted rate 0.0% 25.0% Interim evaluation 0/7 (0%) 1/17 (6%) First incidence (days)

) 458 (I) Life table test P=0.677 Logistic regression test P=0.677 Fisher exact test P=0.655Lesions in Male Stop-Exposure Rats 299



### T ABLE 

### E2b Statistical Analysis of Primar y Neoplasms in Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation (continued)

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

All Organs: Benign Neoplasms Overall rate 10/10 (100%) 15/20 (75%) Adjusted rate 100.0% 93.4% Interim evaluation 7/7 (100%) 14/17 (82%) First incidence (days) 414 423 Life table test P=0.045N Logistic regression test P=0.100N Fisher exact test P=0.109N

All Organs: Malignant Neoplasms Overall rate 8/10 (80%) 19/20 (95%) Adjusted rate 80.0% 100.0% Interim evaluation 5/7 (71%) 16/17 (94%) First incidence (days) 414 400 Life table test P=0.608 Logistic regression test P=0.203 Fisher exact test P=0.251

All Organs: Benign or Malignant Neoplasms Overall rate 10/10 (100%) 20/20 (100%) Adjusted rate 100.0% 100.0% Interim evaluation 7/7 (100%) 17/17 (100%) First incidence (days) 414 400 Life table test P=0.332N Logistic regression test

) e
Fisher exact test P=1.000

(I) Interim evaluation

a Number of lesion-bearing animals/number of animals examined. De nominator is number of animals examined microscopically for kid ney, liver, pituitary gland, testes, and urinary bladder; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between both exposed groups. The li fe table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regard s these lesions as nonfatal. The Fisher exact test compares directly the overall incidence rates. For all tests, a lower incidence in an exposure group is indicated by N

. d Not applicable; no neoplasms in animal group

e Value of the statistic cannot be computed.300 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone a

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)

Disposition Sum

mary Animal s initial

ly i n study 70 10 30
9-Month interim eval

uation 10 10 Early deaths
Moribund 3 2 Natural death 1 Survivors 7 17

Animals examined microscopi cally 10 10 20



### 9-Month Interim Evaluation b Alimentary System Intestine large, colon (10) (10)
Parasite metaz

oan 2 (20%)
Inte stine l arge , rect um (10) (10)
Inte stine l arge , cecum (9)
Parasite metaz

oan (10) 1 (11%)
Liver (10) (10) Basophil ic focu s 6 (60%)
Clear cell focus 4 (40%) Cytomegaly 2 (20%) Fatty change 3 (30%) Inflammation, chronic ac tive 1 (10%)
Mixed c ell

focus 1 (10%) Necrosis, coagulative 1 (10%) 2 (20%) Pigmentation 10 (100%)

Bile d uct, hy perpl asia 1 (10%) 7 (70%) Periportal, inflammation , chronic acti ve 9 (90%) 10 (100%)
Pancreas (10) (10)
Atro phy 2 (20%) Ectopic tissue 1 (10%) Infiltration cellular, mononuclear cell 3 (30%) 1 (10%) Infiltration cellular, mixed cell 1 (10%)
Inf lammat ion, chr onic 1 (10%) Inflammation, chronic ac tive 1 (10%) Necrosis, coagulative 2 (20%) Pigmentation 1 (10%) 1 (10%)
Acinus , atrop

hy 1 (10%)
Saliv ary g lands (10) (10) Subling ual gl and, atro phy 1 (10%) Sublingual gland, pigmentation 1 (10%)

Cardiovascu

lar System

Heart (10) (10)
Cardiom yopa thy 9 (90%) 6 (60%)Lesion s in Male Stop-Exp osure

Rats 301

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 9-Month Interim Evaluation (conti nued) Endocrine System

Adrenal co rtex (10) (10) Angie ctasis 1 (10%)
Adrenal medulla (10) (10) Hype rplas ia 1 (10%)
Pituitar y gl and (9) (10) Pars distalis, cy

st 1 (11%)
Pars di stalis , hype

rplas ia 3 (33%) 4 (40%) Thyr oid gl and (10) (10)

Genital System Preput ial gl and (9) (10)
Inf lammat ion, chr onic 2 (22%) 2 (20%) Inflammation, chronic ac tive 7 (78%) 8 (80%)
Pros tate (10) (10)
Inf lammat ion, acut e 1 (10%) 6 (60%) Inflammation, chronic ac tive 1 (10%)
Testes (10) (10)
Atro phy 1 (10%)
Infarct 1 (10%) Inflammation, chronic ac tive 1 (10%)
Inte rstitia l cel l, hy perpl asia 1 (10%) 2 (20%)

Hematop oietic System Ly mph node (2) (3)
Mediastina l, hemorrhage 1 (50%) 1 (33%)
Mediastina l, pigmentation 1 (50%) 1 (33%) Pancreatic, pi gmentation 1 (33%) Renal, hemorrhage 1 (50%) Renal, pigmentati on 1 (50%) Lymph no de, mandibular (10) (10) Hemorrhage 9 (90%) 1 (10%)
Inf iltrat ion cel lular , histio cyte 1 (10%) Ly mph node , mese

nteric (10) (10) Hemorrhage 1 (10%)
Inf iltrat ion cel lular , histio cyte 10 (100%) 10 (100%) Pigmentation 8 (80%) Spleen (10) (10) Thym us (9) (10) Depl etion l

ympho id 3 (33%) 8 (80%) Hemorrhage 1 (10%)

Integumen

tary System Mammary

gland (8) (5) Hype rplas ia 7 (88%) 5 (100%)302 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 9-Month Interim Evaluation (conti nued) Respiratory System Lung (10) (10)
Inf iltrat ion cel lular , histio cyte 1 (10%) Artery, mineralization 6 (60%) 4 (40%)
Nose (10) (10)
Glan ds, infl ammatio n, acute 1 (10%) Nasolacrimal duct, i nflammation, ch ronic active 1 (10%)

Special Senses System

Ear (1) Inflammation, chronic ac tive 1 (100%)
Ulcer 1 (100%)

Urinary System Kidney (10) (10)
Granuloma 1 (10%) Infiltration cellular, mononuclear cell 7 (70%) Infiltration cellular, mixed cell 1 (10%)
Inf lammat ion, chr onic 4 (40%)
Renal tubule , dege

nerati on, hy

aline 10 (100%) Renal tubule, pigmen tation 10 (100%) Renal tubule, regeneration 10 (100%) 10 (100%)
Urinar y bladder (10) (9) Calculus microscopi c observation only 2 (20%) 2 (22%) Serosa, mineralization 1 (10%)

Systems Exam ined With No Lesions Observed General Body System Musculoskeletal System Nervous System



### 15-Month Evaluation c Alimentary System Intestine large, colon (10) (10) (20)

Parasite metaz

oan 4 (40%) 1 (5%)
Inte stine l arge , rect um (9) (10) (20)
Parasite metaz

oan 1 (11%) 1 (10%)
Inte stine l arge , cecum (10) (10) (19) Inflammation, chronic ac tive 1 (5%)
Parasite metaz

oan 3 (30%) 3 (30%)
Inte stine s

mall , ile um (19) Inflammation, chronic ac tive 1 (5%)Lesion s in Male Stop-Exp osure

Rats 303

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 15-Month Evaluation (conti nued) Alimentary System

(conti nued)
Liver (10) (10) (20)
Aniso kary osis 1 (10%) Basophil ic focu s 1 (10%) 4 (40%) 13 (65%)
Clear cell focus 6 (60%) 13 (65%) Degeneration 2 (20%) 4 (20%)
Eosino philic f ocus 1 (10%) 2 (10%) Fatty change 6 (60%) 9 (90%) 10 (50%) Hemmorrhage 1 (5%) Hematopoietic cell proliferation 2 (20%) Hepatodiaph ragmatic nodu le 1 (10%)
Inf lammat ion, chr onic, g ranulo matous 2 (20%) Inflammation, chronic ac tive 6 (60%) 2 (10%) Necrosis, coagulative 6 (60%) 1 (10%) 2 (10%) Pigmentation 8 (80%) 18 (90%)
Bile d uct, hy perpl asia 10 (100%) 7 (70%) 19 (95%) Perip ortal , infla

mmatio n, chro nic 10 (100%) 7 (70%) 17 (85%) Periportal, inflammation , chronic acti ve 1 (5%) Mesentery (1) (1) Hemorrhage 1 (100%)
Inf lammat ion, chr onic 1 (100%) Inflammation, chronic ac tive 1 (100%) Necrosis, coagulative 1 (100%)
Thro mbosis 1 (100%)
Pancreas (10) (10) (20)
Atro phy 4 (40%) 2 (20%) 4 (20%) Hype rplas ia 1 (5%)
Inf lammat ion, chr onic 8 (80%) 5 (50%) 11 (55%) Inflammation, chronic ac tive 1 (5%)
Vacuol izatio n cyto plasmi c 1 (10%) Artery, inflammation, chronic a ctive 1 (5%)
Saliv ary g lands (19) Duct, parotid gland, mineralization 1 (5%) Stomach, fore stomach (10) (10) (20)
Acantho sis 2 (10%) Hype rkera tosis 2 (20%) 1 (5%) Hyperplasia, basal cell 2 (20%) 3 (15%) Hype rplas ia, squam

ous 2 (20%) Inflammation, chronic ac tive 1 (10%) 1 (5%)
Ulcer 2 (20%) Muscularis, mi neralization 1 (10%) Stomach, glandular (10) (10) (20)
Inf lammat ion, chr onic 1 (10%) 1 (10%) 1 (5%) Inflammation, chronic ac tive 1 (10%) Arteriole, mineralization 1 (5%) Muscularis, mi neralization 1 (10%) 1 (5%)304 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 15-Month Evaluation (conti nued)
Cardiovascu

lar System

Heart (10) (10) (20)
Cardiom yopa thy 10 (100%) 8 (80%) 17 (85%)

Endocrine System

Adrenal co rtex (10) (10) (20) Hype rplas ia 1 (10%) 1 (10%)
Bilate ral, v acuoli zatio n cyto plasmi c 1 (10%) 6 (30%)
Adrenal medulla (9) (10) (20)
Bilate ral, hy perpl asia 1 (11%) 1 (5%)
Isl ets, pa ncreat ic (4) (3) Hemorrhage 1 (25%)
Pituitar y gl and (8) (10) (19) Pars distalis, cy

st 1 (10%) 3 (16%)
Pars di stalis , hype

rplas ia 8 (100%) 6 (60%) 16 (84%)
Pars nervo sa, cyst 1 (5%)
Pars ne rvos a, hype

rplas ia 1 (5%) Thyr oid gl and (10) (10) (20) Ultimobranchial cy

st 1 (10%) 1 (5%)
C-cel l, hy perpl asia 1 (10%) 1 (10%) 1 (5%)

General Body System

None

Genital System Epididy mis (10) (10) (20)
Inf lammat ion, chr onic 1 (5%)
Vacuol izatio n cyto plasmi c 1 (10%) Preput ial gl and (9) (10) (19)
Inf lammat ion, chr onic 5 (56%) 2 (20%) 6 (32%) Inflammation, chronic ac tive 4 (44%) 7 (70%) 9 (47%)
Pros tate (10) (10) (20)
Inf lammat ion, acut e 1 (10%) 4 (40%) 5 (25%)
Inf lammat ion, chr onic 1 (5%) Inflammation, chronic ac tive 6 (60%) 4 (40%) 6 (30%) Epithel ium, hy perpl asia 1 (5%)
Testes (10) (10) (20)
Abscess 1 (10%)
Absce ss, chr

onic 1 (5%)
Inf lammat ion, chr onic 2 (10%)
Inte rstitia l cel l, hy perpl asia 10 (100%) 8 (80%) 17 (85%)
Semini fero

us tubule , atrop

hy 2 (20%) 3 (30%) 7 (35%) Seminiferous tubule, min eralization 2 (20%)Lesion s in Male Stop-Exp osure

Rats 305

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 15-Month Evaluation (conti nued) Hematop oietic System

Bone marrow (10) (10) (20) Myel ofibr osis 1 (10%) Ly mph node (2) (3)
Mediastina l, hemorrhage 1 (50%) 2 (67%) Pancreatic, hemorrha ge 1 (50%)
Pancre atic, inf

iltrat ion cel lular , histio cyte 1 (50%) 2 (67%) Pancreatic, pi gmentation 2 (67%) Lymph no de, mandibular (10) (8) (17) Hemorrhage 3 (30%) 1 (13%) 3 (18%) Hyperplasia, plasma cell 1 (6%)
Inf iltrat ion cel lular , histio cyte 1 (6%) Pigmentation 1 (6%) Ly mph node , mese

nteric (10) (9) (20) Depl etion l

ympho id 1 (10%) Hemorrhage 1 (10%) 1 (5%)
Inf iltrat ion cel lular , histio cyte 10 (100%) 9 (100%) 20 (100%) Pigmentation 9 (90%) 7 (78%) 15 (75%) Spleen (20) Depl etion l

ympho id 1 (5%) Thym us (10) (9) (16) Cyst 1 (10%) Depl etion l

ympho id 4 (44%) 12 (75%)

Integumen

tary System Mammary

gland (4) (7) (10) Hype rplas ia 2 (50%) 7 (100%) 9 (90%)
Skin (19)
Inf lammat ion, chr onic 1 (5%)

Musculoskeletal System

None

Nervous System

None

Respiratory System Lung (10) (10) (20)
Inf iltrat ion cel lular , histio cyte 2 (20%) 2 (20%) 7 (35%)
Alv eol ar epit helium , hype

rplas ia 3 (30%) 1 (5%) Alveolus, mineralization 1 (10%) 1 (5%) Artery, mineralization 8 (80%) 7 (70%) 9 (45%)306 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE E3 Summary of t he Inci dence of Nonneoplas tic Lesi ons i n M ale Rats in t he Stop- Exposure Evaluati on of 1-Ami no-2,4- dibromoant hraquinone (conti nued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)



### 15-Month Evaluation (conti nued) Respiratory System

(conti nued)

Nose (10) (10) (20)
Glan ds, infl ammatio n, acute 3 (30%) 3 (30%) 7 (35%) Glands, inflammation , chronic acti ve 1 (10%) 3 (15%)
Lume n, infl ammatio

n, acute 1 (10%) Nasolacrimal duct, i nflammation, ch ronic active 1 (10%) Respir atory epithe lium, m

etapl asia, sq

uamous 1 (5%)

Special Senses System Eye (1) Conjuncti va, inflammation, chronic acti ve 1 (100%)
Corne a, fibr

osis 1 (100%) Cornea, neovascularization 1 (100%)

Urinary System Kidney (10) (10) (20)
Autol ysis 1 (5%) Cyst 1 (10%) Nephr opathy 10 (100%) 10 (100%) 20 (100%)
Renal tubule , hype

rplas ia 1 (10%) 1 (5%) Renal tubule, inflamm ation, chroni c active 1 (5%) Renal tubule, pigmen tation 9 (90%) 20 (100%)
Trans itional

epithe lium, hy

perpl asia 1 (10%) 11 (55%)
Urinar y bladder (10) (10) (19)
Inf lammat ion, chr onic 1 (5%) Fat, proliferation 1 (5%) Muscularis, mi neralization 1 (5%)
Trans itional

epithe lium, hy

perpl asia 9 (47%)

a Number of animal s examined micro scopically at site and number of animals with le sion (includes inter im and moribund animals) b Contro ls fr

om the 9-mont h inter im eva luatio n of the 2-ye ar cor e study wer e use d for c ompari son.

c Controls fr

om the 15-month interim evaluation of

the 2-ye ar core study

were used for com

parison.307



# APPENDIX F
SUMMARY OF LESIONS IN FEMALE RATS
IN THE STOP-EXPOSURE EVALUATION OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### F1 Summary of the Incidence of Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 T ABLE 

### F2a Statistical Analysis of Prim ary Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 T ABLE 

### F2b Statistical Analysis of Prim ary Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . 317308 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### F1 Summary of the Incidence of Neoplasms in Fe male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Disposition Summary Animals initially in study 70 10 30 9-Month interim evaluation 10 10 Early deaths Moribund 1 5 Natural deaths 3 Survivors 9 11 Missexed 1

Animals examined microscopically 10 10 18 b



### 9-Month Interim Evaluation c Alimentary System Liver (10) (10) Hepatocellular carcinoma 1 (10%) Hepatocellular adenoma 2 (20%)

Endocrine System Pituitary gland (10) (10) Pars distalis, adenoma 1 (10%)

Urinary System Kidney (10) (10) Adenoma 1 (10%)



### Systems Examined With No Neoplasms Observed Cardiovascular System General Body System Genital System Hematopoietic System Integumentary System Musculoskeletal System Nervous System Respiratory System Special Senses System



### 15-Month Evaluation d Alimentary System Intestine large, rectum (10) (10) (17) Polyp adenomatous 5 (50%) 2 (12%) Polyp adenomatous, multiple 1 (6%)Lesions in Female Stop-Exposure Rats 309



### T ABLE 

### F1 Summary of the Incidence of Neoplasms in Fe male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Alimentary System (continued) Liver (10) (10) (18) Hepatocellular carcinoma 3 (30%) 7 (39%) Hepatocellular carcinoma, multiple 3 (30%) 8 (44%) Hepatocellular adenoma 5 (50%) 3 (17%) Hepatocellular adenoma, multiple 1 (10%) 7 (39%)

Cardiovascular System

None

Endocrine System Pituitary gland (10) (10) (18) Pars distalis, adenoma 2 (20%) 2 (20%) 1 (6%) Thyroid gland (10) (10) (18) Adenoma 1 (10%)

General Body System

None

Genital System Uterus (10) (10) (18) Polyp stromal 1 (10%) 1 (10%) 1 (6%)

Hematopoietic System

None

Integumentary System Mammary gland (7) (7) Fibroadenoma 1 (14%)

Musculoskeletal System

None

Nervous System

None

Respiratory System Lung (10) (10) (18) Hepatocellular carcinoma, metastatic, liver 1 (10%) 1 (6%)310 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### F1 Summary of the Incidence of Neoplasms in Fe male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Special Senses System

None

Urinary System Kidney (10) (10) (18) Renal tubule, adenoma 3 (30%) 1 (6%) Renal tubule, adenoma, multiple 1 (6%) Urinary bladder (10) (10) (18) Squamous cell, carcinoma 4 (22%) Squamous cell, papilloma 1 (6%) Transitional epithelium, carcinoma 1 (6%) Transitional epithelium, papilloma 1 (6%)

Neoplasm Summary Total animals with primary neoplasms e 2 9 17 Total primary neoplasms 4 25 39 Total animals with benign neoplasms 2 9 13 Total benign neoplasms 4 18 19 Total animals with malignant neoplasms 6 16 Total malignant neoplasms 7 20 Total animals with metastatic neoplasms 1 1 Total metastatic neoplasms 1 1

a Number of animals examined microscopically at the site and the number of animals with neoplasm (includes interim and moribund a nimals) b One animal not examined microscopically

c Controls from the 9-month interim evaluation of the 2-year core study were used for comparison. d Controls from the 15-month interim evaluation of the 2-year core study were used for comparison.

e Primary neoplasms: all neoplasms except metastatic neoplasmsLesions in Female Stop-Exposure Rats 311



### T ABLE 

### F2a Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

Kidney (Renal Tubule): Adenoma
Overall rate a 0/10 (0%) 3/10 (30%) 2/18 (11%) Adjusted rate b 0.0% 40.7% 24.2% Interim evaluation 0/10 (0%) 3/9 (33%) 2/11 (18%) First incidence (days)

) d 462 (I) 462 (I) Life table test c P=0.189 P=0.093 P=0.256 Logistic regression test c P=0.189 P=0.093 P=0.256 Cochran-Armitage test c P=0.345 Fisher exact test c P=0.105 P=0.405

Large Intestine (Rectum): Adenomatous Polyp Overall rate 0/10 (0%) 5/10 (50%) 3/18 (17%) Adjusted rate 0.0% 61.1% 20.5% Interim evaluation 0/10 (0%) 5/9 (56%) 1/11 (9%) First incidence (days)

) 462 (I) 306 Life table test P=0.136 P=0.015 P=0.186

Logistic regression test P=0.320 P=0.015 P=0.367

Cochran-Armitage test P=0.266 Fisher exact test P=0.016 P=0.249

Liver: Hepatocellular Adenoma Overall rate 0/10 (0%) 6/10 (60%) 10/18 (56%) Adjusted rate 0.0% 65.0% 100.0% Interim evaluation 0/10 (0%) 5/9 (56%) 9/11 (82%) First incidence (days)

) 456 306 Life table test P<0.001 P=0.009 P<0.001 Logistic regression test P=0.002 P=0.015 P=0.003

Cochran-Armitage test P=0.005 Fisher exact test P=0.005 P=0.003

Liver: Hepatocellular Carcinoma Overall rate 0/10 (0%) 6/10 (60%) 15/18 (83%) Adjusted rate 0.0% 66.7% 100.0% Interim evaluation 0/10 (0%) 6/9 (67%) 11/11 (100%) First incidence (days)

) 462 (I) 426 Life table test P<0.001 P=0.005 P<0.001

Logistic regression test P<0.001 P=0.005 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P=0.005 P<0.001

Liver: Hepatocellular Adenoma or Carcinoma Overall rate 0/10 (0%) 8/10 (80%) 16/18 (89%) Adjusted rate 0.0% 80.0% 100.0% Interim evaluation 0/10 (0%) 7/9 (78%) 11/11 (100%) First incidence (days)

) 456 306 Life table test P<0.001 P<0.001 P<0.001

Logistic regression test P<0.001 P=0.002 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P<0.001 P<0.001312 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### F2a Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation (continued)

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

Pituitary Gland (Pars Distalis): Adenoma Overall rate 2/10 (20%) 2/10 (20%) 1/18 (6%) Adjusted rate 20.0% 25.0% 8.3% Interim evaluation 2/10 (20%) 1/9 (11%) 0/11 (0%) First incidence (days) 462 (I) 456 461 Life table test P=0.387N P=0.670 P=0.456N Logistic regression test P=0.319N P=0.539N P=0.427N

Cochran-Armitage test P=0.222N Fisher exact test P=0.709N P=0.284N

Urinary Bladder: Papilloma Overall rate 0/10 (0%) 0/10 (0%) 2/18 (11%) Adjusted rate 0.0% 0.0% 22.2% Interim evaluation 0/10 (0%) 0/9 (0%) 1/11 (9%) First incidence (days)

) ) 426 Life table test P=0.174 P=0.295 Logistic regression test P=0.215 P=0.357 Cochran-Armitage test P=0.220 Fisher exact test P=0.405

Urinary Bladder: Carcinoma Overall rate 0/10 (0%) 1/10 (10%) 5/18 (28%) Adjusted rate 0.0% 11.1% 48.7%

Interim evaluation 0/10 (0%) 1/9 (11%) 4/11 (36%) First incidence (days)

) 462 (I) 299 Life table test P=0.022 P=0.479 P=0.044 Logistic regression test P=0.061 P=0.479 P=0.105 Cochran-Armitage test P=0.051 Fisher exact test P=0.500 P=0.087

All Organs: Benign Neoplasms Overall rate 2/10 (20%) 9/10 (90%) 13/18 (72%) Adjusted rate 20.0% 90.0% 100.0% Interim evaluation 2/10 (20%) 8/9 (89%) 10/11 (91%) First incidence (days) 462 (I) 456 306 Life table test P<0.001 P=0.004 P<0.001

Logistic regression test P=0.001 P=0.007 P=0.004 Cochran-Armitage test P=0.008 Fisher exact test P=0.003 P=0.011

All Organs: Malignant Neoplasms Overall rate 0/10 (0%) 6/10 (60%) 16/18 (89%) Adjusted rate 0.0% 66.7% 100.0% Interim evaluation 0/10 (0%) 6/9 (67%) 11/11 (100%) First incidence (days)

) 462 (I) 299 Life table test P<0.001 P=0.005 P<0.001

Logistic regression test P<0.001 P=0.005 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P=0.005 P<0.001Lesions in Female Stop-Exposure Rats 313



### T ABLE 

### F2a Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquino ne: 15-Month Interim Evaluation Control Group versus 9-Month and 15-Month 20,000 ppm Groups at the 15-Month Evaluation (continued)

0 ppm 20,000 ppm 20,000 ppm (15-month (9-month stop-exposure) (15-month exposure) interim evaluation)

All Organs: Benign or Malignant Neoplasms Overall rate 2/10 (20%) 9/10 (90%) 17/18 (94%) Adjusted rate 20.0% 90.0% 100.0% Interim evaluation 2/10 (20%) 8/9 (89%) 11/11 (100%) First incidence (days) 462 456 299 Life table test P<0.001 P=0.004 P<0.001 Logistic regression test P<0.001 P=0.007 P<0.001

Cochran-Armitage test P<0.001 Fisher exact test P=0.003 P<0.001

(I) Interim evaluation

a Number of lesion-bearing animals/number of animals examined (inc ludes interim and moribund animals). Denominator is number of animals examined microscopically for kidney, liver, pituitary gland, and urinary bladder; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the controls and that expose d group. The life table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regressio n test regards these lesions as nonfatal. The Fisher exact test compares directly the overall incidence rates. For all tests, a lower incidence in an exposure group is indicated b y N

. d Not applicable; no neoplasms in animal group314 1-Amino-2,4-Dibromoanthraquinone, NTP TR 383



### T ABLE 

### F2b Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Kidney (Renal Tubule): Adenoma
Overall rates a 3/10 (30%) 2/18 (11%) Adjusted rates b 40.7% 24.2% Interim evaluation 3/9 (33%) 2/11 (18%) First incidence (days) 462 (I) 462 (I) Life table tests c P=0.400N Logistic regression tests c P=0.400N Fisher exact test c P=0.228N

Large Intestine (Rectum): Adenomatous Polyp Overall rates 5/10 (50%) 3/18 (17%) Adjusted rates 61.1% 20.5% Interim evaluation 5/9 (56%) 1/11 (9%) First incidence (days) 462 (I) 306 Life table tests P=0.209N Logistic regression tests P=0.057N Fisher exact test P=0.077N

Liver: Hepatocellular Adenoma Overall rates 6/10 (60%) 10/18 (56%) Adjusted rates 65.0% 100.0% Interim evaluation 5/9 (56%) 9/11 (82%) First incidence (days) 456 306 Life table tests P=0.305 Logistic regression tests P=0.595 Fisher exact test P=0.570N

Liver: Hepatocellular Carcinoma Overall rates 6/10 (60%) 15/18 (83%) Adjusted rates 66.7% 100.0% Interim evaluation 6/9 (67%) 11/11 (100%) First incidence (days) 462 (I) 426 Life table tests P=0.014 Logistic regression tests P=0.010 Fisher exact test P=0.181

Liver: Hepatocellular Adenoma or Carcinoma Overall rates 8/10 (80%) 16/18 (89%) Adjusted rates 80.0% 100.0% Interim evaluation 7/9 (78%) 11/11 (100%) First incidence (days) 456 306 Life table tests P=0.063 Logistic regression tests P=0.126 Fisher exact test P=0.452Lesions in Female Stop-Exposure Rats 315



### T ABLE 

### F2b Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation (continued)

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Pituitary Gland (Pars Distalis): Adenoma Overall rates 2/10 (20%) 1/18 (6%) Adjusted rates 25.0% 8.3% Interim evaluation 1/9 (11%) 0/11 (0%) First incidence (days) 456 461 Life table tests P=0.423N Logistic regression tests P=0.385N Fisher exact test P=0.284N

Urinary Bladder: Papilloma Overall rates 0/10 (0%) 2/18 (11%) Adjusted rates 0.0% 22.2% Interim evaluation 0/9 (0%) 1/11 (9%) First incidence (days)

) d 426 Life table tests P=0.308 Logistic regression tests P=0.357 Fisher exact test P=0.405

Urinary Bladder: Carcinoma Overall rates 1/10 (10%) 5/18 (28%) Adjusted rates 11.1% 48.7% Interim evaluation 1/9 (11%) 4/11 (36%) First incidence (days) 462 (I) 299 Life table tests P=0.154 Logistic regression tests P=0.292 Fisher exact test P=0.277

All Organs: Benign Neoplasms Overall rates 9/10 (90%) 13/18 (72%) Adjusted rates 90.0% 100.0% Interim evaluation 8/9 (89%) 10/11 (91%) First incidence (days) 456 306 Life table tests P=0.412 Logistic regression tests P=0.539N Fisher exact test P=0.277N

All Organs: Malignant Neoplasms Overall rates 6/10 (60%) 16/18 (89%) Adjusted rates 66.7% 100.0% Interim evaluation 6/9 (67%) 11/11 (100%) First incidence (days) 462 (I) 299 Life table tests P=0.010 Logistic regression tests P=0.015 Fisher exact test P=0.098316 1-Amino-2,4-Dibromoanthraquinone, NTP TR 383



### T ABLE 

### F2b Statistical Analysis of Primar y Neoplasms in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone: 9-Month 20,000 ppm Stop-Exposure Group versus 15-Month 20,000 ppm Exposure Group at the 15-Month Evaluation (continued)

20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

All Organs: Benign or Malignant Neoplasms Overall rates 9/10 (90%) 17/18 (94%) Adjusted rates 90.0% 100.0% Interim evaluation 8/9 (89%) 11/11 (100%) First incidence (days) 456 299 Life table tests P=0.082 Logistic regression tests P=0.396 Fisher exact test P=0.595

(I) Interim evaluation

a Number of lesion-bearing animals/number of animals examined (inc ludes interim and moribund animals). Denominator is number of animals examined microscopically for kidney, liver, pituitary gland, and urinary bladder; for other tissues, denominator is number of animals necropsied. b Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality

c Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between both exposed groups. The li fe table analysis regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regard s these lesions as nonfatal. The Fisher exact test compares directly the overall incidence rates. For all tests, a lower incidence in an exposure group is indicated by N

. d Not applicable; no neoplasms in animal groupLesions in Female Stop-Exposure Rats 317



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)

Disposition Summary Animals initially in study 70 10 30 9-Month interim evaluation 10 10 Early deaths Moribund 1 5 Natural deaths 3 Survivors 9 11 Missexed 1

Animals examined microscopically 10 10 18 b



### 9-Month Interim Evaluation c Alimentary System Intestine large, colon (10) (10) Parasite metazoan 1 (10%) Intestine large, rectum (10) (10) Parasite metazoan 2 (20%) Intestine large, cecum (10) (10) Liver (10) (10) Angiectasis 2 (20%) Basophilic focus 1 (10%) 3 (30%) Clear cell focus 1 (10%) Fatty change 7 (70%) Inflammation, chronic active 2 (20%) Necrosis, coagulative 8 (80%) Pigmentation 10 (100%) Bile duct, hyperplasia 1 (10%) 9 (90%) Periportal, inflammation, chronic active 6 (60%) 10 (100%) Pancreas (10) (10) Atrophy 1 (10%) 2 (20%) Infiltration cellular, mononuclear cell 3 (30%) 4 (40%) Salivary glands (10) (10) Stomach, forestomach (10) (10) Muscularis, mineralization 1 (10%) Stomach, glandular (10) (10) Muscularis, mineralization 1 (10%) 1 (10%)

Cardiovascular System Heart (10) (10) Cardiomyopathy 7 (70%) 5 (50%)

Endocrine System Adrenal cortex (10) (10) Capsule, fibrosis 1 (10%) Capsule, inflammation, chronic 1 (10%)318 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383

T ABLE F3 Summary of the Incidence of Nonneoplastic Lesi ons in Female Rats in the Stop-Exposure Eval uation of 1-Amino-2,4-di bromoanthraquinone (continued)

0 ppm 20,00 0 ppm 20,00 0 ppm (9-month s

top-exposure) (15-month exposu re)

9-Month Interi m Evaluation (continued)
End ocr ine Sy ste m (continued)
Pituit ary gland (10) (10)

Pa rs d ista lis, a ng iecta sis 1 (1 0%) Pars distalis, cyst 2 (2 0%) 3 (3 0%)
Pars distal is, hemorrhage 1 (1 0%)

Pa rs d ista lis, h yp erp lasi a 1 (1 0%) Pars intermedia, cyst 1 (1 0%) 1 (1 0%) Thyroid gland (10) (10)
Inf iltrat ion cellu lar, m on on ucl ear cell 1 (1 0%)

Genital System

Clitora l gland (10) (9)
Inf iltrat ion cellu lar, m on on ucl ear cell 1 (1 0%)
Inf lam ma tion , ch ron ic 3 (3 0%) 1 (1 1%) Inflammation, chronic act ive 2 (2 0%) 2 (2 2%)
Ov ary (10) (10)
Periovari an tissue 1 (1 0%)
Uterus (10) (10) Hy drom etra 1 (1 0%) 1 (1 0%)

Hematop oietic System Lymph node (3) (5) Mediastinal, hemorrhage 3 (1 00%) Mediastinal, pigmentati on 3 (6 0%)

Pa ncr eatic , inf iltrat ion cellu lar, h istio cyt e 4 (8 0%)
Pancreati c, pigmentat ion 5 (1 00%) Lymp h node, m andibular (9) (10) Hemorrhage 7 (7 8%) 2 (2 0%) Hy per pla sia 1 (1 0%)
Inf iltrat ion cellu lar, h istio cyt e 1 (1 0%) Ly mp h n od e, m ese nte ric (10) (10) Hemorrhage 5 (5 0%)
Inf iltrat ion cellu lar, h istio cyt e 10 (1 00%) 10 (1 00%) Pigmentation 1 (1 0%) Spleen (10) (10) Congestion 1 (1 0%) Thymus (10) (9) Congestion 1 (1 0%) Hemorrhage 1 (1 0%) 2 (2 2%)

Respiratory System Lung (10) (10)
Inf iltrat ion cellu lar, h istio cyt e 1 (1 0%) 5 (5 0%) Artery, mineral izati on 4 (4 0%) 2 (2 0%)Lesions in Female Stop-Exposure Rats 319



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluaton of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 9-Month Interim Evaluation (continued) Special Senses System Eye (1) Anterior chamber, inflammation, acute 1 (100%) Posterior chamber, inflammation, acute 1 (100%) Retina, degeneration 1 (100%) Harderian gland (1) Hyperplasia 1 (100%)

Urinary System Kidney (10) (10) Fibrosis, focal 1 (10%) Hydronephrosis 1 (10%) Infiltration cellular, mononuclear cell 7 (70%) Inflammation, chronic 4 (40%) Mineralization 3 (30%) 1 (10%) Mineralization, focal 1 (10%) Renal tubule, pigmentation 10 (100%) Renal tubule, regeneration 4 (40%) 7 (70%) Transitional epithelium, mineralization 1 (10%) Urinary bladder (10) (10) Transitional epithelium, hyperplasia 4 (40%)



### Systems Examined With No Lesions Observed General Body System Integumentary System Musculoskeletal System Nervous System



### 15-Month Evaluation d Alimentary System Intestine large, colon (10) (10) (18) Parasite metazoan 2 (20%) 1 (10%) Intestine large, rectum (10) (10) (17) Parasite metazoan 1 (10%) Intestine large, cecum (10) (10) (18) Parasite metazoan 2 (20%) 2 (20%)320 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Alimentary System (continued) Liver (10) (10) (18) Basophilic focus 8 (80%) 6 (60%) 13 (72%) Clear cell focus 5 (50%) 13 (72%) Cyst 1 (10%) Cytomegaly 1 (10%) 2 (11%) Cytoplasmic alteration 1 (10%) Eosinophilic focus 2 (20%) 2 (20%) 3 (17%) Fatty change 10 (56%) Hematopoietic cell proliferation 1 (6%) Hepatodiaphragmatic nodule 2 (20%) Inflammation, chronic, granulomatous 6 (60%) 2 (20%) 1 (6%) Inflammation, chronic active 1 (10%) 1 (6%) Mixed cell focus 2 (20%) 3 (17%) Necrosis, coagulative 1 (10%) 4 (22%) Pigmentation 1 (10%) 10 (100%) 17 (94%) Bile duct, hyperplasia 7 (70%) 10 (100%) 18 (100%) Periportal, inflammation, chronic 10 (100%) 9 (90%) 11 (61%) Periportal, inflammation, chronic active 1 (10%) 7 (39%) Mesentery (1) Inflammation, chronic, granulomatous 1 (100%) Necrosis, coagulative 1 (100%) Pancreas (10) (10) (18) Atrophy 1 (10%) 3 (30%) 3 (17%) Ectopic tissue 1 (10%) 1 (6%) Inflammation, chronic 9 (90%) 6 (60%) 6 (33%) Inflammation, chronic, active 2 (11%) Salivary glands (10) (10) (18) Duct, submandibular gland, dilatation 1 (6%) Parotid gland, atrophy 1 (6%) Parotid gland, inflammation, chronic 2 (11%) Parotid gland, inflammation, chronic active 2 (11%) Submandibular gland, atrophy 1 (6%) Submandibular gland, inflammation, chronic 1 (6%) Submandibular gland, inflammation, chronic
active 5 (28%) Submandibular gland, metaplasia, squamous 2 (11%) Submandibular gland, pigmentation 1 (6%) Stomach, forestomach (10) (10) (18) Acanthosis 1 (6%) Erosion 1 (6%) Hyperkeratosis 1 (6%) Hyperplasia, basal cell 6 (33%) Hyperplasia, squamous 2 (11%) Ulcer 1 (6%) Stomach, glandular (10) (10) (18) Inflammation, chronic 1 (6%) Mineralization 1 (10%) Artery, mineralization 1 (6%) Muscularis, mineralization 1 (10%) 1 (6%)Lesions in Female Stop-Exposure Rats 321



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Cardiovascular System
Blood vessel (1) Aorta, inflammation, chronic 1 (100%) Heart (10) (10) (18) Cardiomyopathy 10 (100%) 10 (100%) 13 (72%)

Endocrine System Adrenal cortex (10) (10) (17) Angiectasis 5 (50%) 2 (20%) 3 (18%) Hyperplasia 1 (10%) Vacuolization, cytoplasmic 1 (6%) Adrenal medulla (10) (10) (17) Hyperplasia 1 (10%) 2 (12%) Parathyroid gland (10) (10) (15) Hyperplasia 1 (10%) Pituitary gland (10) (10) (18) Pars distalis, angiectasis 5 (50%) Pars distalis, cyst 8 (80%) 6 (60%) 8 (44%) Pars distalis, hyperplasia 1 (10%) 2 (11%) Pars intermedia, cyst 3 (30%) 1 (10%) 3 (17%) Thyroid gland (10) (10) C-cell, hyperplasia 4 (40%)

General Body System

None

Genital System Clitoral gland (10) (10) (18) Cyst 1 (6%) Inflammation, chronic 7 (70%) 6 (60%) 3 (17%) Inflammation, chronic active 1 (10%) 1 (10%) 8 (44%) Ovary (18) Periovarian tissue, cyst 3 (17%) Uterus (10) (10) (18) Hydrometra 2 (20%) 2 (20%) 5 (28%) Inflammation, chronic active 1 (6%) Endometrium, hyperplasia 1 (10%) 1 (10%) 1 (6%) Submucosa, hyperplasia 1 (6%)

Hematopoietic System Lymph node (1) (1) (4) Mediastinal, hemorrhage 1 (100%) 1 (100%) Mediastinal, pigmentation 1 (100%) Pancreatic, infiltration cellular, histiocyte 1 (100%) 4 (100%) Pancreatic, pigmentation 1 (100%) 2 (50%)322 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Hematopoietic System (continued) Lymph node, mandibular (7) (9) (17) Hemorrhage 1 (14%) 3 (33%) 1 (6%) Infiltration cellular, hystiocyte 6 (35%) Lymph node, mesenteric (10) (9) (18) Depletion lymphoid 1 (6%) Hemorrhage 2 (22%) Infiltration cellular, histiocyte 10 (100%) 9 (100%) 18 (100%) Pigmentation 10 (100%) 8 (89%) 11 (61%) Spleen (18) Fibrosis 1 (6%) Thymus (10) (10) (13) Cyst 1 (10%) Depletion lymphoid 1 (10%) 6 (46%) Hemorrhage 1 (10%)

Integumentary System Mammary gland (7) (7) (14) Hyperplasia 6 (86%) 4 (57%) 5 (36%)

Musculoskeletal System Bone (10) (10) Osteopetrosis 1 (10%)

Nervous System

None

Respiratory System Lung (10) (10) (18) Congestion 1 (10%) Hemorrhage 1 (10%) Infiltration cellular, histiocyte 6 (60%) 7 (70%) 12 (67%) Inflammation, chronic active 1 (6%) Alveolus, mineralization 1 (10%) Artery, mineralization 4 (40%) 5 (50%) 10 (56%) Nose (10) (10) (18) Inflammation, chronic active 2 (11%) Submucosa, inflammation, chronic 1 (10%)

Special Senses System

NoneLesions in Female Stop-Exposure Rats 323



### T ABLE 

### F3 Summary of the Incidence of Nonne oplastic Lesions in Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 20,000 ppm 20,000 ppm (9-month stop-exposure) (15-month exposure)



### 15-Month Evaluation (continued) Urinary System Kidney (10) (10) (18) Fibrosis 1 (6%) Inflammation, chronic active 1 (6%) Nephropathy 10 (100%) 10 (100%) 18 (100%) Papilla, necrosis, coagulative 2 (11%) Pelvis, inflammation, chronic active 1 (6%) Renal tubule, hyperplasia 2 (20%) 2 (11%) Renal tubule, inflammation, chronic active 1 (10%) Renal tubule, mineralization 1 (10%) Renal tubule, pigmentation 10 (100%) 18 (100%) Transitional epithelium, hyperplasia 3 (30%) 1 (10%) 5 (28%) Transitional epithelium, mineralization 2 (20%) 1 (10%) Urinary bladder (10) (10) (18) Hemorrhage 4 (22%) Necrosis 1 (6%) Fat, proliferation 2 (11%) Transitional epithelium, hyperplasia 4 (40%) 17 (94%) Transitional epithelium, metaplasia, squamous 3 (17%)

a Number of animals examined microscopically at site and number of animals with lesion (includes interim and moribund animals) b One animal not examined microscopically

c Controls from the 9-month interim evaluation of the 2-year core study were used for comparison. d Controls from the 15-month interim evaluation of the 2-year core study were used for comparison.324 1-Amino-2,4-dibromoanthraquinone, NTP TR 383325



# APPENDIX G
GENETIC TOXICOLOGY

S

ALMONELLA TYPHIMURIUM 

### M UTAGENICITY 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 C HINESE 

### H AMSTER 

### O VARY 

### C ELL 

### C YTOGENETICS 

### P ROTOCOLS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 R ESULTS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 T ABLE 

### G1 Mutagenicity of 1-Amino-2,4-dibromoanthraquinone in Salmonella typhimurium . . . . . . . 329 T ABLE 

### G2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 T ABLE 

### G3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332326 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



# GENETIC TOXICOLOGY

S ALMONELLA TYP

HIMURIUM 

# M UTAGENICITY 

# T EST 

# P ROTOCOL

Testin g was per formed as report ed by Hawort h et al. (1983). 1-Amino-2,4- dibromoanthraquinone was sent to the laboratory as a co ded aliquot from Radian Co rporation (A ustin, TX). It was incub ated with
the Salmon ell

a ty phimur ium tester str ains TA 98, TA 100, T A15 35, and TA 1537 e ither in bu ffer or S 9 mix (metabol ic act ivat ion en zymes a nd cofac tors f rom Aroclor 12 54-in duced mal e Sprag ue-Dawl ey rat or Syria n hamster

liv er) fo r 20 minu tes at 37° C. Top agar supple mented wi th l -his tidi ne and d -biotin was added, and the cont ents of the tube s were mixed and poured onto t he surfaces of mini mal glucose agar plate s. His tidi ne-i ndepend ent muta nt colo nies arisi ng on the se pla tes wer e counte d foll owing
incu bati on fo r 2 da

ys a t 37° C.

Each tri al cons iste d of tr ipli cate pl ates o f concur rent po siti ve and n egati

ve contr ols an d of fi

ve dose s of 1-amino-2,4-di

bromoanthraquinone. The high dose was limited t o 10,000 

### : g/plate. A ll positive t rials were repeated unde r the conditi ons that eli cited the posi tive response. If no positi ve responses were

seen, all negat ive t rial s were r epeat ed.

In this assay, a positive response is defined as a repro ducible, dose-related increase in histidineindepen dent (r evertan t) colo nies in any one str ain/activa tion co mbin ation. A n equiv ocal res pons e is
defi ned as a n incr ease i n rever tants that i s not dos e rel ated, not rep roduci ble, or is of ins uffi cient magnitu de to su pport a deter minati on of muta genic ity. A negati ve res ponse was obtai ned when no increase i n revertant col onies was observed f ollowing chemical

treatment. There was no minimum percentage or fold increase req uired for a chemical to the judg ed positive or weakly positive.



# C HINESE 

# H AMSTER 

# O VARY 

# C ELL 

# C YTOGENETICS 

# P ROTOCOLS Testing was perfor med as reported by Loveday et al. (1990). 1-Amino-2,4- dibromoanthraquinone was
sent to each of t

wo testing la

boratories as a coded aliquot by Radian Corporati on. The aliquots were tested in cultured Chinese ham ster ovary (CH O) cells for induction of sister ch romatid exchan ges (SCEs) and chromo somal aberrations (Abs), bo th in the presence and absen ce of Aroc lor 1254-induced male Spr ague- Dawley r at li ver S9 a nd cofac tor mix. Cultur es were handle d under g old li ghts to preven t photoly sis of

bromodeoxyu ridi ne-sub stit uted DNA. Each t est con sist ed of con current solve nt and positive controls and o f at least three doses of 1-amino-2,4-dibr omoanth raquinone. A single flask per
dose was used, and te sts yi eldi ng equi vocal o r posit ive r esult s were r epeat ed.

Sister Chro mati d Ex cha nge T est: In the SCE test without S9, CHO cells were incubated for 26 hours
with 1-amin o-2,4 -dibr omoanthra quinone in Mc Coy 's 5A medium supplemented with fetal bovine

ser um, l -glutamine, and antibioti cs. Bromodeoxyur idin e (Br dU) was added 2 hours after culture
init iati on. After 26 hours, the medium contai ning 1-amin o-2,4 -dibr omoanthra quinone was removed
and replaced with fresh medium plus BrdU and Colcemid

, and incuba tion was continued for 2 hours.
Cells were then harvested by mitot ic shake-off, fixe d, and stained with Hoechst 33258 and Giemsa.

In the SCE test with S9, cells were incubated with 1-amino-2,4-dibrom oanthraquino ne, serum-free
medi um, and S9 for 2 hours. The medium was then removed and replaced with medium contai ning

serum and BrdU and no 1-amin o-2,4 -dibr omoanthra quinone and incuba tion proceeded for an additional 26 hours, with Colce mid presen t for the final 2 hours. Harvesti ng and stai ning were the

same as for cells treated without S9. All slides were scored blind and those from a single test were read by the same person. Usually, 50 second- divi sion metaphase cells were scored for freque ncy of
SCE s/cell from each dose level. The high dose was limited by toxicity.Geneti c Toxicolo gy 327

Statistical analyse s were condu cted on the slopes of the dose-resp onse cu rves an d the individu al dose p oints (Galloway et al. , 1987). An SC E frequen cy 20% above the concurrent solvent control value w as chosen as a

stat ist ical ly c onserv ati ve posi tiv e resp onse. The probabi lit y of t his l evel of

dif fere nce oc curri ng by c hance a t one dose point is l ess t han 0. 01; t he proba bil ity for such a c hance o ccurr ence at two dose point s is less than

0.001. An incr ease of 20% or great er at any si ngle dose was c onsi dered weak evide nce of acti vit y; i ncrea ses at two or more doses result ed in a deter mination that the tr ial was posi tive. A statist icall y signi ficant

trend (P 

### # 0.005 ) in the abse nce of any respon ses reach ing 20% above backgro und led to a call of equ ivocal.



### Chr om oso mal Aber ratio ns Te st: In the A bs test witho ut S9, cells w ere incu bated in M cCoy 's 5A m edium with 1-amino-2,4-dibrom oanthraquinone for 8 to 10 hours; Co lcemid was add ed and incubation continued for 2
hours. The cells were then harv ested by mitotic shake-off, f ixed, and stained with Giem sa. For the Abs test with S9, c ells were t reated wit h 1-amino-2,4- dibromoanthraquinone and S9 f or 2 hours, af

ter which t he

treatm ent m ediu m w as re mo ved and th e cells incub ated for 10 to 11 hou rs in fre sh m ediu m, w ith C olcem id present f

or the f inal 2 hours. Cells were harvest ed in the s ame m anner as for the tr eatment without S9. The high dose was limit ed to 100 

### : g/mL.

Cells were selec ted for scoring on the basi s of good morphology and completeness of karyotype (2 1 ± 2 ch ro mos ome s)

. Al l s li de s w er e s co re d b li nd an d t ho se f ro m a s in gl e t es t wer e r ea d b y t he sa me person. Two hundred fi rst- divisi on metaphase cells were scored at each dose level . Classes of aberrati ons included si mple (breaks and terminal deleti ons), c omplex (rearrangements and t ranslocat ions), and other (pulverized cells, despiraliz ed chromo somes, and cells containing 10 or m ore aberrations).

Chromosomal aberr ati on dat a are pr esent ed as pe rcent age of cell s wit h aberr ati ons. To arri ve at a stat ist ical
call for a tri al, analyses were conducted on bo th the dose response curve and individual dose points. For a single trial , a statist ically sig nificant (P 

### # 0.05) difference for one dose point and a significant t rend (P 

### # 0.015)
are conside red weak evidence f or a posit ive response; signi ficant diff erences f

or two or more doses indi cated the trial was positive. A positive trend test in the absence of a statisti cally signif icant increase at any one dose
resulted i n an equivocal c all (Gall oway et al. , 1987). Ulti mately, the t rial call s were bas ed on a c onsi derat ion
of the statist ical analyses as well as the biological i nformation available to the reviewers

.



# R ESULTS

1 -Amino-2,4-di bromoanthraquinone (100 to 10,000 

### : g/plat e) was tested for induc tio n of gene mutations in

four strai ns of Sa lm on ella typ himurium in a prei ncubat ion proto col with and wit hout Aroclor 1254-induced

ma le Sprague-Dawley rat or Syri an hamster liver S9 (Tab le G1; Ha worth et al.

, 1983). 1-Amino2,4-dibr omoanthraquinone was posit ive in the absence of S9 in the fram eshift strai ns TA98 and TA1537;
with S9, an equivoc al response was obtained in TA1537, and TA98 was negative. In TA100, 1-amino2,4-dibr omoanthraquinone gave equivoc al responses with and wit hout S9, and all trials were negat ive in
TA1535. The equivoc al calls were the results of posit ive or wea kly posit ive responses that were not duplicat ed in a second trial. Preci pit ati on of 1-amino-2,4- dibromoanthraquinone occurred at

concentrat ions of 100 

### : g/plate and above, and this may have been a fact or in the nonrep rodu cibility of the
results.

1-Amino-2,4-dibr omoanthraquinone was tested in two laboratori es for induc tio n of SCEs and Abs in cultured

CHO cells, with and wit hout Aroclor 1254-induced ma le Sp rag ue- Dawl ey rat liver S9. In the SCE test, Environment al He alth Research & Testing observed a signi ficant increase in SCEs only in the absence of S9,
while Bioassa y Sy st ems Corporat ion recorded posit ive responses with and wit hout S9 (Tab le G2; Loveday et al.

, 1990). The discrepancy with S9 cannot be explained by a di fference i n dose levels employed at the two laboratori es because posit ive responses were seen at 3, 10, 15, and 30 

### : g/ mL at Bioassa y Sy st ems Corporation,
whereas negat ive trials resulted from testi n g doses u p t o 100 

### : g / m L a t328 1-Ami no-2,4-d ibrom oanth raqu inone , NTP TR 383



### Environmental Health Research & Testing. In the Abs test, Environmental Health Research &
Testin g observed a weakly positive response only in the absence of S9 (Tab le G3). Bioassay Sys tem s Corporat ion obtained a positive response in the first trial without S9 but did not duplicate the positive

response in the second trial (Tab le G3), and the overall call without S9 was concluded to be equivocal (Loveday et al.

, 1990). Neither laboratory observed an increase in Abs with 1-amin o2,4-d ibromoant hraqui none in the presen ce of S9.329 1-Am i no-2,4- d ibro m oant h raq u inon e , NTP TR 383

T ABLE G1

M utageni cit y of 1-Ami no-2,4- dibromoant hraquinone in Salmonell a typhi murium a

Revert ants/p late

b

Strain Dose

! S9 +10 ham ster S9 +10 rat S9

: g/plate Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 0 119 ± 4.2 170 ± 2.6 112 ± 6.7 138 ± 10.6 121 ± 7.8 156 ± 12.0

100 115 ± 5.0 146 ± 6.2 c 143 ± 11.8 157 ± 10.2 202 ± 8.0 182 ± 3.5
333 136 ± 7.5 c 140 ± 13.0 c 136 ± 2.9 c 218 ± 11.8 194 ± 3.8 c 184 ± 10.7 1,000 154 ± 6.4 c 177 ± 6.4 c 151 ± 8.5 c 209 ± 7.2 c 191 ± 4.4 c 181 ± 9.1 c 3,333 158 ± 6.2c 184 ± 8.8 c 190 ± 18.4 c 194 ± 12.9 c 185 ± 7.8 c 159 ± 5.4 c 10,000 218 ± 24.5c 233 ± 27.0 c 178 ± 12.5 c 215 ± 10.7 c 205 ± 14.0 c 184 ± 11.0 c

Trial sum mary Weakly Positive Equivocal Weakly Positive Equivocal Equivocal Negative
Positive cont rol d 420 ± 7.0 365 ± 4.7 1,027 ± 43.5 1,978 ± 31.5 612 ± 14.7 1,703 ± 202 .1

TA1535 0 29 ± 0.9 26 ± 6.4 11 ± 1.8 16 ± 0.6 13 ± 0.6 16 ± 0.9

100 22 ± 2.1 25 ± 3.8 c 9 ± 2.3 14 ± 2.7 11 ± 3.0 13 ± 2.7
333 27 ± 1.5 c 22 ± 3.1 c 10 ± 0.9 c 11 ± 0.9 13 ± 1.7 c 16 ± 3.0 1,000 28 ± 5.6 c 30 ± 7.6 c 13 ± 2.1 c 14 ± 0.7 c 8 ± 1.5 c 12 ± 1.9 c 3,333 25 ± 3.5 c 30 ± 3.5 c 11 ± 1.7 c 14 ± 3.0 c 10 ± 2.0 c 13 ± 2.4 c 10,000 26 ± 6.0 c 25 ± 5.0 c 10 ± 2.3 c 11 ± 0.9 c 7 ± 1.5 c 11 ± 0.9 c

Trial sum mary Negative Negative Negative Negative Negative Negative

Positive cont rol 554 ± 6.7 359 ± 12.5 377 ± 16.4 606 ± 23.6 346 ± 24.7 528 ± 24.8

TA1537 0 5 ± 0.7 5 ± 1.2 9 ± 1.8 16 ± 2.1 8 ± 1.0 12 ± 3.2

100 11 ± 1.2 9 ± 1.2 c 8 ± 0.9 13 ± 3.2 10 ± 1.2 12 ± 2.5
333 11 ± 0.6 c 8 ± 1.2 c 15 ± 2.0 c 13 ± 1.2 16 ± 2.7 c 19 ± 4.5 1,000 13 ± 1.8 c 8 ± 1.2 c 17 ± 2.7 c 12 ± 2.6 c 20 ± 1.7 c 18 ± 2.5 c 3,333 19 ± 5.3 c 17 ± 2.3 c 21 ± 2.9 c 21 ± 2.1 c 24 ± 3.8 c 19 ± 2.7 c 10,000 28 ± 0.7 c 32 ± 0.9 c 30 ± 3.0 c 27 ± 4.0 c 28 ± 0.3 c 30 ± 3.5 c

Trial sum mary Positive Positive Positive Negative Positive Equivocal
Positive cont rol 141 ± 3.2 147 ± 18.7 324 ± 8.8 367 ± 4.4 234 ± 14.0 308 ± 36.0

TA98 0 23 ± 4.9 19 ± 1.5 28 ± 2.0 29 ± 0.7 36 ± 3.3 36 ± 3.2

100 20 ± 3.2 18 ± 3.6 c 24 ± 3.2 30 ± 1.8 40 ± 1.5 44 ± 7.4
333 19 ± 2.1 c 29 ± 1.5 c 33 ± 8.3 c 21 ± 3.2 41 ± 3.5 c 36 ± 6.1 1,000 33 ± 1.7 c 24 ± 4.6 c 30 ± 4.6 c 33 ± 3.8 c 38 ± 4.2 c 46 ± 3.3 c 3,333 46 ± 6.0 c 37 ± 1.2 c 43 ± 4.4 c 47 ± 1.8 c 38 ± 4.4 c 50 ± 2.7 c 10,000 73 ± 10.6 c 86 ± 9.5 c 53 ± 3.8 c 46 ± 0.7 c 45 ± 2.9 c 41 ± 2.9 c

Trial sum mary Positive Positive Equivocal Negative Negative Negative
Positive cont rol 536 ± 43.2 591 ± 76.8 1,048 ± 40.5 1,503 ± 69.9 507 ± 21.0 1,080 ± 15.6

a The detail ed protocol and t hese data are presented i n Hawo rth et al . (19 83). b Revertants are presented as m ean ± the standard error from three plates.

c Pre cip itate on pla te d The positive c ontrols i n the absence of metabol ic act ivati on were sodium azide (TA1535 and TA 100), 9-aminoacri dine (TA1537), and 4-nitr oo -phenylenediam

ine (TA98 ). The positive control for metabolic activation with all strains was 2-am inoanthracene.Genet i c T o x i co l ogy 3 30



### T ABLE 

### G2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by 1-Amino-2,4-dibromoanthraquinone a

No. of SCEs/ Relative Dose Total Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/ Compound ( 

###  g/mL) Cells somes SCEs some Cell in BrdU Chromosome b (%)

Study Performed at Environmental Health Research & Testing



###  S9

Trial 1 Summary: Positive

Dimethylsulfoxide 50 1,049 435 0.41 8.7 26.0

Mitomycin-C 0.0008 50 1,039 591 0.56 11.8 26.0 37.17
0.0050 10 210 194 0.92 19.4 26.0 122.77

1-Amino-2,4-dibromoanthraquinone 1.6 50 1,043 516 0.49 10.3 26.0 19.30 5.0 50 1,041 526 0.50 10.5 26.0 21.85* 16.0 50 1,048 565 0.53 11.3 26.0 30.01*
50.0 14 c 290 171 0.58 12.2 26.0 42.19*

P<0.001 d

Trial 2 Summary: Weakly Positive

Dimethylsulfoxide 50 1,047 468 0.44 9.4 26.0

Mitomycin-C 0.0005 50 1,043 574 0.55 11.5 26.0 23.12
0.0050 10 210 273 1.30 27.3 26.0 190.83

1-Amino-2,4-dibromoanthraquinone 1.6 50 1,046 515 0.49 10.3 26.0 10.15 5.0 50 1,044 538 0.51 10.8 26.0 15.29 16.0 50 1,047 554 0.52 11.1 26.0 18.38 50.0 13 c 267 147 0.55 11.3 26.0 23.17*

P=0.001 +S9

Summary: Negative

Dimethylsulfoxide 50 1,051 432 0.41 8.6 26.0

Cyclophosphamide 0.1 50 1,046 546 0.52 10.9 26.0 26.99
0.6 10 210 193 0.91 19.3 26.0 123.59

1-Amino-2,4-dibromoanthraquinone 5.0 50 1,049 412 0.39 8.2 26.0

 4.45 16.0 50 1,050 465 0.44 9.3 26.0 7.74 50.0 50 1,040 489 0.47 9.8 26.0 14.39 100.0 50 1,050 480 0.45 9.6 26.0 11.22

P=0.003

* Significant positive response (P  0.01)

a A detailed description of the protocol and these data are presented by Loveday et al . (1990). SCE = sister chromatid exchange; BrdU = bromodeoxyuridine.33 1 1-A m i no-2 , 4-d i bro m oanthraq u i none, N T P T R 383



### T ABLE 

### G2 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary Cells by 1-Amino-2,4-dibromoanthraquinone (continued)

No. of SCEs/ Relative Dose Total Chromo- No. of Chromo- SCEs/ Hrs Change of SCEs/ Compound ( 

###  g/mL) Cells somes SCEs some Cell in BrdU Chromosome (%)

Study Performed at Bioassay Systems Corporation



###  S9

Summary: Weakly Positive

Dimethylsulfoxide 50 1,046 335 0.32 6.7 26.0

Mitomycin-C 0.002 50 1,041 567 0.54 11.3 26.0 70.07
0.010 10 208 243 1.16 24.3 26.0 264.78

1-Amino-2,4-dibromoanthraquinone 2.5 50 1,037 355 0.34 7.1 26.0 6.89 5.0 50 1,040 395 0.37 7.9 26.0 18.59 10.0 50 1,043 436 0.41 8.7 26.0 30.53*

P<0.001 +S9

Trial 1 Summary: Positive

Dimethylsulfoxide 50 1,047 420 0.40 8.4 26.0

Cyclophosphamide 0.5 50 1,038 642 0.61 12.8 26.0 54.18
2.5 10 206 291 1.41 29.1 26.0 252.15

1-Amino-2,4-dibromoanthraquinone 3.01 50 1,044 528 0.50 10.6 26.0 26.08* 10.10 50 1,047 581 0.55 11.6 26.0 38.33* 30.10 50 1,048 597 0.56 11.9 26.0 42.01*

P<0.001 Trial 2 Summary: Weakly Positive

Dimethylsulfoxide 50 1,032 402 0.38 8.0 26.0

Cyclophosphamide 0.5 50 1,044 593 0.56 11.9 26.0 45.82
2.5 10 207 248 1.19 24.8 26.0 207.57

1-Amino-2,4-dibromoanthraquinone 7.5 50 1,041 440 0.42 8.8 26.0 8.51 10.0 50 1,041 452 0.43 9.0 26.0 11.47 15.0 50 1,043 501 0.48 10.0 26.0 23.31*

P<0.001

b SCEs/chromosome in treated cells versus SCEs/chromosome in solvent control cells

c Decreased number of metaphases available for evaluation due to the cytostatic nature of 1-amino-2,4-dibromoanthraquinone d Significance of relative SCEs/chromosome tested by the linear regression trend test vs. log of the doseGenet i c T o x i co l ogy 33 2



### T ABLE 

### G3 Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by 1-Amino-2,4-dibromoanthraquinone a



###  S9 +S9 Dose Total No. of Abs/ Cells with Dose Total No. of Abs/ Cells with ( 

###  g/mL) Cells Abs Cell Abs (%) ( 

###  g/mL) Cells Abs Cell Abs (%)

Study Performed at Environmental Health Research & Testing

Trial 1

 Harvest Time: 12.0 hours Harvest Time: 13.0 hours Summary: Weakly Positive Summary: Negative

Dimethylsulfoxide Dimethylsulfoxide
200 3 0.02 1.5 200 4 0.02 2.0

Mitomycin-C Cyclophosphamide
0.0625 200 23 0.12 10.0 5.0 200 15 0.08 7.0 0.2500 50 18 0.36 32.0 7.5 50 19 0.38 36.0

1-Amino-2,4-dibromoanthraquinone 1-Amino-2,4-dibromoanthraquinone
5 200 7 0.04 3.5 16 200 3 0.02 0.5 16 200 9 0.05 4.0 50 200 2 0.01 1.0 50 200 12 0.06 5.5* 100 200 4 0.02 2.0

P=0.017 b P=0.467

Trial 2

 Harvest Time: 12.0 hours Summary: Weakly Positive

Dimethylsulfoxide
200 4 0.02 2.0

Mitomycin-C
0.0625 200 25 0.13 12.5 0.2500 50 21 0.42 36.0

1-Amino-2,4-dibromoanthraquinone
16 200 12 0.06 5.0 30 200 9 0.05 4.5 50 200 13 0.07 6.0

P=0.039333 1-Am i no-2,4- d ibro m oant h raq u inon e , NTP TR 383

T ABLE G3

Indu ction of C hro mos oma l Ab erra tions in C hine se H ams ter O var y C ells by 1-Amino- 2,4-di bromoanthraqui none (continued)

! S9 +S9
Dose Total No. of Abs/ Cells with Dose Total No. of Abs/ Cells with ( : g/mL) Cells Abs Cell Abs (%) ( : g/mL) Cells Abs Cell Abs (%)

Study Performed at Bioass ay Systems Corporation

Trial 1

) Har

v est Ti m e: 10.5 h ours Har v est Ti m e: 12.0 h ours Summ ary: Posit ive Summ ary: Negative

Dimethylsulfoxide Dimethylsulfoxide

200 0 0.00 0.0 200 4 0.02 2.0

Mito myc in-C Cyclophosphamide
1 200 51 0.26 20.5 50 50 25 0.50 34.0
5 50 47 0.94 56.0 1-Amino-2,4-dibromoanthraquinone 1-Amino-2,4-dibromoanthraquinone 3.02 200 9 0.05 4.0

3.02 200 7 0.04 3.0* 10.10 200 13 0.07 5.5
10.10 200 5 0.03 2.5* 30.20 200 7 0.04 3.5
30.20 200 4 0.02 1.5
P=0.153
P=0.164

Trial 2

) Harv

est Tim e: 10.0 h ours Summ ary: Negative

Dimethylsulfoxide
200 5 0.03 2.0

Mito myc in-C
1 200 171 0.86 40.5
5 50 41 0.82 56.0

1-Amino-2,4-dibromoanthraquinone
1 200 12 0.06 5.0
3 200 18 0.09 6.0
10 200 11 0.06 4.5
30 200 6 0.03 2.0

P=0.550

* Significan

t positive res

pons e (P< 0.05)

a The detail ed protocol and t hese data are presented i n Loveday et al . (1990). Ab s = aberrations. b Significance of percent cells with aberrations tested by the linear regression trend test vs. log of the dos eGenet i c T o x i co l ogy 33 4335



# APPENDIX H
ORGAN WEIGHTS
AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS

T ABLE 

### H1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . 336 T ABLE 

### H2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 9-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 T ABLE 

### H3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats at the 15-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 T ABLE 

### H4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . 340 T ABLE 

### H5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice at the 15-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342336 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### H1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,500 ppm 5,000 ppm 10,000 ppm 25,000 ppm 50,000 ppm

Male

n 10 10 9 9 10 7

Necropsy body wt 359 ± 4 327 ± 4** 332 ± 4** 305 ± 2** 232 ± 3** 161 ± 6**

Brain Absolute 1.924 ± 0.021 1.942 ± 0.014 1.945 ± 0.016 1.914 ± 0.019 1.798 ± 0.016** 1.708 ± 0.015** Relative 5.36 ± 0.08 5.95 ± 0.06** 5.86 ± 0.07** 6.26 ± 0.05** 7.77 ± 0.07** 10.69 ± 0.37** Heart Absolute 0.953 ± 0.019 0.964 ± 0.042 0.923 ± 0.026 0.895 ± 0.019 0.683 ± 0.008** b 0.579 ± 0.050** Relative 2.65 ± 0.05 2.95 ± 0.12 2.78 ± 0.08 2.90 ± 0.05 2.94 ± 0.06 b 3.59 ± 0.26** R. Kidney Absolute 1.102 ± 0.017 1.094 ± 0.023 1.121 ± 0.018 1.096 ± 0.015 c 0.892 ± 0.011** 0.708 ± 0.026** Relative 3.07 ± 0.04 3.35 ± 0.06** 3.37 ± 0.06** 3.58 ± 0.03** c 3.86 ± 0.04** 4.40 ± 0.09** Liver Absolute 12.513 ± 0.288 14.374 ± 0.381** 16.172 ± 0.309** 16.295 ± 0.370** 13.661 ± 0.206 12.333 ± 0.562 Relative 34.84 ± 0.71 43.96 ± 0.95** 48.64 ± 0.67** 52.67 ± 1.02** 59.03 ± 0.76** 76.58 ± 1.70** Lung Absolute 1.322 ± 0.042 1.338 ± 0.026 1.329 ± 0.047 1.259 ± 0.011 1.067 ± 0.038** 0.861 ± 0.014** Relative 3.68 ± 0.12 4.10 ± 0.07 4.00 ± 0.14 4.13 ± 0.05* 4.62 ± 0.18** 5.40 ± 0.22** R. Testis Absolute 1.468 ± 0.020 b 1.506 ± 0.020 1.508 ± 0.014 1.479 ± 0.020 1.390 ± 0.026* 1.082 ± 0.037** Relative 4.08 ± 0.08 b 4.61 ± 0.05** 4.54 ± 0.06** 4.82 ± 0.07** 6.01 ± 0.11** 6.73 ± 0.12** Thymus Absolute 0.235 ± 0.014 0.193 ± 0.009* 0.163 ± 0.018** 0.146 ± 0.008** 0.112 ± 0.010** b 0.065 ± 0.010** Relative 0.65 ± 0.04 0.59 ± 0.03 0.49 ± 0.05* 0.48 ± 0.03** 0.48 ± 0.04** b 0.42 ± 0.07**Organ Weight Analyses 337



### T ABLE 

### H1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,500 ppm 5,000 ppm 10,000 ppm 25,000 ppm 50,000 ppm

Female

n 10 10 10 10 10 9

Necropsy body wt 209 ± 3 195 ± 3** 188 ± 3** 184 ± 2** 157 ± 2** 129 ± 4**

Brain Absolute 1.809 ± 0.019 1.757 ± 0.014* 1.727 ± 0.016** b 1.710 ± 0.012** 1.683 ± 0.018** 1.598 ± 0.017** Relative 8.66 ± 0.13 9.02 ± 0.19 9.20 ± 0.10 b 9.32 ± 0.11* 10.71 ± 0.15** 12.49 ± 0.39** Heart Absolute 0.616 ± 0.010 0.605 ± 0.015 0.580 ± 0.016 0.557 ± 0.014** 0.473 ± 0.012** b 0.439 ± 0.016** Relative 2.94 ± 0.03 3.10 ± 0.09 3.09 ± 0.08 3.04 ± 0.07 3.03 ± 0.06 b 3.42 ± 0.11** R. Kidney Absolute 0.629 ± 0.013 0.684 ± 0.015 0.667 ± 0.012 0.656 ± 0.014 0.590 ± 0.007* 0.532 ± 0.011** Relative 3.01 ± 0.04 3.51 ± 0.07** 3.55 ± 0.04** 3.57 ± 0.05** 3.75 ± 0.06** 4.15 ± 0.15** Liver Absolute 6.561 ± 0.095 7.674 ± 0.202** 8.335 ± 0.213** 8.509 ± 0.159** 8.090 ± 0.158** 8.243 ± 0.236** Relative 31.39 ± 0.41 39.28 ± 0.77** 44.35 ± 1.06** 46.33 ± 0.60** 51.45 ± 0.77** 64.12 ± 1.45** Lung Absolute 1.001 ± 0.018 1.010 ± 0.043 b 0.937 ± 0.029 0.930 ± 0.020 0.855 ± 0.023** b 0.702 ± 0.022** Relative 4.79 ± 0.06 5.15 ± 0.17 b 4.98 ± 0.12 5.07 ± 0.11 5.47 ± 0.16** b 5.46 ± 0.12** Thymus Absolute 0.221 ± 0.007 0.179 ± 0.008** b 0.155 ± 0.006** 0.145 ± 0.008** 0.130 ± 0.016** b 0.062 ± 0.006** Relative 1.06 ± 0.03 0.91 ± 0.04 b 0.82 ± 0.03** 0.79 ± 0.04** 0.84 ± 0.11** b 0.48 ± 0.04**

* Significantly different (P # 0.05) from the control group by Williams' or Dunnett's test ** P # 0.01

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios ar e given as mg organ weight/g body weigh t (mean ± standard error) b n=9

c n=8338 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### H2 Organ Weights and Organ-Weight-to-Body-Weight Rati os for Rats at the 9-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm 20,000 ppm (15-month exposure)

Male

n 10 10 10 10 10

Necropsy body wt 435 ± 11 424 ± 9 398 ± 4** 388 ± 7** 372 ± 10**

R. Kidney Absolute 1.247 ± 0.040 1.362 ± 0.047 1.300 ± 0.022 1.259 ± 0.026 1.294 ± 0.044 Relative 2.87 ± 0.06 3.21 ± 0.07** 3.27 ± 0.06** 3.25 ± 0.04** 3.47 ± 0.05** Liver Absolute 13.934 ± 0.391 16.876 ± 0.506** 16.578 ± 0.441** 18.009 ± 0.594** 17.925 ± 0.901** Relative 32.04 ± 0.54 39.80 ± 0.72** 41.60 ± 0.85** 46.37 ± 0.80** 48.06 ± 1.92**

Female

n 10 10 10 10 10

Necropsy body wt 251 ± 7 236 ± 4* 229 ± 5** 218 ± 3** 208 ± 3**

R. Kidney Absolute 0.710 ± 0.014 b 0.786 ± 0.020* 0.770 ± 0.019 0.775 ± 0.016* 0.739 ± 0.016 Relative 2.90 ± 0.06 b 3.35 ± 0.09** 3.37 ± 0.07** 3.56 ± 0.06** 3.56 ± 0.09** Liver Absolute 7.378 ± 0.400 8.753 ± 0.240** 9.361 ± 0.253** 9.313 ± 0.124** 9.651 ± 0.241** Relative 29.17 ± 0.95 37.20 ± 0.88** 40.89 ± 0.74** 42.83 ± 0.43** 46.38 ± 0.94**

* Significantly different (P # 0.05) from the control group by Williams' or Dunnett's test ** P # 0.01

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios ar e given as mg organ weight/g body weigh t (mean ± standard error). b n=9Organ Weight Analyses 339



### T ABLE 

### H3 Organ Weights and Organ-Weight-to-Body-Weight Rati os for Rats at the 15-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm 20,000 ppm (15-month exposure) (9-month stop-exposure)

Male

n 10 10 10 7

Necropsy body wt 481 ± 6 394 ± 7** 361 ± 11** 374 ± 22**

R. Kidney Absolute 1.431 ± 0.032 1.472 ± 0.044 1.543 ± 0.041 1.319 ± 0.032 Relative 2.98 ± 0.07 3.73 ± 0.07** 4.34 ± 0.25** 3.61 ± 0.26** Liver Absolute 16.348 ± 0.522 20.110 ± 1.296 26.448 ± 2.340** 25.495 ± 5.628* Relative 34.00 ± 1.01 50.88 ± 2.84 75.40 ± 8.94** 74.37 ± 20.34**

Female

n 10 10 8 9

Necropsy body wt 318 ± 6 234 ± 11** 216 ± 4** 260 ± 9**

R. Kidney Absolute 0.941 ± 0.028 0.971 ± 0.035 0.994 ± 0.037 0.917 ± 0.035 Relative 2.96 ± 0.06 4.18 ± 0.08** 4.67 ± 0.17** 3.55 ± 0.13** Liver Absolute 9.394 ± 0.276 11.986 ± 0.644** 13.738 ± 0.358** 10.804 ± 0.679** Relative 29.59 ± 0.85 51.31 ± 1.29** 64.66 ± 2.34** 41.65 ± 2.43**

* Significantly different (P # 0.05) from the control group by Williams' or Dunnett's test ** P # 0.01

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios ar e given as mg organ weight/g body weigh t (mean ± standard error).340 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### H4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 2,500 ppm 5,000 ppm 10,000 ppm 25,000 ppm 50,000 ppm

Male

n 10 10 9 10 9 10

Necropsy body wt 30.0 ± 0.6 30.2 ± 0.5 30.0 ± 0.7 31.2 ± 0.5 30.4 ± 0.6 30.5 ± 0.4

Brain Absolute 0.454 ± 0.005 b 0.450 ± 0.006 0.452 ± 0.004 0.446 ± 0.007 0.446 ± 0.007 0.437 ± 0.017 b

Relative 15.26 ± 0.28 b 14.98 ± 0.38 15.12 ± 0.32 14.33 ± 0.28 14.68 ± 0.27 14.35 ± 0.56 b

Heart Absolute 0.146 ± 0.004 0.141 ± 0.005 0.148 ± 0.004 0.147 ± 0.004 0.139 ± 0.003 0.148 ± 0.004 Relative 4.88 ± 0.10 4.68 ± 0.10 4.92 ± 0.10 4.70 ± 0.10 4.57 ± 0.06 4.85 ± 0.10 R. Kidney Absolute 0.266 ± 0.005 0.257 ± 0.010 0.250 ± 0.004 0.251 ± 0.008 0.231 ± 0.006* 0.250 ± 0.008* Relative 8.90 ± 0.20 8.51 ± 0.28 8.35 ± 0.17 8.03 ± 0.20** 7.59 ± 0.17** 8.19 ± 0.24** Liver Absolute 1.593 ± 0.041 1.738 ± 0.039 1.765 ± 0.052* 2.011 ± 0.077** b 2.002 ± 0.069** 2.381 ± 0.061** Relative 53.11 ± 0.94 57.60 ± 0.65* 58.94 ± 1.59** 63.92 ± 1.94** b 65.73 ± 1.79** 78.05 ± 1.84** Lung Absolute 0.215 ± 0.006 0.201 ± 0.006 b 0.195 ± 0.005 0.196 ± 0.008 0.197 ± 0.004 0.205 ± 0.011 Relative 7.17 ± 0.10 6.71 ± 0.21 b 6.55 ± 0.25 6.26 ± 0.21 6.47 ± 0.09 6.75 ± 0.40 R. Testis Absolute 0.114 ± 0.002 0.113 ± 0.008 b 0.110 ± 0.002 0.110 ± 0.003 b 0.108 ± 0.002 c 0.111 ± 0.002 b

Relative 3.81 ± 0.11 3.74 ± 0.22 b 3.69 ± 0.08 3.51 ± 0.09 b 3.53 ± 0.04 c 3.66 ± 0.09 b
Thymus Absolute 0.031 ± 0.001 0.038 ± 0.003 d 0.031 ± 0.004 0.034 ± 0.003 0.033 ± 0.002 0.029 ± 0.002 Relative 1.05 ± 0.04 1.24 ± 0.07 d 1.05 ± 0.14 1.09 ± 0.07 1.09 ± 0.06 0.96 ± 0.06Organ Weight Analyses 341



### T ABLE 

### H4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 13-Week Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 2,500 ppm 5,000 ppm 10,000 ppm 25,000 ppm 50,000 ppm

Female

n 10 10 10 9 10 10

Necropsy body wt 23.8 ± 0.3 24.6 ± 0.6 24.0 ± 0.7 24.5 ± 0.4 23.1 ± 0.3 24.2 ± 0.4

Brain Absolute 0.461 ± 0.005 0.456 ± 0.006 0.452 ± 0.004 0.446 ± 0.005 0.450 ± 0.004 0.438 ± 0.006** Relative 19.37 ± 0.24 18.65 ± 0.36 19.01 ± 0.62 18.20 ± 0.31 19.50 ± 0.30 18.14 ± 0.31 Heart Absolute 0.109 ± 0.002 0.112 ± 0.002 0.111 ± 0.003 0.117 ± 0.003 0.110 ± 0.002 0.116 ± 0.003 Relative 4.56 ± 0.12 4.59 ± 0.07 4.63 ± 0.08 4.75 ± 0.12 4.78 ± 0.10 4.81 ± 0.11 R. Kidney Absolute 0.152 ± 0.004 0.166 ± 0.004* 0.156 ± 0.004 0.163 ± 0.003 0.158 ± 0.003 0.158 ± 0.003 Relative 6.36 ± 0.11 6.77 ± 0.13 6.51 ± 0.15 6.64 ± 0.15 6.84 ± 0.14* 6.55 ± 0.07 Liver Absolute 1.146 ± 0.022 1.453 ± 0.053** 1.529 ± 0.055** 1.666 ± 0.030** 1.613 ± 0.043** 1.904 ± 0.043** Relative 48.16 ± 1.27 59.10 ± 1.30** 63.92 ± 2.11** 67.94 ± 1.16** 69.83 ± 1.49** 78.72 ± 1.02** Lung Absolute 0.184 ± 0.008 0.175 ± 0.007 0.186 ± 0.006 0.185 ± 0.009 0.174 ± 0.006 0.187 ± 0.007 Relative 7.73 ± 0.35 7.15 ± 0.32 7.81 ± 0.31 7.52 ± 0.30 7.55 ± 0.25 7.77 ± 0.32 Thymus Absolute 0.036 ± 0.001 0.037 ± 0.002 0.038 ± 0.003 0.038 ± 0.002 0.029 ± 0.002 0.034 ± 0.003 Relative 1.51 ± 0.06 1.52 ± 0.08 1.60 ± 0.11 1.55 ± 0.08 1.25 ± 0.09 1.39 ± 0.12

* Significantly different (P # 0.05) from the control group by Williams' or Dunnett's test ** P # 0.01

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios ar e given as mg organ weight/g body weigh t (mean ± standard error). b n=9

c n=7 d n=8342 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### H5 Organ Weights and Organ-Weight-to-Body-Weight Rati os for Mice at the 15-Month Interim Evaluation in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone a

0 ppm 10,000 ppm 20,000 ppm

Male

n 10 9 10

Necropsy body wt 42.9 ± 1.2 39.9 ± 0.9 35.5 ± 1.1**

R. Kidney Absolute 0.334 ± 0.014 0.289 ± 0.011* 0.288 ± 0.011* Relative 7.81 ± 0.32 7.26 ± 0.23 8.18 ± 0.40 Liver Absolute 1.979 ± 0.146 2.022 ± 0.051 1.932 ± 0.092 Relative 46.69 ± 4.43 50.85 ± 1.59 54.45 ± 2.13

Female

n 10 10 10

Necropsy body wt 38.5 ± 1.5 34.5 ± 1.9 33.6 ± 0.9*

R. Kidney Absolute 0.215 ± 0.005 0.209 ± 0.005 0.208 ± 0.006 b

Relative 5.66 ± 0.25 6.21 ± 0.36 6.24 ± 0.23 b

Liver Absolute 1.373 ± 0.038 1.657 ± 0.042** 1.805 ± 0.043** Relative 35.96 ± 1.06 48.92 ± 2.20** 53.86 ± 1.13**

* Significantly different (P # 0.05) from the control group by Williams' or Dunnett's test ** P # 0.01

a Organ weights and body weights are given in grams; organ-weight-to-body-weight ratios ar e given as mg organ weight/g body weigh t (mean ± standard error). b n=9343



# APPENDIX I
CHEMICAL CHARACTERIZATION AND
DOSE FORMULATION STUDIES

P ROCUREMENT AND 

### C HARACTERIZATION OF 

### 1-A MINO 

### -2,4- DIBROMOANTHRAQUINONE 

### . . . . . . . . . . . . . . . . . 344 P REPARATION AND 

### A NALYSIS OF 

### D OSE 

### F ORMULATIONS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 F IGURE 

### I1 Infrared Absorption Spectrum of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . 346 F IGURE 

### I2 Nuclear Magnetic Resonance Spectrum of 1-Amino-2,4-dibromoanthraquinone . . . . . . . 347 T ABLE 

### I1 Preparation and Storage of Dose Formulations in the Feed Studies of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 T ABLE 

### I2 Results of Analysis of Dose Formulat ions Administered to Rats and Mice in the 13-Week Feed Studies of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . 349 T ABLE 

### I3 Results of Analysis of Dose Formul ations Administered to Rats and Mice in the 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . 350 T ABLE 

### I4 Results of Referee Analysis of Dose Formulations in the 13-Week and 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . 352344 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



# CHEMICAL CHARACTERIZATION AND
DOSE FORMULATION STUDIES

P ROCUREMENT AND 

# C HARACTERIZATION

OF 

# 1-A MINO 

# -2,4- DIBROMOANTHRAQUINONE 1-Amino-2,4-dibromoanthraquinone was obtained from Amer ican Color and Chemical Corporation (Charlotte, NC) (lot 1076-C) and Mobay Corporation (Pittsburgh, PA). Lot 1076-C was used in the 13-week studies and for 2 months of the 2-year studies. For tracking purposes, the lot from Mo bay Corporation was assigned the number M061583 and was used throughout the remainder of the 2-year studies. Identity, pu rity, and stability analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (MRI) (Kansas City, MO). MRI reports on analyses performed in support of the 1-amino-2,4-dibromoanthraquinone stu dies are on file at the National Institute of Environmental Health Sciences.

The two lots of the study chemical, a reddish brown to orange powder, were identified as 1- amino-2,4-dibromoanthraquinone by infrared, ultraviolet/visible, and nuclea r magnetic resonance (NMR) spectroscopy. All spectra were consistent with those expected for the structure or with the literatu re spectra, as shown in Figures I1 and I2.

The purity of both lots was determined by elemental analysis , Karl Fischer water analysis, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). TLC wa s performed with two systems: A) aluminum oxide F-254 Type E plates using a solvent system of hexan es:diethylether (50:50) and B) Whatman RP KC 18 

### F-254 plates using methanol as the solvent. Visualization was accomplished with visibl e light and shortwave (254 nm) ul traviolet light for both lots; longwave (366 nm) ultraviolet light was also used for visu alization of lot M061583. HPLC was performed with a Waters



### : Bondapak C 18 

### column with ultraviolet detection at 254 nm using a so lvent system of water: tetrahydrofuran (65:35 for lot 1076-C and 61:39 for lot M061583) and a flow rate of 1.0 mL/minute.

For lot 1076-C, elemental analyses for hydr ogen and nitrogen were in agreement with theoretical values; the elemental analysis for carbon was high and the an alysis for bromine was low. Karl Fisc her water analysis indicated 0.21% ± 0.05% water. TLC indicated a major spot and two slight trace impu rities by system A and a major spot and one trace impurity by system B. HPLC indicated a major peak and six impurities, with a total impurity area of approximately 20% relative to the major peak area.

The impurities in lot 1076-C were further ide ntified and quantified using HPLC and direct-inlet mass spectrometry. A filtered solution of 0.57 mg/mL 1-amino- 2,4-dibromoanthraquinone in N,N -dimethylformamide was analyzed by HPLC with a Waters



### : Bondapak C 18 

### column with a solvent system of water:acetonitrile ( 52:48) at a flow rate of 1.0 mL/minute. A major peak and eight impurities with areas of 0.1% or larger were detect ed by ultraviolet light (254 nm ). No additional impurities were observed when the ratio of the solvent was increased linearly from 48% to 100%. Three impurities had areas larger than 1% of the total peak area. Aliquots of 5.0 mg/mL 1-amino-2,4-dibromoanthraquinone in N,N -dimethylformamide were analyzed using the same HPLC system as was used in the impurity pr ofile analysis, but with a solv ent ratio of 53:47. The fractions were then evaporated to dryness, reconstituted in 5 mL n -hexane, and concentrated under purified nitrogen to a volume of
200 

### : L. The samples were then analyzed using direct-inlet mass spectrometry. The three impurities with areas greater than 1% of the major peak area were identified as anthraquinone, a monoamino-monobromoanthraquinone, and an isomer of the major component. Further analysis of the second and th ird of these impurities using proton Fourier transform NMR spectroscopy indicated that the structure of the second impu rity, a monoamino-monobromoanthr aquinone, was consistent with that of 1-amino-2-bromoanthraquinone, and that the isomer was probably 2-amino-1,3-dib romoanthraquinone. Quantitation of the impurity identified as anthraquinone using HPLC with a Dupont Zorbax ODS column using a solvent system of water: acetonitrile (30:70) and 0.3 mg/m L butyrophenone as an internal standard in dicated approximately 5.0% anthraquinone. Quantitation of the impurities identified as 1-amino-2-bromoant hraquinone and an isomer of the major component by the HPLC system described for the impurity profile analysis in dicated approximately 4.3% 1-amino-2-bromoanthraquinone and approximately 2.2% isomer. The overall purity of lot 1076-C was determined to be approximately 87%.

For lot M061583, elemental analyses for carb on, hydrogen, nitrogen, and bromine were in agreement with theoretical values. Karl Fischer water analysis indicated 0. 32% ± 0.04% water. TLC indicated a major spot and one trace and one slight trace impurity by system A and a major spot and one slight trace im purity by system B. HPLC indicated a major peak and six impurities with the same retention times as found for lot 1076- C and a total impurity area of 3% relative to the major peak area. Additionally, when the tetrahydrofuran in the solvent wa s increased to 60%, two impurity peaks with areas less than 1% of the peak area were observed. A concomitant analysis of lot 1076-C with lot M061583 w ith the same high-performance liquid chromatography system described for the purity analyses , but with a solvent ratio of 50:50 and with octanophenone as an internal standard, indicated a 1-am ino-2,4-dibromoanthraquinone concentra tion of approximately 112% in lot M061583 relative to lot 1076-C. The overall purity of l ot M061583 was determined to be approximately 97%.

Stability studies were performed using the same HPLC system described for the purity determination of lot 1076-C but with a solvent ratio of 55:45 and with octanophe n one added as an internal standard. The studies indicated that 1-amino-2,4-Chemical Characterization and Dose Formulations 345



### dibromoanthraquinone, when stored protected from light, was st able as a bulk chemical for 2 weeks at temperatures up to 60° C. To ensure stability, the bulk chemical was stored in th e dark at 4° ± 3° C throughout the studies. During the 2-year studies, the stability of the bulk chemical was monitored per iodically by the study laboratory using the same HPLC system; no degradation of 1-amino-2,4-dibromoanthraqu inone was seen throughout the studies.



# P REPARATION AND 

# A NALYSIS OF 

# D OSE 

# F ORMULATIONS The dose formulations were prepared by mixing appropriate qu antities of 1-amino-2,4-dibromoanthraquinone with feed (Table I1). Dosed feed formulations for chronic toxicity testing we re made by preparing a 1-amino-2,4-dibromoanthraquinone/feed premix by hand, which was then blended with plain feed in a Patterson-Kelly twin-shell blender for 15 minutes using an intensifier bar. Dose formulations were prepared weekly.

Homogeneity and stability analyses of the dosed feed preparation s were conducted by the analytical chemistry laboratory. For the homogeneity analyses, the formulations were extracted with 100 mL of acetonitrile, centrifu ged, and then further diluted with acetonitrile. The absorbance of the samples was determin ed versus acetonitrile using ultraviolet spectroscopy at 249 nm. For the stability studies, 2,000 ppm feed samples were extr acted with 100 mL of an acetonitrile hydrochloride solution (999:1) and centrifuged; the extracts were then mixed with 0.5 mg/mL octanophenone (internal standard) in aceto-nitrile and further diluted with acetonitrile. The samples we re injected into an HPLC system equipped with a 

### : Bondapak C 18 

### column and a 254 nm filtered detector, with a mobile phase of water: acetonitrile (57:43) at 1 mL/minute. Homogeneity of these formulations was confirmed; stability was established for at le ast 2 weeks when dose formulations were stored in the dark at temperatures up to 25° C.

Periodic analyses of the dose formulations of 1-amino-2,4-dibromoanthraquinone were conducted at the study laboratory and at the analytical chemistry laboratory us ing ultraviolet/visible spectroscopy at 460 nm. For the 13-week studies, dose formulations were analyzed at the beginning, midpoint, and end of the studies (Table I2). During the 2-year studies, the first set of dose formulations and one randomly selected prepara tion every 8 weeks were analyzed (Table I3). All dose formulations for rats and mice were within 10% of the target concentrations during the 13-week and 2-year studies. Results of periodic referee analyses performed by the analytical ch emistry laboratory indicated good agreement with the results obtained by the study laboratory (Table I4).346 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### F IGURE 

### I1 Infrared Absorption Spectrum of 1-Amino-2,4-dibromoanthraquinoneChemical Characterization and Dose Formulations 347



### F IGURE 

### I2 Nuclear Magnetic Resonance Spectrum of 1-Amino-2,4-dibromoanthraquinone348 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### I1 Preparation and Storage of Dose Formulations in th e Feed Studies of 1-Amino-2,4-dibromoanthraquinone

13-Week Studies 2-Year Studies

Preparation A premix with feed and 1-amino-2,4-dibromoanthraquinone was prepared with a mortar and pestle; premix and remainder of feed were layered into a blender with a inte nsifier bar and mixed for 15 minutes with the bar on. Doses were prepared weekly. Same as 13-week studies

Chemical Lot Number
1076-C 1076-C and M061583

Maximum Storage Time 14 days 14 days

Storage Conditions In double, clear plastic bags, in the dark, at 4° C In double, clear plastic bags, in the dark, at 4° ± 3° C

Study Laboratory EG&G Mason Research Institute (Worcester, MA) EG&G Mason Research Institute (Worcester, MA)

Referee Laboratory Midwest Research Institute (Kansas City, MO) Midwest Research Institute (Kansas City, MO)Chemical Characterization and Dose Formulations 349



### T ABLE 

### I2 Results of Analysis of Dose Formulations Administer ed to Rats and Mice in the 13-Week Feed Studies of 1-Amino-2,4-dibromoanthraquinone

Target Determined % Difference Date Prepared Date Analyzed Concentration Concentration a from Target (ppm) (ppm)

14 April 1982 16 April 1982 2,500 b 2,500 0 2,500 c 2,620 +5 2,500 d 2,400

! 4 5,000 4,780

! 4

20 April 1982 10,000 10,200 +2 25,000 24,800

! 1 50,000 b 50,100 0 50,000 c 49,500

! 1 50,000 d 48,600

! 3

9 June 1982 11 June 1982 2,500 2,500 0 5,000 4,690

! 6 10,000 9,700

! 3 25,000 25,000 0 50,000 50,400 +1

21 July 1982 22 July 1982 2,500 2,400

! 4 5,000 4,670

! 7 10,000 10,000 0 25,000 24,500

! 2 50,000 50,200 0

a Results of duplicate analyses b Sample selection from top left of twin-shell blender

c Sample selection from top right of twin-shell blender d Sample selection from bottom of twin-shell blender350 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### I3 Results of Analysis of Dose Formulations Administer ed to Rats and Mice in the 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone

Target Determined % Difference Date Prepared Date Analyzed Concentration Concentration a from Target (ppm) (ppm)

13 June 1983 14 June 1983 b 10,000 9,920

! 1 20,000 19,700

! 2

6 July 1983 7 July 1983 2,000 1,980

! 1 5,000 4,950

! 1 10,000 9,760

! 2 20,000 20,100 +1

15 August 1983 16 August 1983 2,000 1,990

! 1 5,000 5,000 0 10,000 9,900

! 1 20,000 19,600

! 2

17 October 1983 19 October 1983 2,000 1,910

! 5 5,000 4,920

! 2 10,000 9,800

! 2 20,000 20,000 0

19 December 1983 21 December 1983 2,000 1,980

! 1 5,000 4,870

! 3 10,000 9,800

! 2 20,000 20,700 +4

21 February 1984 22 February 1984 2,000 1,960

! 2 5,000 4,780

! 4 10,000 10,100 +1 20,000 19,900

! 1

9 April 1984 10 April 1984 2,000 1,900

! 5 5,000 4,980 0 10,000 9,800

! 2 20,000 19,400

! 3

29 May 1984 30 May 1984 2,000 1,970

! 2 5,000 4,870

! 3 10,000 10,100 +1 20,000 20,000 0

9 July 1984 10 July 1984 2,000 1,880

! 6 5,000 4,930

! 1 10,000 10,000 0 20,000 20,100 +1

17 September 1984 18 September 1984 2,000 1,910

! 5 5,000 5,120 +2 10,000 10,100 +1 20,000 19,800

! 1Chemical Characterization and Dose Formulations 351



### T ABLE 

### I3 Results of Analysis of Dose Formulations Administer ed to Rats and Mice in the 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone (continued)

Target Determined % Difference Date Prepared Date Analyzed Concen tration Concentration from Target (ppm) (ppm)

19 November 1984 20 November 1984 2,000 1,910

! 5 5,000 4,900

! 2 10,000 10,200 +2 20,000 19,900

! 1

21 January 1985 23 January 1985 2,000 1,930

! 4 5,000 4,970

! 1 10,000 10,200 +2 20,000 20,100 +1

1 April 1985 2 April 1985 2,000 1,920

! 4 5,000 5,150 +3 10,000 10,200 +2 20,000 20,200 +1

3 June 1985 4 June 1985 2,000 1,970

! 2 5,000 4,830

! 3 10,000 10,100 +1 20,000 19,500

! 3

a Results of duplicate analyses b Doses mixed on this date were administered to mice only.352 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### I4 Results of Referee Analysis of Dose Formulat ions in the 13-Week and 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone

Determined Concentration (ppm)
Target Concentration Study Referee Date Prepared (ppm) Laboratory a Laboratory b

13-Week Studies

14 April 1982 5,000 4,780 5,050 ± 130

2-Year Studies

13 June 1983 10,000 9,920 10,500 ± 100 19 December 1983 2,000 1,980 1,960 ± 20 9 April 1984 20,000 19,400 19,600 ± 100 17 September 1984 5,000 5,120 5,250 ± 110 1 April 1985 2,000 1,920 2,030 ± 40

a Results of duplicate analyses b Results of triplicate analyses (mean ± standard error)353



# APPENDIX J
FEED AND COMPOUND CONSUMPTION
IN THE 2-YEAR FEED STUDIES OF 1-AMINO-2,4-DIBROMOANTHRAQUINONE

T ABLE 

### J1 Feed and Compound Consumption by Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
T ABLE 

### J2 Feed and Compound Consumption by Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
T ABLE 

### J3 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
T ABLE 

### J4 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
T ABLE 

### J5 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
T ABLE 

### J6 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359354 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### J1 Feed and Compound Consumption by Male Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone

20,000 ppm (9-month stop-exposure) 20,000 ppm (15-month exposure) Feed Body Dose/ Feed Body Dose/ (g/day) a Weight Day b (g/day) Weight Day Week (g) (mg/kg) (g) (mg/kg)

1 11.2 130 1,718 10.9 136 1,605 2 15.1 144 2,096 15.9 149 2,136 4 22.8 169 2,700 20.0 173 2,303 5 17.7 169 2,095 17.0 177 1,922 8 20.3 211 1,924 20.8 217 1,919 12 20.2 267 1,511 18.5 277 1,336 13 17.8 267 1,328 17.7 271 1,308 14 19.6 284 1,381 18.9 292 1,292 17 16.9 305 1,110 17.8 318 1,117 21 17.9 327 1,092 18.9 334 1,133 25 14.1 341 827 14.5 351 829 29 15.8 338 932 15.9 355 898 33 16.8 351 957 15.4 364 844 37 18.0 352 1,019 18.9 364 1,038 41 c 17

. 6 374 0 15

. 6 377 825 45 16.0 384 0 15.2 381 799 49 17.3 395 0 15.4 383 805 53 15.5 398 0 16.3 389 840 57 15.3 393 0 15.7 377 833 61 14

. 9 379 0 15

. 1 370 817 65 15.3 364 841

Mean for weeks 1-13 17.9 194 1,910 17.3 200 1,790 14-40 17.0 328 1,045 17.1 340 1,022 41-61 or 65 16.1 387 0 15.5 377 823

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per day

c 9-Month stop-exposure animal s switched to undosed feedFeed and Compound Consumption 355



### T ABLE 

### J2 Feed and Compound Consumption by Female Rats in the Stop-Exposure Evaluation of 1-Amino-2,4-dibromoanthraquinone

20,000 ppm (9-month stop-exposure) 20,000 ppm (15-month exposure) Feed Body Dose/ Feed Body Dose/ (g/day) a Weight Day b (g/day) Weight Day Week (g) (mg/kg) (g) (mg/kg)

1 8.1 90 1,810 7.3 94 1,543 2 10.6 93 2,259 10.0 97 2,079 4 14.6 113 2,591 14.6 114 2,563 5 13.2 117 2,240 13.8 121 2,296 8 14.0 141 1,982 14.7 150 1,955 9 12.9 146 1,764 11.6 157 1,474 12 15.1 168 1,794 12.8 171 1,497 13 14.6 172 1,688 14.2 175 1,628 14 15.0 177 1,694 15.8 179 1,764 17 18.8 188 2,000 16.8 190 1,770 21 17.1 194 1,771 16.4 196 1,666 25 14.5 198 1,462 12.9 200 1,290 29 16.5 200 1,653 14.9 203 1,469 33 21.0 201 2,083 21.0 203 2,071 37 10.9 207 1,052 11.0 211 1,048 41 c 9

. 7 207 0 10

. 9 210 1

, 040 49 12.1 231 0 10.2 213 959 53 11.0 248 0 11.6 219 1,062 57 11.9 252 0 11.2 220 1,022 61 11.1 258 0 10.3 217 953 65 10.9 220 992

Mean for weeks 1-13 12.9 130 2,016 12.4 135 1,879 14-40 16.3 195 1,673 15.5 197 1,582 41-61 or 65 11.2 239 0 10.9 217 1,005

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per day

c 9-Month stop-exposure animal s switched to undosed feed356 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### J3 Feed and Compound Consumption by Mal e Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm Feed Body Feed Body Dose/ Feed Body Dose/ Feed Body Dose/ (g/day) a Weight (g/day) Weight Day b (g/day) Weight Day (g/day) Weight Day Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg)

1 20.1 139 16.2 136 238 15.0 136 549 12.6 134 937 2 17.2 161 16.6 165 202 16.5 164 504 14.9 155 961 4 21.4 235 20.3 225 180 20.7 222 466 19.3 204 943 5 17.5 240 16.9 233 145 17.0 230 369 15.9 215 742 8 22.5 302 20.7 285 145 21.3 280 380 20.0 260 769 12 18.5 338 18.8 323 116 20.2 318 317 16.9 300 563 13 17.9 332 17.8 321 111 17.8 309 288 17.6 298 589 14 22.4 356 19.9 339 117 18.5 330 281 17.7 314 561 17 22.9 387 20.1 363 111 19.3 350 275 19.7 335 589 21 20.9 406 20.8 382 109 18.7 368 254 17.3 356 486 25 19.0 423 18.0 398 91 15.8 383 206 15.8 369 427 29 20.8 435 18.5 403 92 17.9 384 232 19.5 376 520 33 19.9 445 17.6 413 85 16.8 398 210 16.9 383 441 37 21.8 453 21.6 420 103 21.9 401 273 19.9 388 514 41 17.1 468 16.7 433 77 16.2 415 196 15.5 397 392 45 16.2 473 16.0 440 73 16.1 424 190 15.7 403 389 49 15.5 479 17.4 452 77 16.7 428 195 15.6 409 382 53 16.7 489 17.8 457 78 17.7 439 201 16.6 415 399 57 17.4 486 16.9 453 75 16.8 430 195 15.9 409 389 61 16.3 484 15.3 445 69 15.4 430 179 14.3 405 352 65 15.8 484 16.8 448 75 15.5 426 182 16.5 408 405 69 15.6 479 15.1 442 68 15.7 417 188 14.6 398 368 72 16.0 472 15.1 429 70 14.8 407 182 15.8 389 407 77 15.1 460 13.8 412 67 14.3 392 183 15.2 381 400 80 15.3 462 14.9 418 71 14.5 390 187 14.7 373 393 85 14.3 467 14.8 419 71 14.2 388 183 13.9 365 382 89 13.8 455 13.8 405 68 14.7 378 194 14.8 357 414 93 14.4 445 14.9 396 75 14.6 363 202 16.0 347 462 97 12.9 432 15.7 380 82 16.4 355 231 16.9 332 508 101 14.5 422 16.2 380 85 15.6 343 227 17.0 301 565 103 14.7 406 14.7 349 84 16.6 341 244 18.6 283 656

Mean for weeks
1-13 19.3 249 18.2 241 163 18.4 237 411 16.7 224 786 14-52 19.6 433 18.7 404 93 17.8 388 231 17.4 373 470 53-103 15.2 460 15.4 417 74 15.5 393 198 15.8 369 436

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per dayFeed and Compound Consumption 357



### T ABLE 

### J4 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 2,000 ppm 5,000 ppm 10,000 ppm Feed Body Feed Body Dose/ Feed Body Dose/ Feed Body Dose/ (g/day) a Weight (g/day) Weight Day b (g/day) Weight Day (g/day) Weight Day Week (g) (g) (mg/kg) (g) (mg/kg) (g) (mg/kg)

1 12.4 93 10.2 93 219 9.3 94 493 8.5 94 911 2 14.6 114 11.8 110 216 12.2 107 571 11.1 102 1,091 4 15.5 147 14.0 141 198 14.5 136 534 14.3 128 1,119 5 15.7 157 14.5 151 192 15.0 146 514 13.4 137 984 8 16.4 180 15.2 174 175 18.4 167 552 13.9 158 882 9 16.9 186 11.8 178 133 12.5 173 362 12.5 167 749 12 17.0 203 13.3 194 137 13.3 188 352 13.5 181 747 13 19.2 208 14.6 195 149 15.2 192 396 14.0 185 758 14 19.2 212 14.8 201 147 14.6 196 372 14.5 189 767 17 20.0 222 14.7 209 141 16.7 205 409 16.9 200 846 21 17.1 228 15.0 216 139 14.7 213 346 14.8 205 723 25 15.3 237 11.0 219 100 12.1 216 280 11.3 208 545 29 18.6 246 13.7 223 123 13.7 219 312 13.1 212 619 33 19.8 251 18.7 227 165 19.3 222 434 20.1 213 943 37 10.4 258 10.3 233 89 12.9 230 282 10.7 218 492 41 9.8 265 9.6 233 82 9.1 227 201 9.2 217 425 45 10.6 272 10.4 239 87 10.0 231 216 10.2 221 461 49 11.8 284 11.4 246 93 10.8 237 228 10.7 227 474 53 11.0 299 11.3 257 88 10.7 245 218 10.8 232 467 57 12.4 311 11.5 264 88 11.0 249 222 10.9 238 457 61 12.0 315 12.0 267 90 11.4 251 228 10.9 238 456 65 12.6 328 11.6 276 84 11.6 257 226 10.9 242 452 68 12.0 333 11.8 277 85 11.5 257 224 11.4 241 471 73 12.4 343 12.4 286 87 12.1 263 230 11.8 245 481 77 11.7 347 11.2 289 78 11.2 269 208 10.9 243 449 81 11.9 351 11.7 296 79 11.2 270 206 10.9 239 457 85 11.7 354 11.2 295 76 10.8 268 201 10.8 234 463 89 12.3 354 11.8 298 79 11.2 262 213 11.5 229 500 93 11.6 356 10.9 299 73 10.4 251 208 11.6 224 518 97 11.9 358 12.5 298 84 11.7 250 233 11.6 213 543 100 11.7 361 12.0 293 82 11.5 243 237 12.3 202 607 103 12.3 362 12.6 290 87 11.6 234 249 12.9 194 663

Mean for weeks
1-13 16.0 161 13.2 154 177 13.8 150 472 12.7 144 905 14-52 15.3 248 13.0 225 117 13.4 219 308 13.2 211 629 53-103 12.0 341 11.8 285 83 11.3 255 222 11.4 230 499

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per day358 1-Am ino-2, 4-dibromoanthraqu

inone, NTP T

R 383

T ABLE J5 Feed and Compound C

onsumption by Male Mice in the 2-Year Feed Study of 1-Amino-2,4-di bromoanthraquinone

0 ppm 10,000 ppm 20,000 ppm
Feed Body Feed Body Dose/ Feed Body Dose/ (g/day) a W

eight (g/day) W eight Day

b (g/day) W eight Day
W eek (g) (g) (mg/kg) (g) (mg/kg)

1 6.6 21.2 6,260
2 5.8 23.5 5.9 23.2 2,525 6.0 23.9 5,000
5 6.0 27.3 6.2 26.9 2,297 6.0 27.3 4,380
9 6.2 30.2 5.8 30.1 1,916 5.4 30.5 3,561
13 5.7 33.7 5.6 32.5 1,732 5.5 33.0 3,326
17 5.9 36.3 5.8 34.4 1,689 5.7 34.4 3,339
21 5.8 38.8 6.4 34.0 1,890 5.7 34.6 3,292
25 6.0 39.2 6.1 37.0 1,653 6.1 35.6 3,445
29 5.5 39.9 5.8 35.6 1,617 5.5 36.2 3,064
33 5.7 41.3 5.8 38.5 1,496 5.0 36.4 2,757
37 5.3 42.1 5.1 39.6 1,281 5.1 37.9 2,697
41 5.3 42.8 4.9 39.8 1,242 5.2 38.2 2,703
45 5.9 42.1 5.9 39.5 1,492 5.8 37.4 3,097
49 5.5 43.5 5.5 40.1 1,373 6.1 38.9 3,119
53 6.2 44.4 5.1 41.8 1,215 5.5 39.6 2,792
57 5.4 44.0 5.4 41.6 1,294 5.1 39.3 2,630
61 6.6 44.8 5.8 42.3 1,383 6.4 40.2 3,165
65 6.8 45.2 5.5 41.6 1,323 6.0 39.4 3,046
69 6.5 43.6 6.1 40.7 1,501 6.5 37.7 3,468
73 4.1 42.1 6.4 39.8 1,600 6.0 39.2 3,052
77 6.3 44.2 6.0 40.0 1,507 6.3 38.7 3,257
81 5.1 43.0 6.0 39.5 1,530 6.1 38.4 3,200
85 5.6 44.1 6.2 38.9 1,584 6.3 38.0 3,306
89 6.2 44.0 6.4 37.7 1,694 6.7 37.1 3,600
93 4.7 42.3 6.3 38.1 1,656 5.8 36.2 3,191
97 7.8 43.8 9.4 37.4 2,505 9.5 36.8 5,179
101 8.3 43.7 8.9 37.4 2,394 8.5 36.7 4,668
104 9.1 43.6 11.2 37.0 3,016 9.6 36.3 5,315

M ean for weeks
1-13 5.9 28.7 5.9 28.2 2,117 5.9 27.2 4,506
14-52 5.6 40.7 5.7 37.6 1,526 5.6 36.6 3,057
53-104 6.3 43.8 6.7 39.6 1,702 6.7 38.1 3,519

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per dayFeed and Com pound Consum ption 359

T ABLE J6 Feed and Compound C

onsumption by Female Mice in the 2-Year Feed Study of 1-Amino-2,4-di bromoanthraquinone

0 ppm 10,000 ppm 20,000 ppm
Feed Body Feed Body Dose/ Feed Body Dose/ (g/day) a W

eight (g/day) W eight Day

b (g/day) W eight Day
W eek (g) (g) (mg/kg) (g) (mg/kg)

2 5.4 18.1 4.8 18.4 2,628 4.8 18.0 5,385
5 6.4 20.8 5.5 21.3 2,597 5.7 20.8 5,487
9 5.9 22.9 5.9 22.9 2,579 5.1 23.3 4,402
13 6.0 24.8 5.0 24.8 2,023 6.7 25.0 5,339
17 5.8 26.0 5.8 26.6 2,190 6.2 26.0 4,789
21 5.6 27.7 5.1 27.3 1,866 5.7 27.3 4,189
25 6.0 29.2 5.6 28.6 1,941 6.3 28.1 4,507
29 6.4 30.2 5.8 28.8 2,002 6.8 28.9 4,737
33 6.0 32.1 5.7 30.7 1,854 5.3 28.8 3,681
37 6.5 32.9 5.5 31.9 1,726 6.1 30.5 4,029
41 6.7 34.2 6.3 33.0 1,910 6.6 30.1 4,365
45 5.9 35.2 5.5 32.8 1,687 5.6 31.2 3,588
49 6.8 36.6 6.4 34.5 1,844 6.7 32.5 4,124
53 5.3 36.8 4.9 35.0 1,389 5.2 32.5 3,190
56 6.7 38.2 6.8 36.3 1,881 6.7 33.1 4,086
61 6.9 39.1 6.2 36.2 1,704 6.4 33.8 3,796
65 6.7 38.4 6.2 36.1 1,722 6.3 33.5 3,754
69 6.6 37.6 6.4 35.4 1,798 6.6 32.4 4,075
73 5.1 37.3 7.4 35.7 2,062 7.2 32.7 4,374
77 5.1 39.3 5.7 35.8 1,581 5.7 33.1 3,427
81 5.5 39.9 5.7 36.4 1,575 6.1 33.8 3,597
85 6.3 39.5 6.4 36.7 1,754 6.6 33.6 3,905
89 7.0 41.1 7.2 36.1 1,985 7.3 33.7 4,347
93 7.8 41.4 7.5 35.3 2,137 8.5 33.3 5,121
97 8.3 40.0 6.8 33.6 2,019 7.8 31.9 4,902
101 8.6 38.5 9.6 34.1 2,833 10.2 31.5 6,521
104 9.1 39.2 10.2 33.6 3,021 11.1 31.9 6,985

M ean for weeks
1-13 5.9 21.7 5.3 21.9 2,457 5.6 21.8 5,153
14-52 6.2 31.6 5.5 30.5 1,798 6.2 29.3 4,223
53-104 6.8 39.0 6.8 35.5 1,935 7.2 32.9 4,415

a Grams of feed consumed per animal per day b Milligrams of compound consumed per kilogram body weight per day360 1-Amino-2,4-dibromoanthraquinone, NTP TR 383361



# APPENDIX K INGREDIENTS, NUTRIENT COMPOSITION,
AND CONTAMINANT LEVELS
IN NIH-07 RAT AND MOUSE RATION

T ABLE 

### K1 Ingredients of NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 T ABLE 

### K2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . 362 T ABLE 

### K3 Nutrient Composition of NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . 363 T ABLE 

### K4 Contaminant Levels in NIH-07 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . 364362 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### K1 Ingredients of NIH-07 Rat and Mouse Ration a

Ingredients b Percent by Weight

Ground #2 yellow shelled corn 24.50 Ground hard winter wheat 23.00 Soybean meal (49% protein) 12.00 Fish meal (60% protein) 10.00 Wheat middlings 10.00 Dried skim milk 5.00 Alfalfa meal (dehydrated, 17% protein) 4.00 Corn gluten meal (60% protein) 3.00 Soy oil 2.50 Dried brewer's yeast 2.00 Dry molasses 1.50 Dicalcium phosphate 1.25 Ground limestone 0.50 Salt 0.50 Premixes (vitamin and mineral) 0.25

a NCI, 1976; NIH, 1978 b Ingredients were ground to pass through a U.S. Standard Screen No. 16 before being mixed.



### T ABLE 

### K2 Vitamins and Minerals in NIH-07 Rat and Mouse Ration a

Amount Source

Vitamins

A 5,500,000 IU Stabilized vitamin A palmitate or acetate D 3 4,600,000 IU D-activated animal sterol
K 3 2.8 g Menadione d

- " -Tocopheryl acetate 20,000 IU Choline 560.0 g Choline chloride Folic acid 2.2 g Niacin 30.0 g d

-Pantothenic acid 18.0 g d -Calcium pantothenate Riboflavin 3.4 g Thiamine 10.0 g Thiamine mononitrate B 12 4,000 : g Pyridoxine 1.7 g Pyridoxine hydrochloride Biotin 140.0 mg d -Biotin

Minerals

Iron 120.0 g Iron sulfate Manganese 60.0 g Manganous oxide Zinc 16.0 g Zinc oxide Copper 4.0 g Copper sulfate Iodine 1.4 g Calcium iodate Cobalt 0.4 g Cobalt carbonate

a Per ton (2,000 lb) of finished productFeed Analyses 363



### T ABLE 

### K3 Nutrient Composition of NIH-07 Rat and Mouse Ration

Mean ± Standard Nutrient Deviation Range Number of Samples

Protein (% by weight) 22.29 ± 0.86 21.00

) 24.30 22 Crude fat (% by weight) 5.38 ± 0.59 4.40

) 6.30 22 Crude fiber (% by weight) 3.72 ± 0.47 3.10

) 5.40 22 Ash (% by weight) 6.67 ± 0.29 5.96

) 7.27 22

Amino Acids (% of total diet)

Arginine 1.308 ± 0.060 1.210

) 1.390 8 Cystine 0.306 ± 0.084 0.181

) 0.400 8 Glycine 1.150 ± 0.047 1.060

) 1.210 8 Histidine 0.576 ± 0.024 0.531

) 0.607 8 Isoleucine 0.917 ± 0.029 0.881

) 0.944 8 Leucine 1.946 ± 0.055 1.850

) 2.040 8 Lysine 1.270 ± 0.058 1.200

) 1.370 8 Methionine 0.448 ± 0.128 0.306

) 0.699 8 Phenylalanine 0.987 ± 0.140 0.665

) 1.110 8 Threonine 0.877 ± 0.042 0.824

) 0.940 8 Tryptophan 0.236 ± 0.176 0.107

) 0.671 8 Tyrosine 0.676 ± 0.105 0.564

) 0.794 8 Valine 1.103 ± 0.040 1.050

) 1.170 8

Essential Fatty Acids (% of total diet)

Linoleic 2.393 ± 0.258 1.830

) 2.570 7 Linolenic 0.280 ± 0.040 0.210

) 0.320 7

Vitamins

Vitamin A (IU/kg) 10,459 ± 4,285 4,200

) 17,800 22 Vitamin D (IU/kg) 4,450 ± 1,382 3,000

) 6,300 4

"

)

Tocopherol (ppm) 37.95 ± 9.406 22.5

) 48.90 8 Thiamine (ppm) 20.73 ± 5.08 12.0

) 37.0 22 Riboflavin (ppm) 7.92 ± 0.87 6.10

) 9.00 8 Niacin (ppm) 103.38 ± 26.59 65.0

) 150.0 8 Pantothenic acid (ppm) 29.54 ± 3.60 23.0

) 34.0 8 Pyridoxine (ppm) 9.55 ± 3.48 5.60

) 14.0 8 Folic acid (ppm) 2.25 ± 0.73 1.80

) 3.70 8 Biotin (ppm) 0.25 ± 0.04 0.19

) 0.32 8 Vitamin B 12 (ppb) 38.45 ± 22.01 10.6

) 65.0 8 Choline (ppm) 3,089 ± 328 2,400

) 3,430 8

Minerals

Calcium (%) 1.20 ± 0.14 0.91

) 1.43 22 Phosphorus (%) 0.94 ± 0.06 0.84

) 1.10 22 Potassium (%) 0.883 ± 0.078 0.772

) 0.971 6 Chloride (%) 0.526 ± 0.092 0.380

) 0.635 8 Sodium (%) 0.313 ± 0.390 0.258

) 0.371 8 Magnesium (%) 0.168 ± 0.010 0.151

) 0.181 8 Sulfur (%) 0.280 ± 0.064 0.208

) 0.420 8 Iron (ppm) 360.54 ± 100 255.0

) 523.0 8 Manganese (ppm) 91.97 ± 6.01 81.70

) 99.40 8 Zinc (ppm) 54.72 ± 5.67 46.10

) 64.50 8 Copper (ppm) 11.06 ± 2.50 8.09

) 15.39 8 Iodine (ppm) 3.37 ± 0.92 1.52

) 4.13 6 Chromium (ppm) 1.79 ± 0.36 1.04

) 2.09 8 Cobalt (ppm) 0.681 ± 0.14 0.490

) 0.780 4364 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### K4 Contaminant Levels in NIH-07 Rat and Mouse Ration a

Mean ± Standard Deviation b Range Number of Samples

Contaminants

Arsenic (ppm) 0.56 ± 0.19 0.18

) 0.80 22 Cadmium (ppm) c 0.11 ± 0.03 0.10

) 0.20 22 Lead (ppm) 0.57 ± 0.19 0.24

) 1.00 22 Mercury (ppm) <0.05 22 Selenium (ppm) 0.33 ± 0.06 0.23

) 0.45 22 Aflatoxins (ppb) <5.00 22 Nitrate nitrogen (ppm) d 11.57 ± 5.85 2.50

) 22.0 22 Nitrite nitrogen (ppm) d 0.69 ± 1.42 <0.10

) 6.10 22 BHA (ppm) e <2 22 BHT (ppm) e 2.36 ± 1.00 <1.00

) 4.00 22 Aerobic plate count (CFU/g) 144,259 ± 157,664 6,200

) 443,800 22 Coliform (MPN/g) 317 ± 567 <3.00

) 2,400 22 Escherichia coli (MPN/g) f 9.73 ± 31.33 <3.00

) 150 22 Escherichia coli (MPN/g) g 3.04 ± 0.22 <3.00

) 4.0 21 Total nitrosoamines (ppb) h 6.44 ± 6.26 0.80

) 30.30 22 N

) Nitrosodimethylamine (ppb) h 5.91 ± 6.21 0.50

) 30.00 22 N

) Nitrosopyrrolidine (ppb) h 0.53 ± 0.58 0.30

) 2.70 22 Pesticides (ppm)

"

) BHC <0.01 22 $

) BHC <0.02 22

(

) BHC <0.01 22 *

) BHC <0.01 22 Heptachlor <0.01 22 Aldrin <0.01 22 Heptachlor epoxide <0.01 22 DDE <0.01 22 DDD <0.01 22 DDT <0.01 22 HCB <0.01 22 Mirex <0.01 22 Methoxychlor <0.05 22 Dieldrin <0.01 22 Endrin <0.01 22 Telodrin <0.01 22 Chlordane <0.05 22 Toxaphene <0.1 22 Estimated PCBs <0.2 22 Ronnel <0.01 22 Ethion <0.02 22 Trithion <0.05 22 Diazinon <0.1 22 Methyl parathion <0.02 22 Ethyl parathion <0.02 22 Malathion i 0.32 ± 0.68 0.05

) 3.20 22 Endosulfan I <0.01 22 Endosulfan II <0.01 22 Endosulfan sulfate <0.03 22Feed Analyses 365



### T ABLE 

### K4 Contaminant Levels in NIH-07 Rat and Mouse Ration (continued)

a CFU = colony forming units, MPN = most probable number , BHC is hexachlorocyclohexane or benzene hexachloride b For values less than the limit of detection, the detection limit is given as the mean.

c Three lots milled 22 February 1984, 14 March 1984, and 9 May 1984 cont ained 0.20 ppm. All other lots measured less than or equ al to the detection limit. d Sources of contamination: alfalfa, grains, and fish meal

e Sources of contamination: soy oil and fish meal f Mean, standard deviation, and range include one high value of 150 MPN/g from the lot milled on 17 October 1984. g Mean, standard deviation, and range exclude the high value of 150 MPN/g from the lot milled on 17 October 1984. h All values were corrected for percent recovery. i Mean, standard deviation, and range include one high value of 3.20 ppm.366 1-Amino-2,4-dibromoanthraquinone, NTP TR 383367



# APPENDIX L
SENTINEL ANIMAL PROGRAM

M ETHODS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 T ABLE 

### L1 Murine Virus Antibody Determinations for Rats in the 13-Week and 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone . . . . . . . . . . . . . . . . . . . . 370368 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



# SENTINEL ANIMAL PROGRAM

M ETHODS Rodents used in the Carcinogenesis Program of the National To xicology Program are produced in optimally clean facilities to eliminate potential pathogens that may affect study results. Th e Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxic ologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sen tinel) animals in the study rooms. These animals and the study animals are all subject to identical environmental conditions. Th e sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds.

Serum samples were collected from randomly selected rats and mice during the 13-week and 2-year studies. Blood from each animal was collected, allowed to clot, and the serum separate d. The samples were processed appropriately and sent to Microbiological Associates, Inc. (Bethesd a, MD) for determination of antibody titers. The laboratory serology methods and viral agents for which testing was performed are tabulated below; the times at which blood was collected during the studies are also listed.

Method of Analysis Time of Analysis



## Rats 13-Week Study Complement Fixation RCV (rat coronavirus) Study termination Sendai Study termination

Hemagglutination Inhibition H-1 (Toolan's H-1 virus) Study termination KRV (Kilham rat virus) Study termination PVM (pneumonia virus of mice) Study termination

2-Year Study ELISA Mycoplasma arthritidis 18 and 24 months Mycoplasma pulmonis 18 and 24 months PVM 18 and 24 months RCV/SDA (rat coronavirus/ sialodacryoadenitis virus) 6, 12, 18, and 24 months Sendai 18 and 24 months

Hemagglutination Inhibition
H-1 6, 12, 18, and 24 months KRV 6, 12, 14, 15, 18, and 24 months PVM 6 and 12 months Sendai 6 and 12 monthsSentinel Animal Program 369



## Mice 13-Week Study Complement Fixation LCM (lymphocytic choriomeningitis virus) Study termination Mouse adenoma virus Study termination Sendai Study termination

ELISA MHV (mouse hepatitis virus) Study termination

Hemagglutination Inhibition Ectromelia virus Study termination GDVII (mouse encephalomyel itis virus) Study termination MVM (minute virus of mice) Study termination PVM Study termination Polyoma virus Study termination Reovirus 3 Study termination

2-Year Study Complement Fixation LCM 6, 12, 18, and 24 months Mouse adenoma virus 6 and 12 months

ELISA Ectromelia virus 18 and 24 months GDVII 12, 18, and 24 months Mouse adenoma virus 18 and 24 months MHV 6, 12, 18, and 24 months M. arthritidis 18 and 24 months M. pulmonis 18 and 24 months PVM 18 and 24 months Reovirus 3 18 and 24 months Sendai 18 and 24 months

Hemagglutination Inhibition Ectromelia virus 6 and 12 months GDVII 6 months K (papovavirus) 18 and 24 months MVM 6, 12, 18, and 24 months PVM 6 and 12 months Polyoma virus 6, 12, 18, and 24 months Reovirus 3 6 and 12 months Sendai 6 and 12 months

Immunofluorescence Assay EDIM (epizootic diarrhea of infant mice) 18 and 24 months

Results of serology tests for rats are presented in Table L1. All test results for mice were negative.370 1-Amino-2,4-dibromoanthraquinone, NTP TR 383



### T ABLE 

### L1 Murine Virus Antibody Determinations for Rats in the 13-Week and 2-Year Feed Studies of 1-Amino-2,4-dibromoanthraquinone

Interval Incidence of Antibody Positive Serologic in Sentinel Animals Reaction for

13-Week Study

Study termination 0/10 None positive

2-Year Study

Male

6 Months 0/5 None positive 12 Months 0/5 None positive 18 Months 0/4 None positive 24 Months 0/5 None positive

Female

6 Months 5/5 Sendai 12 Months 1/5 KRV 5/5 Sendai

14 Months 1/5 KRV 15 Months 0/10 None positive 18 Months 1/5 KRV 5/5 Sendai 24 Months 5/5 Sendai