# FINAL

Report on Carcinogens Background Document for



# 1-Amino-2,4- dibromoanthraquinone

September 19, 2002

Prepared for the: U.S. Department of Health and Human Services
Public Health Service National Toxicology Program Research Triangle Park, NC 27709

Prepared by: Technology Planning and Management Corporation Canterbury Hall, Suite 310 4815 Emperor Blvd Durham, NC 27703
Contract Number N01-ES-854219/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## Foreword

The Report on Carcinogens (RoC) is prepared in response to Section 301 of the Public
Health Service Act as amended. The RoC contains a list of all substa nces (i) that either are known to be human carcinogens or may reasonably be anticipated to be human carcinogens; and (ii) to which a significan t number of persons residing in the United States are exposed. The Secretary, Departme nt of Health and Human Services (DHHS) has delegated responsibility for preparation of the RoC to the National Toxicology Program (NTP) who prepares the Report with assistance from other Federal health and regulatory agencies and non-government institutions.

Nominations for listing in or delisting from the RoC are reviewed by a formal process that includes a multi-phased, scientific peer review and multiple opportunities for public comment. The review groups evaluate each nom ination according to specific RoC listing criteria. This Background Document was prep ared to assist in the review of the nomination of 1-amino-2,4-dibr omoanthraquinone. The scientific information in this document comes from publicly available, peer reviewed sources. Any interpretive conclusions, comments or statistical calcul ations, etc made by the authors of this
document that are not contained in the original citation are identified in brackets [ ]. If any member(s) of the scientific peer revi ew groups feel this Background Document does not adequately capture and present the relevant information they will be asked to write a commentary for this Background Document that will be included as an addendum to the document. In addition, a meeting summary th at contains a brief discussion of the respective review group’s revi ew and recommendation for the nomination will be added to the Background Document, also as an addendum.

A detailed description of the RoC nomination re view process and a list of all nominations under consideration for listing in or delisti ng from the RoC can be obtained by accessing the NTP Home Page at http://ntp-server.niehs.nih.gov

. The most recent RoC, the 9 th 

### Edition, was published in May, 2000 and may be obtained by contacting the NIEHS
Environmental Health Inform ation Service (EHIS) at http://ehis.niehs.nih.gov (800-315- 3010).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## CONTRIBUTORS

NIEHS/NTP Staff

C W Jameson, Ph.D. Head, Report on Carcinogens, Environmental Toxicology Program, NIEHS

Ruth M Lunn, Dr. P.H. Report on Carcinogens Group, Environmental Toxicology Program, NIEHS

Shawn Jeter, B.S. Report on Carcinogens Group, Environmental Toxicology Program, NIEHS

AnnaLee Sabella-Mosley Re port on Carcinogens Group, Environmental Toxicology Program, NIEHS

Support to the National Toxicology Program for the preparation of this background
document was provided by Technology Planni ng and Management Corporation through NIEHS Contract Number NO1-ES-85421

Ronald Thomas, Ph.D., Pr incipal Investigator

Sanford Garner, Ph.D. Co-P rincipal Investigator

Stanley Atwood, M.S., DABT, Senior Scientist

Susan Goldhaber, M.S. Senior Scientist

Greg Pazianos, B.S., Scientist

Ashlee Duncan, M.S. Scientist

Support staff

Angie Fralick, B.S

Tracy Saunders, B.S.

Consultants

Elizabeth Delzell, MSPH, S.D. Professor, De partment of Epidemiology and International Health, University of Alabama at Birmi ngham, Birmingham, Alabama (contributed to Section 3)

Richard Di Giulio, Ph.D. Pr ofessor, Nicholas School of the Environment, Duke University, Durham, NC (document review)9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Criteria for Listing Agents, Substances or Mixtures in the Report on Carcinogens

U.S. Department of Health and Human Services National Toxicology Program

Known to be Human Carcinogens:

There is sufficient evidence of carcinogeni city from studies in humans, which indicates a causal relationship between exposure to the agent, substance or
mixture and human cancer.

Reasonably Anticipated to be Human Carcinogens:

There is limited evidence of carcinogenic ity from studies in humans, which indicates that causal interpretation is cr edible but that alternative explanations such as chance, bias or confounding factor s could not adequately be excluded; or

There is sufficient evidence of carcinogenicity from studies in experimental
animals which indicates there is an incr eased incidence of malignant and/or a combination of malignant and benign tumors: (1) in multiple species, or at multiple tissue sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site or type of tumor or age at onset; or

There is less than sufficient evidence of carcinogenicity in humans or laboratory animals, however; the agent, substance or mixture belongs to a well defined, structurally-related class of substances whose members are listed in a previous Report on Carcinogens as either a known to be human carcinogen, or reasonably anticipated to be human carcinogen or there is convincin g relevant information that the agent acts through mechanisms i ndicating it would likely cause cancer in
humans.

Conclusions regarding carcinog enicity in humans or experimental animals are based on scientific judgment, with consideration given to all relevant information. Relevant information includes, but is not limited to dose response, route of exposure, chemical structure, metabolism, pharmacokinetics, sens itive sub populations, genetic effects, or other data relating to mechanism of action or factors that may be unique to a given substance. For example, there may be subs tances for which there is evidence of carcinogenicity in laboratory animals but ther e are compelling data indicating that the agent acts through mechanisms which do not operate in humans and would therefore not reasonably be anticipated to cause cancer in humans.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

iv9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## Executive Summary

Introduction 1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone vat dye that is used in the textile industry. It was nominated by the National Institute of Environmental
Health Sciences based on the results of the National Toxicolo gy Program two-year feeding studies, which concluded that ADBAQ was carcinogenic in rats and mice.

Human Exposure
Use . ADBAQ is used as a dye and as a dye in termediate. Anthraquinones are widely used as starting material for the manu facture of vat dyes, which are used in fibers and textiles, typically for cotton, wool, and cellulose acetate.

Production . ADBAQ is prepared from 1-aminoant hraquinone by bromination in dilute
mineral acids and is available from two vendors in the United States.

Environmental Exposure . Because ADBAQ is not found naturally in the environment, any environmental exposure will be a result of releases from facilities where ADBAQ is produced or is used as an intermediate for production of other anthraquinone dyes. A dibromoaminoanthraquinone was found in ra w wastewater from a dye manufacturing plant in four of eight sample s at concentrations of 92 to 170 ppb but was not observed in
final effluent.

Occupational Exposure . Dermal exposure is the main source of exposure to ADBAQ. Because ADBAQ is not very volatile, inhala tion exposure likely is limited to solid particulates. No specific occupationa l exposure information for ADBAQ or anthraquinone dyes in general was found in cu rrent literature. Epid emiological studies, however, indicate occupational e xposures to anthraquinone dyes in a New Jersey dye and resin manufacturing plant.

Human Cancer Studies Only two groups of workers exposed to anth raquinone dyes and intermediates have been investigated. All of the studies of these workers have limitati ons that make detection of causal relationships difficult, including small size, potential exposure to multiple agents in the workplace, and lack of quantitative exposure data. In addition, the follow-up studies lack information on nonoccupational can cer risk factors. The results of the pertinent research are not consistent. Th e study of Scottish workers reported small increases in esophageal and prostate cancer and found no excess of respiratory cancer (64 observed vs. 66.2 expected) or brain cancer (4/3.5 ). In contrast, the studies of New Jersey workers reported that work in anthraqui none dye operations was positively associated with lung cancer and with CNS tumors. In th e latter investigations , the results for CNS tumors were based on a very small number of observations. The data on anthraquinone dye operations and lung cancer were more precise, and no confounding factor was identified that could explain the positive association. Thus, th e increase in lung cancer in anthraquinone dye workers at the New Jersey plant may have been due to occupational9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### exposure. However, the specific agent(s) responsible for the excess have not been
identified.

Studies in Experimental Animals In two-year dietary studie s with ADBAQ, male and female F344/N rats exhibited significantly increased incidences of neoplasms of the liver, large intestine, kidney, and urinary bladder. Significant increases in the incidence of tumors of the liver and large intestine (females only) also were observ ed in a 15-month stop-exposure study in rats.
B6C3F 1 

### mice consuming ADBAQ-containing di ets for two years had increased incidences of hepatocellular adenoma and carcinoma, squamous-cell papilloma and carcinoma of the forestomach, and alveolar/b ronchiolar adenoma and carcinoma of the lung. The NTP report concluded that “under the conditions of these 2-year feed studies,
there was clear evidence for carcinogenic activity of 1-amino-2,4-dibromoanthraquinone
in male and female F344/N rats based on incr eased incidences of neoplasms in the liver, large intestine, kidney, and urinary bladder. There was clear evidence for carcinogenic activity of 1-amino-2,4-dibromoanthraquinone in male and female B6C3F 1 

### mice based on increased incidences of neoplasms in the liver, forestomach, and lung.”

Genotoxicity ADBAQ was mutagenic in S. typhimurium strains that revert by frameshift mutations, and the mutagenicity was decreased or elimin ated in the presence of induced hamster or rat liver S9 metabolic activation. ADBAQ did not induce detectable mutations at the tk locus in mouse lymphoma cells; however, the concentrations used (20 or 25 μ g/mL) were

less than those used for the S. typhimurium assays (100 to 10,000 μ g/mL) and slightly

less than those used to asse ss clastogenicity (up to 100 μ g/mL of ADBAQ). In CHO cells, ADBAQ induced chromosomal aberrations and SCE without metabolic activation; however, the results were inconsistent be tween different trials and laboratories. Forestomach and lung tumors induced by ADBAQ had a higher frequency of ras mutations than did spontaneous tumors. In particular, CAA to CTA transversions were common in ADBAQ-induced tumors but were ab sent in spontaneous tumors. The results obtained with ADBAQ must be viewed in the light of the limited solubility of this molecule and the uncertain purity of the preparations tested.

Other Relevant Data Absorption, excretion, and metabolism. In rats, ADBAQ is rapidly absorbed from the gastrointestinal tract and distributed ra pidly and widely to most soft tissues. Approximately 97% of ADBAQ is metabolized , as demonstrated by the small amount (less than 3%) of radiolabel recovered as the parent compou nd from either blood or urine; however, the metabolites of ADBAQ have not been identified. ADBAQ and its metabolites are excreted primar ily in the feces and urine.

Potential mechanisms. ADBAQ and other anthraquinones are classified with a large number of other quinone molecu les that can be derived from aromatic molecules such as benzene, naphthalene, and anthracene. R eactive oxygen species generated by metabolism of a variety of quinones may be associated with DNA damage or activation of signaling pathways involved in initiation, promotion, a nd progression of carcinoge nesis. Four other anthraquinone vat dyes evalua ted by the NTP have been listed in the RoC as reasonably9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

vii 

### anticipated to be human carcinogens. In addition, a high percentage (36/80) of phenolic anthraquinones have been reported to be mutagenic in Salmonella

.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

viii9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table of Contents

Executive Summary.............................................................................................................. .......... v

1 Introduction................................................................................................................. .............. 1

1.1 Chemical identification.............................................................................................. 1 1.2 Physical-chemical properties ..................................................................................... 1 2 Human Exposure............................................................................................................... ........ 5

2.1 Use ........................................................................................................................ ..... 5

2.2 Production................................................................................................................. . 5 2.3 Analysis................................................................................................................... ... 5

2.4 Environmental occurrence ......................................................................................... 5

2.5 Environmen tal fate..................................................................................................... 5

2.5.1 Air............................................................................................................... 5

2.5.2 Water........................................................................................................... 6

2.5.3 Soil.............................................................................................................. 6 2.6 Environmental exposure ............................................................................................ 6 2.7 Occupational exposure............................................................................................... 6 2.8 Biological indices of exposure................................................................................... 6 2.9 Regulations ................................................................................................................ 6

3 Human Cancer Studies......................................................................................................... ..... 7
3.1 Human epidemiological studies................................................................................. 7 3.1.1 Study of Scottish anthraquinone dyestuffs workers ................................... 7 3.1.2 Studies of dye and resin manufact uring workers at a plant in New Jersey .......................................................................................................... 8 3.2 Summary.................................................................................................................. 13

4 Studies of Cancer in Experimental Animals........................................................................... 17

4.1 Rats ....................................................................................................................... ... 17 4.1.1 Thirteen-week study ................................................................................. 17 4.1.2 Two-year study ......................................................................................... 18 4.1.3 Fifteen-month stop-exposure study .......................................................... 21

4.2 Mice ....................................................................................................................... .. 24 4.2.1 Thirteen-week study ................................................................................. 24 4.2.2 Two-year study ......................................................................................... 25 4.3 Summary.................................................................................................................. 28 5 Genotoxicity................................................................................................................. ........... 29
5.1 Prokaryotic systems: Salmonella typhimurium ........................................................ 29 5.2 Mammalian systems................................................................................................. 29 5.2.1 Mouse lymphoma L5178YTK +/- 

### cells ...................................................... 29

5.2.2 Chromosomal aberrations......................................................................... 30 5.2.3 Sister chromatid exchange........................................................................ 309/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### 5.2.4 Ras mutations in tumors ........................................................................... 31 5.3 Summary.................................................................................................................. 32

6 Other Relevant Data.......................................................................................................... ...... 33
6.1 Absorption, distribution, meta bolism, and excretion............................................... 33 6.1.1 Absorption ................................................................................................ 33

6.1.2 Distribution and metabolism .................................................................... 33

6.1.3 Excretion................................................................................................... 33

6.2 Liver focus model .................................................................................................... 34 6.3 Carcinogenicity and mutagenicity of other quinones and anthraquinones .............. 35 6.4 Summary.................................................................................................................. 41

7 References................................................................................................................... ............ 43
Appendix A: NTP TR 383 (1996). Toxicology and Carcinogenesis Studies of 1-Amino2,4-Dibromoanthraquinone in F344/N Rats and B6C3F1 Mice (Feed Studies). PP A-1
– A-86. ........................................................................................................................ ............ 47
Appendix B: Tissues examined for histopathol ogy in mice and rats in the NTP bioassays of ADBAQ....................................................................................................................... ....... 49

List of Tables

Table 1-1. Physical and chem ical properties of ADBAQ............................................................... 2
Table 1-2. Anthraquinone and some anthraquinone-based compounds ......................................... 3

Table 3-1. Human cancer studies on anthraquinone dye workers ................................................ 14
Table 4-1. Survival and body weight in F 344/N rats following dietary exposure to ADBAQ for 13 weeks............................................................................................................. 18
Table 4-2. Survival, body weight, and tumor in cidence in F344/N rats following dietary exposure to ADBAQ for two years......................................................................................... 20
Table 4-3. Survival, body weight, and tumor in cidences in F344/N rats in the 15-month stop-exposure study of ADBAQ (20,000 ppm) ...................................................................... 23
Table 4-4. Occurrence of altered hepatocellu lar foci in female F344/N rats following dietary exposure to ADBAQ for 15 months ........................................................................... 24
Table 4-5. Survival and body wieght in B6C3F 1 

### mice following dietary exposure to ADBAQ for 13 weeks............................................................................................................. 25
Table 4-6. Survival, body weight, and tumor incidence in B6C3F 1 

### mice following dietary exposure to ADBAQ for two years......................................................................................... 27
Table 5-1. Mutagenic ity of ADBAQ in S. typhimurium a 

### ............................................................. 29

Table 5-2. Induction of chromosomal abe rrations and SCE in CHO cells by ADBAQ a 

### ............. 31
Table 5-3. Frequency and spectra of ras mutations in forestomach and lung tumors from
B6C3F 1 

### mice exposed to ADBAQ for two years a 

### .................................................................. 32
Table 6-1. Occurrence of altered hepatocellu lar foci neonatal rats following dietary exposure to ADBAQ for 75 or 300 days a 

### ............................................................................... 349/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 6-2. Carcinogenicity and mutagenicity of anthraquinone and some anthraquinonederived dyes (data from NTP and IARC) ............................................................................... 37
Table 6-3. Genotoxicity of some anthraquinone dyes a 

### ................................................................. 40

List of Figures

Figure 1-1. Struct ure of ADBAQ................................................................................................. ... 1
Figure 4-1. Exposure protocol for the two- year study of ADBAQ in F344/N rats ...................... 19
Figure 4-2. 15 Month st op exposure study ................................................................................... 21
Figure 4-3. Exposure protocol for the two-year study of ADBAQ in B6C3F 1 

### mice.................... 269/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 1 Introduction

1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone vat dye that is used in the textile industry and is a member of a class of insoluble dyes that are impregnated into textile fibers. ADBAQ was nominated by the National Institute of Environmental Health Sciences (NIEHS) for possible listi ng in the Report on Carcinogens (RoC) based on a National Toxicology Program (NTP) bioa ssay (TR-383) reporting clear evidence of carcinogenicity in rats and mice. The NTP st udied the carcinogenicity of ADBAQ as part of a class study with five other anthraqui none-derived dyes with representative and diverse structures, for the pur pose of predicting the carcinogeni city of chemicals in this
class. The conclusions from the NTP’s two- year feeding study of ADBAQ were that
there was clear evidence of carcinogenic activity in ma le and female F344/N rats (neoplasms in the liver, large intestine, kidney, and urinary bladder) and in male and
female B6C3F 1 

### mice (neoplasms in the liver, forestomach, and lung).

1.1 Chemical identification
ADBAQ (C 14 

### H 7 

### Br 2 

### NO 2 

### , mol wt 381.02, CASRN 81- 49-2) is a substituted anthraquinone with a single primary amine located in th e 1-position and two bromine atoms located ortho and para to the amino group. Synony ms for ADBAQ include 1-amino-2,4-dibromo9,10-anthracenedione; 1-amino-2,4-dibroman thrachinon; anthraquinone, 1-amino-2,4- dibromo-; and 2,4-dibromo-1-anthraquinonyl amine. Its RTECS number is CB5500000. The structure of ADBAQ is shown in Figure 1-1.

Figure 1-1. Structure of ADBAQ

1.2 Physical-chemical properties ADBAQ is a red powder at room temperature a nd melts at 226°C. It is sparingly soluble in ether and benzene; soluble in hot nitrobenzene, chloroform, acetic acid, pyridine, and concentrated sulfuric acid; and insoluble in wate r. It is sensitive to long-term air and light exposure. The physical and chemical propertie s of ADBAQ are summarized in Table 1-1. O NH 2

O Br Br9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 1-1. Physical and chem ical properties of ADBAQ

Property Information Reference

Molecular weight 381.02 ChemFinder 2001

Color red ChemFinder 2001

Odor odorless NTP Chemical Repository 2001

Physical state powder ChemFinder 2001

Melting point (°C) 226 ChemFinder 2001

Flash point (°C) > 200 (> 392°F) NTP Chemical Repository 2001

Solubility (at 23 o C):

water

acetone

DMSO

95% ethanol

toluene < 0.1 g/100 mL
< 1 mg/mL
1–10 mg/mL
< 1 mg/mL
< 1 mg/mL ChemFinder 2001
NTP Chemical Repository 2001
NTP Chemical Repository 2001
NTP Chemical Repository 2001
NTP Chemical Repository 2001



### Anthraquinone (TR-494) and the following six other anthraqui none-based compounds (identified in Table 1-2) have been tested in two-year bioassays by the NTP or have been
listed in the RoC: 2-aminoanthr aquinone (TR-144, RoC), 1-amino-2- methylanthraquinone (TR-111, RoC), 1,8-dihydr oxyanthraquinone (danthron) (RoC), 2- methyl-1-nitroanthraquinone (TR-029), 1,4,5,8- tetraaminoanthraquinone (disperse blue 1) (TR-299, RoC), and emodin (TR-493). Carcinoge nicity and genetic toxicology data for
these seven molecules are summarized in Table 6-2. Other anthraquinone-derived compounds that are listed in the NTP database but have not been tested in two-year bioassays include the follo wing: chrysophanic acid, CA SRN 481-74-3; rhein, CASRN 478-43-3; 1,8-dihydroxy-4,5- dinitroanthraquinone, CASRN 81 -55-0; C.I. disperse blue 27, CASRN 15791-78-3; C.I. disperse red 60, CASRN 17418-58-5; disperse blue 7, CASRN 3179-90-6; D&C viol et no. 2, CASRN 81-48-1; D&C green 5, CASRN 4403- 90-1; carminic acid, CASRN 1260-17 -9; and vat blue 4, CASRN 81-77-6.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 1-2. Anthraquinone and some anthraquinone-based compounds

Compound name Cas # Chemical formula Molecular weight Structure

Anthraquinone 84-65-1 C 14 H 8 O 2 208.22

O

O

2-Aminoanthraquinone 117-79-3 C 14 H 9 NO 2 223.23

O

O H 2 N

1-Amino-2-methylanthraquinone 82-28-0 C 15 H 11 NO 2 237.26

O

O H 3 C NH 2

1,8-Dihydroxyanthraquinone (danthron) 117-10-2 C 14 H 8 O 4 240.22

O OH OH

O2-Methyl-1-nitroanthraquinone 129-15-7 C 15 H 9 NO 4 267.24

O

O H 3 C NO 2

1,4,5,8-
Tetraaminoanthraquinone (disperse blue 1) 2475-45-8 C 14 H 12 N 4 O 2 268.27

O

O NH 2 NH 2

NH 2 NH 2

Emodin 518-82-1 C 15 H 10 O 5 270.24

O

O OH OH

OH H 3 C9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

49/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 2 Human Exposure

2.1 Use ADBAQ is used as a dye and as a dye inte rmediate (NTP Chemical Repository 2001). Anthraquinones are widely used as starting ma terial for the manufacture of vat dyes. Vat dyes are a class of water-insoluble dyes that can easily be reduced to a water-soluble and usually colorless form. In this form, they can readily be impregnated into fibers and textiles. Oxidation then produces an insoluble colored form that is remarkably fast to washing, light, and chemicals. Vat dyes typica lly are used for cotton, wool, and cellulose

acetate.

2.2 Production ADBAQ is prepared from 1-aminoanthraquinon e by bromination in dilute mineral acids (HSDB 2000). ADBAQ was found to be availabl e from two vendors in the United States: Pfaltz and Bauer, in Connecticut, and Alfa Aesar, in Massachusetts (ChemFinder 2001). U.S. production of vat dyes totaled 14 million kilograms (30.8 million pounds) in 1991 (NTP 1996).

2.3 Analysis ADBAQ has been analyzed in various sample s by gas chromatography/flame ionization detection and gas chromatography/mass spec trometry (Games and Hites 1977). Recently, several other analytic al methods have been describe d for detecting and quantifying anthraquinone-type dyes (liquid chromat ography/nuclear magnetic resonance, liquid chromatography/mass spectrometry, and high-performance liquid chromatography [HPLC]); however, ADBAQ has not been analyzed by these methods (Preiss et al. 2000,
Novotná et al. 1999).

2.4 Environmental occurrence ADBAQ is not known to be naturally forme d. Production of ADBAQ an d its subsequent
use as an intermediate in th e production of anthraquinone dyes may result in releases into the environment (HSDB 2000). No information was found in the U.S. Environmental Protection Agency’s Toxic Release I nventory regarding ADBAQ (TRI 2001).

2.5 Environmental fate ADBAQ is sensitive to long-term exposure to light and air (NTP Chemical Repository 2001). Although ADBAQ was found in raw wastew ater of a dye manu facturing plant, it was not present in the final e ffluent (effluent into the Cooper River, which flows into Charleston Bay of the Atlantic Ocean) (Games and Hites 1977). This may indicate that ADBAQ was either biodegraded or adsorbed to sludge during treatment (HSDB 2000).

2.5.1 Air



### Based on estimated vapor pre ssure, ADBAQ should exist as a solid particulate in the ambient atmosphere. Particulate-phase ADBAQ would be removed from the atmosphere by dry deposition (HSDB 2000).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

6 2.5.2 Water



### ADBAQ may adsorb to suspended solids and sediment in water. The amino group in ADBAQ also may bind covalently with active site s in sediment and particulate matter. It is not expected to volatilize from water surf aces. The estimated partition coefficients and a recommended regression-derived equation we re used to calculate a bioconcentration factor of 6,400 for ADBAQ. This suggests th at ADBAQ will bioconcentrate in aquatic organisms (HSDB 2000).

2.5.3 Soil



### ADBAQ is expected to be immobile in so il; its mobility may be limited because the amino group may bind to active si tes in the soil. Vo latilization is not expected to occur
from either moist or dr y soil surfaces (HSDB 2000).

2.6 Environmental exposure Because ADBAQ is not found naturally in the environment, any environmental exposure
will be a result of releases from facilities where ADBAQ is produced or is used as an intermediate for production of other anthra quinone dyes. A dibromoaminoanthraquinone was found in raw wastewater from a dye manuf acturing plant in four of eight samples at concentrations of 92 to 170 ppb but was not observed in final effluent (HSDB 2000).

2.7 Occupational exposure Dermal exposure is the main source of exposure to ADBAQ. Because ADBAQ is not very volatile, inhalation e xposure likely is limited to solid particulates. Ikeda et al. (1977) found that workers exposed to dyes (as evid enced by diazo-positive metabolites in their urine) had many dye stains on their hands, wrists , and forearms. Analyses of the air in the
workroom environments were not conducted.

No specific occupational exposure informa tion for ADBAQ or anthraquinone dyes in general were found in current literature. Epidemiological studies, however, indicate potential occupational exposures to anthraqu inone dyes in a New Jersey dye and resin manufacturing plant. A cohor t study followed more than 3,000 workers who may have been exposed to anthraquinone dyes (Sathi akumar and Delzell 2000). Results of this study are described in Section 3.

2.8 Biological indices of exposure No indices of ADBAQ exposure in humans or animal models were found in current
literature.

2.9 Regulations No regulations specifically for ADBAQ were identified.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 3 Human Cancer Studies

No study has evaluated the relationship betw een human cancer and specific exposure to ADBAQ; however, exposure to anthraquinone dy es as a class has been studied. Since the late 1880s, the dye manufacturing industry has used a large number of anthraquinones and aromatic amines (arylamines) to synthe size industrial colorants and has used nitro aromatic amines to make hair dyes. Re latively little is known about the human carcinogenicity of anthraquinone dyes. The NTP classifies 2-am inoanthraquinone, 1- amino-2-methylanthraquinone, 1,8-dihydroxyanthraquinone, and 1,4,5,8- tetraaminoanthraquinone as reasonably anticipated to be human carcinogens. The International Agency for Research on Can cer (IARC) lists 1,8-dihydroxyanthraquinone, 2-methyl-1-nitroanthraquinone, and 1,4,5,8- tetraaminoanthraquinone as possibly carcinogenic to humans (Group 2B) and consid ers 2-aminoanthraqui none and 1-amino-2- methylanthraquinone not classifiable as to ca rcinogenicity to humans (Group 3). Neither the NTP nor IARC has evaluated the carcinogeni city of anthraquinone dyes as a chemical category or anthraquinone dye manufact uring as an exposure circumstance.

3.1 Human epidemiological studies Data on the carcinogenicity to humans of dye s and chemicals used in synthesizing dyes come mainly from studies of workers in ma nufacturing plants. These groups of workers typically were exposed to many different ch emicals and classes of chemicals, often making it difficult to attribute observed carcinoge nic effects to a single agent. Only five epidemiologic studies of workers potentia lly exposed to anthraquinone dyes and anthraquinone dye intermediates are available. One of these investigations evaluated mortality among workers at a plant in Scot land that mainly made anthraquinone dyes (Gardiner et al. 1982). Each of the other four studies pertains to the same group of workers at a plant in New Jersey (Barbone et al. 1992, 1994, Delzell et al. 1989, Sathiakumar and Delzell 2000). The New Jersey plant’s manufacturing operations were more diverse than those of the plant in Scotland and included the production of anthraquinone, anthraquinone dye interm ediates and dyes, epichlorohydrin, azo dye intermediates and dyes, epoxy resins , and a variety of additives.

3.1.1 Study of Scottish anthraquinone dyestuffs workers



### Gardiner et al . (1982) evaluated mortality from various causes among 1,975 men employed at an anthraquinone dyestuffs plan t for at least six months between 1956 and
the end of 1965. Personnel records were used to identify all subjects who had worked in jobs that entailed potential exposure to s ubstituted anthraquinones. The study excluded men who had worked exclusively in cler ical, accounting, and personnel management positions. Vital status and cause of death as of the end of June 1980 were determined for about 97% of the subjects through the use of plant records and da ta from the National Records Offices of Edinburgh and Newcastle upon Tyne. Analyses compared workers’ mortality rates with those of the general male population of Sc otland in 1975, adjusting for age and using the standardized mortality ratio (SMR) (ratio of observed to expected numbers of deaths, multiplied by 100) as th e measure of association. An SMR of 100 implies that the mortality rate of workers wa s the same as that of the general population, after analytic adjustment for any difference in the age distributions of these two groups.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Gardiner et al . (1982) found that workers at the plan t had about 23% fewer deaths from all causes combined than expected on the basis of general population rates (470 observed vs. 611.5 expected deaths, SMR = 76.9). Workers also had fewer deaths than expected from cerebrovascular disease (36/66.5, SM R = 54.1), cardiovascular disease (188/235.6, SMR = 79.8), and all cancers combined (129/ 149.7, SMR = 86.2). There were at least 5 observed or expected deaths for eight specif ic types of cancer. Among these, there were more deaths than expected for cancer of the esophagus (6/4.3) and fewer deaths than expected for cancer of the stomach (14/18.1), in testinal tract (7/11.0) , pancreas (4/6.8), bladder (4/5.6), respiratory system (64/66.2), prostate (4/8.0), and rectum (6/6.8). None
of these differences was statistically significant.

The mortality rate for all cancer combined wa s higher than expected for workers in some areas of the plant. Gardiner et al . (1982) did not present de tailed results for specific cancers by plant area but noted statisitcally significant excesses of cancer of the esophagus in the engineering department (all deaths from this cancer were among men in this department) and cancer of the prostate in one particular process area. Workers’ respiratory cancer rates were not statistically significantly greater than expected in any plant area. Work activities in the engineeri ng department were not thoroughly described but presumably included main tenance operations. Gardiner et al . (1982) indicated that engineering workers constituted 45% of the study group, that they worked mainly in proximity to plant process areas , and that their plant exposur es probably were similar to those of process workers. The authors could no t interpret their results for prostate cancer. They stated that cadmium, a suspected cause of prostate cancer, was not used at the plant.

Gardiner et al . (1982) stated that their research did not provide any evidence of “an abnormal pattern of mortality” in the group of anthraquinone dye workers studied. Because of its methodologic weaknesses, the st udy provided little evidence either for or against the hypothesis that anthraquinones cause cancer in humans. Limitations were the small numbers of deaths for most specific types of cancer, the l ack of quantitative estimates of exposure to anthraquinones or other chemicals, and the lack of data on nonoccupational factors. Moreover, the very low SMR for all causes of death combined, especially in older age gr oups, which probably contained a high proportion of inactive workers, suggests that ascertainment of deaths in the study group may have been incomplete or that the use of comparison rates derived from a single year (1975) was inappropriate. If so, all SMRs may have been underestimated.

3.1.2 Studies of dye and resin manufacturing workers at a plant in New Jersey



### Delzell et al . (1989) conducted a retrospective fo llow-up study of mortality from 1952 through 1985 among 2,642 white men who had been employed for at least six months at a dye and resin plant in New Jersey. Barbone and colleagues carried out nested casecontrol studies of lung cancer (Barbone et al. 1992) and central nervous system (CNS) tumors (Barbone et al. 1994) occurring among these work ers. In a late r investigation, Sathiakumar and Delzell (2000) expanded th e original retrospective follow-up study (Delzell et al. 1989) to include 3,266 male and female workers of all races and evaluated their mortality patterns during the period 1952 through 1995.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### The manufacturing operations of the New Jers ey plant took place in three major areas. Operations in the “south dye area” (ant hraquinone dye/epichlorohydrin area) included anthraquinone production (1952 to 1983), anthraquinone dye production (1952 to 1983), anthraquinone dye intermediates produc tion (1952 to 1983), formulation of anthraquinone dyes (1952 to late 1980s), and epichlorohydrin production (1961 to 1965). The “north dye area” produced azo dyes and intermediates from 1959 to the late 1980s. The plastics and additives area made epoxy re sins, fabric softeners, water-repellent agents, optical brighteners, and various additives from 1959 to the late 1980s. Raw
materials and intermediates used in each ar ea included many chemicals, some of which are known carcinogens (e.g., benzene, benz idine, ethylene oxid e) or suspected carcinogens (e.g., epichlorohydrin, formaldehyde, dimethyl sulfate). Chemicals used in the anthraquinone dye/epi chlorohydrin (AQ/ECH) ar ea included anthracene, anthraquinones, anthraquinone sulfonates , anthraquinone intermediates, aniline, substituted aniline, benzene, nitrobenzene, chlorobenzenes, chlorotoluenes, pyridine, tetrachloroethylene, chlorine, ammonia, ar senic acid, methanol, sulfuric acid, and

mercury.

3.1.2.1 Original retrospective follow-up study


### The first investigation of workers at th e New Jersey plant used company personnel records to identify all white men with at least six months of employme nt as of the end of 1984 and to develop information on each subj ect’s jobs at the facility (Delzell et al. 1989). Plant records and linkages with several national and state databases established the vital status of 96% of the 2,642 eligible subjects as of the end of 1985. Death certificates provided information on deceased subjects’ cau ses of death. Comparisons of workers’ mortality rates with those for the white male general population of the United States yielded observed and expected numbers of deaths for the overall study group and subgroups of hourly workers classified by the process area(s) in which they had worked for at least one year.

The overall study group had 357 deaths from all causes combined, compared with 440 expected deaths (SMR = 81, 95% confidence interval [CI] = 73 to 90), and 106 cancer deaths, compared with 97 expected can cer deaths (SMR = 109, 95% CI = 89 to 132).
There were more deaths from CNS cancer than expected (5/3.3), not including the deaths of two employees from this cancer while livi ng in Europe, and there were 3 deaths each from bladder cancer (expected = 2.3) and ki dney cancer (expected = 2.5). The paper did not include results for other specific types of cancer for the overall study group.

The study included 588 hourly employees who had worked in the AQ/ECH area for at least one year. These men had fewer deaths than expected from all causes combined (65/97) and from cancer (20/22) . For all specific types of can cer, observed and expected
numbers of deaths were under 10 and were too small to yield st atistically stable results. Hourly AQ/ECH workers had 6 lung cancer deaths (expected = 8). Although only 44 workers in this area had been employed in epichlorohydrin operations, and most of these also had worked longer in anthraquinone dye operations, this gr oup had statistically significantly more lung cancer deaths than expected (4/0.91, P = 0.03).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Hourly maintenance workers (N = 514), many of whom were employed throughout the plant, had more deaths than expected from all cancers combined (37/25, SMR = 148, 95% CI = 103 to 202), from lung cancer (1 8/9.2, SMR = 196, 95% CI = 116 to 309), and from liver cancer (3/0.46).

The investigators concluded that the excess of lung cancer among maintenance workers
could have been due to an unidentified o ccupational exposure. They noted that the association they observed between epichlor ohydrin and lung cancer had not been found in research on other, larger groups of workers exposed to this chemical.

The study was limited by the small number of subjects and of deaths in the AQ/ECH work group. Other weaknesses were the absen ce of quantitative exposure estimates or data on nonoccupational risk factors. However, even if such data had been available, the study would have had little pow er to assess dose response, confounding, or interaction for specific forms of cancer.

3.1.2.2 Case-control study of lung cancer.



### Barbone et al . (1992) conducted a nested case-contro l study of lung cancer that included as cases 51 white male employees of the New Jersey plant who had died of or developed primary lung cancer by October 1988. Cont rols were 102 individually matched employees chosen randomly from among other white male employees who were born in the same year and were alive as of the case subject’s death or dia gnosis date. Plant job records identified each pro cess area and building in whic h a subject had carried out production-related work. The AQ/ECH area c onsisted of five buildings with the following operations: (1) anthraquinone produc tion, (2) production of anthraquinone dye intermediates, (3) anthraquinone dye synthesis, (4) anthraquinone dye standardization, and (5) epichlorohydrin producti on. Interviews with long-term employees (not subjects) provided more detailed work history informa tion on building-specific work, as well as an ordinal score for subjects’ pot ential exposure to asbestos an d epichlorohydrin. Interviews with subjects or family members furnishe d data on smoking and on jobs and exposures outside the New Jersey plant and were comple ted for 69% of cases and 74% of controls. Plant medical records, available for 67% of cases and 68% of controls, supplied information on smoking and exposures to chemi cals that had been re ported to the plant medical department. The odds ratio (OR) (the ra tio of the rates of lung cancer in subjects exposed and not exposed to a given factor) was used as the measure of association.

As expected, smoking was positively and consis tently associated with lung cancer. Lung cancer also was positively associated with ever having worked in the AQ/ECH area (OR = 2.4, 95% CI = 1.1 to 5.2), based on 21 exposed cases and 24 exposed controls. This association was limited to subjects who had worked in the area for at least 10 years (smoking-adjusted OR = 4.6, 95% CI = 0.9 to 23), but there was no tr end of increasing OR with increasing time spent in the area. Lung cancer was positively associated with employment in each of the five building- specific operations in the area, but the association was statistically significant only for anthraqu inone production (OR = 12, 95% CI = 1.4 to 99) and was of borderline stat istical significance for anthraquinone dye standardization (OR = 3.3, 95% CI = 1.0 to 11). The positive association between work in the AQ/ECH area and lung cancer did not appear to be due to exposure to asbestos or9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### epichlorohydrin or to smoking. Results for other process areas we re not striking or statistically significant.

Workers in the AQ/ECH area potentially were exposed to chlorine and to epichlorohydrin, in addition to anthraquinone dyes and intermediates. The study reported a positive association between acute exposure to chlori ne and lung cancer (OR = 5.7, 95% CI = 1.1 to 30). Adjustment for smoking ra ised the OR for chlorine to 27, but these results were based on only 6 exposed cases and 3 exposed controls. Chlorine was encountered in the AQ/ECH area by 3 of the 6 chlorine-exposed case subjects and 2 of the 3 chlorine-exposed control subjects. A pos itive [but not statistically significant] association between lung cancer and exposur e to epichlorohydrin (OR = 1.7, 95% CI = 0.7 to 4.1) was reported, based on 12 exposed cas es and 18 exposed controls. The latter
association was restricted to workers with short-term and relatively low exposure to epichlorohydrin. Only 3 of the 21 case s ubjects in the AQ/ECH area had spent any appreciable time in epichlorohydrin operations.

Barbone et al . (1992) noted that the positive association between lung cancer and anthraquinone dye operations could have been due to occupational exposure (to anthraquinone or other chemicals used in the area) or to chance. The study lacked data on cumulative exposure to anthraquinones. If anthraquinones cause lung cancer, the lack of
an association between durati on of employment in the vari ous anthraquin one areas and lung cancer in this study could have been due to poor correlati on between duration of work in the areas and amount of exposure. Th e study also did not have quantitative or comprehensive data on exposure to chlo rine and had only crude estimates of epichlorohydrin exposure. Thus, the impact of these agents on the association between anthraquinone work areas and lung cancer may not have been accurately assessed.
Barbone et al . (1992) noted that the only other da ta available on human exposure to anthraquinones, the study by Gardiner et al . (1982), did not find an excess of lung cancer among workers with po tential exposure.

3.1.2.3 Case-control study of central nervous system tumors


### The case-control study of CNS tumors include d as cases 11 white male employees who were known to have died of or developed a primary CNS tumor by October 1988 (7 with astrocytoma or glioblastoma, 2 with meni ngioma, and 2 with a benign tumor) (Barbone et al. 1994). Controls were 44 individually matc hed subjects chosen randomly from among other white male employees who were born in th e same year and were alive as of the case subject’s death or diagnosis date. Procedures for obtaining and clas sifying work history data, estimating exposure to epichlorohydrin, conducting interviews, and obtaining data from plant medical records were similar to those used in the cas e-control study of lung cancer (Barbone et al. 1992). Interviews, completed for 73% of cases and 84% of controls, asked about non-pl ant jobs and occupational e xposures, smoking habits, and history of head trauma, irradi ation to the head, epilepsy, and use of antiepileptic drugs.

The occurrence of CNS tumors was statistica lly significantly and positively associated with anthraquinone dye intermediate producti on, but only 3 case subj ects and no controls had worked in this process area. Positive asso ciations also were found with work in the epoxy resin process area and in the azo dye area and with estimated exposure to9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### epichlorohydrin. Barbone et al . (1994) reported that the a ssociation with anthraquinone dye intermediate production was “nonindepende nt” of the association with exposure to epichlorohydrin and concluded that the sma ll study size precluded fi rm conclusions about the causes of CNS tumors in the plant workfo rce. All of the results were based on small numbers of exposed subjects and were quite imprecise. The study also did not include quantitative estimates of exposure to anthraqu inone or other chemicals and was not able to evaluate anthraquinone dose response or to adequately assess the possibility that the association between anthraquinone dye inte rmediate production and CNS tumors was confounded by the other positive associations observed, or vice versa.

3.1.2.4 Retrospective follow-up study update


### Sathiakumar and Delzell (2000) expanded and updated the original study by Delzell et al

. (1989), adding women and nonwhite men to the study group and extending follow-up through the end of 1995. The expanded study compared the mortality rates for 3,266 employees with the age-, race-, gender-, a nd time-period-specific rates for the general population of New Jersey.

The total study group had 728 observed and 809.8 expected deaths from all causes combined (SMR = 90, 95% CI = 83 to 97), 225 observed and 231.7 expected total cancer deaths (SMR = 97, 95% CI = 85 to 111), 89 observed and 73.2 expected lung cancer deaths (SMR = 122, 95% CI = 98 to 150), a nd 8 observed and 6.0 expected CNS cancer deaths (SMR = 134, 95% CI = 58 to 264). White male employees who had worked in the AQ/ECH area (N = 842) had an excess of lung cancer, based on 32 observed and 19.1 expected deaths (SMR = 168, 95% CI = 115 to 237), and had 3 deat hs from CNS cancer (1.5 expected, SMR = 205, 95% CI = 42 to 598). Maintenance workers at the plant also had more deaths from lung cancer than e xpected (40/26.2, SMR = 153, 95% CI = 109 to 208).

The lung cancer excess among workers in the AQ/ECH area was not consistently larger in subgroups with long duration of employment (5 years or more) or long potential induction time in the area (20 years or mo re); however, small numbers limited the
conclusions that could be drawn from these su bgroup analyses. In analyses that compared the lung cancer mortality rate of AQ/ECH area workers with that of other plant employees, adjusting for age, calendar time, and employment in maintenance operations, the OR was 1.7 (95% CI = 1.1 to 2.6) for the AQ/ECH area, confirming the increased lung cancer mortality for this area seen in the SMR analysis.

This study, like the study of the same workforce by Barbone et al . (1992), found a positive association between employment in anthraquinone dye operations and lung
cancer. There was no discernible trend of increasing lung cancer SMR or OR with increasing length of employment in anthra quinone dye operations, but this could be explained by poor correlation between durati on of employment and cumulative exposure to anthraquinones or other chemicals used in the area. Sathiakuma r and Delzell (2000)
also found an increase in CNS cancer among workers potentially exposed to anthraquinones, based on small numbers, consistent with the report by Barbone et al

. (1994).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Compared with the previous follow-up study of this group of workers, the update had 72% more person-years and twice as many observed deaths. The median length of follow-up was 27 years, and 22% of the work ers were deceased. However, numbers of deaths from specific types of cancer were sm all in the work-area-specific analyses, and the update lacked direct exposure estimate s and data on nonoccupational risk factors. These limitations impede the detection of causal associations.

3.2 Summary Only two groups of workers exposed to anth raquinone dyes and intermediates have been investigated. All of the studies of these workers have limitati ons that make detection of causal relationships difficult, including small size, potential exposure to multiple agents in the workplace, and lack of quantitative exposure data. In addition, the follow-up studies lack information on nonoccupational can cer risk factors. The results of the pertinent research are not consistent. Th e study of Scottish workers reported small increases in esophageal and prostate cancer and found no excess of respiratory cancer (64 observed vs. 66.2 expected) or brain cancer (4/3.5 ). In contrast, the studies of New Jersey workers reported that work in anthraqui none dye operations was positively associated with lung cancer and with CNS tumors. In th e latter investigations , the results for CNS tumors were based on a very small number of observations. The data on anthraquinone dye operations and lung cancer were more precise, and no confounding factor was identified that could explain the positive association. Thus, th e increase in lung cancer in anthraquinone dye workers at the New Jersey plant may have been due to occupational exposure. However, the specific agent(s) responsible for the excess have not been
identified. Table 3-1 summarizes the human cancer studies on anthraquinone dye
workers.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone14Table 3-1. Human cancer studies on anthraquinone dye workersReferenceStudydesignPopulationExposureEffectsCommentsGardineretal.1982ScotlandCohort study1,975 men employed atanthraquinone dyestuffplant for at least 6months from 1956–1965,followed up until June1980Potential exposure tosubstituted anthraquinoneswas assessed from personnelrecords.SMR (observed/expected deaths)all deaths 76.9 (470/611.5)all cancers 86.2 (129/149.7)esophagus 139.5 (6/4.3)stomach 77.3 (14/18.1)intestinal tract 63.6 (7/11.0)pancreas 58.8 (4/6.8)bladder 71.4 (4/5.6)respiratory 96.7 (64/66.2)prostate 50.0 (4/8.0)rectum 88.2 (6/6.8)Smoking and alcoholconsumption were notascertained.Delzellet al.1989New Jersey,USARetrospectivecohort study2,642 white menemployed at a dye andresin plant for at least 6months from 1952–1985588 hourly workersemployed in theAQ/ECH area for at leastone yearExposure was asssessedfrom personnel records.Three manufacturing areas:AQ/ECH area:anthraquinone, AQ dye andAQ dye intermediates, andECH production andformulation of AQ dyesNorth dye area:azo dyesand intermediatesPlastics and additive areaObserved/expectedAQ/ECH area workers:all deaths 65/97all cancers 20/22lung cancer 6/8Epichlorohydrin workers:(1961–1965) (N = 44)lung cancer 4/0.91;P= 0.03Workers in the AQ/ECHarea also were exposedto other chemicals,including aniline- andbenzene-relatedcompounds,tetrachloroethylene,chlorine, sulfuric acid,and mercury.Nonoccupational riskfactors were notacertained.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone15ReferenceStudydesignPopulationExposureEffectsCommentsBarboneet al.1992New Jersey,USANested casecontrol study(Delzellet al.1989)Lung cancerCases:51 white maleemployees who died ofor developed lung cancerby 10/1988Controls:102 employeesrandomly selected andmatched for age andalive as of case’s deathor diagnosis dateThe AQ/ECH area consistedof 5 buildings:(1) AQ production(2) production of AQintermediates(3) AQ dye synthesis(4) AQ dye standardization(5) ECH productionOR (95% CI); no. of exposed casesWorked in AQ/ECH area:ever 2.4 (1.1–5.2); 21> 10 years 4.6 (0.9–23)Building in AQ/ECH area:AQ production 12 (1.4–99); 6AQ dye/standardization3.3 (1.0–11); 8ECH production:3 exposed cases, 0 exposed controls;OR not calculatedORs were computedwith and withoutadjustment for smokingand other job exposures.Only smoking proved tobe a confounder and waskept in analyses if itmodified the strength ordirection of anassociation.Lung cancer waspositively associatedwith chlorine exposure.Barboneet al.1994New Jersey,USANested casecontrol study(Delzellet al.1989)CNS tumorsCases:11 white maleswho died of ordeveloped a primaryCNS tumor by 10/1988Controls:44 employeesmatched for age andalive as of the case’sdeath or diagnosis dateSame as Delzellet al.(1989) and Barboneet al.(1992)OR (95% CI); no. of exposed casesAQ dye intermediates:production∞(1.7–∞); 3routine potential exposure to ECH4.2 (0.7–26); 4Three of four casesubjects with potentialexposure to ECH alsoworked in either the AQdye intermediates or azodyes areas.Sathiakumarand Delzell2000New Jersey,USACohort studyExpansion ofDelzellet al.(1989)3,266 employees (menand women) at a dye andresin plant, followedthrough 1995728 deathsSame as Delzellet al.(1989)SMR (95% CI); observed/expectedEntire cohort:all deaths 90 (83–97); 728/809.8all cancers 97 (85–111); 225/231.7South dye area, white males(N = 842):lung cancer 168 (115–237); 32/19.1Nonoccupational riskfactors (includingsmoking) were notascertained.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 4 Studies of Cancer in Experimental Animals

The NTP (1996) carried out studies in male and female rats and mice in which ADBAQ was administered in the diet continuously fo r either 13 weeks or two years. In addition, the NTP conducted a 15-month high-dose stop-ex posure study in rats (9 months of exposure to ADBAQ followed by 6 months on a control diet). Data from the 13-week
studies in rats and mice we re reported by Fleischman et al. (1986), and results from the two-year studies in rats were reported by Harada et al. (1989) and Maronpot et al. (1989a). The results of the NTP studies and additional experiments by Maronpot et al. (1989b) are summarized below.

The NTP obtained two lots of ADBAQ for th e studies and confirme d the identity and purity of each lot by chemical analysis. Fleischman et al . (1986) reported that the study material was technical-grade ADBAQ of 80% to 85% purity, determined by HPLC. The major impurities were identified as anthraquinone (9.3%) and either 2-aminodibromoanthraquinone or a different 1-aminodibromoanthraq uinone (4.7%). The
same material was used for the first 2 months of the two-year studi es. The study material used for the remaining 22 months of the two- year studies was found to be approximately 97% pure, with six unidentified impurities.

Little information is available on the potential carcinogenicity of the three contaminants identified in the initial ADBAQ sample. An thraquinone has not been reviewed in an IARC monograph. It has been the subject of an NTP rodent bioassay (TR-494), which
concluded that there was some evidence of carcinogenic activity in male F344/N rats (kidney and urinary bladder), clear evidence of carcinogenic activity in female F344/N rats (liver, kidney, and urinary bladder), a nd clear evidence of ca rcinogenic activity in
male and female B6C3F 1 

### mice (liver). All of the reporte d sites for anthraquinone-induced neoplastic lesions also are ta rget sites for ADBAQ. No information was available on the potential carcinogenicity of the two other contaminants.

4.1 Rats
The NTP (1996) conducted three studies of ADBAQ in rats: a 13-week study, a two-year (104-week) study, and a 15-month stop-exposur e study. Male and female F344/N rats were administered ADBAQ in their diet at concentrations ranging from 1,000 to 50,000 ppm.

4.1.1 Thirteen-week study



### Groups of F344/N rats (10 of each sex) were fed diets containing ADBAQ at a concentration of 0, 2,500, 5,000, 10,000, 25,000, or 50,000 ppm for 13 weeks. After the 13-week exposure, necropsies were performe d, in which tissues were collected and processed for histologic examination (a comple te list of tissues and organs examined is included in Appendix B). Survival to st udy termination and mean final body weight (b.w.) as a percent of m ean control body weight ar e shown in Table 4-1. The nonneoplastic lesions of the liver included ba sophilic foci, clear-c ell foci, eosinophilic foci, cytomegaly, bile duct hyperplasia, in flammation, fibrosis, necrotizing cholangitis, vacuolar degeneration, and pigmentation. The incidence of hepato cellular cytomegaly9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### (hypertrophy) was significantly increased ( P < 0.01, Fisher’s exact test) in both male and female rats at 25,000 and 50,000 ppm and in fema le rats at 10,000 ppm. The incidences
of inflammation around bile ducts and bile duct hyperplasia also were significantly increased ( P< 0.01, Fisher’s exact test) in male and female rats in the 25,000- and 50,000-ppm groups. The kidney lesions includ ed renal tubule pigmentation (in both males and females in all exposure groups) and hyaline droplet accumulation (in males only in the 2,500-, 5,000-, and 10,000-ppm grou ps). Data for other exposure-related lesions in all groups are su mmarized in the NTP report (Appendix A, p. A-26, Table 4).

Table 4-1. Survival and body weight in F 344/N rats following dietary exposure to ADBAQ for 13 weeks

Exposure concentration (ppm)

End point 0 2,500 5,000 10,000 25,000 50,000

Males

Survival to study termination 0 10/10 9/10 10/10 10/10 7/10

Final mean b.w. ± SD a (g)

% of control mean b.w. 358 ± 3

100 325 ± 3

91** 328 ± 3

92** 310 ± 3

86** 232 ± 3

65** 164 ± 6

46**

Females

Survival to study termination 10/10 10/10 10/10 10/10 10/10 9/10

Final mean b.w. ± SD (g)

% of control mean b.w. 211 ± 3

100 197 ± 3

93** 188 ± 3

89** 185 ± 2

88** 159 ± 2

75** 130 ± 4

61**

a SD = standard deviation.

**Significantly different ( P ≤ 0.01) from the control group by Williams’s or Dunnett’s test.



### Fleischman et al . (1986) reported results from the NT P’s 13-week toxicology studies of ADBAQ in Fischer 344/N rats. Both male and female rats in the 25,000- and 50,000-ppm groups were emaciated and lethargic, and their body weights were significantly lower ( P < 0.01, Williams’s or Dunnett’s test) than thos e of controls. Pathological findings of nonneoplastic lesions of the liver and kidney we re the same as those reported in NTP (1996). The uterus was atrophied in fema les fed ADBAQ at a concentration of 10,000 ppm or higher, and the thymus was atroph ied in males at 5,000 ppm or higher and in females at 25,000 ppm or higher. A consistent finding in both sexes was anemia, with a significant reduction ( P < 0.01, Student’s t test) in the number of red blood cells at all
concentrations tested. The au thors concluded that ADBAQ was “markedly toxic in rats.”

4.1.2 Two-year study



### Dietary concentrations of ADBAQ for the two-year study in rats were based on the results of the 13-week study. Exposure-related lesions of the liver, kidney, and spleen were present mainly in the 25,000- and 50,000-ppm groups in the 13-week studies. Exposure levels of 0, 2,000, 5,000, and 10,000 ppm we re selected for the two-year study. Although the 10,000-ppm exposure level could be considered slightly high, based on body-weight effects (Table 4-1), this dietary concentration wa s predicted not to adversely
affect the health or survival of the animals because it did not cause liver toxicity in the9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### 13-week study. A higher exposure level (20,000 ppm) was chosen for the stop-exposure study.

Male and female F344/N rats were obtaine d at approximately six weeks of age and quarantined for 12 to 14 days (males) or 9 da ys (females) before the study began. Groups of rats (70 of each sex) received ADBAQ in the diet at a concentration of 0, 5,000, or 10,000 ppm, and 50 rats of each sex received ADBAQ at 2,000 ppm. A subset from each exposure level (10 of each sex) was designate d for an interim evaluation after 9 months.
Additional subsets from the control a nd 10,000-ppm groups (10 of each sex) were evaluated after 15 months. Major tissues were collected for histol ogical examination. The exposure protocol is su mmarized in Figure 4-1.

Concentration Duration (sample size)

0 ppm 9 months (N = 10)

(control) 15 months (N = 10)

24 months (103–104 weeks) (N = 50)

2,000 ppm 9 months (N = 10)

24 months (103–104 weeks) (N = 40)

5,000 ppm 9 months (N = 10)

24 months (103–104 weeks) (N = 60)

10,000 ppm 9 months (N = 10)

15 months (N = 10)

24 months (103–104 weeks) (N = 50)

Shaded bars indicate ADBAQ-contai ning diet, and open bars indicate control diet. The total length of each bar indicates the time to study terminati on and the sacrifice of animals in that group.



### Figure 4-1. Exposure protocol for the two- year study of ADBAQ in F344/N rats

Survival to study termination, final mean body weight as a percentage of mean control body weight, and neoplastic lesi on incidence at two years ar e summarized in Table 4-2. Neoplastic lesions were observed in the liver, large intestine, kidney, urinary bladder, and forestomach. Neoplasms of the liver include d both single and multiple adenomas and single and multiple carcinomas. The large intestine contained single and multiple adenomatous polyps and single and multiple carcinomas. Renal lesions included renal tubule adenoma and carcinoma, and urinary bladder lesions included transitional-cell papilloma and carcinoma. Lesions of the fo restomach included squamous-cell papilloma and carcinoma, but the increased incidences did not reach statis tical significance. Significant increases were seen at all exposure levels for tumors of the liver, large intestine, and kidney. The inci dence of urinary bladder tumors also was significantly increased in high-dose males and mid- and high-dose females.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 4-2. Survival, body weight, and tumo r incidence in F344/N rats following dietary exposure to ADBAQ for two years

Exposure concentration (ppm)

End point 0 2,000 5,000 10,000

Males

Survival to study termination 26/50 24/40 21/60 10/50***
Final mean b.w. (g) (% of control mean b.w.) 406 (100) 349 (86) 341 (84) 283 (70)

Neoplastic lesions (two-year data only)

Liver N = 50 N = 40 N = 59 N = 50

Adenoma 1 20*** 40*** 34***

Carcinoma 1 12*** 55*** 46***

Adenoma or carcinoma 2 25*** 57*** 47***

Large intestine N = 50 N = 40 N = 59 N = 50

Adenomatous polyp 0 13** 51** 40**

Carcinoma 0 1 11** 17**

Kidney (renal tubule) N = 50 N = 40 N = 59 N = 50

Adenoma 2 10** 11* 14***

Carcinoma 0 0 2 1

Adenoma or carcinoma 2 10** 13** 15***

Urinary bladder (transitional cell) N = 50 N = 38 N = 58 N = 50
Papilloma 0 1 2 8**

Carcinoma 0 0 1 4*
Papilloma or carcinoma 0 1 3 12***

Females

Survival to study termination 38/50 32/40 38/60 12/50***

Final mean b.w. (% of control mean b.w.) 362 (100) 290 (80) 234 (65) 194 (54)

Neoplastic lesions (two-year data only)

Liver N = 50 N = 40 N = 60 N = 48

Adenoma 0 28*** 47*** 29***

Carcinoma 0 12*** 57*** 45***

Adenoma or carcinoma 0 33*** 59*** 47***

Large intestine N = 50 N = 40 N = 60 N = 49

Adenomatous polyp 0 28** 53** 43**

Carcinoma 0 2 21** 8**

Kidney (renal tubule) N = 50 N = 40 N = 60 N = 48

Adenoma 0 3* 16*** 16***

Carcinoma 0 0 0 2

Adenoma or carcinoma 0 3* 16*** 16***

Urinary bladder (transitional cell) N = 50 N = 40 N = 60 N = 46

Papilloma 0 2 7* 9**9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

21 Exposure concentration (ppm)

End point 0 2,000 5,000 10,000

Carcinoma 0 0 8** 16***

Papilloma or carcinoma 0 2 17*** 26***

*Significantly different ( P ≤ 0.05) from the control group by the logistic regression test.

**Significantly different ( P ≤ 0.01) from the control group by the logistic regression test.
***Significantly different ( P ≤ 0.001) from the control group by lifetable pairwise comparisons (survival data) or the logistic regression test (tumor data).



### Hepatocellular, large intestine, kidney, and bl adder tumors also were found in rats that received ADBAQ at 10,000 ppm fo r 15 months (interim sacrif ice). The incidences of hepatocellular adenoma and carcinoma in 10/10 male and 9/10 female rats were significantly greater ( P < 0.01, Fisher’s exact test) than in controls. A similar incidence of
liver tumors was seen in the 15-mont h stop-exposure study (discussed below).

4.1.3 Fifteen-month stop-exposure study



### The NTP conducted a stop-exposure study to ev aluate the potential for progression or regression of ADBAQ-induced lesions. Male and female F344/N rats were obtained at approximately six weeks of age and quarantin ed for 12 to 14 days (males) or 9 days (females) before the study began. Groups of rats (10 of each sex) were fed a diet containing ADBAQ at a concentr ation of 20,000 ppm and were s acrificed after 9 months (9-month exposure group). Additional groups (10 of each sex) were fed a diet containing ADBAQ at 20,000 ppm for 9 months , followed by the control diet for 6 months (9-month stop-exposure group). The final group (20 of each sex) received only the diet containing ADBAQ at 20,000 ppm for 15 m onths (15-month exposure group). The exposure protocol for the stop-exposure studie s is summarized in Figure 4-2.

Concentration Duration (sample size)

0 ppm 9 months (N = 10)

(control) 15 months (N = 10)

20,000 ppm 9 months (N = 10)

9 months (N = 10)

15 months (N = 20)

Shaded bars indicate ADBAQ-containing diet, and open bars indicate control diet. The total length of each bar indicates the time to study termination and the sacrifice of animals in that group.



### Figure 4-2. 15 Month stop exposure study9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Survival to study termination, final mean body weight as a percentage of mean control body weight, and neoplastic lesion incidence are summarized in Table 4-3. Neoplastic
lesions were found in the liver of male and fe male rats at the 15-month evaluation in both the 9-month stop-exposure group and the 15-m onth exposure group. The large intestines contained adenomatous polyps of the rectum in exposed males and females; however, the
tumor incidence was statistica lly significantly increased ( P < 0.05, Fisher’s exact test) only in males in the 15-month exposure group and females in the 9-month stop-exposure group. The incidences of tumors of the kidney and urinary bladder we re not statistically significantly increased, but the numerical increas es in these tumors are concordant with those observed in the two-year bioassay (see Table 4-2).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 4-3. Survival, body weight, and tumor incidences in F344/N rats in the 15- month stop-exposure study of ADBAQ (20,000 ppm)

9-month evaluation 15-month evaluation

End point Control a 9-mo.

exposure Control a 9-mo. stopexposure 15-mo.

exposure

Males

Survival to study termination 70/70 30/30 57/60 7/10 17/20
Final mean b.w. (g)

% of control mean b.w. 453

100 b 364

80 484

100 388

80 b 364

75

Neoplastic lesions

N = 10 N = 10 N = 10 N = 10 N = 20

Liver (hepatocellular adenoma or

carcinoma) 0 2 0 9** 20**

Large intestine, rectum

(adenomatous polyp) 0 0 c 0 3 7*

Kidney (renal tubule adenoma) 0 0 0 3 2
Urinary bladder:

squamous-cell carcinoma 0 0 d 0 0 1 e

transitional epithelial papilloma 0 1 d 0 0 3 e

transitional epithelial carcinoma 0 0 d 0 0 1 e

Females

Survival to study termination 69/70 29/30 59/60 10/10 12/19
Final mean b.w. (g)

% of mean control b.w. 258

100 b 211

82 328

100 258

79 b 220

67

Neoplastic lesions

N = 10 N = 10 N = 10 N = 10 N = 18

Liver (hepatocellular adenoma or

carcinoma) 0 2 0 8** 16**

Large intestine, rectum

(adenomatous polyp) 0 0 0 5* 3 f

Kidney (renal tubule adenoma) 0 0 0 3 2 f

Urinary bladder:

squamous-cell carcinoma 0 0 0 1 4
transitional epithelial papilloma 0 0 0 0 1 g

transitional epithelial carcinoma 0 0 0 0 1

a Control animals are those from the two-year study.

b Mean body weights are for all animals at 37 weeks in control and 15-month exposure groups.

c A single adenomatous polyp of the colon was observed in this group.

d N = 9; e N = 19; f N = 17.

g A single squamous-cell papilloma of the urinar y bladder also was observed in this group.

*Significantly different ( P ≤ 0.05) from the control group by Fisher’s exact test.

**Significantly different ( P ≤ 0.01) from the control group by Fisher’s exact test.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Maronpot et al. (1989a) analyzed the incidence of hepa tic foci that eith er stained positive

for γ -glutamyl transpeptidase (GGT) or were identified as altered hepatocellular foci (AHF) in hematoxylin and eosin–stained sec tions of livers from female F344/N rats fed ADBAQ at a concentration of 10,000 or 20,000 pp m for 15 months (in the NTP two-year and stop-exposure studies). The number of foci per cubic centimeter and the focus volume fraction (percent of liver volume occupi ed by foci) were significantly increased in both the low- and high-dose groups, a nd the mean focus volume was significantly increased in the high-dose group (Table 4- 4). The authors concluded, however, that increases in AHF in the two-year carcinoge nicity study were insufficient evidence of hepatocarcinogenicity in the abse nce of a liver tumor response.

Table 4-4. Occurrence of altered hepatocellular foci in female F344/N rats following dietary exposure to ADBAQ for 15 months

Exposure concentration (ppm) (N = 10) Foci/cm 3 (mean ± SE) Volume fraction (mean % ± SE) Focus volume (mean mm 3 ± SE)

0 61.27 ± 37.6 0.19 ± 0.16 0.010 ± 0.006

10,000 1,005.76 ± 254.7** 2.70 ± 0.57* 0.024 ± 0.007

20,000 1,217.53 ± 287.0 b ** 5.88 ± 0.79*** 0.072 ± 0.021**

Source: Maronpot et al. 1989a.
*Significantly different ( P < 0.05) from the control group (test not specified).
**Significantly different ( P < 0.01) from the control group (test not specified).
***Significantly different ( P < 0.001) from the control group (test not specified).



### Harada et al. (1989) performed qualitative and qu antitative assessm ent of altered hepatocellular foci in histological material from the NTP two-year and 15-month stopexposure studies of ADBAQ in rats. Liver sa mples collected at 9-, 15-, and 24-month intervals were included in the analysis. The authors separated both basophilic and eosinophilic AHF into common and atypical ty pes. No exposure-related changes in number, size, or volume fraction were f ound for commonly occurring AHF. However, these parameters were significantly increased for atypical AHF and were correlated with the level and duration of exposure to ADBAQ in both male and female rats. The numbers of AHF in the two higher-dose groups ( 10,000 and 20,000 ppm) tended to decrease over time, and this trend correlated with the occurr ence of liver tumors in the animals selected for AHF analysis.

4.2 Mice
The NTP (1996) conducted two studies of ADBAQ in male and female B6C3F 1 

### mice: a 13-week study and a two- year (104-week) study.

4.2.1 Thirteen-week study



### Groups of B6C3F 1 

### mice (10 of each sex) were fed diets containing ADBAQ at a concentration of 0, 2,500, 5,000, 10,000, 25,000, or 50,000 ppm for 13 weeks. After the 13-week exposure, necropsies were performe d, in which tissues of all animals were collected, weighed, and processed for histologi c examination (a complete list of tissues9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

25 

### and organs examined is included in Appendix B). Survival to study termination and final mean body weight as percent of control mean body weight are summarized in Table 4-5. Nonneoplastic lesions of the liver were obs erved, including pigmentation in males (5/10 at 2,500 ppm and 8/10 to 10/10 at 10,000 ppm or higher) and females (1/10 at 25,000 and 50,000 ppm) and centrilobular hypertrophy in ma les only (8/10 to 10/10 at 10,000 ppm or higher). Data for exposure-related nonneoplastic lesions in all groups are summarized in the NTP report (Appendix A, p. A-57, Table 20).

Table 4-5. Survival and body weight in B6C3F 1 

### mice following dietary exposure to ADBAQ for 13 weeks

Exposure concentration (ppm)

End point 0 2,500 5,000 10,000 25,000 50,000

Males

Survival to study termination 10/10 10/10 9/10 10/10 9/10 10/10

Final mean b.w. ± SD (g)

% of control mean b.w. 30.5 ± 0.6

100 30.6 ± 0.6

100 30.7 ± 0.7

101 32.1 ± 0.4

105 30.9 ± 0.6

101 31.5 ± 0.4

103

Females

Survival to study termination 10/10 10/10 10/10 9/10 10/10 10/10

Final mean b.w. ± SD (g)

% of control mean b.w. 24.0 ± 0.2

100 24.7 ± 0.6

103 25.0 ± 0.6

104 25.0 ± 0.4

104 23.6 ± 0.3

98 24.7 ± 0.4

103



### Fleischman et al . (1986) also reported the results from the NTP’s 13-week study of ADBAQ in B6C3F 1 

### mice. The effects of exposure to ADBAQ for 13 weeks were less pronounced in mice than in rats. Body weight was not affected in either sex at any exposure level (Table 4-5). Dose-related lesions of the liver were observed in male mice and included centrilobular gl ycogen depletion at 10,000 ppm or higher and pigmentation at all exposure levels. The authors concl uded that “at comparable doses, ADBAQ was considered to be markedly toxic in rats a nd of minimal, non-life-th reatening toxicity in
mice.”

4.2.2 Two-year study



### Dietary concentrations of ADBAQ for the two-year study in mi ce were based on the results of the 13-week study, specifically, on th e frequency and severi ty of centrilobular hypertrophy of the liver observed in male mi ce. Because only lesions of mild severity were observed in groups exposed to ADB AQ at 10,000 or 25,000 ppm, and lesions of moderate severity were observed in th e 50,000-ppm group, exposure concentrations for the two-year study were set at 0, 10,000, and 20,000 ppm. Other consid erations included consistency among males and females (as female s could have been given higher exposure concentrations) and correspondence to the conc entrations selected for the stop-exposure study in rats (see Appendix A, p. A-57).

Male and female B6C3F 1 

### mice were obtained at five week s of age and quarantined for 15 days before the study began. Groups of mice ( 60 of each sex) received diets containing9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

26 

### ADBAQ at a concentration of 0, 10,000, or 20,000 ppm. A subset of 10 mice from each group was sacrificed at 15 months, and the remaining 50 mice were sacrificed at 24 months. The exposure protocol is shown in Figure 4-3.

Concentration Duration (sample size)

0 ppm 15 months (N = 10)

(control) 24 months (103–104 weeks) (N = 50)

10,000 ppm 15 months (N = 10)

24 months (103–104 weeks) (N = 50)

20,000 ppm 9 months (N = 10)

24 months (103–104 weeks) (N = 50)

Shaded bars indicate ADBAQ-contai ning diet, and open bars indicate control diet. The total length of each bar indicates the time to study terminati on and the sacrifice of animals in that group.



### Figure 4-3. Exposure protocol for the two-year study of ADBAQ in B6C3F 1 

### mice

The results of the two-year study are summa rized in Table 4-6. Feed consumption by exposed mice was generally similar to that of controls; however, final mean body weight was lower for exposed mice than for controls. Survival of exposed male mice was significantly lower than that of controls, but survival of exposed females was similar to that of controls. Hepatoce llular adenoma or carcinoma, forestomach squamous-cell papilloma, and alveolar/bronc hiolar adenoma were observed in exposed mice but not in
controls at the 15-month interim sacrifice. Incidences of hepa tocellular adenoma or carcinoma in high-dose males and females, s quamous-cell papilloma in high-dose males and low-dose females, and alveolar/bronc hiolar adenoma in high-dose and low-dose males were significantly higher than in c ontrols. At the end of the two-year study, significant increases were observed in the incidences of heptatocellular multiple adenomas and multiple carcinomas in both male and female mice at both exposure levels. In addition, squamous-cell papilloma a nd carcinoma of the forestomach were significantly increased in both males and fema les at both exposure levels. The incidence
of alveolar/bronchiolar adenoma or carci noma of the lung also was significantly
increased in both males and females.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### Table 4-6. Survival, body weight, and tumor incidence in B6C3F 1 

### mice following dietary exposure to ADBAQ for two years

Exposure concentration (ppm)

End point 0 10,000 20,000

Males

Survival to study termination 40/50 22/51*** 23/50***

Final mean b.w. (g) (% of control mean b.w.) 43.6 (100) 37.0 (85) 36.3 (83)

15-mo. 2-year 15-mo. 2-year

Liver (hepatocellular tumors) N = 50 N = 9 N = 51 N = 10 N = 50

Adenoma 10 2 38*** 4* 39***

Carcinoma 9 1 18* 0 21**

Adenoma or carcinoma 18 3 43*** 4* 42***

Hepatoblastoma 0 0 3 0 5*

Forestomach (squamous-cell tumors) N = 50 N = 9 N = 51 N = 10 N = 50

Papilloma 0 0 13*** 5* 16***

Carcinoma 0 0 12*** 0 13***

Papilloma or carcinoma 0 0 19*** 5* 27***

Lung (alveolar/bronchiolar tumors) N = 50 N = 9 N = 51 N = 10 N = 50

Adenoma 7 3 26*** 5* 24***

Carcinoma 3 0 4 0 1

Adenoma or carcinoma 10 3 28*** 5* 25**

Females

Survival to study termination 39/50 34/50 33/50

Final mean b.w. (g) (% of control mean b.w.) 39.2 (100) 33.6 (86) 31.9 (81)

15-mo. 2-year 15-mo. 2-year

Liver (hepatocellular tumors) N = 50 N = 10 N = 50 N = 10 N = 50

Adenoma 6 2 45*** 7** 49***

Carcinoma 0 0 23*** 1 27***

Adenoma or carcinoma 6 2 46*** 8** 50***

Hepatoblastoma 0 0 0 0 2

Forestomach (squamous-cell tumors) N = 50 N = 10 N = 50 N = 10 N = 50

Papilloma 2 4* 16*** 2 27***

Carcinoma 0 0 12*** 0 11***

Papilloma or carcinoma 2 4* 25*** 2 34***

Lung (alveolar/bronchiolar tumors) N = 50 N = 10 N = 50 N = 10 N = 49

Adenoma 4 3 17*** 2 13*

Carcinoma 0 0 0 0 2

Adenoma or carcinoma 4 3 17*** 2 15**

*Significantly different ( P < 0.05) from the control group by logistic regression test.

**Significantly different ( P < 0.01) from the control group by the logistic regression test.

*** Significantly different ( P < 0.001) from the control group by lifetable pairwise comparison (survival data) or the logistic regression te st (tumor data).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### 4.3 Summary
ADBAQ was toxic to male and female F344/N ra ts at dietary concentrations of 2,500 to 50,000 ppm consumed over a 13-week period, as evidenced by decreased body weight, nonneoplastic lesions in the liver and kidney, a nd anemia. Exposure of male and female
B6C3F 1 

### mice to ADBAQ at the same dietary con centrations resulted in lower apparent toxicity, as mice gained we ight at all ADBAQ exposure levels, and fewer types of nonneoplastic lesions of the liv er and kidney were observed.

In two-year dietary studie s with ADBAQ, male and female F344/N rats exhibited significantly increased incidences of neoplasms of the liver, large intestine, kidney, and urinary bladder. Significant increases in the incidences of tumors of the liver and large intestinge (females only) also were obser ved in a 15-month stop- exposure study in rats.
B6C3F 1 

### mice consuming ADBAQ-containing di ets for two years had increased incidences of hepatocellular adenoma and carcinoma, squamous-cell papilloma and carcinoma of the forestomach, and alveolar/b ronchiolar adenoma and carcinoma of the lung. The NTP report concluded that “under the conditions of these 2-year feed studies,
there was clear evidence for carcinogenic activity of 1-amino-2,4-dibromoanthraquinone
in male and female F344/N rats based on incr eased incidences of neoplasms in the liver, large intestine, kidney, and urinary bladder. There was clear evidence for carcinogenic activity of 1-amino-2,4-dibromoanthraquinone in male and female B6C3F 1 

### mice based on increased incidences of neoplasms in the liver, forestomach, and lung.”9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 5 Genotoxicity

Limited data are available on the genotoxic ity of ADBAQ. The few studies that have attempted to ascertain the mu tagenicity of ADBAQ have enc ountered solubility problems that make the assays difficult to execute and that complicate interpretation of the results.

5.1 Prokaryotic systems: Salmonella typhimurium
Salmonella typhimurium is the only prokaryote in whic h the mutagenicity of ADBAQ has been assessed. Several strains, including those that revert by base pair substitutions (TA100 and TA1535) and those that revert by frameshift mutati ons (TA98, TA1537, and TA1538), were used in the ADBAQ mutagenicity tests.

Haworth et al . (1983) showed that without rat or hamster liver microsomal (S9) metabolic activation, ADBAQ induced reverse mutation in strains TA98 and TA1537 (Table 5-1). In the presence of S9, reverse mutation was not induced in strain TA98, and strain TA1537 gave a variable response. ADB AQ was weakly mutagenic in strain TA100 (with or without S9 activation) and nonmutagenic in strain TA1535. The ADBAQ preparation used in these studi es had a labeled purity of 91.7 % and an analyzed purity of 79.7%. The researchers reported the pr esence of precipitate when ADBAQ concentrations were in the range of 333 to 10,000 μ g/plate (the highest concentration tested). The NTP (1996) noted that the posit ive or weakly positiv e mutagenic responses
in the Haworth et al . (1983) study were observed in stra ins in which reversion occurs by
frameshift mutations.

Table 5-1. Mutagenicity of ADBAQ in S. typhimurium a

Without S9 Hamster S9 Rat S9

Strain Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

TA100 + (weak) –/+ + (weak) –/+ –/+ –

TA1535 – – – – – –

TA1537 + + + – + –/+

TA98 + + –/+ – – –

Data were presented by Haworth et al . (1983) and NTP (1996). Trial summaries were obtained from Table G1 of NTP (1996).

a + = positive (a reproducible dose-related increase) ; – = negative; –/+ = equivocal (positive or weakly positive responses that were not replicated in a second trial).



### 5.2 Mammalian systems

Rodent cells were used to determine the mutagenicity and DNA-damaging potential of ADBAQ. No reports of in vivo mammalian genotoxicity assays were found in the
literature.

5.2.1 Mouse lymphoma L5178YTK +/- cells



### Harrington-Brock et al . (1991) carried out a limited ev aluation of the ability of ADBAQ to induce mutations at the thymidine kinase ( tk ) locus, but they were unable to test for9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

30 

### micronucleus formation because of ADBAQ’s limited solubility. ADBAQ was not mutagenic in the absence or presence of i nduced-rat liver S9 at the highest testable concentrations (25 μ g/mL without S9 and 20 μ g/mL with S9). The authors reported that the sample of ADBAQ was from the same batch evaluated in the NTP rodent carcinogenesis assay, and that the purity of the sample was 79.7%.

5.2.2 Chromosomal aberrations



### Chinese hamster ovary (CHO) cells were expos ed to various concen trations of ADBAQ and then analyzed for chromosomal aberrations (Loveday et al. 1990). The authors reported the presence of a precipitat e in the flasks c ontaining ADBAQ at 50 μ g/mL (the highest concentration tested). Two separate trials without S9 metabolic activation gave equivocal results (Table 5-2). In one trial, exposure to ADBAQ at 3.02 or 10.10 μ g/mL significantly increased the per centage of cells with chromo somal aberrations (Dunnett’s
test, P < 0.05). In the second trial, the percentage of cells wi th chromosomal aberrations was not significantly increased by ADBA Q at concentrations of 1.0, 3.0, 10.0, or 30.0

μ g/mL. In the presence of S9, the percentage of cells with chromosomal aberrations was not significantly increased by e xposure to ADBAQ at 3.02, 10.10, or 30.20 μ g/mL.

The NTP (1996) reported additional result s obtained in two trials performed by Environmental Health Research and Testing, In c. (Research Triangle Park, NC). In the absence of S9, the percentage of cells with chromosomal aberrations was significantly increased by exposure to ADBAQ at 50 μ g/mL in the first trial, but increases observed at 16, 30, or 50 μ g/mL in the second trial were not sta tistically significant. In the presence of S9, the percentage of cells with chro mosomal aberrations was not significantly increased by ADBAQ at 16, 50, or 100 μ g/mL. Based on these st udies, the NTP (1996) concluded that ADBAQ induced chromosomal aberrations only in the absence of S9.

5.2.3 Sister chromatid exchange



### Loveday et al . (1990) examined the effect of ADBAQ on the induction of sister chromatid exchange (SCE) in CHO cells. The chemical purity of ADBAQ was reported as greater than 79.7%, but the impurities were not identified. A trend test was used to describe the strength of evid ence for SCE induction. Results we re designated as negative, questionable, weakly positive, or positive, based on the trend test and the number of exposure levels resulting in at least a 20% incr ease over controls. Three exposure levels were used for each trial, and at least two ha d to produce a 20% increase for the result to be considered positive. In the presence or absence of induced-rat liver S9, ADBAQ induced SCE in a dose-dependent manner. In the absence of S9, ADBAQ induced at least
a 20% increase in the number of SCEs at a concentration of 10 μg/mL, but not at 5 or 2.5 μg/mL. In the presence of S9, ADBAQ induced at least a 20% incr ease in the number of SCEs at 3.01, 10.1, or 30.1 μ g/mL in trial 1 and at th e highest concentration (15

μ g/mL) in trial 2. The authors classified ADB AQ as weakly positive for SCE induction in the absence of S9 and positive (trial 1) or w eakly positive (trial 2) in the presence of S9 (Table 5-2).

The NTP (1996) also reported results from the Environmental Health Research and Testing Laboratory, which found that ADBAQ te sted at concentrations ranging from 5 to9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

31 

### 50 μg/mL induced SCE in the absence of S9 (results were positiv e in one trial and weakly positive in another). In the presence of S9, ADBAQ at 5, 16, 50, or 100 μ g/mL (one trial) did not induce SCE. The NTP (1996) noted that the discrepancy in SCE induction in the presence of S9 could not be explained by differences between the ADBAQ concentrations used by Loveday et al. (1990) and the NTP (1996).

Table 5-2. Induction of chromosomal ab errations and SCE in CHO cells by ADBAQ a

Metabolic activation Chromosomal aberrations Sister chromatid exchanges

Loveday et al.
1990 NTP 1996 b Loveday et al.
1990 NTP 1996 b

Trial 1 + + (weak)* + (weak) + -S9 Trial 2 – + (weak) NR + (weak)

Trial 1 – – + – +S9 Trial 2 NR NR + (weak) NR

a + = positive; – = negative; NR = not reported.

b Reported by the NTP (1996) as results from the Envi ronmental Health Research and Testing Laboratory.

* P < 0.05 for 50 μ g/mL concentration.

5.2.4 Ras mutations in tumors



### Hayashi et al. (2001) examined 8 forestomach squa mous-cell papillomas, 24 squamouscell carcinomas, 19 alveolar/bronchiolar adenomas, and 4 alveolar/bronchiolar
carcinomas for mutations in the H- ras and/or K- ras genes. These tumors were obtained
from male and female B6C3F 1 

### mice that were fed di ets containing ADBAQ at a concentration of 10,000 or 20,000 ppm for two years (NTP 1996). Ras codons 12, 13, and 61, which are hot spots for mutations in human and rodent tumors, were examined in tumors from exposed mice, in spontaneous tumors from control mice, and in histologically normal forestomach and l ung tissues. The ADBAQ-induced tumors had a higher frequency of ras mutations than did spontaneous tumors, and the mutations were found in both benign and malignant tumors (T able 5-3). The mutation spectra for the low- and high-dose groups were the same. Mutations in ras were not detected in any of the normal tissues. The predominant mutations in ADBAQ-induced tumors were CAA to
CTA transversions and CAA to CG A transitions in codon 61 of K- ras (lung tumors) or

H- ras (forestomach tumors). No A to T transver sions at the second base were detected in spontaneous tumors. Mutations observed in codons 12 or 13 genera lly occurred in both ADBAQ-induced and spontaneous tumors. The authors conclude d that alterations in the

ras gene might be an early event in ADBAQ-induced carcinogenesis.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

32 

### Table 5-3. Frequency and spectra of ras mutations in forestomach and lung tumors
from B6C3F 1 

### mice exposed to ADBAQ for two years a

K- ras codons H- ras codon

12 13 61 61

Group Total activated

ras (%) Total Total A → T A → G A → T A → G C → A

Forestomach tumors

Control b

ADBAQ

SCP

SCC 4/11 (36)

23/32 (72)

5/8 (63)

18/24 (75) 3

5

2

3 1

3

0

3 0

0

0

0 0

0

0

0 0

14

4

10 1

6

0

6 0

3

1

2

Lung tumors

Control c

ADBAQ

ABA

ABC 26/86 (30)

16/23 (70)

14/19 (74)

2/4 (50) 16

7

7

0 3

1

1

0 0

6

6

0 2

5

3

2 NT

NT

NT

NT NT

NT

NT

NT NT

NT

NT

NT

a SCP = squamous-cell papilloma; SCC = squamous-cell carcinoma; ABA = alveolar/bronchiolar adenoma; ABC = alveolar/bronchiolar carcinoma; NT = not tested.

b Spontaneous forestomach tumors in B6C3F 1 mice.

c Study controls (N = 4) combined with historical spontaneous lung tumors (N = 82) of B6C3F 1 mice.



### 5.3 Summary ADBAQ was mutagenic in S. typhimurium strains that revert by frameshift mutations, and the mutagenicity was decreased or elimin ated in the presence of induced hamster or rat liver S9 metabolic activation. ADBAQ did not induce detectable mutations at the tk locus in mouse lymphoma cells; however, the concentrations used (20 or 25 μ g/mL) were

less than those used for the S. typhimurium assays (100 to 10,000 μ g/mL) and slightly

less than those used to asse ss clastogenicity (up to 100 μ g/mL of ADBAQ). In CHO cells, ADBAQ induced chromosomal aberrations and SCE without metabolic activation; however, the results were inconsistent be tween different trials and laboratories. Forestomach and lung tumors induced by ADBAQ had a higher frequency of ras mutations than did spontaneous tumors. In particular, CAA to CTA transversions were common in ADBAQ-induced tumors but were ab sent in spontaneous tumors. The results obtained with ADBAQ must be viewed in the light of the limited solubility of this molecule and the uncertain purity of the preparations tested.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

33 

## 6 Other Relevant Data

6.1 Absorption, distribution, metabolism, and excretion The NTP (1996) reported studies on the ab sorption, distribution, metabolism, and excretion of ADBAQ. Male F344/ N rats were administered [ 14 

### C]ADBAQ orally or intravenously (i.v.), and disposition and elim ination of radioactiv ity (parent compound and/or metabolites) was monitored for up to 72 hours. The speci fic location of the radioactive carbon atom(s) in the AD BAQ molecule was not identified.

6.1.1 Absorption



### ADBAQ was readily absorbed from the gastrointe stinal tract. The percentage of the dose absorbed was inversely proportional to dose leve l. When rats were administered single oral doses of 2, 23, 118, 814, or 1,473 mg/kg b.w., 90% of the lowest dose (2 mg/kg) was absorbed, but only 2% of the 814-mg/kg dose (NTP 1996).

6.1.2 Distribution and metabolism



### ADBAQ was rapidly and widely distributed after i.v. admini stration. Radioactivity in blood was located primarily in plasma, as re flected by a whole blood:plasma ratio of 0.5. Fifteen minutes after i.v. administration (0.4 mg/kg b.w.), radioactivity was detected in most soft tissues, with the hi ghest concentrations in excretory organs, lung, kidney, small intestine, liver, adipose tissu e, and adrenal glands. At 15 minutes, tissue:blood ratios of radioactivity for these tissues were 3.0 or gr eater. Tissue:blood rati os declined during the 72 hours after administration, remaining greate r than 1.0 only in liv er and kidney at 72 hours. No quantitative data on tissue distribution were reported (NTP 1996).

Two hours after administration, only 3% of circ ulating radioactivity was in the form of unchanged parent compound, indicating that ADBAQ was quickly metabolized. Unchanged ADBAQ was deposited in adipose tissue, where it remained largely
unmetabolized. As late as 24 hours after admi nistration, the radioactivity remaining in adipose tissue was still unchanged ADBAQ, wher eas that in liver, muscle, and skin was primarily ADBAQ metabolites (which were not identified) (NTP 1996). The metabolites of ADBAQ are unknown. Other anthraquinone-bas ed dyes (Section 1, Table 1-2) were examined by IARC (1982) and the NTP (1978a, 1978b, 1978c, 1986), but their metabolites also are unknown. The NTP (1996) st udy did find that very little (less than 3%) of [ 14 

### C]ADBAQ was excreted as unmetabolized ADBAQ.

6.1.3 Excretion



### In the three days afte r administration of [ 14 

### C]ADBAQ, approximately 50% of ADBAQassociated radioactivity was recovered from feces, 15% from urine, and 6% from expired air. Consistent with extensive metabolism, le ss than 3% of total radioactivity excreted represented unchanged ADBAQ. Terminal half-l ives of administered radioactivity in liver and kidney were estimated to be 40 and 90 hours, respectivel y, and the elimination half-life from adipose tissue was 11 hours.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

34 

### 6.2 Liver focus model
Maronpot et al. (1989b) tested ADBAQ in a neonata l rat liver focus model for its potential both as an initiator and as a promoter. In the te st for initiation potential, 0.07 mg/g b.w. of ADBAQ was injected intrap eritoneally (i.p.) within 24 hours after birth, and phenobarbital (500 pp m) was used as the promo ting agent over the succeeding 75 or 300 days. The authors found no eviden ce of initiating activity for ADBAQ.

In the test for promotion potential, each neonatal rat was injected with a “subcarcinogenic” dose (4 μ g/g b.w.) of the initiator, di ethylnitrosamine (DEN), within 24 hours of birth. After weaning, rats r eceived ADBAQ at either 5,000 ppm or 10,000 ppm in the diet for either 75 or 300 days pr ior to sacrifice. Li vers were examined microscopically and graded with respect to number of atypical hepatocellular foci, mean focus volume, and tumor incidence. The qualitative results are summarized in Table 6-1. Equivocal results were reported for number of foci and the mean focus volume. The
authors concluded that the lack of a clear a ssociation between the e quivocal stereological response and positive hepatocarcinogenicity in the two-year study (see Table 4-2) may have resulted from unpalatability of the feed containing ADBAQ. The neonatal rats initiated with DEN may not have consumed a sufficient dose of ADBAQ to produce an unequivocal AHF response.

Table 6-1. Occurrence of altered hepatocellula r foci neonatal rats following dietary exposure to ADBAQ for 75 or 300 days a

Exposure concentration (ppm) Mean foci/cm 3 Mean focus
volume Tumor incidence

75 days

Males

5,000 NS ↑ P < 0.01

⎯ 10,000 NS NS NR

Females

5,000 ↓ P < 0.05 NS NR

10,000 ↓ P < 0.05 NS NR

300 days

Males

5,000 NS NS NS

10,000 NS ↑ P < 0.01 NS

Females

5,000 ↑ P < 0 .01 ↑ P < 0.01 NS

10,000 NS NS NS

Source: Maronpot et al. 1989b

a NS: no significant difference from control; ↑ = significantly higher than control; ↓ = significantly lower than control; NR = not reported.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

35 

### 6.3 Carcinogenicity and mutagenicity of other quinones and anthraquinones ADBAQ and other anthraquinones belong to a ge neral class of quinone molecules that
can be derived from aromatic molecules such as benzene, naphthalene, and anthracene. Quinone molecules can be reduced to a rela tively stable hydroquinone, which usually is not associated with oxidative stress, or they may be reduced in a one-electron reduction to semiquinone free radicals that give rise to superoxide anions , hydrogen peroxide, and other reactive oxygen species (Parkinson 1996). Bolton et al. (2000) recently reviewed the role of quinones in toxicology, including carcinogenesis. Quinones may be produced from benzene, polycyclic aromatic hydrocar bons, estrogens, and catecholamines and give rise to reactive oxygen species that can damage DNA and ot her cellular macromolecules and activate signaling pathways. These molecu lar events may be associated with the initiation, promotion, and progres sion of carcinogenesis.

The benzoquinone metabolite of benzene has been proposed as a proximate carcinogenic product (Golding and Watson 1999); however, the IARC (1999) overall evaluation was that 1,4-benzoquinone is not classifiable as to its carcinogenicity to humans (Group 3). In a review of the role of quinoids in estrogen carcinogenesis, Bolton et al. (1998) suggested that the catechol metabolites of estrogens th at are implicated in the carcinogenic effects
of these steroids could be metabolized to electrophilic and redox- active quinoids that could damage DNA. Their conclusion, howev er, was that the ultimate carcinogenic metabolites of estrogens have not been unequivocally established.

Anthraquinones comprise a la rge class of diverse molecu les which may be naturally occurring or synthetic in origin. Sendel bach (1989) reviewed the toxicity and carcinogenicity of anthraquinone derivatives w ith phenolic, amino, or nitro substitutions to the anthraquinone ring structure. The au thor’s general conclusion concerning amino
substitutions was that addition of an amino group at either the 1- or 2-position of the anthraquinone ring appeared to consistently produce renal lesions in rats. The relevance of this conclusion to ADBAQ, which has bot h amino and halogen substitutions to the anthraquinone ring, is not clear. However, anthraquinone also produces renal tumors. Renal tubule adenomas and carcinomas (com bined) were significantly increased ( P < 0.05) by dietary exposure to ADBAQ at 5,000 ppm in female rats and at 10,000 ppm in both male and female rats (see Table 4-2).

A number of anthraquinone-derived vat dye s have been reviewed by both IARC and the NTP. ADBAQ produces tumors at some of the same sites as other anthraquinones.
Table 6-2 summarizes the IARC and NTP evaluations of several anthraquinone
derivatives. Information on a number of other anthraquinone derivatives at various stages of testing by the NTP can be accessed through the NTP Web site (http://ntpserver.niehs.nih.gov/main_page s/NTP_ALL_STDY_PG.html).

As described in Sect ion 5.2.1, Harrington-Brock et al . (1991) carried out a limited evaluation of the ability of ADBAQ to i nduce mutations at the thymidine kinase ( tk )
locus of L5178/TK +/- 

### mouse lymphoma cells, but they were unable to test for
micronucleus formation because of ADB AQ’s limited solubility. An additional 15 anthraquinones were tested in the same a ssays. Testing of 8 of these substances was incomplete because of their limited sol ubility. For these 15 compounds, Table 6-39/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

36 

### summarizes the results of tests of mutagenicity at the L5178/TK +/- 

### locus in two or three experiments and micronucleus analysis from one experiment per compound. Table 6-3 also summarizes the mutagenicity of these compounds in S. typhimurium, as reviewed by Harrington-Brock et al. (1991).

Brown (1980) reviewed the gene tic effects of approximately 80 phenolic anthraquinones and reported that a high percentage of these hydroxyanthraquinones (36, or 45%) were mutagenic in S. typhimurium strains TA1537, TA1538, and TA98, which are sensitive to mutagens that cause frameshifts. A sma ll portion (~10%) of the hydroxyanthraquinones contained either amino or halogen (chlorine or bromine) substitutions; however, the relevance of these findings to the possibl e mutagenicity of ADBAQ is not clear.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone37Table 6-2. Carcinogenicity and mutagenicityof anthraquinone and some anthraquinone-derived dyes (data from NTP and IARC)PropertyAnthraquinone2-Aminoanthraquinone1-Amino-2-methylanthraquinone1,8-Dihydroxyanthraquinone(danthron)2-Methyl-1-nitroanthraquinone1,4,5,8-Tetraaminoanthraquinone(disperse blue 1)EmodinLong-termcarcinogenicityin miceLiver neoplasms inmale and femaleB6C3F1miceHepatocellularcarcinomas in maleand femaleB6C3F1mice(NTP)Malignanthematopoieticlymphomas in highdose female mice(NTP)Negative in malemice (NTP)Hepatocellularcarcinomas andneoplastic livernodules in femaleB6C3F1mice(NTP)Hepatocellularadenomas andcarcinomas andadenomatoushyperplasia of thececum inC3H/HeN malemice (IARC)Hemangiosarcomasin male and femaleB6C3F1mice(NTP)Hepatocellularadenomas andcarcinomas(combined) in maleB6C3F1mice (NTP)Alveolar/bronchiolaradenomas andcarcinomas(combined) in maleB6C3F1mice (NTP)Uncommonrenal tubuleneoplasms inmale B6C3F1miceLong-termcarcinogenicityin ratsRenal tubuleneoplasms in maleand femaleF344/N ratsTransitionalepithelialpapillomas of theurinary bladder inmale F344/N ratsHepatocellularcarcinomas in maleF344 rats (NTP)Inadequate studyin female F344 rats(poor survival)(NTP)Hepatocellularcarcinomas inmale and femaleF344 rats (NTP)Adenocarcinomasof the colon inmale ACI rats(IARC)Hepatocellularcarcinomas in maleF344 rats (NTP)Subcutaenousfibromas in maleand female F344rats (NTP)Transitional-cellneoplasms of theurinary bladder inmale and femaleF344 rats (NTP)Squamous-cellneoplasms in maleF344 rats (NTP)Leiomyomas andleiomyosarcomas(combined) of theurinary bladder infemale F344 rats(NTP)Zymbal’s glandcarcinomas infemale F344/Nrats9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone38PropertyAnthraquinone2-Aminoanthraquinone1-Amino-2-methylanthraquinone1,8-Dihydroxyanthraquinone(danthron)2-Methyl-1-nitroanthraquinone1,4,5,8-Tetraaminoanthraquinone(disperse blue 1)EmodinGenetictoxicologyPositive formutagenicity inS.typhimuriumTA98and TA100 withand without S9Positive formicronucleatednormochromaticerythrocytes inmale and femalemice (14-weekdietary study)Negative formicronuclei inmouse bonemarrow (i.p.injection)Positive forchromosomalaberrationsPositive for SCEPositive formutagenicity inS.typhimurium(NTP)Positive forchromosomalaberrationsPositive for SCEPositive formutagenicity inS. typhimurium(NTP)Positive forchromosomalaberrationsNot tested forSCENegative formicronuclei [cellsnot specified]Weakly positivefor mutagenicityinS. typhimurium(NTP)Positive forchromosomalaberrationsPositive for SCENegative forreciprocaltranslocation inDrosophilaPositive formutagenicity inS.typhimuriumPositive forchromosomalaberrationsPositive for SCEPositive formutagenicity inmouse lymphomacellsNegative formicronuclei [cellsnot specified]Positive formutagencity inS.typhimuriumPositive formutagenicity inS. typhimurium(TA100) withS9; negative inTA98 with orwithout S9Positive forchromosalaberrationsNegative formicronuclei inmouse bonemarrow (i.p.injection)Weakly postivefor micronucleiin female mice(14-weekdietary study)[cells notspecified]9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone39PropertyAnthraquinone2-Aminoanthraquinone1-Amino-2-methylanthraquinone1,8-Dihydroxyanthraquinone(danthron)2-Methyl-1-nitroanthraquinone1,4,5,8-Tetraaminoanthraquinone(disperse blue 1)EmodinRoC listing orNTP evaluationNTP: someevidence ofcarcinogenicactivity in maleF344/N rats; clearevidence ofcarcinogenicactivity in femaleF344/N rats; clearevidence ofcarcinogenicactivity in maleand femaleB6C3F1miceRoC: reasonablyanticipated to be ahuman carcinogenRoC: reasonablyanticipated to bea humancarcinogenRoC: reasonablyanticipated to be ahuman carcinogenNTP: found to becarcinogenic inmale and femalerats and miceRoC: reasonablyanticipated to be ahuman carcinogenNTP: equivocalevidence ofcarcinogenicactivity infemale F344/Nrats andB6C3F1miceIARCevaluationaNot listed Group 3(IARC 1987)Group 3(IARC 1987)Group 2B(IARC 1990)Group 2B(IARC 1987)Group 2B(IARC 1990)Not listedaGroup 2B = possibly carcinogenic to humans, based on limited evidence of carcinogenicity in humans, less than sufficient evidence of carcinogenicity in animals.Group 3 = not classifiable as to its carcinogenicity in humans, based on inadequate or limited evidence of carcinogenicity in animals and inadequate evidence ofcarcinogenicity in humans.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone40Table 6-3. Genotoxicity of some anthraquinone dyesaAnthraquinoneCASRNPurity(%)Mutagenicity in mouselymphoma cellsMicronucleiin mouselymphoma cellsReverse Mutation inS. typhimurium1-Amino-2-methylanthraquinone82-28-0 99 + w/o S9 + w/o S9 +2-Aminoanthraquinone 117-79-3 86.9 + w/o S9 + w/o S9 +D&C green 5 4403-90-1 60 limited solubility limited solubility –/+Disperse blue 1 2475-45-8 50 limited solubility limited solubility +Disperse blue 3 2475-46-9 42 + w/o S9 + w/o S9 +Disperse blue 7 3179-90-6 60 + w/o S9 + +Disperse red 11 2872-48-2 95 + w/o S9 + w/o S9 +Disperse red 60 17418-58-5 92 limited solubility limited solubility NR1-Nitro-2-methylanthraquinone129-15-7 95.9 limited solubility(–/+ w S9)limited solubility(–/+ w S9)+Reactive blue 19 2580-78-1 36 + (weak) w/o S9increased from 9/1,000 to19/1,000 cellsw/o S9NRVat blue 4 81-77-6 48 limited solubility limited solubility NRVat blue 20 116-71-2 26 limited solubility limited solubility NRVat brown 1 2475-33-4 56 limited solubility limited solubility NRVat brown 3 131-92-0 85 limited solubility limited solubility –Vat yellow 4 128-66-5 18.2 + w S9 slight increase w S9 –Adapted from Harrington-Brocket al.1991 and references therein.a+ = positive; – = negative; –/+ equivocal; NR = not reported.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

41 

### 6.4 Summary In rats, ADBAQ is rapidly absorbed from the ga strointestinal tract a nd distributed rapidly and widely to most soft tissues. Approxi mately 97% of ADBAQ is metabolized, as demonstrated by the small amount (less than 3% ) of radiolabel recovered as the parent compound from either blood or urine; howev er, the metabolites of ADBAQ have not been identified. ADBAQ and its metabolites are ex creted primarily in the feces and urine.

ADBAQ and other anthraquinones ar e classified with a large number of other quinone
molecules that can be derived from aromatic molecules such as benzene, naphthalene, and anthracene. Reactive oxygen species ge nerated by metabolism of a variety of quinones may be associated with DNA damage or activation of signaling pathways involved in initiation, promotion, and progr ession of carcinogenesis. Four other anthraquinone vat dyes evalua ted by the NTP have been listed in the RoC as reasonably anticipated to be human carcinogens. In addition, a high percentage (36/80) of phenolic anthraquinones have been reported to be mutagenic in Salmonella

.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

429/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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## 7 References

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14. Haworth, S., T. Lawlor, K. Mortel mans, W. Speck, and E. Zeiger. 1983. Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5:1-142.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

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### 15. Hayashi, S., H.H. Hong, K. Toyoda, T.V. Ton, T.R. Devereux, R.R. Maronpot, J. Huff, and R.C. Sills. 2001. High frequency of ras mutations in forestomach and lung tumors of B6C3F1 mice exposed to 1-amino-2,4-dibromoanthraquinone for 2

years. Toxicol Pathol 29:422-429.

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24. Maronpot, R.R., H.C. Pitot, and C. Peraino. 1989b. Use of rat liver altered focus models for testing chemicals that have completed two-year carcinogenicity studies.
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### 25. Novotná, P., V. Pacáková, Z. Bosáková, and K. Štulík. 1999. High-performance liquid chromatographic determination of some anthraquinone and naphthoquinone dyes occurring in historical textiles. J Chromatogr A 863:235-241.

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29. NTP. 1986. Toxicology and Carcinogenesis St udies of C.I. Disperse Blue 1 (A Commercial Dye Containing Approximately 50% 1,4,5,8- Tetraaminoanthraquinone, and 20% Water) (CAS No. 2475-45-8) in F344/N Rats and B6C3F1 Mice (Feed Studies). Nationa l Toxicology Program Technical Report
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### mice (feed studies). National Toxicology Program Technical Report Series No 383.

31. NTP Chemical Repository. 2001. 1-ami no-2,4-dibromoanthraquinone. Revised August 13, 2001. National Toxicology Progr am. NTP Chemical Repository.Available at http://ntpserver.niehs.nih.gov/cgi/iH_Indexes/ ALL_SRCH/iH_ALL_SRCH_Frames.html
and search 81-49-2.

32. Parkinson, A. 1996. Biotransformation of Xenobiotics. In Casarett and Doull's Toxicology: The Basic Science of Pois ons, Fifth Edition. Klaassen, C.D., M.O. Amdur and J. Doull, eds. McGraw-Hill, New York. pp. 113-186.

33. Preiss, A., U. Sänger, N. Karfich, K. Levsen, and C. Mugge. 2000. Characterization of dyes and other pollutants in the efflue nt of a textile company by LC/NMR and
LC/MS. Anal Chem 72:992-998.

34. Sathiakumar, N. and E. Delzell. 2000. An updated mortality study of workers at a dye and resin manufacturing plant. J Occup Environ Med 42:762-771.

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36. TRI. 2001. Toxic Release Inventory. Re vised 2001. Environmental Protection Agency. Available at (http://toxnet. nlm.nih.gov/cgi-bin/sis/htmlgen?TRI).9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

469/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

47 

## Appendix A: NTP TR 383 (1996). Toxicology and Carcinogenesis Studies of 1-Amino-2,4- Dibromoanthraquinone in F344/N Rats and B6C3F1 Mice (Feed Studies). PP A-1 – A-86.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

485



# 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 eMaterials 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 EvaluationResults 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 YearsResults 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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49 

## Appendix B: Tissues examined for histopathology in mice and rats in the NTP bioassays of ADBAQ9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

509/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

51 

### Tissues examined for histopathology in mi ce and rats in the NTP bioassays of ADBAQ

13-Week studies 2-Year studies Stop-exposure evaluation

Complete histopathologic examinations were performed on all animals that died during the study, control animals, and 50,000 ppm animals. In addition to tissue masses, gross lesions, and associated lymph nodes, the tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, and uterus. The kidney, liver, spleen (rats), thymus (rats), and uterus (rats) of all other exposed animals
were examined. Complete histopathologic examinations were performed on all animals. In addition to tissue masses, gross lesions, and associated lymph nodes, the tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, gallbladder (mice), heart, kidney, large intestine (cecum, colon, rect um), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, and

uterus. Complete histopathologic examinations were performed on all animals. In addition to tissue masses, gross lesions, and associated lymph nodes, the tissues examined included: adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, and

uterus.9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone

52