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-85421 

9/19/02 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 substances (i) that either 

are known to be human carcinogens or may reasonably be anticipated to be human 

carcinogens; and (ii) to which a significant number of persons residing in the United 

States are exposed. The Secretary, Department 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 nomination according to specific RoC listing 

criteria. This Background Document was prepared to assist in the review of the 

nomination of 1-amino-2,4-dibromoanthraquinone. The scientific information in this 

document comes from publicly available, peer reviewed sources. Any interpretive 

conclusions, comments or statistical calculations, 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 review 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 that contains a brief discussion of the 

respective review group’s review and recommendation for the nomination will be added 

to the Background Document, also as an addendum. 

A detailed description of the RoC nomination review process and a list of all nominations 

under consideration for listing in or delisting from the RoC can be obtained by accessing 

the NTP Home Page at http://ntp-server.niehs.nih.gov. The most recent RoC, the 9th

Edition, was published in May, 2000 and may be obtained by contacting the NIEHS 

Environmental Health Information Service (EHIS) at http://ehis.niehs.nih.gov (800-315-

3010). 
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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 Report on Carcinogens Group, 

Environmental Toxicology Program, NIEHS 

Support to the National Toxicology Program for the preparation of this background 

document was provided by Technology Planning and Management Corporation 

through NIEHS Contract Number NO1-ES-85421

Ronald Thomas, Ph.D., Principal Investigator 

Sanford Garner, Ph.D. Co-Principal 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, Department of Epidemiology and International 

Health, University of Alabama at Birmingham, Birmingham, Alabama (contributed to 

Section 3) 

Richard Di Giulio, Ph.D. Professor, Nicholas School of the Environment, Duke 

University, Durham, NC (document review) 
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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 carcinogenicity 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 carcinogenicity from studies in humans, which 

indicates that causal interpretation is credible but that alternative explanations 

such as chance, bias or confounding factors could not adequately be excluded; or

There is sufficient evidence of carcinogenicity from studies in experimental 

animals which indicates there is an increased 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 convincing relevant information 

that the agent acts through mechanisms indicating it would likely cause cancer in 

humans. 

Conclusions regarding carcinogenicity 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, sensitive 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 substances for which there is evidence of 

carcinogenicity in laboratory animals but there 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. 
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9/19/02 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 Toxicology 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 intermediate. Anthraquinones are widely used 

as starting material for the manufacture of vat dyes, which are used in fibers and textiles, 

typically for cotton, wool, and cellulose acetate. 

Production. ADBAQ is prepared from 1-aminoanthraquinone 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 raw wastewater from a dye manufacturing 

plant in four of eight samples 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, inhalation exposure likely is limited to solid 

particulates. No specific occupational exposure information for ADBAQ or 

anthraquinone dyes in general was found in current literature. Epidemiological studies, 

however, indicate occupational exposures to anthraquinone dyes in a New Jersey dye and 

resin manufacturing plant. 

Human Cancer Studies 

Only two groups of workers exposed to anthraquinone dyes and intermediates have been 

investigated. All of the studies of these workers have limitations 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 cancer risk factors. The results of the 

pertinent research are not consistent. The 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 anthraquinone dye operations was positively associated 

with lung cancer and with CNS tumors. In the 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, the increase in lung cancer in 

anthraquinone dye workers at the New Jersey plant may have been due to occupational 
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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 studies 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 observed in a 15-month stop-exposure study in rats. 

B6C3F1 mice consuming ADBAQ-containing diets for two years had increased 

incidences of hepatocellular adenoma and carcinoma, squamous-cell papilloma and 

carcinoma of the forestomach, and alveolar/bronchiolar 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 increased 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 B6C3F1 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 eliminated 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 μg/mL) were 

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

less than those used to assess clastogenicity (up to 100 μg/mL of ADBAQ). In CHO 

cells, ADBAQ induced chromosomal aberrations and SCE without metabolic activation; 

however, the results were inconsistent between 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 absent 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 rapidly 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 compound from either blood or urine; 

however, the metabolites of ADBAQ have not been identified. ADBAQ and its 

metabolites are excreted primarily in the feces and urine. 

Potential mechanisms. ADBAQ and other anthraquinones are 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 generated by metabolism 

of a variety of quinones may be associated with DNA damage or activation of signaling 

pathways involved in initiation, promotion, and progression of carcinogenesis. Four other 

anthraquinone vat dyes evaluated by the NTP have been listed in the RoC as reasonably 
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anticipated to be human carcinogens. In addition, a high percentage (36/80) of phenolic 

anthraquinones have been reported to be mutagenic in Salmonella. 
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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 Environmental 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 manufacturing 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........................................................................ 30 
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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, metabolism, 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 histopathology in mice and rats in the NTP bioassays 

of ADBAQ.............................................................................................................................. 49 

List of Tables 

Table 1-1. Physical and chemical 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 F344/N rats following dietary exposure to 

ADBAQ for 13 weeks............................................................................................................. 18 

Table 4-2. Survival, body weight, and tumor incidence in F344/N rats following dietary 

exposure to ADBAQ for two years......................................................................................... 20 

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) ...................................................................... 23 

Table 4-4. Occurrence of altered hepatocellular foci in female F344/N rats following 

dietary exposure to ADBAQ for 15 months ........................................................................... 24 

Table 4-5. Survival and body wieght in B6C3F1 mice following dietary exposure to 

ADBAQ for 13 weeks............................................................................................................. 25 

Table 4-6. Survival, body weight, and tumor incidence in B6C3F1 mice following dietary 

exposure to ADBAQ for two years......................................................................................... 27 

Table 5-1. Mutagenicity of ADBAQ in S. typhimuriuma ............................................................. 29 

Table 5-2. Induction of chromosomal aberrations and SCE in CHO cells by ADBAQa ............. 31 

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

B6C3F1 mice exposed to ADBAQ for two yearsa

.................................................................. 32 

Table 6-1. Occurrence of altered hepatocellular foci neonatal rats following dietary 

exposure to ADBAQ for 75 or 300 daysa ............................................................................... 34 
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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 dyesa

................................................................. 40 

List of Figures 

Figure 1-1. Structure 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 stop exposure study ................................................................................... 21 

Figure 4-3. Exposure protocol for the two-year study of ADBAQ in B6C3F1 mice.................... 26 
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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 listing in the Report on Carcinogens (RoC) based 

on a National Toxicology Program (NTP) bioassay (TR-383) reporting clear evidence of 

carcinogenicity in rats and mice. The NTP studied the carcinogenicity of ADBAQ as part 

of a class study with five other anthraquinone-derived dyes with representative and 

diverse structures, for the purpose of predicting the carcinogenicity 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 male and female F344/N rats 

(neoplasms in the liver, large intestine, kidney, and urinary bladder) and in male and 

female B6C3F1 mice (neoplasms in the liver, forestomach, and lung). 

1.1 Chemical identification 

ADBAQ (C14H7Br2NO2, mol wt 381.02, CASRN 81-49-2) is a substituted anthraquinone 

with a single primary amine located in the 1-position and two bromine atoms located 

ortho and para to the amino group. Synonyms for ADBAQ include 1-amino-2,4-dibromo9,10-anthracenedione; 1-amino-2,4-dibromanthrachinon; anthraquinone, 1-amino-2,4-

dibromo-; and 2,4-dibromo-1-anthraquinonylamine. 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 and 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 water. It is sensitive to long-term air and light 

exposure. The physical and chemical properties of ADBAQ are summarized in Table 1-1. 

NH2 O

Br O

Br
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Table 1-1. Physical and chemical 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 23oC): 

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 anthraquinone-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-aminoanthraquinone (TR-144, RoC), 1-amino-2-

methylanthraquinone (TR-111, RoC), 1,8-dihydroxyanthraquinone (danthron) (RoC), 2-

methyl-1-nitroanthraquinone (TR-029), 1,4,5,8-tetraaminoanthraquinone (disperse blue 

1) (TR-299, RoC), and emodin (TR-493). Carcinogenicity 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 following: chrysophanic acid, CASRN 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 violet 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.
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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 C14H8O2 208.22 O

O

2-Aminoanthraquinone 117-79-3 C14H9NO2 223.23 O

O

H2N

1-Amino-2-

methylanthraquinone 

82-28-0 C15H11NO2 237.26 

O

O

H3C

NH2

1,8-Dihydroxyanthraquinone 

(danthron) 

117-10-2 C14H8O4 240.22 

OH OH O

O

2-Methyl-1-nitroanthraquinone 129-15-7 C15H9NO4 267.24 

O

O

H3C

NO2

1,4,5,8-

Tetraaminoanthraquinone 

(disperse blue 1) 

2475-45-8 C14H12N4O2 268.27 O

O

NH2 NH2

NH2 NH2

Emodin 518-82-1 C15H10O5 270.24 

O

O

OH OH

H3C OH
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2 Human Exposure 

2.1 Use 

ADBAQ is used as a dye and as a dye intermediate (NTP Chemical Repository 2001). 

Anthraquinones are widely used as starting material 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 typically are used for cotton, wool, and cellulose 

acetate. 

2.2 Production 

ADBAQ is prepared from 1-aminoanthraquinone by bromination in dilute mineral acids 

(HSDB 2000). ADBAQ was found to be available 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 samples by gas chromatography/flame ionization 

detection and gas chromatography/mass spectrometry (Games and Hites 1977). Recently, 

several other analytical methods have been described for detecting and quantifying 

anthraquinone-type dyes (liquid chromatography/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 formed. Production of ADBAQ and its subsequent 

use as an intermediate in the 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 Inventory 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 wastewater of a dye manufacturing plant, it 

was not present in the final effluent (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 pressure, 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). 
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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 sites in sediment and particulate matter. It 

is not expected to volatilize from water surfaces. The estimated partition coefficients and 

a recommended regression-derived equation were used to calculate a bioconcentration 

factor of 6,400 for ADBAQ. This suggests that ADBAQ will bioconcentrate in aquatic 

organisms (HSDB 2000). 

2.5.3 Soil 

ADBAQ is expected to be immobile in soil; its mobility may be limited because the 

amino group may bind to active sites in the soil. Volatilization is not expected to occur 

from either moist or dry 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 anthraquinone dyes. A dibromoaminoanthraquinone 

was found in raw wastewater from a dye manufacturing 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 exposure likely is limited to solid particulates. Ikeda et al. (1977) 

found that workers exposed to dyes (as evidenced 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 information for ADBAQ or anthraquinone dyes in 

general were found in current literature. Epidemiological studies, however, indicate 

potential occupational exposures to anthraquinone dyes in a New Jersey dye and resin 

manufacturing plant. A cohort study followed more than 3,000 workers who may have 

been exposed to anthraquinone dyes (Sathiakumar 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. 
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3 Human Cancer Studies 

No study has evaluated the relationship between human cancer and specific exposure to 

ADBAQ; however, exposure to anthraquinone dyes 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 synthesize industrial colorants and has used nitro 

aromatic amines to make hair dyes. Relatively little is known about the human 

carcinogenicity of anthraquinone dyes. The NTP classifies 2-aminoanthraquinone, 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 Cancer (IARC) lists 1,8-dihydroxyanthraquinone, 

2-methyl-1-nitroanthraquinone, and 1,4,5,8-tetraaminoanthraquinone as possibly 

carcinogenic to humans (Group 2B) and considers 2-aminoanthraquinone and 1-amino-2-

methylanthraquinone not classifiable as to carcinogenicity to humans (Group 3). Neither 

the NTP nor IARC has evaluated the carcinogenicity of anthraquinone dyes as a chemical 

category or anthraquinone dye manufacturing as an exposure circumstance. 

3.1 Human epidemiological studies 

Data on the carcinogenicity to humans of dyes and chemicals used in synthesizing dyes 

come mainly from studies of workers in manufacturing plants. These groups of workers 

typically were exposed to many different chemicals and classes of chemicals, often 

making it difficult to attribute observed carcinogenic effects to a single agent. Only five 

epidemiologic studies of workers potentially exposed to anthraquinone dyes and 

anthraquinone dye intermediates are available. One of these investigations evaluated 

mortality among workers at a plant in Scotland 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 intermediates 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 plant 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 substituted anthraquinones. The study excluded 

men who had worked exclusively in clerical, 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 data from the National 

Records Offices of Edinburgh and Newcastle upon Tyne. Analyses compared workers’ 

mortality rates with those of the general male population of Scotland in 1975, adjusting 

for age and using the standardized mortality ratio (SMR) (ratio of observed to expected 

numbers of deaths, multiplied by 100) as the measure of association. An SMR of 100 

implies that the mortality rate of workers was the same as that of the general population, 

after analytic adjustment for any difference in the age distributions of these two groups. 
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Gardiner et al. (1982) found that workers at the plant 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, SMR = 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 specific 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), intestinal 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 was higher than expected for workers in some 

areas of the plant. Gardiner et al. (1982) did not present detailed 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 engineering department were not thoroughly described 

but presumably included maintenance 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 exposures probably were similar to 

those of process workers. The authors could not 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 study 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 lack 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 groups, 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 follow-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 workers. In a later investigation, 

Sathiakumar and Delzell (2000) expanded the 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. 
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The manufacturing operations of the New Jersey plant took place in three major areas. 

Operations in the “south dye area” (anthraquinone dye/epichlorohydrin area) included 

anthraquinone production (1952 to 1983), anthraquinone dye production (1952 to 1983), 

anthraquinone dye intermediates production (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 resins, fabric softeners, water-repellent 

agents, optical brighteners, and various additives from 1959 to the late 1980s. Raw 

materials and intermediates used in each area included many chemicals, some of which 

are known carcinogens (e.g., benzene, benzidine, ethylene oxide) or suspected 

carcinogens (e.g., epichlorohydrin, formaldehyde, dimethyl sulfate). Chemicals used in 

the anthraquinone dye/epichlorohydrin (AQ/ECH) area included anthracene, 

anthraquinones, anthraquinone sulfonates, anthraquinone intermediates, aniline, 

substituted aniline, benzene, nitrobenzene, chlorobenzenes, chlorotoluenes, pyridine, 

tetrachloroethylene, chlorine, ammonia, arsenic acid, methanol, sulfuric acid, and 

mercury. 

3.1.2.1 Original retrospective follow-up study 

The first investigation of workers at the New Jersey plant used company personnel 

records to identify all white men with at least six months of employment as of the end of 

1984 and to develop information on each subject’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’ causes 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 cancer 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 living in Europe, and there were 3 deaths each 

from bladder cancer (expected = 2.3) and kidney 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 cancer, observed and expected 

numbers of deaths were under 10 and were too small to yield statistically 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 group had statistically 

significantly more lung cancer deaths than expected (4/0.91, P = 0.03). 
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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 (18/9.2, 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 occupational exposure. They noted that the 

association they observed between epichlorohydrin 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 absence of quantitative exposure estimates or 

data on nonoccupational risk factors. However, even if such data had been available, the 

study would have had little power 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-control 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. Controls 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 diagnosis date. Plant job 

records identified each process area and building in which a subject had carried out 

production-related work. The AQ/ECH area consisted of five buildings with the 

following operations: (1) anthraquinone production, (2) production of anthraquinone dye 

intermediates, (3) anthraquinone dye synthesis, (4) anthraquinone dye standardization, 

and (5) epichlorohydrin production. Interviews with long-term employees (not subjects) 

provided more detailed work history information on building-specific work, as well as an 

ordinal score for subjects’ potential exposure to asbestos and epichlorohydrin. Interviews 

with subjects or family members furnished data on smoking and on jobs and exposures 

outside the New Jersey plant and were completed 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 chemicals that had been reported to the plant 

medical department. The odds ratio (OR) (the ratio 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 consistently 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 trend 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 anthraquinone production (OR = 12, 95% 

CI = 1.4 to 99) and was of borderline statistical 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 or 
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epichlorohydrin or to smoking. Results for other process areas were 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 chlorine and lung cancer (OR = 5.7, 

95% CI = 1.1 to 30). Adjustment for smoking raised 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 positive [but not statistically significant] 

association between lung cancer and exposure to epichlorohydrin (OR = 1.7, 95% CI = 

0.7 to 4.1) was reported, based on 12 exposed cases 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 subjects 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 duration of employment in the various anthraquinone areas and 

lung cancer in this study could have been due to poor correlation between duration of 

work in the areas and amount of exposure. The study also did not have quantitative or 

comprehensive data on exposure to chlorine 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 data available on human exposure to 

anthraquinones, the study by Gardiner et al. (1982), did not find an excess of lung cancer 

among workers with potential exposure. 

3.1.2.3 Case-control study of central nervous system tumors 

The case-control study of CNS tumors included 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 meningioma, and 2 with a benign tumor) (Barbone et 

al. 1994). Controls were 44 individually matched subjects 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 diagnosis date. Procedures for obtaining and classifying work history 

data, estimating exposure to epichlorohydrin, conducting interviews, and obtaining data 

from plant medical records were similar to those used in the case-control study of lung 

cancer (Barbone et al. 1992). Interviews, completed for 73% of cases and 84% of 

controls, asked about non-plant jobs and occupational exposures, smoking habits, and 

history of head trauma, irradiation to the head, epilepsy, and use of antiepileptic drugs. 

The occurrence of CNS tumors was statistically significantly and positively associated 

with anthraquinone dye intermediate production, but only 3 case subjects and no controls 

had worked in this process area. Positive associations also were found with work in the 

epoxy resin process area and in the azo dye area and with estimated exposure to 
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epichlorohydrin. Barbone et al. (1994) reported that the association with anthraquinone 

dye intermediate production was “nonindependent” of the association with exposure to 

epichlorohydrin and concluded that the small study size precluded firm conclusions about 

the causes of CNS tumors in the plant workforce. 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 anthraquinone or other chemicals and was not able 

to evaluate anthraquinone dose response or to adequately assess the possibility that the 

association between anthraquinone dye intermediate 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-, and 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), and 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 deaths 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 expected (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 more); however, small numbers limited the 

conclusions that could be drawn from these subgroup 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 anthraquinone dye operations, but this could be 

explained by poor correlation between duration of employment and cumulative exposure 

to anthraquinones or other chemicals used in the area. Sathiakumar 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). 
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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 workers were deceased. However, numbers of 

deaths from specific types of cancer were small in the work-area-specific analyses, and 

the update lacked direct exposure estimates and data on nonoccupational risk factors. 

These limitations impede the detection of causal associations. 

3.2 Summary 

Only two groups of workers exposed to anthraquinone dyes and intermediates have been 

investigated. All of the studies of these workers have limitations 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 cancer risk factors. The results of the 

pertinent research are not consistent. The 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 anthraquinone dye operations was positively associated 

with lung cancer and with CNS tumors. In the 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, the 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. 
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Table 3-1. Human cancer studies on anthraquinone dye workers Reference 

Study 

design Population Exposure Effects 

Gardiner et al. 1982 Scotland 

Cohort study 1,975 men employed at 

anthraquinone dyestuff plant for at least 6 months from 1956–1965, followed up until June 1980 

Potential exposure to substituted anthraquinones was assessed from personnel records. 

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) 

Smcoas

Delzell et al.

1989 New Jersey, USA 

Retrospective cohort study 

2,642 white men employed at a dye and resin plant for at least 6 months from 1952–1985 588 hourly workers employed in the AQ/ECH area for at least one year 

Exposure was asssessed from personnel records. Three manufacturing areas: AQ/ECH area: anthraquinone, AQ dye and AQ dye intermediates, and ECH production and formulation of AQ dyes North dye area: azo dyes and intermediates Plastics and additive area 

Observed/expected AQ/ECH area workers: all deaths 65/97 all cancers 20/22 lung cancer 6/8 Epichlorohydrin workers: (1961–1965) (N = 44) lung cancer 4/0.91; P = 0.03 

Wareto incbecotetchanNofacac
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Reference 

Study 

design Population Exposure Effects 

Barbone et al.

1992 New Jersey, USA 

Nested casecontrol study (Delzell et al.1989) Lung cancer 

Cases: 51 white male 

employees who died of or developed lung cancer by 10/1988 Controls: 102 employees 

randomly selected and matched for age and alive as of case’s death or diagnosis date 

The AQ/ECH area consisted of 5 buildings: (1) AQ production (2) production of AQ  intermediates (3) AQ dye synthesis (4) AQ dye standardization (5) ECH production 

OR (95% CI); no. of exposed cases Worked 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); 6 AQ dye/standardization  3.3 (1.0–11); 8 ECH production: 3 exposed cases, 0 exposed controls; OR not calculated 

ORwiadanOnbe kemodirasLupowi

Barbone et al.

1994 New Jersey, USA 

Nested casecontrol study (Delzell et al.1989) CNS tumors 

Cases: 11 white males 

who died of or developed a primary CNS tumor by 10/1988 Controls: 44 employees 

matched for age and alive as of the case’s death or diagnosis date 

Same as Delzell et al.(1989) and Barbone et al.(1992) 

OR (95% CI); no. of exposed cases AQ dye intermediates: production 

∞ (1.7–

∞); 3 

routine potential exposure to ECH  4.2 (0.7–26); 4 

Thsuexwodydy

Sathiakumar and Delzell 2000 New Jersey, USA 

Cohort study Expansion of Delzell et al.(1989) 

3,266 employees (men and women) at a dye and resin plant, followed through 1995 728 deaths 

Same as Delzell et al.(1989) 

SMR (95% CI); observed/expected Entire cohort: all deaths 90 (83–97); 728/809.8 all cancers 97 (85–111); 225/231.7 South dye area, white males (N = 842): lung cancer 168 (115–237); 32/19.1 

Nofacsmas
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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 for either 13 weeks or two years. In addition, 

the NTP conducted a 15-month high-dose stop-exposure 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 were 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 the studies and confirmed 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-aminodibromoanthraquinone (4.7%). The 

same material was used for the first 2 months of the two-year studies. 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. Anthraquinone 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), and clear evidence of carcinogenic activity in 

male and female B6C3F1 mice (liver). All of the reported sites for anthraquinone-induced 

neoplastic lesions also are target 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-exposure 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 performed, in which tissues were collected and 

processed for histologic examination (a complete list of tissues and organs examined is 

included in Appendix B). Survival to study termination and mean final body weight 

(b.w.) as a percent of mean control body weight are shown in Table 4-1. The 

nonneoplastic lesions of the liver included basophilic foci, clear-cell foci, eosinophilic 

foci, cytomegaly, bile duct hyperplasia, inflammation, fibrosis, necrotizing cholangitis, 

vacuolar degeneration, and pigmentation. The incidence of hepatocellular cytomegaly 
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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 female 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 included 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 groups). Data for other exposure-related 

lesions in all groups are summarized in the NTP report (Appendix A, p. A-26, Table 4). 

Table 4-1. Survival and body weight in F344/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. ± SDa (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 NTP’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 those of controls. Pathological findings of 

nonneoplastic lesions of the liver and kidney were the same as those reported in NTP 

(1996). The uterus was atrophied in females fed ADBAQ at a concentration of 10,000 

ppm or higher, and the thymus was atrophied 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 authors 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 were 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 was predicted not to adversely 

affect the health or survival of the animals because it did not cause liver toxicity in the 
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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 obtained at approximately six weeks of age and 

quarantined for 12 to 14 days (males) or 9 days (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 designated for an interim evaluation after 9 months. 

Additional subsets from the control and 10,000-ppm groups (10 of each sex) were 

evaluated after 15 months. Major tissues were collected for histological examination. The 

exposure protocol is summarized 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-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 4-1. 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 lesion incidence at two years are summarized in Table 4-2. 

Neoplastic lesions were observed in the liver, large intestine, kidney, urinary bladder, and 

forestomach. Neoplasms of the liver included 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 forestomach included squamous-cell papilloma 

and carcinoma, but the increased incidences did not reach statistical significance. 

Significant increases were seen at all exposure levels for tumors of the liver, large 

intestine, and kidney. The incidence of urinary bladder tumors also was significantly 

increased in high-dose males and mid- and high-dose females. 
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Table 4-2. Survival, body weight, and tumor 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** 
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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 bladder tumors also were found in rats that 

received ADBAQ at 10,000 ppm for 15 months (interim sacrifice). 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-month stop-exposure study (discussed below). 

4.1.3 Fifteen-month stop-exposure study 

The NTP conducted a stop-exposure study to evaluate 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 quarantined 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 concentration of 20,000 ppm and were sacrificed 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 months (15-month exposure group). The exposure 

protocol for the stop-exposure studies 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 study 
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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 female rats at the 15-month evaluation in both 

the 9-month stop-exposure group and the 15-month exposure group. The large intestines 

contained adenomatous polyps of the rectum in exposed males and females; however, the 

tumor incidence was statistically 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 were not statistically 

significantly increased, but the numerical increases in these tumors are concordant with 

those observed in the two-year bioassay (see Table 4-2). 
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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 Controla

9-mo. 

exposure Controla

9-mo. stopexposure15-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 

100b

364 

80 

484 

100 

388 

80b

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 0c

 0 3 7* 

Kidney (renal tubule adenoma) 0 0 0 3 2 

Urinary bladder: 

squamous-cell carcinoma 0 0d 0 0 1e

transitional epithelial papilloma 0 1d 0 0 3e

transitional epithelial carcinoma 0 0d 0 0 1e

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 

100b

211 

82 

328 

100 

258 

79b

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* 3f

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

Urinary bladder: 

squamous-cell carcinoma 0 0 0 1 4 

transitional epithelial papilloma 0 0 0 0 1g

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; eN = 19; fN = 17. 

g

A single squamous-cell papilloma of the urinary 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. 
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Maronpot et al. (1989a) analyzed the incidence of hepatic foci that either stained positive 

for γ-glutamyl transpeptidase (GGT) or were identified as altered hepatocellular foci 

(AHF) in hematoxylin and eosin–stained sections of livers from female F344/N rats fed 

ADBAQ at a concentration of 10,000 or 20,000 ppm 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 occupied by foci) were significantly increased 

in both the low- and high-dose groups, and 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 carcinogenicity study were insufficient evidence of 

hepatocarcinogenicity in the absence 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/cm3 

(mean ± SE) 

Volume fraction 

(mean % ± SE) 

Focus volume 

(mean mm3 ± 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 ± 287.0b

** 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 quantitative assessment of altered 

hepatocellular foci in histological material from the NTP two-year and 15-month stopexposure studies of ADBAQ in rats. Liver samples 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 types. No exposure-related changes in 

number, size, or volume fraction were found 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 occurrence 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 B6C3F1 mice: a 

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

4.2.1 Thirteen-week study 

Groups of B6C3F1 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 performed, in which tissues of all animals were 

collected, weighed, and processed for histologic examination (a complete list of tissues 
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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 observed, 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 males 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 B6C3F1 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 B6C3F1 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 glycogen depletion at 10,000 ppm or higher and pigmentation 

at all exposure levels. The authors concluded that “at comparable doses, ADBAQ was 

considered to be markedly toxic in rats and of minimal, non-life-threatening toxicity in 

mice.” 

4.2.2 Two-year study 

Dietary concentrations of ADBAQ for the two-year study in mice were based on the 

results of the 13-week study, specifically, on the frequency and severity of centrilobular 

hypertrophy of the liver observed in male mice. Because only lesions of mild severity 

were observed in groups exposed to ADBAQ at 10,000 or 25,000 ppm, and lesions of 

moderate severity were observed in the 50,000-ppm group, exposure concentrations for 

the two-year study were set at 0, 10,000, and 20,000 ppm. Other considerations included 

consistency among males and females (as females could have been given higher exposure 

concentrations) and correspondence to the concentrations selected for the stop-exposure 

study in rats (see Appendix A, p. A-57). 

Male and female B6C3F1 mice were obtained at five weeks of age and quarantined for 15 

days before the study began. Groups of mice (60 of each sex) received diets containing 
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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-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 4-3. Exposure protocol for the two-year study of ADBAQ in B6C3F1 mice 

The results of the two-year study are summarized 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. Hepatocellular adenoma or carcinoma, forestomach squamous-cell 

papilloma, and alveolar/bronchiolar adenoma were observed in exposed mice but not in 

controls at the 15-month interim sacrifice. Incidences of hepatocellular adenoma or 

carcinoma in high-dose males and females, squamous-cell papilloma in high-dose males 

and low-dose females, and alveolar/bronchiolar adenoma in high-dose and low-dose 

males were significantly higher than in controls. 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 and carcinoma of the forestomach were 

significantly increased in both males and females at both exposure levels. The incidence 

of alveolar/bronchiolar adenoma or carcinoma of the lung also was significantly 

increased in both males and females. 
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Table 4-6. Survival, body weight, and tumor incidence in B6C3F1 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 test (tumor data). 
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4.3 Summary 

ADBAQ was toxic to male and female F344/N rats 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, and anemia. Exposure of male and female 

B6C3F1 mice to ADBAQ at the same dietary concentrations resulted in lower apparent 

toxicity, as mice gained weight at all ADBAQ exposure levels, and fewer types of 

nonneoplastic lesions of the liver and kidney were observed. 

In two-year dietary studies 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 observed in a 15-month stop-exposure study in rats. 

B6C3F1 mice consuming ADBAQ-containing diets for two years had increased 

incidences of hepatocellular adenoma and carcinoma, squamous-cell papilloma and 

carcinoma of the forestomach, and alveolar/bronchiolar 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 increased 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 B6C3F1 mice based on 

increased incidences of neoplasms in the liver, forestomach, and lung.” 
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5 Genotoxicity 

Limited data are available on the genotoxicity of ADBAQ. The few studies that have 

attempted to ascertain the mutagenicity of ADBAQ have encountered 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 which 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 mutations (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. ADBAQ was weakly mutagenic in strain TA100 

(with or without S9 activation) and nonmutagenic in strain TA1535. The ADBAQ 

preparation used in these studies had a labeled purity of 91.7% and an analyzed purity of 

79.7%. The researchers reported the presence of precipitate when ADBAQ 

concentrations were in the range of 333 to 10,000 μg/plate (the highest concentration 

tested). The NTP (1996) noted that the positive or weakly positive mutagenic responses 

in the Haworth et al. (1983) study were observed in strains in which reversion occurs by 

frameshift mutations. 

Table 5-1. Mutagenicity of ADBAQ in S. typhimuriuma

 

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 evaluation of the ability of ADBAQ 

to induce mutations at the thymidine kinase (tk) locus, but they were unable to test for 
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micronucleus formation because of ADBAQ’s limited solubility. ADBAQ was not 

mutagenic in the absence or presence of induced-rat liver S9 at the highest testable 

concentrations (25 μg/mL without S9 and 20 μg/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 exposed to various concentrations of ADBAQ 

and then analyzed for chromosomal aberrations (Loveday et al. 1990). The authors 

reported the presence of a precipitate in the flasks containing ADBAQ at 50 μg/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 μg/mL 

significantly increased the percentage of cells with chromosomal aberrations (Dunnett’s 

test, P < 0.05). In the second trial, the percentage of cells with chromosomal aberrations 

was not significantly increased by ADBAQ at concentrations of 1.0, 3.0, 10.0, or 30.0 

μg/mL. In the presence of S9, the percentage of cells with chromosomal aberrations was 

not significantly increased by exposure to ADBAQ at 3.02, 10.10, or 30.20 μg/mL. 

The NTP (1996) reported additional results obtained in two trials performed by 

Environmental Health Research and Testing, Inc. (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 μg/mL in the first trial, but increases observed at 

16, 30, or 50 μg/mL in the second trial were not statistically significant. In the presence 

of S9, the percentage of cells with chromosomal aberrations was not significantly 

increased by ADBAQ at 16, 50, or 100 μg/mL. Based on these studies, 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 evidence for SCE induction. Results were 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% increase over controls. Three exposure levels 

were used for each trial, and at least two had 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% increase in the number 

of SCEs at 3.01, 10.1, or 30.1 μg/mL in trial 1 and at the highest concentration (15 

μg/mL) in trial 2. The authors classified ADBAQ as weakly positive for SCE induction in 

the absence of S9 and positive (trial 1) or weakly 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 tested at concentrations ranging from 5 to 
9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone 

31

50 μg/mL induced SCE in the absence of S9 (results were positive in one trial and 

weakly positive in another). In the presence of S9, ADBAQ at 5, 16, 50, or 100 μg/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 aberrations and SCE in CHO cells by ADBAQa

Metabolic activation Chromosomal aberrations Sister chromatid exchanges 

 

Loveday et al.

1990 NTP 1996b

Loveday et al.

1990 NTP 1996b

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 Environmental Health Research and Testing Laboratory. 

*P < 0.05 for 50 μg/mL concentration. 

5.2.4 Ras mutations in tumors 

Hayashi et al. (2001) examined 8 forestomach squamous-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 B6C3F1 mice that were fed diets 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 lung 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 (Table 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 CGA transitions in codon 61 of K-ras (lung tumors) or 

H-ras (forestomach tumors). No A to T transversions at the second base were detected in 

spontaneous tumors. Mutations observed in codons 12 or 13 generally occurred in both 

ADBAQ-induced and spontaneous tumors. The authors concluded that alterations in the 

ras gene might be an early event in ADBAQ-induced carcinogenesis. 
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Table 5-3. Frequency and spectra of ras mutations in forestomach and lung tumors 

from B6C3F1 mice exposed to ADBAQ for two yearsa

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 

Controlb

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 

Controlc

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 B6C3F1 mice. 

c

Study controls (N = 4) combined with historical spontaneous lung tumors (N = 82) of B6C3F1 mice. 

5.3 Summary 

ADBAQ was mutagenic in S. typhimurium strains that revert by frameshift mutations, 

and the mutagenicity was decreased or eliminated 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 μg/mL) were 

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

less than those used to assess clastogenicity (up to 100 μg/mL of ADBAQ). In CHO 

cells, ADBAQ induced chromosomal aberrations and SCE without metabolic activation; 

however, the results were inconsistent between 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 absent 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. 
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6 Other Relevant Data 

6.1 Absorption, distribution, metabolism, and excretion 

The NTP (1996) reported studies on the absorption, distribution, metabolism, and 

excretion of ADBAQ. Male F344/N rats were administered [14C]ADBAQ orally or 

intravenously (i.v.), and disposition and elimination of radioactivity (parent compound 

and/or metabolites) was monitored for up to 72 hours. The specific location of the 

radioactive carbon atom(s) in the ADBAQ molecule was not identified. 

6.1.1 Absorption 

ADBAQ was readily absorbed from the gastrointestinal tract. The percentage of the dose 

absorbed was inversely proportional to dose level. 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. administration. Radioactivity in 

blood was located primarily in plasma, as reflected 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 highest concentrations in excretory organs, lung, kidney, small 

intestine, liver, adipose tissue, and adrenal glands. At 15 minutes, tissue:blood ratios of 

radioactivity for these tissues were 3.0 or greater. Tissue:blood ratios declined during the 

72 hours after administration, remaining greater than 1.0 only in liver and kidney at 72 

hours. No quantitative data on tissue distribution were reported (NTP 1996). 

Two hours after administration, only 3% of circulating 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 administration, the radioactivity remaining in 

adipose tissue was still unchanged ADBAQ, whereas that in liver, muscle, and skin was 

primarily ADBAQ metabolites (which were not identified) (NTP 1996). The metabolites 

of ADBAQ are unknown. Other anthraquinone-based 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) study did find that very little (less than 

3%) of [14C]ADBAQ was excreted as unmetabolized ADBAQ. 

6.1.3 Excretion 

In the three days after administration of [14C]ADBAQ, approximately 50% of ADBAQassociated radioactivity was recovered from feces, 15% from urine, and 6% from expired 

air. Consistent with extensive metabolism, less than 3% of total radioactivity excreted 

represented unchanged ADBAQ. Terminal half-lives of administered radioactivity in 

liver and kidney were estimated to be 40 and 90 hours, respectively, and the elimination 

half-life from adipose tissue was 11 hours. 
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34

6.2 Liver focus model 

Maronpot et al. (1989b) tested ADBAQ in a neonatal rat liver focus model for its 

potential both as an initiator and as a promoter. In the test for initiation potential, 

0.07 mg/g b.w. of ADBAQ was injected intraperitoneally (i.p.) within 24 hours after 

birth, and phenobarbital (500 ppm) was used as the promoting agent over the succeeding 

75 or 300 days. The authors found no evidence of initiating activity for ADBAQ. 

In the test for promotion potential, each neonatal rat was injected with a 

“subcarcinogenic” dose (4 μg/g b.w.) of the initiator, diethylnitrosamine (DEN), within 

24 hours of birth. After weaning, rats received ADBAQ at either 5,000 ppm or 10,000 

ppm in the diet for either 75 or 300 days prior to sacrifice. Livers 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 association between the equivocal 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 hepatocellular foci neonatal rats following dietary 

exposure to ADBAQ for 75 or 300 daysa

Exposure concentration 

(ppm) Mean foci/cm3

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. 
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35

6.3 Carcinogenicity and mutagenicity of other quinones and anthraquinones 

ADBAQ and other anthraquinones belong to a general class of quinone molecules that 

can be derived from aromatic molecules such as benzene, naphthalene, and anthracene. 

Quinone molecules can be reduced to a relatively 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 hydrocarbons, estrogens, and catecholamines and give 

rise to reactive oxygen species that can damage DNA and other cellular macromolecules 

and activate signaling pathways. These molecular events may be associated with the 

initiation, promotion, and progression 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 that are implicated in the carcinogenic effects 

of these steroids could be metabolized to electrophilic and redox-active quinoids that 

could damage DNA. Their conclusion, however, was that the ultimate carcinogenic 

metabolites of estrogens have not been unequivocally established. 

Anthraquinones comprise a large class of diverse molecules which may be naturally 

occurring or synthetic in origin. Sendelbach (1989) reviewed the toxicity and 

carcinogenicity of anthraquinone derivatives with phenolic, amino, or nitro substitutions 

to the anthraquinone ring structure. The author’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 both amino and halogen substitutions to the 

anthraquinone ring, is not clear. However, anthraquinone also produces renal tumors. 

Renal tubule adenomas and carcinomas (combined) 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 dyes 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_pages/NTP_ALL_STDY_PG.html). 

As described in Section 5.2.1, Harrington-Brock et al. (1991) carried out a limited 

evaluation of the ability of ADBAQ to induce mutations at the thymidine kinase (tk) 

locus of L5178/TK+/-mouse lymphoma cells, but they were unable to test for 

micronucleus formation because of ADBAQ’s limited solubility. An additional 15 

anthraquinones were tested in the same assays. Testing of 8 of these substances was 

incomplete because of their limited solubility. For these 15 compounds, Table 6-3 
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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 genetic 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 small portion (~10%) of the hydroxyanthraquinones 

contained either amino or halogen (chlorine or bromine) substitutions; however, the 

relevance of these findings to the possible mutagenicity of ADBAQ is not clear. 
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Table 6-2. Carcinogenicity and mutagenicity of anthraquinone and some anthraquinone-derived dyes (data fr

Property Anthraquinone 

2-Aminoanthraquinone 

1-Amino-2-

methylanthraquinone 

1,8-Dihydroxyanthraquinone 

(danthron) 

2-Methyl-1-nitroanthraquinone 

1,4,

Tetraa

anthraq

(dispers

Long-term carcinogenicityin mice 

Liver neoplasms in male and female B6C3F1 mice 

Hepatocellular carcinomas in male and female B6C3F1 mice 

(NTP) Malignant hematopoietic lymphomas in high dose female mice (NTP) 

Negative in male mice (NTP) Hepatocellular carcinomas and neoplastic liver nodules in female B6C3F1 mice 

(NTP) 

Hepatocellular adenomas and carcinomas and adenomatous hyperplasia of the cecum in C3H/HeN male mice (IARC) 

Hemangiosarcomas in male and female B6C3F1 mice 

(NTP) 

Hepatocelladenomas carcinoma(combinedB6C3F1 m

Alveolar/badenomas carcinoma(combinedB6C3F1 m

Long-term carcinogenicityin rats 

Renal tubule neoplasms in male and female F344/N rats Transitional epithelial papillomas of the urinary bladder in male F344/N rats 

Hepatocellular carcinomas in male F344 rats (NTP) Inadequate study in female F344 rats (poor survival) (NTP) 

Hepatocellular carcinomas in male and female F344 rats (NTP) 

Adenocarcinomas of the colon in male ACI rats (IARC) 

Hepatocellular carcinomas in male F344 rats (NTP) Subcutaenous fibromas in male and female F344 rats (NTP) 

Transitionaneoplasms urinary blamale and fF344 rats (Squamousneoplasms F344 rats (Leiomyomleiomyosar(combinedurinary blafemale F34(NTP) 
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Property Anthraquinone 

2-Aminoanthraquinone 

1-Amino-2-

methylanthraquinone 

1,8-Dihydroxyanthraquinone 

(danthron) 

2-Methyl-1-nitroanthraquinone 

1,4,

Tetraa

anthraq

(dispers

Genetic toxicology 

Positive for mutagenicity in S. typhimurium TA98 and TA100 with and without S9 Positive for micronucleated normochromatic erythrocytes in male and female mice (14-week dietary study) Negative for micronuclei in mouse bone marrow (i.p. injection) 

Positive for chromosomal aberrations Positive for SCE Positive for mutagenicity in S. typhimurium(NTP) 

Positive for chromosomal aberrations Positive for SCE Positive for mutagenicity in S. typhimurium(NTP) 

Positive for chromosomal aberrations Not tested for SCE Negative for micronuclei [cells not specified] Weakly positive for mutagenicity in S. typhimurium(NTP) 

Positive for chromosomal aberrations Positive for SCE Negative for reciprocal translocation in DrosophilaPositive for mutagenicity in S. typhimurium

Positive fochromosomaberrations Positive foPositive fomutagenicimouse lymcells Negative fmicronuclenot specifiPositive fomutagencittyphimuriu
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Property Anthraquinone 

2-Aminoanthraquinone 

1-Amino-2-

methylanthraquinone 

1,8-Dihydroxyanthraquinone 

(danthron) 

2-Methyl-1-nitroanthraquinone 

1,4,

Tetraa

anthraq

(dispers

RoC listing or NTP evaluation 

NTP: some evidence of carcinogenic activity in male F344/N rats; clear evidence of carcinogenic activity in female F344/N rats; clear evidence of carcinogenic activity in male and female B6C3F1 mice 

RoC: reasonably anticipated to be a human carcinogen 

RoC: reasonably anticipated to be a human carcinogen 

RoC: reasonably anticipated to be a human carcinogen 

NTP: found to be carcinogenic in male and female rats and mice 

RoC: reasoanticipated human car

IARC evaluationa

Not listed Group 3 

(IARC 1987) 

Group 3 

(IARC 1987) 

Group 2B 

(IARC 1990) 

Group 2B 

(IARC 1987) 

Grou

(IARC 

aGroup 2B = possibly carcinogenic to humans, based on limited evidence of carcinogenicity in humans, less than sufficient evidence of carc

Group 3 = not classifiable as to its carcinogenicity in humans, based on inadequate or limited evidence of carcinogenicity in animals and inacarcinogenicity in humans. 
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Table 6-3. Genotoxicity of some anthraquinone dyesa

Anthraquinone CASRN 

Purity 

(%) 

Mutagenicity in mouse 

lymphoma cells 

Micronuclei in mouse 

lymphoma cells 

Reverse 

S. typ

1-Amino-2-methylanthraquinone 

82-28-0 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 1-Nitro-2-methylanthraquinone 

129-15-7 95.9 limited solubility 

(–/+ w S9) 

limited solubility 

(–/+ w S9) 

Reactive blue 19 2580-78-1 36 + (weak) w/o S9 increased from 9/1,000 to 

19/1,000 cells 

w/o S9 

Vat blue 4 81-77-6 48 limited solubility limited solubility Vat blue 20 116-71-2 26 limited solubility limited solubility Vat brown 1 2475-33-4 56 limited solubility limited solubility Vat 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-Brock et al. 1991 and references therein. a+ = positive; – = negative; –/+ equivocal; NR = not reported. 
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6.4 Summary 

In rats, ADBAQ is rapidly absorbed from the gastrointestinal tract and distributed rapidly 

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 

compound from either blood or urine; however, the metabolites of ADBAQ have not 

been identified. ADBAQ and its metabolites are excreted primarily in the feces and urine. 

ADBAQ and other anthraquinones are 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 generated by metabolism of a variety of 

quinones may be associated with DNA damage or activation of signaling pathways 

involved in initiation, promotion, and progression of carcinogenesis. Four other 

anthraquinone vat dyes evaluated 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. 
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and B6C3F1 Mice (Feed Studies). National Toxicology Program Technical Report 

Series No 299. 

30. NTP. 1996. Toxicology and carcinogenesis studies of 1-Amino-2,4-

Dibromoanthraquinone in F344/N rats and B6C3F1 mice (feed studies). National 

Toxicology Program Technical Report Series No 383. 

31. NTP Chemical Repository. 2001. 1-amino-2,4-dibromoanthraquinone. Revised 

August 13, 2001. National Toxicology Program. 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 Poisons, 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 effluent 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. 

35. Sendelbach, L.E. 1989. A review of the toxicity and carcinogenicity of 

anthraquinone derivatives. Toxicology 57:227-240. 

36. TRI. 2001. Toxic Release Inventory. Revised 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 

46
9/19/02 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 

48
5

ABSTRACT

1-AMINO-2,4-DIBROMOANTHRAQUINONE

CAS No. 81-49-2

Chemical Formula: C14H7Br2NO2 Molecular Weight: 381.04

Synonym: ADBAQ 

1-Amino-2,4-dibromoanthraquinoneisananthraquinonederivedvatdye, a member of a class of insolubledyes

that are impregnated into textile fibers. Five

anthraquinone-derived dyes with representative and

diverse structures, as well as the parent chemical,

anthraquinone,wereselectedfortoxicology andcarcinogenesis evaluation. Similartothebenzidinedye initiative, the rationale for selecting these vat dyes was to

generatesufficienttoxicologicdatatopermitmorereliable

predictionsof carcinogenicitytobemadeonotherchemicalsinthisclass,therebyeliminatingorreducingtheneed

to study every anthraquinone dye. 1-Amino2,4-dibromoanthraquinone is the last anthraquinonederived dye in this group to be studied.

Groups of male and female F344/Nrats and B6C3F1

micewere exposed to 1-amino-2,4-dibromoanthraquinone (87% to 97% pure) for 13 weeks or for 9, 15,

or 24 months. Because 1-amino-2,4-dibromoanthraquinonewaspredictedtobe carcinogenic,thesestudies

were designed to evaluate the potential for tumor

progression and regression. Absorptionandexcretion

studieswere carried out in male F344/Nrats. Genetic

toxicity was determined in vitro using Salmonella

typhimurium and cultured Chines e

hamsterovarycells. Extensive chemical analyseswere

performedtoidentifyand characterize impuritiesofthe

1-amino-2,4-dibromoanthraquinoneusedinthesestudies.

13-W EEK S T U DY IN R A T S

Groupsof10maleand10female ratsweregiven0,2,500,

5,000, 10,000, 25,000, or 50,000 ppm 1-amino2,4-dibromoanthraquinone in feed for 13weeks. These

levels correspond to approximately 150 to 3,200 mg

1-amino-2,4-dibromoanthraquinone/kgbodyweightper

dayformales and to approximately170 to 3,200mg/kg

for females. Chemical-relatedmortalitywaslimitedto

onemaleandonefemaleinthe50,000ppmgroups. Final

meanbodyweights andbodyweightgainsof allexposed

groupsofratswere significantlylowerthanthoseofthe

controls. Feed consumptionbyall exposed groupswas

less than that by the controls throughout the study and

generallydecreasedwithincreasingexposureconcentration. Pink-redstainingofthe fur andtailwasobservedin

allexposedgroups. Absoluteandrelative liverweightsof

all exposedgroupsweregenerallysignificantlygreater

than those of the controls.
6 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Chemical-related lesions were present in the liver,

kidney, and spleen of male and female rats. Nonneoplasticlesionsinthe liverincludedfociofhepatocellular

alteration, diffuse hepatocellular hypertrophy (cytomegaly),hepatocellular cytoplasmic vacuolation,bile

duct hyperplasia, inflammation, and pigmentation.

Thesedifferenceswereobservedprimarilyinthe25,000

and 50,000 ppm groups of males and females; the

spectrumofproliferative lesionsofthebileducts(hyperplasia,fibrosis,andnecrotizingcholangitis)inthe25,000

and50,000ppmgroupswasmorphologicallyconsistent

withthe lesiondescribedas cholangiofibrosis. Pigmentationwas present in the renal tubuleepitheliumof all

groups of exposed rats; nuclear enlargement

(karyomegaly) was also present in the renal tubule

epitheliuminsome ofthe exposedrats. Accumulationof

hyaline droplets in the cytoplasm of the renal tubule

epithelium and tubule lumina was present in 2,500,

5,000, 10,000, and 25,000 ppm males. Incidences of

hematopoiesisofthe spleeninexposedgroupsofmales

and females were increased compared to those in the

controls. 

13-WEEK STUDY IN MICE

Groups of 10 male and 10 female mice were given 0,

2,500,5,000,10,000,25,000,or 50,000ppm1-amino2,4-dibromoanthraquinone infeedfor13weeks. These

levelscorrespond to approximately 500 to 10,600mg

1-amino-2,4-dibromoanthraquinone/kgbodyweightper

dayformales and approximately660 to 11,700mg/kg

per day for females. There was no chemical-related

mortality. Feed consumption and final mean body

weights of exposedgroupswere similarto those ofthe

controls. Red staining of the fur was observed in all

exposedgroups. Absolute andrelative liverweightsof

the exposed groups were greater than those of the

controls except for the absolute liver weight of

2,500ppmmales. Absolute andrelativekidneyweights

of25,000and50,000ppmmaleswere lowerthanthose

of the controls. 

Chemical-related lesions were limitedto the livers of

malesandconsistedofpigmentationofhepatocytesatall

exposureconcentrationsandcentrilobularhepatocellular

hypertrophyat10,000,25,000, and50,000ppm. Minimalpigmentwaspresentinthe liverofonefemale inthe

25,000ppmgroupandinone female inthe50,000ppm

group.

2-YEAR STUDY IN RATS

Groupsof70maleand70femaleratsweregiven0,5,000,

or10,000ppm1-amino-2,4-dibromoanthraquinoneinfeed

for 103 weeks. In addition, groups of 50 male and 50

female rats were given 2,000 ppm 1-amino2,4-dibromoanthraquinoneinfeedfor104weeks. These

exposureconcentrationswereapproximatelyequalto90,

240,or490mg1-amino-2,4-dibromoanthraquinone/kg

body weight for males and 110, 285, or 600 mg/kg for

females. Tenanimalsfromeachgroupwereevaluatedfor

histopathology at 9 months. Additional groups of

10 animals from the 0 and 10,000 ppm groups were

evaluated for histopathology at 15 months.

Survival, Body Weights, Feed Consumption,

and Clinical Findings

Inthe2-yearstudy,survivalofthe10,000ppmmalesand

femaleswassignificantlylowerthanthatofthe controls.

Survivalofthe 2,000and5,000ppmgroupswassimilar

to that ofthe controls. During the last year ofthe study,

the mean body weights of exposed males were 80% to

91% those of controls, and the mean body weights of

exposed females were 67% to 84% those of controls.

Feedconsumptionamongexposedgroupswasgenerally

similar, but wasless than that by controls. The fur and

urineof all exposedmaleandfemalegroupswerediscolored. 

Pathology Findings

Inthe2-yearstudy, 1-amino-2,4-dibromoanthraquinone

wasassociatedwithsignificantchemical-relatedincreases

in the incidencesof benign andmalignant neoplasmsin

the liver, large intestine, kidney, and urinarybladder of

males and females. Chemical-related nonneoplastic

proliferative and degenerative lesions occurred in the

liver,kidney, urinarybladder, andforestomachofmales

and females.

The incidences offoci of hepatocellular alteration and

pigmentation in the liver of males and females were

increased at the 9-month interim evaluation, and a

hepatocellular adenoma was presentin one 5,000 ppm

male. At the 15-month interim evaluation,

hepatocellular adenoma or carcinoma (combined)

occurred in all males and nine females in the

10,000 ppm groups. By the end of the 2-yearstudy,

hepatocellular adenoma, carcinoma, cholangioma, or

cholangiocarcinoma were observed in males and

 
1-Amino-2,4-dibromoanthraquinone, NTP TR 383 7

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

2,000ppmgroups,similarliverneoplasmswerepresent

in 63% of themales and in 83%ofthe females. Of the

hepatocellularcarcinomasinthe5,000and10,000ppm

groups ofmales and females,31% to 49% were metastatic to the lungs or other sites. Increases in the incidences offoci of hepatocellular alteration(basophilic,

eosinophilic,andclear cell)andpigmentationofthe liver

were also observed in exposed groups of males and

females. 

Adenomatouspolyps(adenoma)ofthe large intestine

werepresentin six10,000ppmmales at the 15-month

interimevaluation. Incidences of adenomatouspolyp

(adenoma) and carcinoma of the large intestine were

significantlyincreasedinexposedgroupsofmalesand

females after 2 years; multiple benign and malignant

intestinal neoplasms were observed in many of these

rats. 

In the kidney, incidencesofrenaltubule adenoma and

carcinoma were significantly increased in exposed

groupsofmalesandfemalesafter2years. Renaltubule

adenomas were present in two 10,000 ppm males at

15months. Therewerealsochemical-relatedincreases

inthe incidences andseveritiesofrenaltubuleepithelial

hyperplasia,pigmentation,andtransitionalcellhyperplasia in the kidneyofmalesandfemales. Hyalinedroplet

accumulation was present in all exposed male rats at

9 months.

Incidencesoftransitional cellpapilloma andcarcinoma

oftheurinarybladderwereincreasedat2yearsinmales

andfemalesinthe10,000ppmgroups. Transitional cell

hyperplasiawasobservedinexposedmalesandfemales

atthe15-monthinterimevaluation. Othernonneoplastic

lesions observed in the urinary bladder at 2 years included metaplasia of the transitional epithelium and

submucosal stromal tissue.

In the forestomach, the incidences and severities of

inflammation,ulceration,hyperkeratosis, andhyperplasia of the squamous mucosa were increased in all

exposedgroupsofmales andfemalesat2years,butnot

at the 9- or 15-month interim evaluations.

In exposed males and females, the incidences of

mononuclearcellleukemiaweresignificantlydecreased.

The incidencesof atrophyofthe seminal vesiclewere

increased in exposed male rats in the 2-year study.

Stop-Exposure Evaluation in Rats

Groups of 40 male and 40 female rats were given

20,000ppm1-amino-2,4-dibromoanthraquinone infeed

for 9 or 15 months. At 9 months, 10 males and 10 femaleswereevaluatedforhistopathology (9-monthinterim

evaluation groups). After 9 months of exposure, an

additional10males and10femaleswerefedcontroldiet

until the end of the 15-month evaluation (9-month

stop-exposure groups), and 20 males and 20 females

continuedtoreceive20,000ppm1-amino-2,4-dibromoanthraquinoneuntilthe endofthe evaluation(15-month

exposuregroups). The approximatedailyconsumptionof

1-amino-2,4-dibromoanthraquinonewas1,335mg/kgfor

malesand1,790mg/kgforfemalesin the 9-monthstopexposure groups and 1,115 mg/kg for males and

1,435 mg/kg for females in the 15-month exposure

groups.

Survivalwassimilaramonggroupsexceptforthe females

inthe15-monthexposuregroup;thesurvivalofthisgroup

was lower than that of the controls. Lower mean body

weightswererelatedtoincreasedexposureduration. The

meanbodyweightsof exposedmaleswere76%to 82%

that of controls, and themeanbody weights of exposed

females were 73% to 84% that of controls. 

Forthestop-exposureevaluation,similarchemical-related

neoplasmsandnonneoplasticlesionswereobservedinthe

same sites as in the 2-year study: liver, large intestine,

kidney, urinary bladder, and forestomach.

After 9monthsof dietaryexposure toa concentrationof

20,000 ppm 1-amino-2,4-dibromoanthraquinone,

hepatocellularadenoma andcarcinomaoccurredinmales

andfemales. Nonneoplastic chemical-relatedlesionsin

the liver of exposed rats included pigmentation, focal

hepatocellular alteration, and bile duct hyperplasia.

Neoplasms atothersitesinmalesincludedoneadenomatous polyp (adenoma) in the large intestine and one

transitionalcellpapilloma intheurinarybladder. Hyaline

droplet accumulation was present in the kidney of exposed males at 9 months. 

In the stop-exposure groups examined at 15 months,

hepatocellular adenoma and carcinoma werepresentin

most males and females. Adenomatous polyp

(adenoma)ofthe colon,renaltubulecell adenoma,and

urinary bladder transitional cell papilloma and
8 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

carcinoma also occurred in males and females.

Nonneoplasticchemical-relatedlesionsincludedfociof

hepatocellularalteration in the liver andhyperplasiaof

the renaltubuleepitheliumand urinarybladdertransitional epithelium. Hyperplasia,hyperkeratosis,inflammation, and ulceration were observed in the

forestomachsofsomemaleandfemale ratscontinuously

exposed for 15 months.

2-YEAR STUDY IN MICE

Groups of 60 male and 60 female mice were given 0,

10,000, or 20,000 ppm 1-amino -

2,4-dibromoanthraquinone infeedfor104weeks. The

dailycompoundconsumptionwasapproximately1,690

or3,470mg1-amino-2,4-dibromoanthraquinone/kgbody

weightformalesand1,950or4,350mg/kgforfemales.

Ten animals from each group were evaluated for

histopathology at 15 months.

Survival, Body Weights, Feed Consumption,

and Clinical Findings

In the 2-year study, survival of exposed males was

significantlylowerthanthatofthe controls. Survivalof

exposedfemaleswassimilartothatofthe controls. The

finalmeanbodyweightsof exposedmaleswere83%to

85%thatof controls,andthe finalmeanbodyweightsof

exposed females were 81% to 86% that of controls.

Feed consumption by exposed groups was generally

similar to that by controls. Discoloration of the fur,

urine, and feces was observed in all exposed groups. 

Pathology Findings

Inthe2-yearstudy, 1-amino-2,4-dibromoanthraquinone

was associated with significant chemical-related increases in the incidences of benign and malignant

neoplasmsinthe liver,forestomach,and lungofmales

and females.

Incidencesof hepatocellular adenoma and carcinoma

were increased in exposed groups at the 15-month

interimevaluationandat2years. At 2years,therewere

significant increases in the incidences of multiple

hepatocellular adenoma and carcinoma in males and

females and in the incidences of hepatoblastoma in

males. Centrilobular hypertrophy of hepatocytes in

malesandfociofhepatocellularalterationandpigmentation in the liver of males and females were also

chemical-related changes.

Squamous cell papilloma of the forestomach mucosa

occurred in 10,000ppmfemales and 20,000ppmmales

andfemales atthe 15-monthinterimevaluation, andthe

incidences of squamous cell papilloma and carcinoma

were significantlyincreasedinexposedgroupsofmales

andfemales at 2 years. Chemical-relatedhyperplasiaof

forestomachepitheliumwasalsopresentat15monthsand

at 2 years.

Alveolar/bronchiolaradenomaswerepresentonlyinthe

exposedgroups ofmales and females at 15months,and

the incidences of alveolar/bronchiolar adenoma were

significantlyincreased in exposedmalesandfemales at

2years. The incidencesofmultiplealveolar/bronchiolar

adenoma were also increased in exposed males. 

In the kidney, pigmentation was present in the renal

tubules of most mice after 2 years of exposure.

DISPOSITION

AND METABOLISM STUDIES

Adult male F344/N rats were given [14C]-labeled

1-amino-2,4-dibromoanthraquinone as a singleintravenous dose of 0.4 mg/kg body weight or as a single oral

dose of 2, 23, 118, 814, or 1,473 mg/kg. A 6-hour bile

cannulation study was also performed. From day 0

throughday3afterintravenousadministration,about50%

ofthe 14Cwas excretedinthe feces,15%intheurine, and

6% in expired air. Unmetabolized 1-amino2,4-dibromoanthraquinone accountedforlessthan3%of

the excreted14Cafterintravenousadministration. Fororal

doses administered, the amount of the dose that was

absorbed fit the equation: absorbed dose = 6.6 

log(dose). Afterintravenousadministration,themetabolitesof1-amino-2,4-dibromoanthraquinoneinbloodwere

primarilyin the plasma fraction (blood:plasma ratio of

approximately0.5:1). The highest concentrationsof 14C

in tissues 15 minutes after intravenous dosing were in

excretory organs, lung, kidney, small intestine, liver,

adipose tissue, and adrenal gland. 

GENETIC TOXICOLOGY

1-Amino-2,4-dibromoanthraquinone was mutagenic

inSalmonellatyphimuriumstrainsTA98andTA1537in

the absence of S9; with S9, an equivocal response was

observedinTA1537. 1-Amino-2,4-dibromoanthraquinone

resulted in an equivocal response in
1-Amino-2,4-dibromoanthraquinone, NTP TR 383 9

strain TA100 with and without S9, and no mutagenic

activitywas detected with strainTA1535. In cultured

Chinese hamster ovary cells, 1-amino2,4-dibromoanthraquinone induced sister chromatid

exchangeswithandwithoutS9; chromosomal aberrations were induced only in the absence of S9. 

CONCLUSIONS

Underthe conditionsofthese2-yearfeedstudies,there

was clear evidence of carcinogenic activity* of

1-amino-2,4-dibromoanthraquinoneinmaleandfemale

F344/Nratsbasedonincreasedincidencesofneoplasms

inthe liver,large intestine,kidney, andurinarybladder.

There was clear evidence of carcinogenic activity of

1-amino-2,4-dibromoanthraquinoneinmaleandfemale

B6C3F1 mice based on increased incidences of

neoplasms in the liver, forestomach, and lung.

Exposure of male and female rats to 1-amino2,4-dibromoanthraquinonefor2yearswasassociatedwith

basophilic focus (males only), clear cell focus,

eosinophilic focus, and pigmentationin the liver;renal

tubulehyperplasia,renaltubulepigmentation,andtransitional cell hyperplasia in the kidney; transitional cell

hyperplasia,squamousmetaplasia,andstromalmetaplasia

(femalesonly)intheurinarybladder;squamoushyperplasia, hyperkeratosis,ulceration, andinflammationofthe

forestomach mucosa; and seminal vesicle atrophy.

Exposure of male and female mice to 1-amino2,4-dibromoanthraquinonefor2yearswasassociatedwith

centrilobularhepatocellularhypertrophy(malesonly),

basophilic focus, clear cell focus (females only),

eosinophilicfocus,andpigmentationinthe liver;pigmentationinthekidney; andhyperplasia,basal cellhyperplasia,hyperkeratosis, andinflammationofthe forestomach

mucosa. 

__________

*

Explanation of Levels of Evidence of Carcinogenic Activity is on page 13. A summary of the Technical Reports Review

 Subcommittee comments and the public discussion on this Technical Report appears on page 15.
10 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Font Size:11ptSummary of the 2-Year Carcinogenesis and Genetic Toxicology Studies 

of 1-Amino-2,4-dibromoanthraquinone

Male

F344/N Rats

Female

F344/N Rats

Male 

B6C3F1 Mice

Female

B6C3F1 Mice

Doses 0, 2,000, 5,000, or

10,000 ppm

[approximately 

90, 240, or

490 mg/kg/day]

0, 2,000, 5,000, or

10,000 ppm

[approximately 

110, 285, or

600 mg/kg/day]

0, 10,000, or 20,000

ppm

[approximately 

1,690 or

3,470 mg/kg/day]

0, 10,000, or

20,000 ppm

[approximately 

1,950 or

4,350 mg/kg/day]

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

Nonneoplastic

effects

Liver: basophilic

focus (9/50, 12/40,

24/59, 22/50); clear

cell focus (3/50,

26/40, 39/59, 27/50);

eosinophilic focus

(1/50, 13/40, 14/59,

6/50); pigmentation

(3/50, 19/40, 48/59,

39/50)

Kidney: renal tubule

hyperplasia (9/50,

30/40, 25/59, 19/50);

renal tubule

pigmentation (5/50,

40/40, 58/59, 49/50);

transitional cell

hyperplasia (30/50,

40/40, 51/59, 35/50)

Urinary bladder:

transitional cell

hyperplasia (1/50,

5/38, 17/58, 30/50);

squamous metaplasia

(0/50, 0/38, 0/58,

3/50)

Liver: clear cell focus

(3/50, 28/40, 39/60,

17/48); eosinophilic

focus (7/50, 23/40,

12/60, 1/48);

pigmentation (1/50,

19/40, 51/60, 45/48)

Kidney: renal tubule

hyperplasia (1/50,

12/40, 23/60, 27/48);

renal tubule

pigmentation (0/50,

40/40, 60/60, 48/48);

transitional cell

hyperplasia (10/50,

16/40, 44/60, 21/48)

Urinary bladder:

transitional cell

hyperplasia (1/50,

2/40, 41/60, 41/46);

squamous metaplasia

(0/50, 1/40, 4/60,

8/46); stromal

metaplasia (0/50,

0/40, 4/60, 2/46)

Liver: centrilobular

hepatocyte

hypertrophy (0/50,

17/51, 13/50);

basophilic focus

(0/50, 4/51, 3/50);

eosinophilic focus

(0/50, 6/51, 1/50);

pigmentation (1/50,

50/51, 47/50)

Kidney: renal tubule

pigmentation (0/50,

42/51, 43/50)

Forestomach:

hyperplasia (1/50,

9/50, 4/50); basal cell

hyperplasia (0/50,

0/50, 2/50);

hyperkeratosis (1/50,

7/50, 6/50);

inflammation (2/50,

6/50, 13/50)

Liver: basophilic

focus (0/50, 4/50,

5/50); clear cell focus

(0/50, 10/50, 9/50);

eosinophilic focus

(0/50, 4/50, 2/50);

pigmentation (0/50,

44/50, 49/50)

Kidney: renal tubule

pigmentation (0/50,

43/50, 43/50)

Forestomach:

hyperplasia (9/48,

15/50, 19/50); basal

cell hyperplasia (0/48,

7/50, 3/50);

hyperkeratosis (10/48,

14/50, 17/50);

inflammation (7/48,

10/50, 21/50)

(continued)
1-Amino-2,4-dibromoanthraquinone, NTP TR 383 11

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies 

of 1-Amino-2,4-dibromoanthraquinone (continued)

Male

F344/N Rats

Female

F344/N Rats

Male 

B6C3F1 Mice

Female

B6C3F1 Mice

Nonneoplastic

effects

(continued)

Forestomach: 

squamous hyperplasia

(3/49, 19/39, 25/59,

26/49); hyperkeratosis

(5/49, 18/39, 21/59,

20/49); ulcer (3/49,

10/39, 15/59, 16/49);

inflammation (3/49,

12/39, 11/59, 11/49);

Seminal vesicle:

atrophy (1/49, 30/40,

35/59, 23/50)

Forestomach: 

squamous hyperplasia

(2/49, 7/40, 26/60,

33/47); hyperkeratosis

(2/49, 7/40, 23/60,

28/47); ulcer (1/49,

2/40, 7/60, 17/47);

inflammation (0/49,

1/40, 13/60, 10/47)

Neoplastic effects Liver: hepatocellular

adenoma (1/50,

20/40, 40/59, 34/50);

hepatocellular

carcinoma (1/50,

12/40, 55/59, 46/50);

hepatocholangiocarcinoma (0/50,

0/40, 6/59, 2/50)

Large intestine (all

sites): adenomatous

polyp (adenoma)

(0/50, 13/40, 51/59,

40/50); carcinoma

(0/50, 1/40, 11/59,

17/50)

Kidney (renal tubule):

adenoma (2/50,

10/40, 11/59, 14/50);

carcinoma (0/50,

0/40, 2/59, 1/50)

Urinary bladder:

transitional cell

papilloma (0/50, 1/38,

2/58, 8/50);

transitional cell

carcinoma (0/50,

0/38, 1/58, 4/50)

Liver: hepatocellular

adenoma (0/50,

28/40, 47/60, 29/48);

hepatocellular

carcinoma (0/50,

12/40, 57/60, 45/48);

hepatocholangiocarcinoma (0/50,

0/40, 11/60, 13/48)

Large intestine (all

sites): adenomatous

polyp (adenoma)

(0/50, 28/40, 53/60,

43/49); carcinoma

(0/50, 2/40, 21/60,

8/49)

Kidney (renal tubule):

adenoma (0/50, 3/40,

16/60, 16/48);

carcinoma (0/50,

0/40, 0/60, 2/48)

Urinary bladder:

transitional cell

papilloma (0/50, 2/40,

7/60, 9/46);

transitional cell

carcinoma (0/50,

0/40, 8/60, 16/46)

Liver: hepatocellular

adenoma (10/50,

38/51, 39/50);

hepatocellular

carcinoma (9/50,

18/51, 21/50);

hepatoblastoma (0/50,

3/51, 5/50)

Forestomach:

squamous cell

papilloma (0/50,

13/51, 16/50); 

squamous cell

carcinoma (0/50,

12/51, 13/50)

Lung: alveolar/

bronchiolar adenoma

(7/50, 26/51, 24/50)

Liver: 

hepatocellular

adenoma (6/50,

45/50, 49/50);

hepatocellular

carcinoma (0/50,

23/50, 27/50)

Forestomach:

squamous cell

papilloma (2/50,

16/50, 27/50);

squamous cell

carcinoma (0/50,

12/50, 11/50)

Lung: alveolar/

bronchiolar adenoma

(4/50, 17/50, 13/49)

(continued)
12 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies 

of 1-Amino-2,4-dibromoanthraquinone (continued)

Male

F344/N Rats

Female

F344/N Rats

Male 

B6C3F1 Mice

Female

B6C3F1 Mice

Level of evidence

of carcinogenic

activity

Clear evidence Clear evidence Clear evidence Clear evidence

Genetic toxicology

Salmonella typhimurium gene mutation: Equivocal in strain TA100 with and without S9; negative in strain TA1535 with and without S9;

positive in strain TA1537 without S9, equivocal with S9; positive in strain TA98 without S9,

negative with S9 

Sister chromatid exchanges

Cultured Chinese hamster ovary cells in vitro: Positive with and without S9 (combined results from testing in two laboratories) 

Chromosomal aberrations

Cultured Chinese hamster ovary cells in vitro: Weakly positive without S9, negative with S9 
1-Amino-2,4-dibromoanthraquinone, NTP TR 383 13

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the

evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of

neoplasia than control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are

conducted under a limited set of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under

the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations,

such as the International Agency for Research on Cancer, assign a strength of evidence for conclusions based on an examination of all

available evidence, including animal studies such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. 

Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider

analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence

observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain

findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot

be evaluated because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June

1983 and then revised in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual

weight of evidence of carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the

following five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and

not to potency or mechanism.

• Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked

increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to

malignancy.

• Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a

chemical-related increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the

response is less than that required for clear evidence.

• Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal

increase of neoplasms that may be chemical related.

• No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related

increases in malignant or benign neoplasms.

• Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or

quantitative limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic

activity.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the

actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and

current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the

borderline between two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct;

• occurrence of common versus uncommon neoplasia;

• progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions;

• some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present,

it is impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent

course is to assume that benign neoplasms of those types have the potential to become malignant;

• combining benign and malignant tumor incidence known or thought to represent stages of progression in the same

organ or tissue;

• latency in tumor induction;

• multiplicity in site-specific neoplasia;

• metastases;

• supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments

(same lesion in another sex or species);

• presence or absence of dose relationships;

• statistical significance of the observed tumor increase;

• concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm;

• survival-adjusted analyses and false positive or false negative concerns;

• structure-activity correlations; and

• in some cases, genetic toxicology.
14 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on 1-amino2,4-dibromoanthraquinone on June 21, 1994, are listed below. Subcommittee members serve as independent scientists, not as

representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major

responsibilities in reviewing NTP studies:

! to ascertain that all relevant literature data have been adequately cited and interpreted,

! to determine if the design and conditions of the NTP studies were appropriate,

! to ensure that the Technical Report presents the experimental results and conclusions fully and clearly,

! to judge the significance of the experimental results by scientific criteria, and

! to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.

Arnold L. Brown, M.D., Chair

University of Wisconsin Medical School

Madison, WI

Paul T. Bailey, Ph.D.

Environmental and Health Sciences Laboratory

Mobil Oil Corporation

Princeton, NJ

Meryl H. Karol, Ph.D.

Department of Environmental Occupational Health

University of Pittsburgh

Pittsburgh, PA

Curtis D. Klaassen, Ph.D.

Department of Pharmacology and Toxicology

University of Kansas Medical Center

Kansas City, KS

Claudia S. Miller, M.D.

University of Texas Health Sciences Center

San Antonio, TX

Janardan K. Reddy, M.D., Principal Reviewer

Department of Pathology

Northwestern University Medical School

Chicago, IL

Irma Russo, M.D.

Fox Chase Cancer Center

Philadelphia, PA

Louise Ryan, Ph.D.

Division of Biostatistics

Harvard School of Public Health and

Dana-Farber Cancer Institute

Boston, MA

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

Department of Pharmacology

Howard University College of Medicine

Washington, DC

Matthew J. van Zwieten, D.V.M., Ph.D., Principal Reviewer

Department of Safety Assessment

Merck Research Laboratories

West Point, PA

Mary Jo Vodicnik, Ph.D.

Lilly MSG Development Center

Belgium

Jerrold Ward, D.V.M., Ph.D., Principal Reviewer

National Cancer Institute

Frederick, MD 
1-Amino-2,4-dibromoanthraquinone, NTP TR 383 15

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

On June 21, 1994, the draft Technical Report on the

toxicology and carcinogenesis studies of 1-amino--

2,4-dibromoanthraquinonereceivedpublicreviewbythe

National Toxicology Program's Board of Scientific

Counselors'TechnicalReportsReviewSubcommittee.

The reviewmeetingwasheldattheNationalInstituteof

EnvironmentalHealthSciences,ResearchTrianglePark,

NC.

Dr. J.E. Huff, NIEHS, introduced the toxicology and

carcinogenesisstudiesof1-amino-2,4-dibromoanthraquinone bydiscussingthe uses and rationale forstudy,

includingitbeingapartof a classstudyof anthraquinone

derivatives. He described the experimental design,

reportedonsurvivalandbodyweight effects, and commentedonchemical-relatedneoplasms andnonneoplastic

lesionsinmale and femaleratsandmice. The proposed

conclusions for the studies were clear evidence of

carcinogenic activityinmale and female F344/N rats

and in male and female B6C3F1 mice.

Dr.Huffreviewed the carcinogenicresponsesin other

anthraquinonederivativesthathadbeenstudied,noting

thatthe liverseemedtobeamajorsiteandthat1-amino2,4-dibromoanthraquinonewasthemostactive asfar as

thenumberofsites. Interpretive conclusionsthat could

be drawn from the cumulative National Toxicology

Programstudiesonthisclassofinsolubledyeswerethat

anthraquinonesare typicallymutagenicandclastogenic,

theyare carcinogenictomaleandfemale rats andmice,

and theyarepredicted to representlikely carcinogenic

hazardsto humansexposed to these agents, especially

occupationally. Dr.J.R.Bucher,NIEHS,reportedthat

the toxicology and carcinogenesis studies on

anthraquinone were in progress. 

Dr.vanZwieten, a principal reviewer,agreedwiththe

proposedconclusions. He thoughtthereshouldbemore

discussionofthe findingsfromthe stop-exposuregroups

ofrats. (Stop-exposure groupswere evaluatedat 9and

15months as part of an attempt to gain insightintothe

progressionorregressionof chemical-inducedlesions.)

Dr.vanZwietennotedthehigh impuritylevelsinthe first

lotofthe chemicalused forthe 13-weekstudies andfor

the first 2 months of the 2-year studies and said that a

 

statementindicatingthatthisdidnot affectthe integrity

ofthe studiesmight behelpful. Dr.Huffrespondedthat

the impuritieshadbeencharacterized(page 20;Arneson

et al., 1996).

Dr.Ward,the secondprincipalreviewer,agreedwiththe

proposedconclusions. He commented that no hyaline

droplets were reported in the kidney of rats after

9months,andsince1-amino-2,4-dibromoanthraquinone

might cause accumulation of "2:-globulin, the report

shouldindicatethat dropletswere searched for but not

found or found but not reported (page 83). Dr. Ward

objectedtocharacterizingcholangiofibrosisfoundinthe

liverofratsin a 13-week study as "premalignant." He

statedthatthislesionisusuallyinducedbylivercarcinogens but does not typically progress to bile duct neoplasms. Dr.M.R.Elwell,NIEHS,saidthisinterpretation

was from the literature and the wording on neoplastic

potential would be revised to also reflect Dr. Ward's

experience.

Dr.Reddy, the thirdprincipalreviewer,alsoagreedwith

the proposedconclusions. He said it would have been

usefultocharacterize the chemicalnatureofthepigment

that accumulatedinthe liver,kidney, andotherorgans,as

well as in the fur and tail. Dr. Huff responded that,

logically, the pigmentwas eitherthe chemicaloroneof

its metabolites, but the feasibility of going back and

defining it better would have to be determined.

Dr.Russohad observedevidenceof chronicinflammationinoneoftheplates andwonderedwhetherthe liver

lesionswere associatedwithhepatitis. Dr.Karol asked

iftherewasinflammationofthe eosinophilic foci,which

would suggest a hypersensitivity-type reaction.

Dr. Elwellsaidtherewassome inflammation with the

cholangiofibrosis,butthiswasreallylimitedtothe focal

lesionswhere therewasfibrosisandtocysticbileducts,

andtherewasnot aneosinophilicinflammation;the term

"eosinophilic foci" referredtoa focal cellular alteration

of hepatocytes. 

Dr.Baileycited a statement from the use, production,

and human exposure sections that "no individualized

information waslocated regardingamounts produced
16 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

or specific uses of 1-amino-2,4-dibromoanthraquinone," leading him to wonder if this chemical is currently used. Dr. Huffsaid this was a valid

question for 1-amino-2,4-dibromoanthraquinoneand

the other anthraquinone derivatives. He said proprietary information was difficult to obtain, although he

was hopeful that a request to the AmericanPharmaceuticalAssociationconcerninganthraquinonedyesin

over-the-counter or prescription items might yield

some data on human exposure. There was some

discussionthatprimaryexposure tothesedyeswouldbe

from topical application or exposure.

Dr. van Zwieten moved that the Technical Report on

1-amino-2,4-dibromoanthraquinonebeacceptedwiththe

revisions discussed and the conclusions as written for

male and female rats and mice, clear evidence of

carcinogenicactivity. Dr.Reddysecondedthemotion,

which was accepted unanimously with eleven votes.
17

INTRODUCTION

1-AMINO-2,4-DIBROMOANTHRAQUINONE

CAS No. 81-49-2

Chemical Formula: C14H7Br2NO2 Molecular Weight: 381.04

Synonym: ADBAQ 

CHEMICAL AND PHYSICAL PROPERTIES

1-Amino-2,4-dibromoanthraquinone, a reddish brown

to orange powder,isan anthraquinone-derived vatdye.

Anthraquinone (9,10-anthraquinone:CAS No.84-65-

1),whichdoes notoccur naturally,wasfirstsynthesized

by Laurent in 1835 as an oxidation product of anthracene and nitric acid (Chung, 1978). "Anthra" comes

fromtheGreekwordfor coal,from which anthracene

was originally obtained.

USE, PRODUCTION,

AND HUMAN EXPOSURE

Anthraquinone is an important and widelyused starting

materialforthemanufactureof vat dyes(Merck Index,

1989). Classhomologuesof anthraquinonecomprise a

greater numberof dyes having outstanding"fastness"

properties than any other group of commercial dyes

(Chung, 1978;Chung and Farris,1979). No informationwaslocated regarding amounts produced orspecific uses of 1-amino-2,4-dibromoanthraquinone. The

2-alkyl derivatives of anthraquinonewith alkyl chains

rangingfromone to fivecarbons aremost often used in

the dye industry (Chung, 1978).

Vat dyes area classofwater-insoluble dyesthat can be

easily reduced (i.e., vatted) to a water-soluble and

usuallycolorlessleucoforminwhich they can readily

impregnate fibers and textiles. Subsequent oxidation

thenproducesthe insoluble colored formthatisremarkably "fast" to washing, light, and chemicals. The

reducingagentsareusuallyalkalinesolutionsofsodium

hydrosulfite; oxidation takes place in the presence of

air,perborate, dichromate, and other agents(Hawley,

1981). Vat dyes areused typicallyforcotton, wool,and

cellulose acetate. Production of vat dyesin theUnited

States totaled 14,000,000kg (30.8 millionpounds)in

1991 (USITC,1993); these figuresdo not accountfor

the "large" amounts extracted frombotanical species

containing naturally occurring anthraquinones used

therapeutically and for other purposes.

ABSORPTION, DISTRIBUTION,

METABOLISM, AND EXCRETION

No informationonthe absorption,distribution,metabolism, and excretion of 1-amino-2,4-dibromoanthraquinone in experimental animals or in humans was

found in a search of the available literature.
18 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TOXICITY

No information on the toxicity of 1-amino-2,4-

dibromoanthraquinone in experimental animals or in

humanswasfound ina search ofthe available literature.

REPRODUCTIVE

AND DEVELOPMENTAL TOXICITY

No informationonthe reproductive and developmental

toxicity of 1-amino-2,4-dibromoanthraquinone in

experimental animals or in humans was found in a

search of the available literature.

CARCINOGENICITY

Experimental Animals

Chemicalsbelongingto the anthraquinone classofdyes

are carcinogenic to rodents(IARC, 1987;Sendelbach,

1989) and consistently induce neoplasms ofthe liver

(Huff et al., 1991). However, each anthraquinone

derivativeappears to induce cancer in other organs or

tissue sites as well (Huff et al., 1991).

For the five anthraquinonesevaluatedand reported by

NCI/NTP, the 2-year exposure concentrations in the

feed varied from a low of 300 ppm (0.03%)

for 2-methyl-1-nitroanthraquinone to a high of

20,000 ppm(2%)for1-amino-2,4-dibromoanthraquinone (Table 1).

Humans

No information on the potential carcinogenicity of

1-amino-2,4-dibromoanthraquinone in humans was

found in a search of the available literature.

GENETIC TOXICITY

All five anthraquinones evaluated and reported by

NCI/NTP induced mutations in Salmonella

typhimurium (Brown and Brown, 1976; Haworth

et al., 1983; Dunkel et al., 1985; Zeiger et al., 1988).

Each also caused sister chromatid exchanges and

chromosomalaberrationsin culturedChinesehamster

ovary cells (Anderson et al., 1990; Loveday et al.,

1990; NTP, unpublished). S9 activation was not

required for 1-amino-2,4-dibromoanthraquinone to

produce theseeffects. The parentcompound,anthraquinone, is also mutagenic in S.typhimurium;significant

increases in mutant colonies were observed with 

strains TA98andTA100with and without S9 (Zeiger

et al., 1988). In addition, anthraquinone and

1-aminoanthraquinone (250 mg/kg) werereported to

induceDNAstrand breaksin liver and kidneytissueof

male Swiss (CD-1®)mice following intraperitoneal

injection (Cesarone et al., 1982).

STUDY RATIONALE

The NCI selected and evaluated several of the

anthraquinone-derived dyes for a classstudyto determinewhetherthesedyes have anyinherentpotentialfor

carcinogenicity in laboratory rodents and, if so, in

humans aswell. The firstthree studieswereconducted

with 2-aminoanthraquinone (NCI, 1978a),

1-amino-2-methylanthraquinone (NCI, 1978b), and

2-methyl-1-nitroanthraquinone(NCI,1978c). Afourth

substance, 1,4,5,8-tetraaminoanthraquinone (C.I.

DisperseBlue1)wasselectedand evaluatedfor carcinogenicitybytheNTP(NTP,1986a). This Technical

Report addresses the fifth chemical in this class,

1-amino-2,4-dibromoanthraquinone. In addition,the

parent chemical, anthraquinone,has been selectedfor

study to complete the overall effort on these dyes.

Anthraquinone and the five substituted anthraquinones

(Figure1), representative of a large group of amino-,

alkyl-, and nitro-, or halogen-containing anthraquinones,werechosenfortoxicologic characterizationand

to establish some predictivestructure-activityrelationships that could be used on other dyesin this category

ratherthan testingeach and everyone. Other chemical

classes that have been likewise evaluated by the

NCI/NTP to reduce the need for "one-by-one" testing

include benzidine-based dyes (Morgan et al.,1994),

phthalates(Kluwe et al.,1982;Huff and Kluwe, 1984;

Kluwe et al., 1985), benzene and metabolites (Huff,

1992), dioxins (Huff, 1992), anilines (Weisburger

et al., 1984; Lamb et al., 1986), naturally occurring

"gums" (Melnick et al., 1983), chlorinated paraffins

(Bucher et al., 1987), 1,3-butadiene and derivatives

(Melnick and Huff, 1992), pesticides (Yang et al.,

1989; Huff and Haseman,1991), and penicillins and

tetracyclines(Dunnicket al., 1989;Dietzet al., 1991).

The bases for selection of anthraquinones(and other

chemical classes as well) centered mainly on four

criteria: 1) lack of available or adequate data on

carcinogenicity, 2) magnitudeof production and use 
Introduction 19

TABLE 1

Exposure Concentrations in the NCI/NTP 2-Year Feed Studies of Anthraquinone Derivativesa

Anthraquinone Derivative Low Dose High Dose Carcinogenic Response

(ppm) (ppm)

Rats

Male

2-Aminoanthraquinoneb 3,500 6,900 liver

1-Amino-2,4-dibromoanthraquinone 2,000 10,000 liver, large intestine, kidney, urinary

bladder

1-Amino-2-methylanthraquinoneb 1,000 2,000 liver, kidney

2-Methyl-1-nitroanthraquinone 600 1,200 liver, skin

1,4,5,8-Tetraaminoanthraquinone 1,250 5,000 urinary bladder, pancreas

Female

2-Aminoanthraquinonec 2,000

1-Amino-2,4-dibromoanthraquinone 2,000 10,000 liver, large intestine, kidney, urinary

bladder

1-Amino-2-methylanthraquinone 1,000 2,000 liver

2-Methyl-1-nitroanthraquinone 600 1,200 skin

1,4,5,8-Tetraaminoanthraquinone 1,250 5,000 urinary bladder

Mice

Male

2-Aminoanthraquinone 5,000 10,000 liver

1-Amino-2,4-dibromoanthraquinone 10,000 20,000 liver, forestomach, lung

1-Amino-2-methylanthraquinone 600 _ d

2-Methyl-1-nitroanthraquinone 300 600 hemangiosarcoma

1,4,5,8-Tetraaminoanthraquinone 600 2,500 liver, lung

e

Female

2-Aminoanthraquinone 5,000 10,000 liver, lymphoma

1-Amino-2,4-dibromoanthraquinone 10,000 20,000 liver, forestomach, lung

1-Amino-2-methylanthraquinone 600 _ d liver

2-Methyl-1-nitroanthraquinone 300 600 hemangiosarcoma

1,4,5,8-Tetraaminoanthraquinone 600 2,500

a Data from NCI, 1978a, 1978b, 1978c; NTP, 1986a

b Exposure concentrations in this study were time-weighted averages.

c

Inadequate study

d Two dosage regimens were used, but the time-weighted average concentrations were the same.

e

"Equivocal evidence" for both organs
20 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

patterns, 3) awareness of potential and actual human

exposure, and 4)representation of asbroad a spec-trum

ofstructural diversity within this class as possible. 1-

Amino-2,4-dibromoanthraquinonewasselectedfrom a

group of36 environmentallysignificantaryl bromides.

Because everyother anthraquinonederivativetested so

far for carcinogenic activity had been shown to be

carcinogenic in rodents, 1-amino-2,4--

dibromoanthraquinone was also expected to be

carcinogenic in laboratory animals. Thus, the

experimental design, while being consistent with a

"core protocol" (Huff et al., 1988), contains 

severalmodificationssuchas "stop-exposure" groupsto

better characterize this chemical. Additionally,

chemicaldispositionstudieswereaccomplishedpriorto

the 2-year exposures to permit optimal selection of

exposure concentrationsforthis water-insoluble dye.

Because these chemicals may and often do contain

considerable quantitiesoftheparentchemicaland other

anthraquinone derivatives, an extensive chemical

analysis was undertaken on these five chemicals to

quantitate their purity and to identify the major

impurities of 1-amino-2,4-dibromoanthraquinone

(Arneson et al., 1996).

1-Amino-2,4-dibromoanthraquinone

2-Aminoanthraquinone

1-Amino-2-methylanthraquinone 2-Methyl-1-nitroanthraquinone

1,4,5,8-Tetraaminoanthraquinone

FIGURE 1

Five Anthraquinone Derivatives Evaluated by the NCI/NTP
21

MATERIALS AND METHODS

PROCUREMENT

AND CHARACTERIZATION OF

1-AMINO-2,4-DIBROMOANTHRAQUINONE

1-Amino-2,4-dibromoanthraquinone was

obtained from American Color and Chemical

Corporation (Charlotte, NC; lot 1076-C) and

Mobay Corporation (Pittsburgh, PA). The

second lot was procured from Mobay

CorporationsinceAmerican Color and Chemical

Corporation had stopped production.

Lot 1076-C was used in the 13-week studies and

for 2 months of the 2-year studies. The lot from

Mobay Corporation was assigned lot number

M061583 and was used throughout the

remainder of the 2-year studies. Identity, purity,

and stability analyses were conducted by the

analytical chemistry laboratory, Midwest

Research Institute (Kansas City, MO)

(Appendix I). Reports on analyses performed in

suppo r t of the 1-ami n o -

2,4-dibromoanthraquinone studies are on file at

the National Institute of Environmental Health

Sciences (NIEHS).

The two lots of the chemical, a reddish brown to

orange powder, were identified as 1-amino2,4-dibromoanthraquinone by infrared,

ultraviolet/visible, and nuclear magnetic

resonance spectroscopy. The purity of each lot

was determined by elemental analyses, Karl

F ischer water an alysis, thin -laye r

chromatography, and high-performance liquid

chromatography. 

For lot 1076-C, elemental analyses for carbon,

hydrogen, nitrogen, and bromine were in general

agreement with theoretical values for 1-amino2,4-dibromoanthraquinone. Karl Fischer water

analysis indicated approximately 0.21% water.

Thin-layer and high-performance liquid

chromatography indicated a major peak and

eight impurities. Five of the impurities had peak

areas of less than 0.3%. The three major

impurities were identified as anthraquinone,

1-amino-2-bromoanthraquinone, and 2-amino1,3-dibromoanthraquinone. By highperformance liquid chromatography,

anthraquinone was found to be present at a

concentration of approximately 5.0%. 1-Amino2-bromoanthraquinone and 2-amino-1,3-

dibromoanthraquinone were found to be present

at concentrations of approximately 4.3% and

2.2%, respectively. The overall purity of

lot 1076-C was determined to be approximately

87%. 

For lot M061583, elemental analyses for carbon,

hydrogen, nitrogen, and bromine were in general

agreement with theoretical values for 1-amino2,4-dibromoanthraquinone. Karl Fischer water

analysis indicated approximately 0.32% water.

Thin-layer and high-performance liquid

chromatography indicated a major peak and six

impurities with the same retention times as found

for lot 1076-C. A total impurity area of 3% of the

total chromatographic peak area was found. The

overall purity of lot M061583 was determined to

be approximately 97%.

Stability studies performed using highperformance liquid chromatography indicated

that 1-amino-2,4-dibromoanthraquinone, when

stored protected from light, was stable as a bulk

chemical for at least 2 weeks at temperatures up

to 60" C. To ensure stability, the bulk chemical was

stored in the dark at 4"± 3" C throughout the

studies. During the 2-year studies, the stability of the

bulk chemical was monitored periodically by the

study laboratory using high-performance liquid

chromatography; no degradation of 1-amino2,4-dibromoanthraquinone was observed throughout

the studies.

PREPARATION AND ANALYSIS 

OF DOSE FORMULATIONS

The dose formulationswere prepared weekly by

mixing 1-amino-2,4-dibromoanthraquinonewith

feed (Table I1). Homogeneity and at least 2-

week stability at 25

B C were confirmed by the

analytical chemistry laboratory using

spectrophotometry and high-performance liquid

chromatography, respectively. During the

13-week and 2-year feed studies, the dose

formulations were stored in the dark for no

longer than 2 weeks.

The study laboratory conducted periodic

a n alyses o f the 1-amin o -

2,4-dibromoanthraquinone dose formulations

using a spectrophotometric method. For the

13-week feed studies, dose formulations were
22 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

analyzed at the beginning, midpoint, and end of the

studies (Table I2). During the 2-year feed studies, the

dose formulations were analyzed every 6 to 10 weeks

(Table I3). All dose formulations for rats and mice

were within 10% of the target concentrations during the

13-week and 2-year studies. Results of periodic referee

analyses performed by the analytical chemistry

laboratory indicated good agreement with the results

obtained by the study laboratory (Table I4).

13-WEEK STUDIES

The 13-week studies were conducted to evaluate the

cumulative toxic effects of repeated exposure to

1-amino-2,4-dibromoanthraquinone and to determine

the appropriate exposure concentrations to be used in

the 2-year studies.

Male and female F344/N rats and B6C3F1 mice were

obtained from Charles River Breeding Laboratories

(Portage, MI). Upon receipt, the animals were 5 weeks

old. The rats and mice were quarantined for 15 days

before the studies began. 

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 libitum, and feed consumption was

measured weekly. Clinical findings were recorded

twice daily. Animals were weighed at study initiation,

weekly, and at the end of the studies. Further details of

study design and animal maintenance are summarized

in Table 2. 

A necropsy was performed on all animals. The brain,

heart, right kidney, liver, lungs, right testis, and thymus

of all animals were weighed. Tissues for microscopic

examination were fixed and preserved in 10% neutral

buffered formalin, processed and trimmed, embedded in

paraffin, sectioned to a thickness of 4 to 6 :m, and

stained with hematoxylin and eosin. A complete

histopathologic examination was performed on all

animals that died prior to the end of the studies, control

animals, and animals administered 50,000 ppm.

Table 2 lists the tissues and organs examined.

2-YEAR STUDIES

Study Design

Groups of 70 male and 70 female rats received 0, 5,000,

or 10,000 ppm 1-amino-2,4-dibromoanthraquinone in

feed, and a group of 50 male and 50 female rats

received 2,000 ppm 1-amino-2,4-dibromoanthraquinone

in feed for 104 weeks. Ten male and 10 female rats

from the 0, 2,000, 5,000, and 10,000 ppm groups were

designated for an interim evaluation after 9 months.

Ten male and 10 female rats from the 0 and 10,000 ppm

groups were designated for an interim evaluation after

15 months. Groups of 60 male and 60 female mice

received 0, 10,000, or 20,000 ppm 1-amino2,4-dibromoanthraquinone in feed for 104 weeks. Ten

male and 10 female mice per group were evaluated after

15 months.

Stop-Exposure Evaluation

Groups of 40 male and 40 female rats received

20,000ppm 1-amino-2,4-dibromoanthraquinone in feed

for 9 months, when 10 males and 10 females were

evaluated. At 9 months, the dosed feed was replaced

with a control diet for 10 male and 10 female rats,

which were then necropsied and evaluated at

15 months. Twenty male and 20 female rats continued

t o r eceive 20, 000 ppm 1-amino -

2,4-dibromoanthraquinone in feed and were also

evaluated at 15 months. 

Source and Specification of Animals

Male and female F344/N rats and B6C3F1 mice were

obtained from Frederick Cancer Research Facility

(Frederick, MD). Rats were quarantined 12 to 14 days

(males) or 9 days (females) and mice were quarantined

12 days (males) or 15 days (females) before the

beginning of the studies. Five male and five female rats

and mice were selected and evaluated for evidence of

parasites and gross observation of disease. Serology

samples were collected for viral screening. Rats and

mice were approximately 6 weeks old at the beginning

of the 2-year studies. The health of the animals was

monitored during the studies according to the protocols

of the NTP Sentinel Animal Program (Appendix L).

Animal Maintenance

Males and females were housed five per cage. Feed and

water were available ad libitum. Feed consumption

was measured monthly (Appendix J). Cages and
Materials and Methods 23

racks were rotated every 2 weeks during the studies.

Further details of animal maintenance are given in

Table 2. Information on feed composition and

contaminants is provided in Appendix K.

Clinical Examinations and Pathology

All animals were observed twice daily. Clinical

findings were recorded weekly for 14 weeks then

monthly until the end of the studies. Animals were

weighed at study initiation, weekly for 14 weeks, and

monthly thereafter.

Animals were killed with CO2, and a complete

necropsy was performed on all animals. The

right kidney and liver of rats and mice were weighed at

the interim evaluations (Appendix H). At necropsy, all

organs and tissues were examined for gross lesions, and

all major tissues were fixed and preserved in 10%

neutral buffered formalin, processed and trimmed,

embedded in paraffin, sectioned, and stained with

hematoxylin and eosin for microscopic examination.

Histopathologic examinations were performed on all

tissues with grossly visible lesions. For all paired

organs (i.e., adrenal gland, kidney, ovary), samples

from each organ were examined. Tissues examined are

listed in Table 2.

Microscopic evaluations were completed by the study

laboratory pathologist, and the pathology data were

entered into the Toxicology Data Management System.

The microscope slides, paraffin blocks, and residual wet

tissues were sent to the NTP Archives for inventory,

slide/block match, and wet-tissue audit. The slides,

individual animal data records, and pathology tables

were evaluated by an independent pathology quality

assessment laboratory. The individual animal records

and tables were compared for accuracy, the slide and

tissue counts were verified, and the histotechnique was

evaluated by the quality assessment laboratory. The

quality assessment pathologist microscopically

reviewed selected neoplasms or nonneoplastic lesions.

The quality assessment report and slides were submitted

to the NTP Pathology Working Group (PWG) chair,

who reviewed the selected slides and any other tissues

for which a disagreement in diagnosis between the

laboratory and quality assessment pathologists existed.

Representative histopathology slides containing

examples of lesions related to chemical administration,

examples of disagreements in diagnoses between the

laboratory and quality assessment pathologists, or

lesions of general interest were presented by the chair

to the PWG for review. Tissues examined included the

adrenal cortex (female rats), ear (rats), kidney (rats),

large intestine (rats), liver, lung (mice), skin (rats),

forestomach, thyroid gland (rats), and urinary bladder

(rats). The PWG consisted of the quality assessment

pathologist and other pathologists experienced in rodent

toxicologic pathology. This group examined the tissues

without any knowledge of exposure groups or

previously rendered diagnoses. When the PWG

consensus differed from the opinion of the laboratory

pathologist, the diagnosis was changed. Thus, the final

diagnoses represent a consensus of contractor

pathologists and the PWG. Details of these review

procedures have been described by Maronpot and

Boorman (1982) and Boorman et al. (1985). For

subsequent analysis of pathology data, the diagnosed

lesions for each tissue type were evaluated separately or

combined according to the guidelines of McConnell

et al. (1986).

STATISTICAL METHODS

Survival Analyses

The probability of survival was estimated by the

product-limit procedure of Kaplan and Meier (1958)

and is presented in the form of graphs. Animals found

dead of other than natural causes or missexed animals

were censored from the survival analyses; animals

dying from natural causes were not censored.

Statistical analyses for possible dose-related effects on

survival used Cox's (1972) method for testing two

groups for equality and Tarone's (1975) life table test to

identify dose-related trends. All reported P values for

the survival analyses are two sided.

Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions

as presented in Tables A1, A5, B1, B5, C1, C5, D1, D5,

E1, E3, F1, and F3 are given as the number of animals

bearing such lesions at a specific anatomic site and the

number of animals with that site examined

microscopically. For calculation of statistical

significance, the incidences of most neoplasms (Tables

A3, B3, C3, D3, E2a, E2b, F2a, and F2b) and of all

nonneoplastic lesions are given as the numbers of

animals affected at each site examined microscopically.

However, when macroscopic examination was required

to detect neoplasms in certain tissues (e.g., harderian

gland, intestine, mammary gland, and skin) before

microscopic evaluation or when neoplasms 
24 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

had multiple potential sites of occurrence (e.g.,

leukemia or lymphoma), the denominators consist of

the number of animals on which a necropsy was

performed. Tables A3, B3, C3, D3, E2a, E2b, F2a, and

F2b also give the survival-adjusted neoplasm rate for

each group and each site-specific neoplasm, i.e., the

Kaplan-Meier estimate of the neoplasm incidence that

would have been observed at the end of the study in the

absence of mortality from all other competing risks

(Kaplan and Meier, 1958).

Analysis of Neoplasm Incidence

The majority of neoplasms in these studies were

considered to be incidental to the cause of death or not

rapidly lethal. Thus, the primary statistical method

used was logistic regression analysis, which assumed

that the diagnosed neoplasms were discovered as the

result of death from an unrelated cause and thus did not

affect the risk of death. In this approach, neoplasm

prevalence was modeled as a logistic function of

chemical exposure and time. Both linear and quadratic

terms in time were incorporated initially, and the

quadratic term was eliminated if the fit of the model

was not significantly enhanced. The neoplasm

incidences of exposed and control groups were

compared on the basis of the likelihood score test for

the regression coefficient of dose. This method of

adjusting for intercurrent mortality is the prevalence

analysis of Dinse and Lagakos (1983), further described

and illustrated by Dinse and Haseman (1986). When

neoplasms are incidental, this comparison of the

time-specific neoplasm prevalences also provides a

comparison of the time-specific neoplasm incidences

(McKnight and Crowley, 1984).

In addition to logistic regression, other methods of

statistical analysis were used, and the results of these

tests are summarized in the appendixes. These methods

include the life table test (Cox, 1972; Tarone, 1975),

appropriate for rapidly lethal neoplasms, and the Fisher

exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the

overall proportion of lesion-bearing animals.

Tests of significance included pairwise comparisons of

each dosed group with controls and a test for an overall

dose-related trend. Continuity-corrected tests were

used in the analysis of neoplasm incidence, and

reported P values are one sided. The procedures

described in the preceding paragraphs were also used

 to evaluate selected nonneoplastic lesions. For further

discussion of these statistical methods, refer to Haseman

(1984).

Analysis of Nonneoplastic Lesion Incidences

Because all nonneoplastic lesions in this study were

considered to be incidental to the cause of death or not

rapidly lethal, the primary statistical analysis used was

a logistic regression analysis in which nonneoplastic

lesion prevalence was modeled as a logistic function of

chemical exposure and time. For lesions detected at the

interim evaluation, the Fisher exact test, a procedure

based on the overall proportion of affected animals, was

used.

Analysis of Continuous Variables

Organ and body weight data, which have approximately

normal distributions, were analyzed using the

parametric multiple comparison procedures of Dunnett

(1955) and Williams (1971, 1972). Jonckheere's test

(Jonckheere, 1954) was used to assess the significance

of the dose-related trends and to determine whether a

trend-sensitive test (Williams' test) was more

appropriate for pairwise comparisons than a test that

does not assume a monotonic dose-related trend

(Dunnett's test). Prior to analysis, extreme values

identified by the outlier test of Dixon and Massey

(1951) were examined by NTP personnel, and

implausible values were eliminated from the analysis.

Average severity values were analyzed for significance

using the Mann-Whitney U test (Hollander and Wolfe,

1973).

Historical Control Data

Although the concurrent control group is always the

first and most appropriate control group used for

evaluation, historical control data can be helpful in the

overall assessment of lesion incidence in certain

instances. Consequently, neoplasm incidences from the

NTP historical control database (Haseman et al., 1984,

1985) are included in the NTP reports for neoplasms

appearing to show compound-related effects.

QUALITY ASSURANCE METHODS

The 13-week and 2-year studies were conducted in

compliance with Food and Drug Administration Good

Laboratory Practice Regulations (21 CFR, Part 58). In

addition, as records from the 2-year studies were

submitted to the NTP Archives, these
Materials and Methods 25

studies were audited retrospectively by an

independent quality assurance contractor.

Separate audits covering completeness and

accuracy of the pathology data, pathology

specimens, final pathology tables, and a draft of

this NTP Technical Report were conducted.

Audit procedures and findings are presented in

the reports and are on file at NIEHS. The audit

findings were reviewed and assessed by NTP

staff, so all discrepancies had been resolved or

were otherwise addressed during the preparation

of this Technical Report.

GENETIC TOXICOLOGY

The genetic t oxicity o f 1-amino2,4-dibromoanthraquinone was assessed by

testing the ability of the chemical to induce

mutations in various strains of Salmonella

typhimurium cells and sister chromatid

exchanges and chromosomal aberrations in

cultured Chinese hamster ovary cells. The

protocols for these studies and the results are

given in Appendix G.

The genetic toxicity studies of 1-amino2,4-dibromoanthraquinone are part of a larger

effort by the NTP to develop a database that

would permit the evaluation of carcinogenicity

in experimental animals from the structure and

responses of the chemical in short-term in vitro

and in vivo genetic toxicity tests. These

genetic toxicity testswere originally developed to

study mechanisms of chemically induced DNA

damage and to predict carcinogenicity in

animals, based on the electrophilic theory of

chemical carcinogenesis and the somatic

mutation theory (Miller and Miller, 1977;

Straus, 1981; Crawford, 1985).

There is a strong correlation between a

chemical's potential electrophilicity (structural

alert to DNA reactivity), mutagenicity in

Salmonella, and carcinogenicity in rodents. The

combination of electrophilicity and Salmonella

mutagenicity is highly correlated with the

induction of carcinogenicity in rats and mice

and/or at multiple tissue sites (Ashby and

Tennant, 1991). Other in vitro genetic toxicity

tests do not correlate well with rodent

carcinogenicity (Tennant et al., 1987; Zeiger

et al., 1990), although these other tests can

provide information on the types of DNA and

chromosome effects that can be induced by the

chemical being investigated. Data from NTP

studies show that a positive response in

Salmonella is currently the most predictive in

vitro test for rodent carcinogenicity (89% of the

Salmonella mutagenswere rodent carcinogens),

and that there is no complementarity among the

in vitro genetic toxicity tests. That is, no battery

of tests that included the Salmonella test

improved the predictivity of the Salmonella test

alone.
26 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 2

Experimental Design and Materials and Methods in the Feed Studies 

of 1-Amino-2,4-dibromoanthraquinone

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Study Laboratory

EG&G Mason Research Institute (Worcester,

MA)

Same as 13-week studies Same as 13-week studies

Strain and Species

Rats: F344/N

Mice: B6C3F1

Rats: F344/N

Mice: B6C3F1

Rats: F344/N

Animal Source

Charles River Breeding Laboratories

(Portage, MI)

Frederick Cancer Research Facility

(Frederick, MD)

Same as 2-year studies

Time Held 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

Duration of Dosing

13 weeks 104 weeks 9-Month stop-exposure group:

39 weeks (males) or 40 weeks (females)

followed by control feed 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 (males) or 8-10 May (females)

1984

15-Month exposure group:

10-12 October (males) or 7-9 November

(females) 1984
Materials and Methods 27

TABLE 2

Experimental Design and Materials and Methods in the Feed Studies 

of 1-Amino-2,4-dibromoanthraquinone (continued)

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Necropsy Dates

Rats: 21-23 July (males) 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 interim 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 evaluation:

19-21 September (males) or

26-28 September (females) 1984

Terminal:

17-20 June (males) or 27 June - 2 July

(females) 1985

9-Month interim evaluation:

10-13 April (males) or 8-10 May (females)

1984

15-Month terminal:

10-12 October (males) or 7-9 November

(females) 1984

Average Age at Necropsy

20 weeks 9-Month interim evaluation:

45-46 weeks

15-Month interim evaluation:

72 weeks 

Terminal:

110-112 weeks

9-Month interim evaluation:

45-46 weeks

15-Month terminal:

72 weeks

Size of Study Groups

10 males and 10 females Rats: 70 males and 70 females in the

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 Distribution

Animals were caged by 1-gram weight

classes and then distributed into treatment

groups such that within a given sex, all cage

weights were approximately equal (± 2 g).

Animals were distributed randomly into

groups of approximately equal initial mean

body weight.

Same as 2-year studies

Animals per Cage

5 5 5

Method of Animal Identification

Ear punch Ear punch Ear punch
28 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 2

Experimental Design and Materials and Methods in the Feed Studies 

of 1-Amino-2,4-dibromoanthraquinone (continued)

13-Week Studies 2-Year Studies Stop-Exposure

Evaluation

Diet

NIH-07 open formula meal diet (Zeigler

Brothers, Inc., Gardners, PA), Available

ad libitum, changed weekly

Same as 13-week studies Same as 13-week studies

Water

Tap water (City of Worcester) available

ad libitum via automatic watering system

(Edstrom Industries, Inc., Waterford, WI)

Same as 13-week studies Same as 13-week studies

Cages

Polycarbonate cage (Lab Products, Inc.,

Rochelle Park, NJ), changed twice weekly

Same as 13-week studies Same as 13-week studies

Bedding

Aspen Bed® heat-treated hardwood chips

(American Excelsior, Baltimore, MD),

changed twice weekly

Same as 13-week studies; BetaChips®

hardwood chips (Northeastern Products,

Warrensburg, NY) were used if necessary.

Same as 2-year studies

Cage Filters

Nonwoven fiber filters (Lab Products,

Rochelle Park, NJ; or Snow Filtration,

Cincinnati, OH); changed every 2 weeks

Nonwoven fiber filters (Snow Filtration Co.,

Cincinnati, OH), changed every 2 weeks

Same as 2-year studies

Racks

Stainless steel racks (Lab Products, Inc.,

Maywood, NY), changed every 2 weeks

Same as 13-week studies Same as 13-week studies

Animal Room Environment

Average temperature: 

22° to 26° C

Relative humidity: 

24% to 66% (rats),

28% to 66% (mice)

Fluorescent light: 

12 hours/day

Room air:

12 to 15 changes/hour

Average temperature: 

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 studies
Materials and Methods 29

TABLE 2

Experimental Design and Materials and Methods in the Feed Studies 

of 1-Amino-2,4-dibromoanthraquinone (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 libitum

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

10,000 ppm in feed, available ad libitum

Mice: 0, 10,000, or

20,000 ppm in feed, available ad libitum

20,000 ppm in feed, available ad libitum

Type and Frequency of Observation

Observed twice daily; animals weighed

initially, weekly, and at end of studies;

clinical observations recorded twice daily;

feed consumption measured weekly

Observed twice daily; animals weighed

initially, weekly for 14 weeks, and monthly

thereafter; clinical observations recorded

weekly for 14 weeks, then monthly until end

of the studies; feed consumption measured

monthly

Same as 2-year studies

Method of Sacrifice

CO2 asphyxiation CO2 asphyxiation CO2 asphyxiation

Necropsy

Necropsy performed on all animals. Organs

weighed were brain, heart, right kidney, liver,

lungs, right testis, and thymus.

Necropsy was performed on all animals. 

Organs weighed at the 9- and 15-month

interim evaluations were right kidney and

liver.

Necropsy was performed on all animals. 

Organs weighed at 9 months and 15 months

were right kidney and liver.

Histopathology

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, 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.

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.
30 1-Amino-2,4-dibromoanthraquinone, NTP TR 383
31

RESULTS

RATS

13-WEEK STUDY

One male (week 13) and one female (week 8) in the

50,000 ppmgroups died during the study (Table 3).

The deaths of one 5,000 ppmmale (week 4) and two

additional 50,000 ppm males (week 13) were not

chemicalrelated. The final mean body weights and

body weight gains of all exposed rat groups were

significantlylowerthan those of the controls. Feed

consumption by all exposed groups was less than that

by the controls throughout the study and generally

decreased with increasing exposure concentration

(Table 3). The greatest differences in feed consumption fromthat by the controls occurred in the 25,000

and 50,000 ppmmales and females. Feed consumption by these groups ranged from45% to 79% that by

the controls at week 1 and from64% to 82% that by

the controls at week 13. Dietary levels of 2,500,

5,000,10,000,25,000, and 50,000ppmdelivered daily

doses of approximately 150, 300, 620, 1,600, and

3,200mg1-amino-2,4-dibromoanthraquinone/kgbody

TABLE 3

Surviva l, Mean Body Weights, and Feed Consumption of Rats in the 13-Week Feed Study

of 1-Amino-2,4-dibromoanthraquinone

Final Weight

Mean Body Weightb

(g) Relative Feed

Dose SurvivalaInitial Final Change to Controls Consumptionc

(ppm) (%) 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 9/10d

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 7/10e180 ± 3 164 ± 6** -17 ± 6** 46 10.3 11.6

Female

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 9/10f140 ± 2 130 ± 4** -10 ± 4** 61 5.9 11.5

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

a Number of animals surviving at 13 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study.

c

Feed consumption is expressed as grams of feed consumed per animal per day.

d Week of death: 4

e Week of death: 13, 13, 13 (2 were accidental deaths)

f Week of death: 8
32 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

weight to males and 170, 340, 660, 1,500, and

3,200 mg/kg to females. Pink-red staining of the fur and

tail was observed in all exposed groups of rats. The

bedding of all exposed groups except the 2,500 ppm

groups was stained pink-red from day 2 of the study.

Lethargy and emaciation were noted in all 50,000 ppm

males. Female rats in the 25,000 and 50,000 ppm groups

were lethargic and staggered, and 50,000 ppm females

exhibited hunched posture.

The relative liver weights of exposed groups of males

and the absolute and relative liver weights of exposed

groupsoffemalesweresignificantlygreaterthanthose

of the controls(Table H1). The absolute and relative

thymus weights of exposed males and females were

significantly lower than those of controls. The lower

absolute brain, heart, kidney, lung, and testis weights

of exposedmale and female ratswereprobably related

to the lower final mean body weights of these groups.

Observations at necropsyincludedred or pink staining

of the gastrointestinal tract contents and/or mucosa,

kidneys, and urine. In addition, regional lymph nodes

and livers were dark in color, and capsularsurfaces of

the liversweregranularinappearance. These findings

were most common in the 25,000 and 50,000 ppm

groups.

Chemical-related lesions were present in the liver,

kidney, and spleen of male and female rats. In the

liver, a spectrum of nonneoplastic degenerative and

proliferative lesions occurred in males and femalesin

the 25,000 and 50,000 ppm groups (Table 4).

Hepatocellular cytomegaly(hypertrophy) waspresent

in all ratsin the 25,000 and 50,000 ppm groups and in

most females in the 10,000 ppm group. This lesion

consisted of enlarged hepatocytes with eosinophilic

cytoplasmandmarkedvariation in nuclearsize. In the

centrilobular areas of a few ratsfrom exposed groups,

there was a minimal to mild cytoplasmic vacuolation

(vacuolar degeneration). The incidence of vacuolar

degeneration was not dose-related, but at the higher

exposure concentrations, minimal hepatocellular

necrosis was sometimes associated with vacuolar

change. Focal hepatocellular alterations including

basophilic, eosinophilic, or clear cell foci were also

present in the 25,000 and 50,000 ppm groups. In the

periportal region of the hepatic lobules, there was an

increased number of inflammatory cells around the

bile d ucts. Bile duct hyperplasi a

consistedof proliferation of oval cellsinthe periportal

area as well as proliferation of larger bile ducts lined

by hyperchromatic, pleomorphic biliary epithelium.

Focal necrosis of biliary epitheliumand acute inflammation (necrotizing cholangitis) in some hyperplastic

bile ducts were associated with periportal fibrosis.

The spectrumof proliferative bileduct lesions(hyperplasia, necrotizing cholangitis, and fibrosis) was

morphologically consistent with the lesion described

as cholangiofibrosis. Abrown pigment was present in

the cytoplasm of hepatocytes. The pigment was

negative foriron, PAS, bile, and acid-faststaining; did

not polarize light or fluoresce; and was considered to

represent1-amino-2,4-dibromoanthraquinone and/orits

metabolites.

In the kidneyof exposedgroups of males and females,

there were chemical-related increases in the incidences of a brown, granular pigment in the tubule

epithelium (Table 4). This brown pigment had the

same staining features asthe pigment that was present

in the liver. In both males and females, there were

renal tubule cellswithenlargednuclei. Inmales,there

was a hyaline droplet nephropathy characterized by an

increase in eosinophilic protein droplets (hyaline

droplet accumulation) in the cytoplasm of the renal

tubule epithelium as well as in the lumen of the

tubules. There was no evidence of increased severity

of tubule regeneration in males or females.

Chemical-related effects in the spleen of all exposed

groups of males and females consisted of a slight

increase in the amount of hematopoiesis relative to

that normally present in controls.

Other nonspecific changes included lymphoid depletion in the thymus and decreased uterus size. These

findings were attributed to the markedly lower body

weight gain in ratsfrom the higher exposure groups.

Dose SelectionRationale: Based on chemical disposition studies,meanbodyweights,and chemical-related

lesions of the liver, kidney, and spleen present mainly

in the 25,000 and 50,000 ppm groups, exposure concentrations selected for the 2-year feed study of

1-amino-2,4-dibromoanthraquinone in rats were 0,

2,000, 5,000, and 10,000 ppm. Much of the differencesin mean body weightsrecorded for the 13-week

studies were more likely due to decreased feed palatability than to any overt toxicity. Nonetheless, if
Results 33

TABLE 4

Incidences of Selected Nonneoplastic Lesions in Rats in the 13-Week Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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

Male

Livera10 10 10 10 10 10

Basophilic Focusb0 0 0 0 4* (1.5)d9** (2.3)

Clear Cell Focus 0 0 0 0 6** (1.0) 0

Eosinophilic Focus 0 0 0 0 4* (1.5) 0

Cytomegaly 0 0 0 0 10** (3.2) 10** (4.0)

Bile Duct Hyperplasia 0 0 0 0 8** (2.3) 10** (3.1)

Inflammation 0 0 0 0 10** (2.1) 10** (3.0)

Fibrosisc0 0 0 0 10** (1.9) 10** (3.1)

Necrotizing Cholangitisc0 0 0 0 7** (1.3) 10** (2.8)

Vacuolar Degenerationc0 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 Pigmentation 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

Basophilic Focus 0 0 0 0 1 (1.0) 9** (2.6)

Eosinophilic Focus 0 0 0 0 1 (1.0) 0

Cytomegaly 0 0 0 8** (1.0) 10** (2.2) 10** (4.0)

Bile Duct Hyperplasia 0 0 0 4* (1.0) 9** (1.6) 10** (2.4)

Inflammation 0 0 0 0 10** (1.6) 9** (2.3)

Fibrosisc0 0 0 0 2 (2.0) 9** (2.7)

Necrotizing Cholangitisc0 0 0 0 0 9** (2.6)

Vacuolar Degenerationc0 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 Pigmentation 0 10** (1.0) 9** (1.0) 10** (1.2) 10** (1.7) 10** (1.9)

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01

a Number of animals with organ examined microscopically

b Number of animals with lesion

c Data from Fleischman et al., 1986

d Average severity of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

exposure selection werebased onmeanbodyweights

alone formale rats,the10,000ppmexposureconcentration could have been considered slightly high. Moreover,consideringthe lackoflivertoxicityat exposures

of 10,000 ppm and below, this exposure 

concentrationwas predicted notto adverselyaffectthe

health or survivalofthese animals. Higher exposure

concentrations(20,000 ppm)werechosen forthe startstop, progression/regressionexperiments(stop-exposure

evaluation).
34 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

2-YEAR STUDY

Survival

Estimatesof 2-yearsurvivalprobabilitiesformale and

female rats are shown in Table 5 and in the

Kaplan-Meiersurvival curvesinFigure 2. Survivalof

male and female rats in the 10,000 ppm groups was

significantly lower than that of the controls.

Body Weights, Feed and Compound

Consumption, and Clinical Findings

Mean body weights of exposed male and female rats

were lower than those of the controls after week 2

(Tables6 and 7,Figure 3). Finalmeanbodyweights of

exposed males were 14% to 30% lower than that

of the controls; final mean body weights of exposed

females were 20% to 46% lower than that of the

controls. Feed consumption by exposed males and

females was similar among exposed groups and was

slightlylowerthan that by the controls (TablesJ3 and

J4). Dietary levels of 2,000, 5,000, and 10,000 ppm

delivered average daily doses of approximately 90,

240, and 490 mg 1-amino-2,4-dibromoanthraquinone/kg bodyweight to males and 110, 285, and 600

mg/kg to females. Discoloration of the furand urine

was evidentin all exposed groups as earlyas day8 and

was observed throughout the study. Emaciation

occurred in a dose-relatedmannerinmale and female

rats and occurred in over 50% of the rats exposed to

10,000 ppm.

TABLE 5

Survival of Rats in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

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

Male

Animals initially in study 70 50 70 70

9-Month interim evaluationa10 10 10 10

15-Month interim evaluationa10 0 0 10

Moribund 19 15 34 33

Natural deaths 5 1 5 7

Animals surviving to study termination 26e24 21 10

Percent probability of survival at end of studyb53 60 35 20

Mean survival (days)c586 618 615 547

Survival analysesd P<0.001 P=0.467N P=0.141 P<0.001

Female

Animals initially in study 70 50 70 70

9-Month interim evaluationa10 10 10 10

15-Month interim evaluationa10 0 0 10

Missexeda0 0 0 1

Moribund 8 5 15 29

Natural deaths 4 3 7 8

Animals surviving to study termination 38f32 38 12

Percent probability of survival at end of study 76 80 63 25

Mean survival (days) 610 626 643 569

Survival analyses P<0.001 P=0.857N P=0.216 P<0.001

a Censored from survival analyses b Kaplan-Meier determinations based on the number of animals alive on first day of terminal sacrifice

c Mean of all deaths (uncensored, censored, and terminal sacrifice) d The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons (Cox, 1972)

with the controls are in the exposed columns. A lower mortality in an exposure group is indicated by N.

e

Includes three males that died during the last week of the study. f

Includes one female that died during the last week of the study.
Results 35 

FIGURE 2

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

for 2 Years
36 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 6

Mean Body Weights and Survival of Male Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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) controls)Survivors (g) controls)Survivors (g) controls)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

 41a 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

 69a 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 evaluations occurred during week 39 for all groups and week 66 for the 0 and 10,000 ppm groups.
Results 37

TABLE 7

Mean Body Weights and Survival of Female Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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) controls)Survivors (g) controls)Survivors (g) controls)Survivors

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

 41a265 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

 68a 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 evaluations occurred du ring week 40 for all groups and week 66 for the 0 and 10,000 ppm groups.
38 l-Ajnino-2,4-dibromoanthraquinone, NTP TR 383

FIGURE 3

Growth Curves for Rats Administered 1-Amino-2,4-dibromoanthraquinone in Feed for 2 Years
Results 39

Pathology and Statistical Evaluation

This section describes the statistically significant or

biologically noteworthy changes in the incidences of

mononuclear cell leukemia; neoplasms of the liver,

large intestine, kidney, urinary bladder, and other

organs; and nonneoplastic lesionsofthe liver, kidney,

urinarybladder,forestomach, and seminal vesicles of

rats. Summaries of the incidences of neoplasms and

nonneoplastic lesions, individual animal tumor

diagnoses, statistical analyses of primary neoplasms

that occurred with an incidence of atleast5%inatleast

one exposuregroup, and historical incidences for the

neoplasms mentioned in this section are presented in

Appendix A formale rats and Appendix B for female

rats.

Liver: Atthe 9-monthinterimevaluation,the absolute

and relative liverweightsof exposed groups of males

and femalesweresignificantlygreaterthan those ofthe

controls(TableH2). One hepatocellular adenomawas

observed in a 5,000 ppm male at 9 months (Tables 8

and A1). Incidencesoffoci of hepatocellularalteration

were increased inmalesin the 10,000ppmgroup, and

aminimalaccumulationofpigmentinhepatocyteswas

present in males and females from the 10,000 ppm

groups and in females from the 5,000 ppm group

(Tables 8, A5, and B5). At the 15-month interim

evaluation, the absolute and relative liver weights of

exposed groups of females were significantly greater

than those of the controls (Table H3). Incidences of

single and multiple hepatocellular adenomas and

carcinomaswereincreasedat15monthsin10,000 ppm

malesand females(Tables8,A1,andB1). Incidences

offoci of hepatocellular alterationand accumulation of

pigmentin hepatocyteswerealsoincreased in exposed

groups of males and females (Tables 8, A5, and B5).

Atthe end ofthe 2-yearstudy, the incidencesofhepatocellular adenoma, hepatocellular carcinoma, and

hepatocellularadenoma or carcinoma (combined)were

significantlyincreasedin all exposed groups of males

and females (Tables 8, A3, and B3). 

The incidences of multiple hepatocellular adenomas

andmultiple hepatocellular carcinomasinexposedmale

and female groups were greater than those of the

controls (Tables 8, A1, and B1). Incidences of

hepatocellular adenoma or carcinoma (combined)inall

exposed groupsofmales and femalesexceeded theNTP

historical ranges(males: 0%-10%;females: 0%-6%)

forfeed studycontrols (Tables 8, A4a, andB4a). The

majority of the benign and malignantliver neoplasms

consisted ofwell-differentiatedneoplastic hepatocytes

with cellular atypia and increasednumbers of mitoses.

Carcinomashad trabecular,glandular,orsolid growth

patterns(Plate1)withareasofnecrosis,cavitation,and

fibrosis. Metastases were common in the lungs

(Plates2 and 3), butmetastatic fociwerealso presentin

the stomach,pancreas,adrenalgland,lymphnode, and

spleen.

The incidences ofsingle andmultiple hepatocholangiocarcinoma were significantly increased in 5,000ppm

malesand femalesand in 10,000ppmfemales(Tables

8, A3, and B3). These neoplasms consisted of a

mixture of malignant hepatocytes and welldifferentiated cuboidal epithelium forming distinct

ductular structures (Plate4). Both hepatocellular and

biliary components of this neoplasm were present in

metastatic foci. In addition, several other benign

(cholangioma and hepatocholangioma) andmalignant

(cholangiocarcinoma)liverneoplasms occurred only in

exposed groups of males and females (Tables 8, A1,

and B1). 

Duringthe2-yearstudy,the incidences ofpigmentation

and foci of hepatocellular alteration (clear cell,

basophilic,and eosinophilic)wereincreased inexposed

groups of males and females (Tables 8,A5, and B5).

Cellsinsome focihad intensely eosinophilic cytoplasm

and hepatocellular atypia similarto the appearance of

cellsinthehepatocellular neoplasms. The pigmentwas

considered to be1-amino-2,4-dibromoanthraquinone or

a metabolite based on the results ofthe histochemical

procedures performed duringthe13-weekstudyand the

15-month interim evaluation.
40 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 8

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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

Male

9-Month Interim Evaluation

Number Examined 10 10 10 10

Basophilic Focusa0 0 1 (1.0)b1 (1.0)

Clear Cell Focus 0 0 0 4* (1.0)

Eosinophilic Focus 0 0 0 1 (1.0)

Pigmentation 0 1 (1.0) 0 6** (1.0)

Hepatocellular Adenoma 0 0 1 0

15-Month Interim Evaluation

Number Examined 10

-

c

- 10

Basophilic Focus 1 (1.0) 5 (1.0)

Clear Cell Focus 0 7** (1.0)

Eosinophilic Focus 1 (1.0) 0

Pigmentation 0 10** (1.0)

Hepatocellular Adenoma (Multiple) 0 2

Hepatocellular Adenoma

(Single or Multiple) 0 4*

Hepatocellular Carcinoma (Multiple) 0 3

Hepatocellular Carcinoma

(Single or Multiple) 0 7**

Hepatocellular Adenoma or Carcinoma 0 10**

2-Year Study

Number Examined 50 40 59 50

Basophilic Focus 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)

Eosinophilic Focus 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)

Hepatocellular Adenoma (Multiple) 0 10** 23** 24**

Hepatocellular Adenoma (Single or Multiple)

Overall rated1/50 (2%) 20/40 (50%) 40/59 (68%) 34/50 (68%)

Terminal ratee1/26 (4%) 16/24 (67%) 18/21 (86%) 9/10 (90%)

Adjusted ratef3.8% 71.3% 92.3% 97.0%

First incidence (days) 729 (T) 675 521 435

Logistic regression testgP<0.001 P<0.001 P<0.001 P<0.001

Hepatocellular Carcinoma (Multiple) 0 1 43** 37**

Hepatocellular Carcinoma (Single or Multiple)

Overall rate 1/50 (2%) 12/40 (30%) 55/59 (93%) 46/50 (92%)

Terminal rate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%)

Adjusted rate 2.7% 43.5% 100.0% 100.0%

First incidence (days) 666 650 465 436

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

(continued)
Results 41

TABLE 8

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone (continued)

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

Male (continued)

2-Year Study (continued)

Hepatocellular Adenoma or Carcinomah

Overall rate 2/50 (4%) 25/40 (63%) 57/59 (97%) 47/50 (94%)

Terminal rate 1/26 (4%) 19/24 (79%) 21/21 (100%) 10/10 (100%)

Adjusted rate 6.4% 83.1% 100.0% 100.0%

First incidence (days) 666 650 465 435

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Number Examined 50 40 59 50

Hepatocholangioma 0 0 1 1

Hepatocholangiocarcinoma 0 0 6* 2

Cholangioma 0 0 2 0

Cholangiocarcinoma 0 0 0 1

Female

9-Month Interim Evaluation

Number Examined 10 10 10 10

Basophilic Focus 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-Month Interim Evaluation

Number Examined 10

- - 10

Basophilic Focus 8 (1.0) 9 (1.4)

Clear Cell Focus 0 5* (1.6)

Pigmentation 1 (1.0) 10** (1.1)

Hepatocellular Adenoma (Multiple) 0 5*

Hepatocellular Adenoma

(Single or Multiple) 0 8**

Hepatocellular Carcinoma (Multiple) 0 3

Hepatocellular Carcinoma

(Single or Multiple) 0 6**

Hepatocellular Adenoma or Carcinoma 0 9**

2-Year Study

Number Examined 50 40 60 48

Basophilic Focus 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)

Eosinophilic Focus 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)

(continued)
42 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 8

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone (continued)

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

Female (continued)

2-Year Study (continued)

Number Examined 50 40 60 48

Hepatocellular Adenoma (Multiple) 0 18** 39** 22**

Hepatocellular Adenoma (Single or Multiple)

Overall rate 0/50 (0%) 28/40 (70%) 47/60 (78%) 29/48 (60%)

Terminal rate 0/38 (0%) 23/32 (72%) 29/38 (76%) 8/12 (67%)

Adjusted rate 0.0% 75.5% 83.7% 83.6%

First incidence (days)_ -

i

600 575 418

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Hepatocellular Carcinoma (Multiple) 0 7** 51** 41**

Hepatocellular Carcinoma (Single or Multiple)

Overall rate 0/50 (0%) 12/40 (30%) 57/60 (95%) 45/48 (94%)

Terminal rate 0/38 (0%) 12/32 (38%) 37/38 (97%) 12/12 (100%)

Adjusted rate 0.0% 37.5% 98.3% 100.0%

First incidence (days) - 729 (T) 575 460

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Hepatocellular Adenoma or Carcinomaj

Overall rate 0/50 (0%) 33/40 (83%) 59/60 (98%) 47/48 (98%)

Terminal rate 0/38 (0%) 28/32 (88%) 38/38 (100%) 12/12 (100%)

Adjusted rate 0.0% 89.1% 100.0% 100.0%

First incidence (days) - 600 575 418

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Hepatocholangioma 0 0 2 0

Hepatocholangiocarcinoma 0 0 11** 13**

Cholangioma 0 0 0 1

* Significantly different (P#0.05) from the control group by the Fisher exact test (9-month and 15-month interim evaluations) or the logistic

regression test (2-year study)

** P#0.01

(T)Terminal sacrifice

a Number of animals with lesion

b Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Liver not microscopically examined in this group

d Number of animals with neoplasm per number of animals with liver examined microscopically

e Observed incidence in animals surviving until the end of the study

f Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

g

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

h Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 45/1,350 (3.3% ± 3.6%); range,

0%-10% i Not applicable; no neoplasms in animal group

j Historical incidence: 9/1,351 (0.7% ± 1.5%); range, 0%-6%
Results 43

Large Intestine: Adenomatous polyps (adenomas)

were observed in the large intestine of 10,000 ppm

males and females at the 15-month interim evaluation (Tables 9, A1, and B1).

At 2 years, the incidences of adenomatous polyps

(adenomas) in the rectum were significantly

increased in all exposed groups of males and

females (Tables 9, A3, and B3). The incidence of

carcinom a of the 

TABLE 9

Incidences of Neoplasms of the Large Intestine in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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

Male

15-Month Interim Evaluation

Rectuma9

-

c

- 10

Adenomatous Polyp (Adenoma)b0 6**

2-Year Study

Colon

Adenomatous Polyp (Adenoma)

Overall rated0/50 (0%) 1/40 (3%) 1/59 (2%) 3/50 (6%)

Terminal ratee0/26 (0%) 1/24 (4%) 0/21 (0%) 1/10 (10%)

Adjusted ratef0.0% 4.2% 4.3% 19.9%

First incidence (days) -

h

729 (T) 720 590

Logistic regression testgP=0.027 P=0.484 P=0.494 P=0.081 

Carcinomai

Overall rate 0/50 (0%) 0/40 (0%) 1/59 (2%) 4/50 (8%)

Terminal rate 0/26 (0%) 0/24 (0%) 1/21 (5%) 0/10 (0%)

Adjusted rate 0.0% 0.0% 4.8% 20.4%

First incidence (days) - - 729 (T) 590

Logistic regression test P=0.003 - P=0.457 P=0.046

Rectum

Adenomatous Polyp (Adenoma)

Overall rate 0/50 (0%) 13/40 (33%) 51/59 (86%) 40/50 (80%)

Terminal rate 0/26 (0%) 9/24 (38%) 21/21 (100%) 10/10 (100%)

Adjusted rate 0.0% 45.8% 100.0% 100.0%

First incidence (days) - 659 478 352

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Carcinomai

Overall rate 0/50 (0%) 1/40 (3%) 10/59 (17%) 15/50 (30%)

Terminal rate 0/26 (0%) 0/24 (0%) 5/21 (24%) 4/10 (40%)

Adjusted rate 0.0% 3.8% 32.4% 63.0%

First incidence (days) - 718 608 493

Logistic regression test P<0.001 P=0.480 P=0.003 P<0.001

Large Intestine (All Sites) 50 40 59 50

Adenomatous Polyp (Adenoma) (Multiple) 0 1 34** 32**

Adenomatous Polyp (Adenoma) 

(Single or Multiple) 0 13** 51** 40**

Carcinoma (Multiple) 0 0 0 3

Carcinoma (Single or Multiple) 0 1 11** 17**

(continued)
44 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 9

Incidences of Neoplasms of the Large Intestine in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone (continued)

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

Female

15-Month Interim Evaluation

Rectum 10

- - 10 Adenomatous Polyp (Adenoma) 0 2

2-Year Study

Colon 50 40 60 49

Adenomatous Polyp (Adenoma) 0 1 2 2

Carcinomaj0 1 2 1

Rectum

Adenomatous Polyp (Adenoma)

Overall rate 0/50 (0%) 27/40 (68%) 53/60 (88%) 43/49 (88%)

Terminal rate 0/38 (0%) 23/32 (72%) 38/38 (100%) 12/12 (100%)

Adjusted rate 0.0% 75.0% 100.0% 100.0%

First incidence (days) - 616 582 512

Logistic regression test P<0.001 P<0.001 P<0.001 P<0.001

Carcinomaj

Overall rate 0/50 (0%) 1/40 (3%) 19/60 (32%) 7/49 (14%)

Terminal rate 0/38 (0%) 1/32 (3%) 13/38 (34%) 4/12 (33%)

Adjusted rate 0.0% 3.1% 41.7% 41.9%

First incidence (days) - 729 (T) 606 625

Logistic regression test P<0.001 P=0.466 P<0.001 P=0.001

Large Intestine (All Sites) 50 40 60 49

Adenomatous Polyp (Adenoma) (Multiple) 0 18** 46** 32**

Adenomatous Polyp (Adenoma) 

(Single or Multiple) 0 28** 53** 43**

Carcinoma (Multiple) 0 1 1 1

Carcinoma (Single or Multiple) 0 2 21** 8**

** Significantly different (P#0.01) from the control group by the Fisher exact test (15-month interim evaluation) or the logistic regression test

(2-year study)

(T)Terminal sacrifice

a Number of animals with large intestine examined microscopically

b Number of animals with lesion

c Large intestine not microscopically examined in this group

d Number of animals with neoplasm per number of animals necropsied

e Observed incidence in animals surviving until the end of the study

f Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

g

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

h Not applicable; no neoplasms in animal group

i Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 1/1,353 (0.1% ± 0.4%); range,

0%-2% (includes all carcinomas of the large intestine)

j Historical incidence: 0/1,351 (includes all carcinomas of the large intestine)
Results 45

colon was significantly increased in 10,000 ppm males,

and the incidences of rectal carcinoma were significantly

increased in 5,000 and 10,000 ppm males and females

(Tables 9, A3, and B3). The intestinal neoplasms that

occurred in the distal colon and rectum of rats were

morphologically similar.

Adenomatous polyps ( adenomas) con sisted of

pedunculated, exophytic masses (Plate 5) of well-differentiated, columnar epithelium with prominent, hyperchromatic nuclei. Carcinoma (adenocarcinoma) was

generally similar to adenoma, except that invasion of the

stromal stalk of the neoplasm and extension into the

submucosa, and occasionally into the muscular wall, were

evident microscopically. In the malignant neoplasms,

irregular glandular structures or cords of atypical epithelial

cells were present in the submucosa (Plate 6) and were

frequently associated with a scirrhous response. In males,

metastatic colon carcinoma was observed in the lung and

mesenteric lymph nodes, and metastatic rectal carcinoma

was observed in the lumbar lymph nodes and pancreas

(Table A1). Incidences of carcinoma (colon and rectum

combined) in exposed groups of males and females

exceeded the NTP historical ranges (males: 0%-2%;

females: 0%) for feed study controls (Tables 9, A4b, and

B4b).

Kidney: At the 9-month interim evaluation, the relative

kidney weights of exposed groups of males and females

were significantly greater than those of the controls (Table

H2). Pigmentation and hyaline droplet accumulation were

present in the kidneys of all exposed males (Tables 10 and

A5). The sizes of some renal tubule nuclei were minimally increased in males and females from all exposure

groups, and the severity of the nephropathy (tubule

epithelial regeneration, mononuclear inflammation, and

renal tubule dilation with protein casts) was slightly more

severe than that observed in the controls (Table 10). At

the 15-month interim evaluation, the relative kidney

weights of 10,000 ppm males and females were significantly greater than those of the controls (Table H3). The

severity of nephropathy was increased in 10,000 ppm

males and females compared to controls (Table 10). This

was characterized by an increase in the foci of tubule

epithelial regeneration; nuclear enlargement in some

tubule epithelium, similar to that in the 13-week studies

and at the 9-month interim evaluation, was also present.

At 15 months in the 10,000 ppm groups, pigmentation of

renal tubule epithelium was present in all rats; renal tubule

epithelial hyperplasia was observed in two males

and three females; and adenomas were observed in two

males (Tables 10, A1, A5, and B5).

At 2 years, there was a significant dose-related increase in

the incidences of renal tubule adenoma in exposed groups

of males and females (Tables 10, A3, and B3). Multiple

adenomas were observed in all exposed groups of males

and in the 5,000 and 10,000 ppm females. Adenomas

were expansile lesions involving one or more adjacent

tubules and were generally five or more times the diameter

of the normal renal tubule. The cells within the adenomas

were generally similar in morphology to those in the focal

hyperplastic lesions. Carcinomas were larger than adenomas and frequently had more cellular atypia, necrosis,

and local invasion. One carcinoma in a male rat metastasized to the lung, and one in a female rat metastasized to

the adrenal gland. Renal tubule carcinomas occurred in

two 5,000 ppm males, one 10,000 ppm male, and two

10,000 ppm females. The combined incidences of renal

tubule adenoma or carcinoma were significantly increased

in exposed males and females and exceeded the NTP

historical ranges (males: 0%-6%; females: 0%-2%) for

feed study controls (Tables 10, A4c, and B4c).

Incidences of renal tubule hyperplasia were significantly

increased in exposed males and females (Tables 10, A5,

and B5). Hyperplasia consisted of a tubule lined by two

or more layers of renal tubule epithelium; these were most

often located in the cortex or outer stripe of the outer

medulla. Foci of hyperplasia were distinguished from the

more basophilic foci of tubule epithelial regeneration

typically associated with nephropathy. There was no clear

dose-related increase in the incidence or severity of

nephropathy in rats at 2 years (Table 10). The incidences

and severity of transitional cell hyperplasia in the renal

pelvis were increased in exposed groups of males and

females (Tables 10, A5, and B5); there were no increases

in the incidences of transitional cell papilloma or carcinoma of the renal pelvis (Tables 10, A1, and B1). The

incidence of a reddish brown pigment within the renal

tubule epithelium and lumina of exposed rats was increased at the 9- and 15-month interim evaluations and at

2 years. The pigment was characterized in the 13-week

study and at the 15-month interim evaluation as PASnegative; resistant to digestion by diastase; isotropic; and

negative for melanin, hemosiderin, hematoidin, bile,

lipofuscin, or ceroid staining methods. The pigment was

presumed to be 1-amino-2,4-dibromoanthraquinone or one

of its metabolites.
46 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 10

Incidences of Neoplasms and Nonneopla stic Lesions of the Kidney in Rats in the 2-Year Feed Study

of 1-Amino-2,4-dibromoanthraquinone

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

Male

9-Month Interim Evaluation

Number Examined 10 10 10 10 Renal Tubule Hyaline Droplet

Accumulationa0 10** (2.0)b10** (2.0) 10** (1.9)

Renal Tubule Pigmentation 0 10** (1.1) 10** (1.4) 10** (1.9)

15-Month Interim Evaluation

Number Examined 10 _c _ 10

Nephropathy 10 (2.0) 10 (2.5)

Renal Tubule Hyperplasia 0 2 (1.0)

Renal Tubule Pigmentation 0 10** (2.0)

Transitional Cell Hyperplasia 0 4* (1.3)

Renal Tubule Adenoma 0 2

2-Year Study

Number Examined 50 40 59 50

Nephropathy 50 (2.9) 40 (3.6) 59 (2.9) 49 (2.7)

Renal Tubule Hyperplasia 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 Hyperplasia 30 (1.4) 40** (2.1) 51** (1.9) 35* (1.6)

Transitional Cell Papilloma 0 0 1 0

Renal Tubule Adenoma (Multiple) 0 4* 4 5*

Renal Tubule Adenoma (Single or Multiple)

Overall rated2/50 (4%) 10/40 (25%) 11/59 (19%) 14/50 (28%)

Terminal ratee2/26 (8%) 6/24 (25%) 4/21 (19%) 5/10 (50%)

Adjusted ratef7.7% 33.6% 34.6% 68.3%

First incidence (days) 729 (T) 618 636 588

Logistic regression testgP<0.001 P=0.007 P=0.014 P<0.001

Renal Tubule Carcinoma 0 0 2 1

Renal Tubule Adenoma or Carcinomah

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%)

Adjusted rate 7.7% 33.6% 39.4% 69.1%

First incidence (days) 729 (T) 618 636 497

Logistic regression test P<0.001 P=0.007 P=0.005 P<0.001

(continued)
Results 47

TABLE 10

Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone (continued)

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

Female

15-Month Interim Evaluation

Number Examined 10

- - 10

Nephropathy 10 (1.7) 10 (2.5)

Renal Tubule Hyperplasia 0 3 (1.0)

Renal Tubule Pigmentation 0 10** (2.0)

Transitional Cell Hyperplasia 3 (1.0) 4 (1.8)

2-Year Study

Number Examined 50 40 60 48

Nephropathy 50 (1.9) 39 (2.6) 60 (2.7) 46 (2.7)

Renal Tubule Hyperplasia 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)

Transitional Cell Hyperplasia 10 (1.2) 16* (1.8) 44** (1.5) 21** (1.6)

Transitional Cell Papilloma 0 0 0 1

Transitional Cell Carcinoma 0 1 0 0

Renal Tubule Adenoma (Multiple) 0 0 5* 5*

Renal Tubule Adenoma (Single or Multiple)

Overall rate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%)

Terminal rate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%)

Adjusted rate 0.0% 8.0% 36.0% 69.7%

First incidence (days) -

i

600 601 625

Logistic regression test P<0.001 P=0.049 P<0.001 P<0.001

Renal Tubule Carcinoma 0 0 0 2

Renal Tubule Adenoma or Carcinomaj

Overall rate 0/50 (0%) 3/40 (8%) 16/60 (27%) 16/48 (33%)

Terminal rate 0/38 (0%) 1/32 (3%) 11/38 (29%) 6/12 (50%)

Adjusted rate 0.0% 8.0% 36.0% 69.7%

First incidence (days) - 600 601 625

Logistic regression test P<0.001 P=0.049 P<0.001 P<0.001

* Significantly different (P#0.05) from the control group by the Fischer exact test (9-month and 15-month interim evaluations) or the logistic

regression test (2-year study) 

** P#0.01

(T)Terminal sacrifice

a Number of animals with lesion

b Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Kidney not microscopically examined in this group

d Number of animals with neoplasm per number of animals with kidney examined microscopically

e Observed incidence in animals surviving until the end of the study

f Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

g

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

h Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 15/1,350 (1.1% ± 1.7%); range,

0%-6% i Not applicable; no neoplasms in animal group

j Historical incidence: 1/1,348 (0.1% ± 0.4%); range, 0%-2%
48 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Urinary Bladder: Hyperplasiaofthe transitional cell

epithelium of the urinary bladder was observed in

10,000 ppmfemales at 9 months and in 10,000 ppm

males and females at 15months(Tables 11, A5, and

B5). Transitional cellhyperplasiawas presentinmost

5,000 and 10,000 ppm females at 2 years, and the

incidences of this lesion were significantly increased

in 5,000and10,000ppmmales. Hyperplasia consisted

of a diffuse or focal increase in thickness of the

transitional epithelium; minimal cellular

pleomorphismand increased numbersofmitotic cells

were sometimespresent. Other nonneoplastic lesions

that occurredonly in exposed rats included squamous

metaplasia of the transitional epithelium and fatty

metaplasia (fat proliferation) of the stroma of the

bladder wall.

Two transitional cell carcinomas and one papilloma

occurred in 10,000 ppm females at the 15-month

interimevaluation. At 2 years,incidences of transitional cell papilloma, carcinoma, and papilloma or

carcinoma (combined)were significantlyincreased in

10,000ppmmales and 5,000 and 10,000 ppm females

(Tables 11,A3, andB3) andexceeded theNTPhistoricalranges(males: 0%-2%; females: 0%-2%)forfeed

study controls (Tables 11, A4d, and B4d). Transitional cell papilloma consisted of a pedunculated or

broad-basedmass of transitional epitheliumwith a

central fibrovascular stroma; there was squamous

metaplasia of the surface epitheliumin some papillomas. Transitional cell carcinoma was characterized

by an exophytic or endophytic growth pattern and

invasion of the lamina propria or muscularis of the

bladder wall (Plates 7 and 8). There was cellular

atypia and squamous or mucous metaplasia of transitional epithelium in some carcinomas.
Results 49

TABLE 11

Incidences of Neoplasms and Nonneopla stic Lesions of the Urinary Bladder in Rats

in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

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

Male

15-Month Interim Evaluation

Number Examined 10 _b _ 10

Transitional Cell Hyperplasia

a

0 3 (1.3)

c

Metaplasia, Squamous 0 1 (2.0)

2-Year Study

Number Examined 50 38 58 50

Transitional Cell Hyperplasia 1 (2.0) 5 (2.0) 17** (1.9) 30** (2.1)

Metaplasia, Squamous 0 0 0 3 (1.7)

Transitional Cell Papilloma

Overall rated0/50 (0%) 1/38 (3%) 2/58 (3%) 8/50 (16%)

Terminal ratee0/26 (0%) 0/22 (0%) 2/21 (10%) 2/10 (20%)

Adjusted ratef0.0% 3.7% 9.5% 40.3%

First incidence (days) _h700 729 (T) 493

Logistic regression testgP<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%)

Adjusted rate 0.0% 0.0% 4.3% 24.5%

First incidence (days) _ _720 674

Logistic regression test P=0.001 _ P=0.491 P=0.022

Transitional Cell Papilloma or Carcinomai

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%)

Adjusted rate 0.0% 3.7% 13.5% 56.2%

First incidence (days) ) 700 720 493

Logistic regression test P<0.001 P=0.459 P=0.096 P<0.001

Female

9-Month Interim Evaluation

Number Examined 10 10 10 10

Transitional Cell Hyperplasia 0 0 0 2 (1.5)

15-Month Interim Evaluation

Number Examined 10 _ _

10

Transitional Cell Hyperplasia 0 9** (2.6)

Transitional Cell Papilloma 0 1

Transitional Cell Carcinoma 0 2

Squamous Cell Carcinoma 0 2

(continued)
50 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 11

Incidences of Neoplasms and Nonneopla stic Lesions of the Urinary Bladder in Rats

in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone (continued)

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

Female (continued)

2-Year Study

Number Examined 50 40 60 46

Transitional Cell Hyperplasia 1 (1.0) 2 (3.0) 41** (2.0) 41** (2.3)

Metaplasia, Squamous 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%)

Adjusted rate 0.0% 6.3% 17.6% 39.5%

First incidence (days) _729 (T) 691 637

Logistic 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%)

Adjusted rate 0.0% 0.0% 19.5% 55.8%

First incidence (days) _ _670 367

Logistic regression test P<0.001 _ P=0.008 P<0.001

Transitional Cell Papilloma or Carcinomaj

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%)

Adjusted rate 0.0% 6.3% 40.9% 78.1%

First incidence (days) _729 (T) 670 367

Logistic regression test P<0.001 P=0.201 P<0.001 P<0.001

Squamous Cell Papilloma

(Single or Multiple) 0 0 1 2

Squamous Cell Carcinoma 0 0 1 0

** Significantly different (P#0.01) from the control group by the Fisher exact test (9-month and 15-month interim evaluations) or the logistic regression

test (2-year study)

(T)Terminal sacrifice

a Number of animals with lesion

b Urinary bladder not microscopically examined in this group

c Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

d Number of animals with neoplasm per number of animals necropsied or examined microscopically

e Observed incidence in animals surviving until the end of the study

f Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

g

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to pairwise

comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

h Not applicable; no neoplasms in animal group

i Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 3/1,329 (0.2% ± 0.6%); range, 0%-2%

j Historical incidence: 3/1,334 (0.2% ± 0.6%); range, 0%-2%
Results 51

Forestomach: Several proliferative and degenerative

lesions occurredwithincreasedincidencesin the forestomach of exposed males and females(Table 12).

These mucosallesions frequently occurred together

and consisted of thickening (hyperplasia) of the

squamous epithelium and an increase

in the surface keratin layers (hyperkeratosis)

(Tables 12, A5, andB5). Focal areas of hyperplasia

were sometimes adjacentto ulceration and inflammation ofthe squamousmucosa. Therewas no significant increase in incidences of neoplasms of the

forestomach (Tables 12, A1, and B1).

TABLE 12

Incidences of Neoplasms and Nonneopla stic Lesions of the Forestomach in Rats

in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

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

Male

Number Examined 49 39 59 49

Hyperkeratosis

a

5 (2.4)

b

18** (1.8) 21** (2.0) 20** (2.0)

Hyperplasia, Squamous 3 (1.7) 19** (3.0) 25** (3.2) 26** (3.0)

Inflammation, 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)

Squamous Cell Papillomac0 2 0 1

Squamous Cell Carcinoma 0 0 0 1

Squamous Cell Papilloma or Carcinomad0 2 0 2

Female

Number Examined 49 40 60 47

Hyperkeratosis 2 (2.0) 7* (1.4) 23** (2.1) 28** (1.9)

Hyperplasia, Squamous 2 (2.0) 7* (1.9) 26** (2.9) 33** (3.0)

Inflammation, 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)

Squamous Cell Papilloma 0 0 0 1

Squamous Cell Carcinoma 0 1 1 1

Squamous Cell Papilloma or Carcinomae0 1 1 2

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

** P#0.01

a Number of animals with lesion

b Average severity grade of lesions in affected animals: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Number of animals with neoplasm per number of animals necropsied

d Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 4/1,353 (0.3% ± 0.7%); range, 0%-2% e Historical incidence: 2/1,351 (0.2% ± 0.5%); range, 0%-2%

MiscellaneousNeoplasms andNonneoplasticLesions:

In exposed males and females, incidences of

mononuclear cell leukemia occurred with significant

negative trends(Tables 13, A3, and B3). The incidences of pituitary gland adenoma in males and

females (males: 0 ppm, 21/48; 2,000 ppm, 14/40;

5,000ppm, 10/56;10,000ppm, 10/49;females: 32/50,

19/39, 32/60, 13/47; Tables A3 and B3) and the

incidence of mammarygland fibroadenoma (21/50,

10/40, 9/60, 5/49; Table B3)in females also occurred

withsignificantnegative trends. These decreasesmay

have been related to lower body weights.
52 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 13

Incidences of Mononuclear Cell Leukemia in Rats in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone 

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

Male

15-Month Interim Evaluation

Mononuclear Cell Leukemia

a

0/10 _b _2/10

2-Year Study

Mononuclear Cell Leukemia

Overall ratea25/50 (50%) 5/40 (13%) 3/59 (5%) 1/50 (2%)

Terminal ratec9/26 (35%) 4/24 (17%) 2/21 (10%) 0/10 (0%)

Adjusted rated59.0% 18.8% 11.7% 2.9%

First incidence (days) 514 604 650 590

Life table testeP<0.001N P<0.001N P<0.001N P<0.001N

Logistic regression test P<0.001N P<0.001N P<0.001N P<0.001N

Female

2-Year Study

Mononuclear Cell Leukemia

Overall rate 9/50 (18%) 1/40 (3%) 5/60 (8%) 1/49 (2%)

Terminal rate 6/38 (16%) 0/32 (0%) 1/38 (3%) 0/12 (0%)

Adjusted rate 21.5% 3.0% 10.0% 3.7%

First incidence (days) 620 689 601 662

Life table test P=0.112N P=0.026N P=0.177N P=0.162N

Logistic regression test P=0.011N P=0.023N P=0.111N P=0.024N

a Number of animals with neoplasm per number of animals necropsied 

b Animals not microscopically examined in this group

c Observed incidence in animals surviving until the end of the study

d Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

e

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The life table analysis regards neoplasms in animals dying prior to

terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. A negative

trend or a lower incidence in an exposure group is indicated by N.

In males, there was a chemical-related increased

incidence of atrophy of the seminal vesicles (1/49,

30/40, 35/59, 23/50; Table A5). This was not

evident at the 9- or 15-month interim evaluation,

but was present in most males at the end of the 2-

year study. 

Atrophy of the seminal vesicles of exposed males

was characterized by a reduction in the size of the

secretory epithelium from a tall columnar shape to

a low cuboidal shape and by an increase in the

amount of connective tissue stroma in the gland. 
Results 53

STOP-EXPOSURE EVALUATION

Stop-exposure groups of male and female rats were

included in the NTP 2-year study to evaluate the

potential for progression or regression of chemicalrelated liver,largeintestine,kidney, urinarybladder,and

forestomachlesionsduringa recoveryperiod. Tenmale

and 10 female rats were exposed to 20,000 ppm

1-amino-2,4-dibromoanthraquinone infeed for9months

followedbyadministrationofundosed feed untilthe end

ofthe15-monthperiod (9-monthstop-exposuregroups).

In addition, 30 males and 30 femaleswereexposed to

20,000ppm1-amino-2,4-dibromoanthraquinoneinfeed

for15months(15-monthexposuregroups). Tenmales

and 10 females from the 15-month exposure groups

were evaluated at the 9-month interim evaluation

(9-month interim evaluation groups).

Survival

Estimatesof 2-year survivalprobabilitiesformale and

female rats are shown in the Kaplan-Meier survival

curvesin Figure 4. All malessurvived9months;three

malesfromthe 9-monthstop-exposuregroup and three

malesfromthe15-monthexposuregroup died beforethe

end ofthe 15-monthevaluation (Table14). Allfemales

in the 9-monthstop-exposuregroup survived until the

end ofthe 15-monthevaluation. One female from the

15-monthexposuregroup died duringthe first9months;

an additionalseven femalesdied betweenmonth9 and

the end of the 15-month evaluation (Table 15).

Body Weights

and Feed and Compound Consumption

Themeanbodyweightsof male and female ratsin the

9-monthstop-exposure and 15-monthexposuregroups

are compared with the controls from the 2-year core

study in Tables 14 and 15, and the growth curves for

exposed rats in the 15-month exposure groups are

shown in Figure 5. The mean body weights of males

and femalesin the 9-monthstop-exposure groupswere

20% to 22% lower than those of the controls at the

9-monthinterimevaluationofthe 2-yearcore studyand

were20%to 21%lowerthanthose ofthe controlsatthe

15-monthinterimevaluation of the 2-year core study.

The mean body weights of males and females in the

15-month exposure groups were 19% to 21% lower

than those of the controls at the 9-month interim

evaluation of the 2-year core study and were 25% to

33% lower than those ofthe controls at the 15-month

interim evaluation of the 2-year core study. Feed

consumption by9-monthstop-exposure and 15-month

exposuremalesand females was generally lowerthan

that bythe controlsthroughout the study(TablesJ1 and

J2). The dietary level of 20,000 ppm delivered daily

doses of approximately 1,300 mg 1-amino2,4-dibromoanthraquinone/kgbodyweighttomalesand

1,800mg/kg to femalesin the 9-monthstop-exposure

groups and daily doses of approximately1,100mg/kg

tomales and 1,400 mg/kg to femalesin the 15-month

exposure groups.
54 l-Amino-2,4-dibromoanthraquinone, NTP TR 383

FIGURE 4

Kaplan-Meier Survival Curves for Rats Administered l-Amino-2,4-dibromoanthraqninone in Feed 

in the 15-Month Stop-Exposure Evaluation 
Results 55 

FIGURE 5

Growth Curves for Rats Administered l-Amino-2,4-dibromoanthraquinone in Feed 

in the 15-Month Stop-Exposure Evaluation 
56 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 14

Mean Body Weights and Survival of Male Rats in the Stop-Exposure Evaluation 

of 1-Amino-2,4-dibromoanthraquinone

Weeks 0 ppm 20,000 ppm (9-Month Stop-Exposure) 20,000 ppm (15-Month Exposure)

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

 1 139 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 60a 374 80 10b 377 81 20a

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 for weeks

1-13 268 203 76 209 78

14-37 415 328 79 340 82

41-65 480 387 81 377 79

a

Interim evaluation occurred during week 39. b Animals switched to undosed feed
Results 57

TABLE 15

Mean Body Weights and Survival of Female Rats in the Stop-Exposure Evaluation 

of 1-Amino-2,4-dibromoanthraquinone

Weeks 0 ppm 20,000 ppm (9-Month Stop-Exposure) 20,000 ppm (15-Month Exposure)

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 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 59a 207 78 10 10b 210 79 19a

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 for weeks

1-13 165 132 80 137 83

14-37 236 195 83 197 84

41-65 296 239 81 216 73

a

Interim evaluation occurred during week 40. b Animals switched to undosed feed
58 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Pathology and Statistical Analysis of Results

Summariesofthe incidences of neoplasms and nonneoplastic lesionsare shown inTablesE1 andE3 formale

rats and Tables F1 and F3 for female rats. For

statisticalanalyses,the incidencesin the 9-monthstopexposuregroups and the15-monthexposuregroups at

the end of 15months are comparedwith the 15-month

interimevaluation controls ofthe2-year core studyfor

male rats(TableE2a) and female rats(Table F2a). The

incidences in the 15-month exposure groups are

comparedwith the 9-monthstop-exposuregroups after

6 months of recovery for male rats (Table E2b) and

fem ale rats (Table F2b) .

Progression or Regression

of Chemical-Induced Lesions

Liver: Rats in the stop-exposure study exposed to

20,000ppm1-amino-2,4-dibromoanthraquinone inthe

feed for 9 or 15 months had chemical-related 

effects similar to those observed in rats exposed to

concentrations up to 10,000 ppm in the 2-year core

study. The absolute and relative liver weights of

exposedmalesand femalesweresignificantly greater

than those of the controls at both the 9-month interim

and 15-month evaluations of the 15-month exposure

groups (Tables H2 and H3). With respect to both

neoplasms and nonneoplastic lesionsin the liver,there

was no evidence of regression in the incidence or

severityof chemical-relatedpigmentation,focalhepatocellular alteration, or hepatocellular adenoma or

carcinoma when administration of 1-amino--

2,4-dibromoanthraquinone was discontinued after

9 months. The incidences and severityofliver lesions

after 9 months of exposure were similar with and

without a 6-monthrecoveryperiod.The incidences of

liver lesions in the 15-month exposure groups were

greater than in the 9-monthstop-exposure groups but

the severities were comparable (Tables 16, E2a, and

F2a).
Results 59

TABLE 16

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Rats

in the 9-Month, 9-Month with 6-Month Recovery, and 15-Month Exposure Evaluation 

of 1-Amino-2,4-dibromoanthraquinone

9-Month Evaluationa 15-Month Evaluationb

0 ppm 20,000 ppm 0 ppm 20,000 ppm 20,000 ppm

(9-Month (9-Month (15-Month 

Exposure) Exposure Plus Exposure)

6-Month Recovery)

Male

Number Examined 10 10 10 10 20

Basophilic Focus

c

0 6** (1.5)d1 (1.0) 4 (1.0) 13** (1.5)

Clear Cell Focus 0 4* (1.0) 0 6** (1.6) 13** (1.2)

Eosinophilic Focus 0 0 1 (1.0) 0 2 (1.5)

Bile Duct Hyperplasia 1 (1.0) 7** (1.3) 10 (1.1) 7 (1.0) 19 (1.3)

Ch ronic an d Chronic Active

Periportal Inflammation 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)

Hepatocellular Adenoma 0 2 0 7** 8*

Hepatocellular Carcinoma 0 2 0 7** 19**

Hepatocellular Adenoma or 

Carcinoma 0 2 0 9** 20**

Female

Number Examined 10 10 10 10 18

Basophilic Focus 1 (1.0) 3 (1.3) 8 (1.0) 6 (1.2) 13 (1.5)

Clear Cell Focus 0 1 (1.0) 0 5* (1.0) 13** (1.2)

Eosinophilic Focus 0 0 2 (1.5) 2 (1.0) 3 (1.7)

Bile Duct Hyperplasia 1 (1.0) 9** (1.1) 7 (1.1) 10 (1.5) 18* (1.6)

Ch ronic an d Chronic Active

Periportal Inflammation 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)

Hepatocellular Adenoma 0 2 0 6** 10**

Hepatocellular Carcinoma 0 1 0 6** 15**

Hepatocellular Adenoma or

Carcinoma 0 2 0 8** 16**

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01

a

Controls from the 9-month interim evaluation of the 2-year core study were used for statistical comparison.

b

Controls from the 15-month interim evaluation of the 2-year core study were used for statistical comparison.

c Number of animals with lesion

d Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

Large Intestine: The stop-exposure regimen had no

effect on the development of adenomatous polyps

(adenomas) of the large intestine. An adenomatous

polypwasobserved in thecolonofone exposedmale at

9 months. After 15 months, the incidence of

adenomatous polyps of the rectum was significantly

increased in the 9-month stop-exposure

females (Tables 17 and F2a). At the 15-month

evaluation, adenomatous polypswereobserved in the

rectums of three males in the 9-monthstop-exposure

group, seven males in the 15-monthexposure group,

five femalesin the 9-monthstop-exposure group, and

three females in the 15-month exposure group. No

carcinomas of the colon or rectum were observed.
60 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Kidney: Atthe9-monthand 15-monthevaluations,the

relative kidney weights of males and females in the

15-monthexposuregroupsweresignificantly greater

than those ofthe controls (Tables H2 and H3). In the

exposedmalesand females at9months,kidneychanges

included pigmentation and minimal enlargement of

some renal tubule cell nuclei (karyomegaly). In

exposed males, there was also hyaline droplet

accumulation and a slight increase in the severity of

nephropathy. At 15 months, renal tubule 

epithelial pigmentation, karyomegaly, and increased

severityof nephropathyand transitionalcellhyperplasia

ofthe renalpelvis wereobserved in exposed groups of

malesand females. The severities ofthese lesionswere

similar or slightly less severe in the stop-exposure

groups than in those exposed continuously for

15 months. Atthe 15-month evaluation, renal tubule

adenomas wereobserved inthe9-monthstop-exposure

and 15-month exposuregroups ofmales and females

(Tables 17, E1, and F1). In the 9-month 

TABLE 17

Incidences of Neoplasms and Nonneoplastic Lesions of the Large Intestine and Kidney in Rats 

in the 9-Month, 9-Month with 6-Month Recovery, and 15-Month Exposure Evaluation 

of 1-Amino-2,4-dibromoanthraquinone

9-Month Evaluationa15-Month Evaluationb

0 ppm 20,000 ppm 0 ppm 20,000 ppm 20,000 ppm

(9-Month (9-Month (15-Month 

Exposure) Exposure Plus Exposure)

6-Month Recovery)

Male

Large Intestine, Colonc10 10 10 10 20

Aden omatous Polyp (Aden oma)d0 1 0 0 0

Large Intestine, Rectum 10 10 9 10 20

Aden omatous Polyp (Aden oma) 0 0 0 3 7*

Kidney 10 10 10 10 20

Nephropathy 10 (1.0)e10 (1.8) 10 (2.0) 10 (2.0) 20 (2.5)

Transition al Cell Hyperplasia 0 0 0 1 (1.0) 11** (1.3)

Hyaline Droplet Accumulation 0 10** (2.1) 0 0 0

Pigmentation 0 10** (2.0) 0 9** (1.2) 20** (2.0)

Renal Tubule Hyperplasia 0 0 0 1 (1.0) 1 (1.0)

Renal Tubule Adenoma 0 0 0 3 2

Female

Large Intestine, Rectum 10 10 10 10 17

Aden omatous Polyp (Aden oma) 0 0 0 5* 3

Kidney 10 10 10 10 18

Nephropathy 4 (1.0) 7 (1.0) 10 (1.7) 10 (2.2) 18 (2.1)

Transition al Cell Hyperplasia 0 0 3 (1.0) 1 (1.0) 5 (1.6)

Pigmentation 0 10** (2.0) 0 10** (1.9) 18** (2.0)

Renal Tubule Hyperplasia 0 0 0 2 (2.0) 2 (1.5)

Renal Tubule Adenoma 0 0 0 3 2

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01

a

Controls from the 9-month interim evaluation of the 2-year core study were used for statistical comparison.

b

Controls from the 15-month interim evaluation of the 2-year core study were used for statistical comparison.

c Number of animals with organ examined microscopically

d Number of animals with lesion

e Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked
Results 61

stop-exposure and 15-monthexposure groups, renal

tubule hyperplasia and adenomas were observed in

males and females. At 15 months, renal tubule

adenomas were observed in three males and three

femalesinthe9-monthstop-exposuregroups and intwo

males and two females in the 15-month exposure

groups. No renal tubule carcinomas were found.

Urinary Bladder: A transitional cell papilloma was

present in the urinary bladder of one exposed male at

9 months (Tables 18 and E1). When exposure was

discontinued at 9 months, no chemical-related

nonneoplastic lesions or neoplasms were present at

15months. Withcontinuoustreatment,transitionalcell

hyperplasia and neoplasms of the urinary bladder

developed by15months. In females,aminimaltomild

transitional cell hyperplasia wasobserved at 9months

and did not completely regress with the cessation of

exposure; a squamous cell papilloma was observed in

one female at 15 months (Tables 18 and

F1). In contrast, continuous exposure for 15 months

resulted in an increased incidence and severity of

transitional cell hyperplasia, as well as squamous

metaplasia of the transitional epithelium and a fatty

metaplasia (fat proliferation) of the stroma of the

bladder wall.

Forestomach: Chemical-related lesions of the forestomach were not present in males or females at the

9-monthinterim evaluation. Hyperplasia,hyperkeratosis, inflammation, and ulceration were present in

approximately 20% of exposed males, but not in the

controls at 15 months (Table 18). A squamous cell

papillomawaspresentin onemale fromthe 15-month

exposuregroup (TableE1). Infemale rats,forestomach

lesionswerenotpresentinthe controlor 9-monthstopexposuregroups. Hyperplasia, hyperkeratosis,and/or

ulceration were observed in a few females in the

15-monthexposuregroup; no neoplasms werepresent

in the forestomach.
62 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 18

Incidences of Neoplasms and Nonneoplastic Lesions of the Urinary Bladder and Forestomach in Rats

in the 9-Month, 9-Month with 6-Month Recovery, and 15-Month Exposure Evaluation 

of 1-Amino-2,4-dibromoanthraquinone

9-Month Evaluationa15-Month Evaluationb

0 ppm 20,000 ppm 0 ppm 20,000 ppm 20,000 ppm

(9-Month (9-Month (15-Month 

Exposure) Exposure Plus Exposure)

6-Month Recovery)

Male

Urinary Bladder

c

10 9 10 10 19

Fat Proliferationd0 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

Transitional Epithelial Papilloma 0 1 0 0 3

Transitional Epithelial 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)

Inflammation 0 0 0 1 (3.0) 1 (3.0)

Ulceration 0 0 0 2 (2.5) 0

Female

Urinary Bladder 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

Squamou s Metaplasia 0 0 0 0 3 (2.7)

Squamous Cell Papilloma 0 0 0 0 1

Squamous Cell Carcinoma 0 0 0 1 4

Transitional Epithelial Papilloma 0 0 0 0 1

Transitional Epithelial 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 different (P#0.05) from the control group by the Fisher exact test

** P#0.01

a

Controls from the 9-month interim evaluation of the 2-year core study were used for statistical comparison.

b

Controls from the 15-month interim evaluation of the 2-year core study were used for statistical comparison.

c Number of animals with organ examined microscopically

d Number of animals with lesion

e Average severity grade of lesions in affected rats: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked
Results 63

MICE

13-WEEK STUDY

One 25,000 ppmmale (week 11) and one 5,000 ppm

male (week 13) died during the study (Table 19).

Neitherdeathwaschemicalrelated. One 10,000 ppm

femalewasaccidentlykilled. Finalmeanbodyweights

of exposed groups of male and female mice were

similar to those of the controls. Mean body weight

gains of exposed groups of males and females were

generally greater than those of the controls. Feed

consumption by exposed mice was similar to

that by the controls. Dietary levels of 2,500, 5,000,

10,000,25,000,and50,000 ppmdeliveredaveragedaily

doses of approximately500,1,080, 1,850, 6,200,and

10,600mg1-amino-2,4-dibromoanthraquinone/kgbody

weighttomalesand approximately660,1,150,2,600,

5,900, and 11,700mg/kgto females. Reddened furwas

observed in 10,000,25,000, and 50,000 ppm mice as

early as day 4 in males and day 5 in females and was

observed throughout the study. No other clinical

observations were attributed to 1-amino2,4-dibromoanthraquinone.

TABLE 19

Survival, Mean Body Weights, and Feed Consumption of Mice in the 13-Week Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

Final Weight

Mean Body Weightb

 (g) Relative Feed

Dose SurvivalaInitial Final Change to Controls Consumption

c

(ppm) (%) 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 9/10

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 9/10

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 animals surviving at 13 weeks/number initially in group

b Weigh ts an d weigh t changes are given as mean ± standard error. Subsequent calcu lations are based on animals surviving to the end of the study.

c

Feed consumption is expressed as grams of feed consumed per animal per day.

d Week of death: 13

e Week of death: 11

f Week of death: 2 (accidental)
64 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Absolute and relative liver weights of 5,000, 10,000,

25,000, and 50,000 ppm male and female mice were

significantly greater than those of the controls

(Table H4). Absolute and relative kidneyweights of

25,000and 50,000 ppmmalesweresignificantlylower

than those of the controls. Observations at necropsy

included red staining ofthegastrointestinaltract and its

contents in all exposed male mice exceptthose in the

2,500 ppm group and red staining in the kidney and

urine. These findingswereobserved lessfrequentlyin

females than in males.

Chemical-related lesions were present in the liver

(Table20). Therewereincreasedincidences of centrilobular hypertrophy in the 10,000, 25,000, and

50,000ppmmaleswithadose-related increasedseverity. Minimal gold to brown pigment granules were

presentin the cytoplasm of hepatocytesof all exposed

groups ofmales. Pigment was generally located in the

centrilobular portion of the hepatic lobule. Pigment

similar to that in male mice was present in just a few

hepatocytes in the liver of one female in the 25,000-

 ppm group and one 50,000 ppm female.

TABLE 20

Incidences of Nonneoplastic Lesions of the Liver in Mice in the 13-Week Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

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

Male

Number Examined 10 10 10 10 10 10

Centrilobular Hypertrophya0 0 0 8** (1.8)b8** (2.0) 10** (2.8)

Pigmentation 0 5* (1.0) 8** (1.0) 8** (1.0) 8** (1.0) 10** (1.1)

Female

Number Examined 10 10 10 10 10 10

Pigmentation 0 0 0 0 1 (1.0) 1 (1.0)

* Significantly different (P#0.05) from the control group by the Fisher exact test

** P#0.01

a Number of animals with lesion

b Average severity grade of lesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

Dose Selection Rationale: As no 1-amino2,4-dibromoanthraquinone-related adverse effectswere

observed in feed consumption,meanbodyweights,or

survival, exposureconcentrations chosenforthe2-year

study were based mainly on the frequency and especially the severity of centrilobularhypertrophy of the

liver in male mice. Because only lesions of mild

severitywereobserved in the 10,000and 25,000 ppm

groups, and lesionsofmoderate severitywereobserved

in the 50,000 ppm group, and these were 

notlife-jeopardizinglesions,exposureconcentrations

selected for the 2-year study of 1-amino2,4-dibromoanthraquinone inmicewere0,10,000,and

20,000ppm. Other considerationsinclude consistency

among males and females (since females could have

been givenhigherexposureconcentrations) and correspondence to the exposureconcentrationsselectedfor

the start-stop,progression/regressionexperiments(stopexposure evaluation) in rats.
Results 65

2-YEAR STUDY

Survival

Estimatesof 2-yearsurvivalprobabilitiesformale and

femalemiceare shown inTable 21 and in the KaplanMeiersurvivalcurvesinFigure 6. Survivalof exposed

male mice was significantly lower than that of the

controls;survivalof exposed female mice was similar

to that of the controls.

Body Weights, Feed and Compound

Consumption, and Clinical Findings

Mean body weights of exposed groups of male mice

were lowerthan that ofthe controls afterweek9;mean

body weights of exposed groups of females were

lowerthan that ofthe controls afterweek17 (Figure7,

Tables 22 and 23). Final mean body weights of exposed groups of males were 15% to 17% lower than

thatofthe controls;finalmeanbodyweightsof exposed

females were 14% to 19% lower than that of the controls. Feed consumption byexposedmales and females

wasgenerally similar to that bythe controls(TablesJ5

and J6). Dietarylevels of10,000 and 20,000ppmwere

estimatedto deliverdaily doses of approximately 1,700

and 3,500mg1-amino-2,4-dibromoanthraquinone/kg

body weight to males and 2,000 and 4,400 mg/kg to

females. Discolorationof the fur,urine,and feceswas

evident in all exposed groups as early as day 8.

TABLE 21

Survival of Mice in the 2-Year Feed Study of 1-Amino-2,4-dibromoanthraquinone

0 ppm 10,000 ppm 20,000 ppm

Male

Animals initially in study 60 60 60

15-Month interim evaluationa10 9 10

Accidental deatha0 1 0

Moribund 7 23 21

Natural deaths 3 5 6

Animals surviving to study termination 40 22 23

Percent probability of survival at end of studyb81 45 47

Mean survival (days)c656 620 609

Survival analysesdP=0.001 P<0.001 P<0.001

Female

Animals initially in study 60 60 60

15-Month interim evaluationa10 10 10

Moribund 5 11 11

Natural deaths 6 5 6

Animals surviving to study termination 39e34e33

Percent probability of survival at end of study 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 sacrifice

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 control column , and the results of the life table pairwise comparison s (Cox, 1 972 ) with the controls

are in the exposed column s. 

e

Includes one female that died during the last week of the study.
66 l-Amino-2,4-dibromoanthraquinone, NTP TR 383

FIGURE 6

Kaplan-Meier Survival Curves for Mice Administered l-Amino-2,4-dibromoanthraquinone in Feed 

for 2 Years 
Results 67

FIGURE 7

Growth Curves for Mice Administered l-Amino-2,4-dibromoanthraquinone in Feed for 2 Years
68 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 22

Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

Weeks 0 ppm 10,000 ppm 20,000 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 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

69a43.6 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

Mean for weeks

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

Interim evaluation occurred during week 66.
Results 69

TABLE 23

Mean Body Weights and Survival of Female Mice in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone

Weeks 0 ppm 10,000 ppm 20,000 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

69a37.6 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

Mean for weeks

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

Interim evaluation occurred during week 65.
70 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Pathology and Statistical Evaluation

This section describes the statistically significant

or biologically noteworthy changes in the incidences of neoplasms and/or nonneoplastic lesions

of the liver, forestomach, lung, kidney, uterus, and

pituitary gland of mice. Summaries of the incidences of neoplasms and nonneoplastic lesions,

individual animal tumor diagnoses, statistical

analyses of primary neoplasms that occurred with

an incidence of at least 5% in at least one exposure

group, and historical incidences for the neoplasms

mentioned in this section are presented in Appendix C for male mice and Appendix D for female

mice.

Liver: At the 15-month interim evaluation, absolute and relative liver weights of exposed groups

of females were significantly greater than those of

the controls (Table H5). Hepatocellular adenomas

and carcinomas were observed in exposed groups

of males and females; none were present in the

controls (Tables 24, C1, and D1). At the end of

the 2-year study, the incidences of hepatocellular

adenoma and hepatocellular carcinoma in exposed

groups of males and females were significantly

increased (Tables 24, C3, and D3). The incidences of multiple hepatocellular adenomas or

multiple hepatocellular carcinomas in exposed

groups of males and females were also increased

(Tables 24, C1, and D1). Incidences of

hepatocellular adenomas or carcinomas (combined)

 

in exposed groups of males and females exceeded

the NTP historical ranges for feed study controls

(Tables 24, C4a, and D4a). In addition to

hepatocellular adenomas and carcinomas, a small

number of hepatoblastomas occurred in exposed

groups of males and females (Tables 24, C1, and

D1). These malignant hepatocellular neoplasms

contained areas resembling hepatocellular carcinoma; in addition, there were prominent lobules or

nodular foci separated by vascular channels and

composed of undifferentiated cells (Plates 9 and

10). The neoplastic cells were elongated with a

scant amount of darkly staining cytoplasm and

oval hyperchromatic nuclei. Cellular

pleomorphism and mitotic figures were commonly

present.

At the 15-month interim evaluation and at the end

of the 2-year study, the incidences of centrilobular

hypertrophy of hepatocytes in exposed groups of

males were significantly increased, and the incidences of hepatocellular pigmentation were significantly increased in exposed groups of males and

females (Tables 24, C5, and D5). This brown,

granular pig-ment resembled that found in the

13-week studies of 1-amino-2,4-dibromoanthraquinone; histochemical procedures were not

repeated during this 2-year study. The incidences

of clear cell focus in exposed groups of female

mice were significantly increased at the end of the

2-year study.
Results 71

TABLE 24

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Feed Study

of 1-Amino-2,4-dibromoanthraquinone 

0 ppm 10,000 ppm 20,000 ppm

Male

15-Month Interim Evaluation

Number Examined 10 9 10

Centrilobular Hepatocyte Hypertrophya0 9** (2.9)b8** (2.9)

Pigmentation 0 9** (1.1) 10** (1.2)

Hepatocellular Adenoma 0 2 4*

Hepatocellular Adenoma or Carcinoma 0 3 4*

2-Year Study

Number Examined 50 51 50

Basophilic Focus 0 4 (1.0) 3 (1.0)

Centrilobular Hepatocyte Hypertrophy 0 17** (2.0) 13** (2.0)

Clear Cell Focus 4 (1.3) 4 (1.0) 2 (1.0)

Eosinophilic Focus 0 6** (1.5) 1 (1.0)

Pigmentation 1 (3.0) 50** (1.1) 47** (1.4)

Hepatocellular Adenoma (Multiple) 6 29** 31**

Hepatocellular Adenoma (Single or Multiple)

Overall ratec10/50 (20%) 38/51 (75%) 39/50 (78%)

Terminal rated9/40 (23%) 20/22 (91%) 21/23 (91%)

Adjusted ratee24.3% 94.7% 95.0%

First incidence (days) 723 451 484

Logistic regression testfP<0.001 P<0.001 P<0.001

Hepatocellular Carcinoma (Multiple) 1 3 9**

Hepatocellular Carcinoma (Single or Multiple)

Overall rate 9/50 (18%) 18/51 (35%) 21/50 (42%)

Terminal rate 7/40 (18%) 10/22 (45%) 9/23 (39%)

Adjusted rate 21.1% 58.1% 58.4%

First incidence (days) 445 505 535

Logistic regression test P=0.002 P=0.017 P=0.003

Hepatocellular Adenoma or Carcinomag

Overall rate 18/50 (36%) 43/51 (84%) 42/50 (84%)

Terminal rate 15/40 (38%) 21/22 (95%) 22/23 (96%)

Adjusted rate 41.7% 97.7% 97.7%

First incidence (days) 445 451 484

Logistic regression test P<0.001 P<0.001 P<0.001

Hepatoblastoma 0 3 5*

(continued)

* Significantly different (P#0.05) from the control group by the Fisher exact test (15-month interim evaluation) or the logistic regression test

(2-year study)

** P#0.01

(T)Terminal sacrifice

a Number of animals with lesion

b Average severity grade of lesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Number of animals with neoplasm per number of animals with liver examined microscopically
72 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 24

Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Feed Study

of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Female

15-Month Interim Evaluation

Number Examined 10 10 10

Pigmentation 0 10** (1.0) 9** (1.0)

Hepatocellular Adenoma (Multiple) 0 1 4*

Hepatocellular Adenoma (Single or Multiple) 0 2 7**

Hepatocellular Adenoma or Carcinoma 0 2 8**

2-Year Study

Number Examined 50 50 50

Basophilic Focus 0 4 (1.3) 5* (1.2)

Clear Cell Focus 0 10** (1.2) 9** (1.6)

Eosinophilic Focus 0 4* (1.5) 2 (2.5)

Pigmentation 0 44** (1.1) 49** (1.6)

Hepatocellular Adenoma (Multiple) 0 40** 45**

Hepatocellular Adenoma (Single or Multiple)

Overall rate 6/50 (12%) 45/50 (90%) 49/50 (98%)

Terminal rate 6/39 (15%) 32/34 (94%) 33/33 (100%)

Adjusted rate 15.4% 95.7% 100.0%

First incidence (days) 729 (T) 442 501

Logistic regression test P<0.001 P<0.001 P<0.001

Hepatocellular Carcinoma (Multiple) 0 13** 13**

Hepatocellular Carcinoma (Single or Multiple)

Overall rate 0/50 (0%) 23/50 (46%) 27/50 (54%)

Terminal rate 0/39 (0%) 17/34 (50%) 16/33 (48%)

Adjusted rate 0.0% 57.2% 60.8%

First incidence (days) )

h

503 538

Logistic regression test P<0.001 P<0.001 P<0.001

Hepatocellular Adenoma or Carcinomai

Overall rate 6/50 (12%) 46/50 (92%) 50/50 (100%)

Terminal rate 6/39 (15%) 33/34 (97%) 33/33 (100%)

Adjusted rate 15.4% 97.9% 100.0%

First incidence (days) 729 (T) 442 501

Logistic regression test P<0.001 P<0.001 P<0.001

Hepatoblastoma 0 0 2

d Observed incidence in animals surviving until the end of the study

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

f

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

g Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 531/1,466 (36.2% ± 14.1%);

range, 10%-68% h Not applicable; no neoplasms in animal group

i Historical incidence: 247/1,462 (16.9% ± 10.7%); range, 3%-42%
Results 73

Forestomach: Squamouscellpapillomaswereobserved

in 20,000 ppm males and in 10,000 and 20,000 ppm

females atthe 15-monthinterimevaluation(Tables 25,

C1, and D1). At the end of the 2-year study, the incidences ofsquamouscellpapilloma and squamous cell

carcinoma inexposedgroupsofmalesandfemaleswere

significantly increased (Tables 25, C3, and D3). The

incidences of multiple squamous cell papilloma in

20,000ppmmales and femaleswere also significantly

increased in the 2-year study(Tables 25, C1, and D1).

Incidences of squamous cell papilloma or carcinoma

(combined)inexposedgroupsofmalesandfemaleswere

significantly greater than those in the controls and

exceeded the NTP historical ranges for feed study

controls(Tables 25, C4b, and D4b). Compared to the

exophyticmasses with well-differentiated squamous

epitheliumtypicalofthe squamouscellpapillomas,the

squamous cell carcinomas were locally invasive

neoplasms that sometimes resulted in perforation of

the forestomach(Plate11). Frequently, a squamouscell

carcinoma appeared to arise atthe base of a squamous

cell papilloma. Metastatic neoplasms arising from

squamous cell carcinomas of the forestomach were

observedin the coagulatingglands, colon, duodenum,

epididymis,gallbladder,glandular stomach,jejunum,

kidney, liver, lung, ovary, pancreas, prostate gland,

spleen,testis, and thymus of exposed mice (TablesC1

and D1).

Nonneoplastic lesions of the forestomach included

acanthosis,hyperkeratosis,and basal cell hyperplasia

(Tables 25, C5, and D5). At the 15-month interim

evaluation,therewereexposure-relatedincreasesinthe

incidences and severities of acanthosis and

hyperkeratosisinexposedgroupsofmalesandfemales.

At the end of the 2-year study, the incidences and

severitiesoftheselesionsinexposedgroupsofmalesand

femalesweregenerallygreaterthanthoseinthe controls.
74 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

TABLE 25

Incidences of Neoplasms and Nonneoplastic Lesions of the Forestomach in Mice in the 2-Year Feed Study

of 1-Amino-2,4-dibromoanthraquinone 

0 ppm 10,000 ppm 20,000 ppm

Male

15-Month Interim Evaluation

Number Examined 9

9 10

Acanthosis (Hyperplasia)a0 2 (1.0)b3 (2.0)

Basal Cell Hyperplasia 0 1 (1.0) 1 (1.0)

Hyperkeratosis 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 Examined 50 50 50

Acanthosis 1 (1.0) 9** (1.1) 4 (2.0)

Basal Cell Hyperplasia 0 0 2 (1.5)

Hyperkeratosis 1 (1.0) 7* (1.0) 6 (1.8)

Inflammation 2 (1.5) 6 (1.2) 13** (1.5)

Squamous Cell Papilloma (Multiple) 0 2 5*

Squamous Cell Papilloma (Single or Multiple)

Overall ratec0/50 (0%) 13/51 (25%) 16/50 (32%)

Terminal rated0/40 (0%) 10/22 (45%) 11/23 (48%)

Adjusted ratee0.0% 51.0% 55.6%

First incidence (days) )

g

613 606

Logistic regression testfP<0.001 P<0.001 P<0.001

Squamous Cell Carcinoma

Overall rate 0/50 (0%) 12/51 (24%) 13/50 (26%)

Terminal rate 0/40 (0%) 4/22 (18%) 4/23 (17%)

Adjusted rate 0.0% 36.5% 37.7%

First incidence (days) ) 505 523

Logistic regression test P<0.001 P<0.001 P<0.001

Squamous Cell Papilloma or Carcinomah

Overall rate 0/50 (0%) 19/51 (37%) 27/50 (54%)

Terminal rate 0/40 (0%) 11/22 (50%) 14/23 (61%)

Adjusted rate 0.0% 61.2% 73.9%

First incidence (days) ) 505 523

Logistic regression test P<0.001 P<0.001 P<0.001

(continued)

* Significantly different (P#0.05) from the control group by the Fisher exact test (15-month interim evaluation) or the logistic regression test

(2-year study)

** P#0.01

(T)Terminal sacrifice
Results 75

TABLE 25

Incidences of Neoplasms and Nonneoplastic Lesions of the Forestomach in Mice in the 2-Year Feed Study

of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Female

15-Month Interim Evaluation

Number Examined 10 10 10

Acanthosis (Hyperplasia) 0 1 (1.0) 8** (2.0)

Basal Cell Hyperplasia 0 0 2 (1.5)

Hyperkeratosis 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 Examined 48 50 50

Acanthosis (Hyperplasia) 9 (1.7) 15 (1.7) 19* (1.6)

Basal Cell Hyperplasia 0 7* (1.4) 3 (1.7)

Hyperkeratosis 10 (1.4) 14 (1.4) 17 (1.5)

Inflammation 7 (1.4) 10 (1.4) 21** (1.7)

Squamous Cell Papilloma (Multiple) 0 4 14**

Squamous Cell Papilloma (Single or Multiple)

Overall rate 2/50 (4%) 16/50 (32%) 27/50 (54%)

Terminal rate 2/39 (5%) 12/34 (35%) 23/33 (70%)

Adjusted rate 5.1% 41.7% 72.4%

First incidence (days) 729 (T) 671 538

Logistic regression test P<0.001 P<0.001 P<0.001

Squamous Cell Carcinoma

Overall rate 0/50 (0%) 12/50 (24%) 11/50 (22%)

Terminal rate 0/39 (0%) 8/34 (24%) 5/33 (15%)

Adjusted rate 0.0% 30.9% 27.3%

First incidence (days) ) 587 501

Logistic regression test P=0.002 P<0.001 P<0.001

Squamous Cell Papilloma or Carcinomai

Overall rate 2/50 (4%) 25/50 (50%) 34/50 (68%)

Terminal rate 2/39 (5%) 18/34 (53%) 25/33 (76%)

Adjusted rate 5.1% 60.7% 80.5%

First incidence (days) 729 587 501

Logistic regression test P<0.001 P<0.001 P<0.001

a Number of animals with lesion

b Average severity grade of lesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Number of animals with neoplasm per number of animals necropsied

d Observed incidence in animals surviving until the end of the study

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

f

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal.

g Not applicable; no neoplasms in animal group

h Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 22/1,474 (1.5% ± 2.0%); range,

0%-6% i Historical incidence: 33/1,470 (2.2% ± 3.1%); range, 0%-14%
76 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Lung: Alveolar/bronchiolar adenomas were

observed in exposed groups of males and females

at the 15-month interim evaluation (Tables 26, C1,

and D1). During the 2-year study, the incidences

of alveolar/bronchiolar adenomas in exposed

groups of males and females were significantly

increased (Tables 26, C3, and D3). In male mice,

the incidences of multiple alveolar/bronchiolar

adenomas in exposed groups were significantly

greater than that in the controls (Tables 26, C1,

and D1). The incidences of alveolar/ 

bronchiolar adenoma in exposed groups of males

and females exceeded the NTP historical ranges

for feed study controls (Tables 26, C4c, and D4c).

The alveolar/bronchiolar adenomas were generally

well-circumscribed, expansile masses that slightly

compressed the surrounding normal pulmonary

alveolar tissue (Plate 12). Well-differentiated

cuboidal to columnar epithelial cells formed

papillary structures or solid foci that filled alveolar

spaces.

TABLE 26

Incidences of Neoplasms of the Lung in Mice in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone 

0 ppm 10,000 ppm 20,000 ppm

Male

15-Month Interim Evaluation 

Number Examined 10 9 10

Alveolar/bronchiolar Adenomaa0 3 5*

2-Year Study

Number Examined 50 51 50

Alveolar/bronchiolar Hyperplasia 1 (1.0)b0 4 (1.3)

Alveolar/bronchiolar Adenoma (Multiple) 0 6** 9**

Alveolar/bronchiolar Adenoma (Single or Multiple)

Overall ratec7/50 (14%) 26/51 (51%) 24/50 (48%)

Terminal rated6/40 (15%) 12/22 (55%) 12/23 (52%)

Adjusted ratee16.8% 71.0% 66.5%

First incidence (days) 445 578 248

Logistic regression testfP<0.001 P<0.001 P<0.001

Alveolar/bronchiolar Carcinoma

Overall rate 3/50 (6%) 4/51 (8%) 1/50 (2%)

Terminal rate 2/40 (5%) 2/22 (9%) 0/23 (0%)

Adjusted rate 6.9% 15.9% 3.0%

First incidence (days) 393 673 648

Logistic regression test P=0.259N P=0.512 P=0.251N

Alveolar/bronchiolar Adenoma or Carcinomag

Overall rate 10/50 (20%) 28/51 (55%) 25/50 (50%)

Terminal rate 8/40 (20%) 13/22 (59%) 12/23 (52%)

Adjusted rate 23.3% 75.0% 67.5%

First incidence (days) 393 578 248

Logistic regression test P<0.001 P<0.001 P=0.002

(continued)
Results 77

TABLE 26

Incidences of Neoplasms of the Lung in Mice in the 2-Year Feed Study 

of 1-Amino-2,4-dibromoanthraquinone (continued)

0 ppm 10,000 ppm 20,000 ppm

Female

15-Month Interim Evaluation

Number Examined 10 10 10

Alveolar/bronchiolar Adenoma 0 3 2

2-Year Study

Number Examined 50 50 49

Alveolar/bronchiolar Hyperplasia 0 0 1 (1.0)

Alveolar/bronchiolar Adenoma (Multiple) 0 2 1

Alveolar/bronchiolar Adenoma (Single or Multiple)

Overall rate 4/50 (8%) 17/50 (34%) 13/49 (27%)

Terminal rate 3/39 (8%) 14/34 (41%) 9/33 (27%)

Adjusted rate 9.8% 45.6% 33.5%

First incidence (days) 685 587 538

Logistic regression test P=0.017 P=0.001 P=0.015

Alveolar/bronchiolar Carcinoma 0 0 2

Alveolar/bronchiolar Adenoma or Carcinomah

Overall rate 4/50 (8%) 17/50 (34%) 15/49 (31%)

Terminal rate 3/39 (8%) 14/34 (41%) 10/33 (30%)

Adjusted rate 9.8% 45.6% 37.9%

First incidence (days) 685 587 538

Logistic regression test P=0.006 P=0.001 P=0.005

* Significantly different (P#0.05) from the control group by the Fisher exact test (15-month interim evaluation) or the logistic regression test

(2-year study)

** P#0.01

a Number of animals with lesion

b Average severity grade of lesions in affected mice: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked

c Number of animals with neoplasm per number of animals with lung examined microscopically

d Observed incidence in animals surviving until the end of the study

e Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality.

f

In the control column are the P values associated with the trend test. In the exposed group columns are the P values corresponding to

pairwise comparisons between the controls and that exposure group. The logistic regression test regards these lesions as nonfatal. A negative

trend or a lower incidence in an exposure group is indicated by N.

g Historical incidence for 2-year NTP feed studies with untreated control groups (mean ± standard deviation): 265/1,469 (18.0% ± 7.6%);

range, 4%-32% h Historical incidence: 110/1,469 (7.5% ± 5.0%); range, 2%-26%
78 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

Kidney: Pigmentationwaspresentin the kidneys ofmost

mice after 2 years of exposure to 1-amino2,4-dibromoanthraquinone (males: 0 ppm, 0/50;

10,000ppm, 42/51; 20,000ppm, 43/50;females: 0/50,

43/50,43/50;TablesC5andD5). Thisbrown,granular

pigmentinthe renaltubuleepitheliumandtubulelumina

resembledthepigmentdescribedinthe liver.Therewere

no other chemical-related lesions in the kidney.

Uterus: There was a significant, but not exposurerelated, increase in the incidence of uterine polyps or

sarcomas(combined)(0/50,5/50,0/50;TableD1)inthe

10,000ppmfemales. Althoughthisincidence (10%)was

above the average for historical controls (3.5%), the

combined incidence was within the historical control

range (0%-16%;TableD4d). Thismarginalincreasewas

not considered to be chemical related. There were no

chemical-relatednonneoplasticlesionsinthe reproductive tract (Table D5).

PituitaryGland: Asignificant,but not exposure-related,

increase in the incidence of adenoma of the pituitary

gland (pars distalis) was observed in the 10,000 ppm

females(1/43,9/45, 4/43;TableD1). The incidenceof

adenoma (20%) in the 10,000 ppm females is slightly

above the average forhistorical controls(15.2%),butis

withinthe historicalrange (2%-36%;TableD4e). This

marginal increase was not considered to be chemical

related. The incidenceofhyperplasiaoftheparsdistalis

ofthe pituitaryglandwas also increased in the 10,000-

 ppm females (7/43, 22/45, 7/43; Table D5).

DISPOSITION

AND METABOLISM STUDIES

AdultmaleF344/Nratsreceived[

14C]-labeled1-amino2,4-dibromoanthraquinoneas a singleintravenousdose

of0.4mg1-amino-2,4-dibromoanthraquinone/kg body

weight or as a single oral dose of 2, 23, 118, 814, or

1,473mg/kg. After excretawerecollectedfor72hours,

the animalswerekilled, and tissues were removed for

analysis. Additionalanimalsthatreceivedintravenous

dosesof1-amino-2,4-dibromoanthraquinonewerekilled

0.25, 0.75, 2, 6, or 24 hours after chemical administration, and their tissues were analyzed. A 6-hour bile

cannulation study was also performed.

Fromday0 throughday3afterintravenousadministrationof[

14C]-1-amino-2,4-dibromoanthraquinone, about

50% of the 14C was excreted in the feces, 15% in the

urine, and 6%in expired air. Unmetabolized1-amino2,4-dibromoanthraquinoneaccountedforlessthan3%of

the excreted 14Cafter intravenousadministration. The

amountof anoraldosethatwasabsorbedwas calculated

fromthepercentofthedose thatwas excretedinexpired

airorurineafteroral administrationversusthepercentof

the dose excreted after intravenous administration.

Excretionof 14Cinexpired air yieldedthemost consistent results. For oral doses greater than or equal to

2mg/kg,the amountofthedose thatwasabsorbedfitted

the equation: absorbed dose = 6.6 log(dose), with

both doses expressed in mg/kg. While 90% of the

2mg/kg dose was absorbed,only2%ofthe 814mg/kg

dose was absorbed.

Twohoursafterintravenousadministration,lessthan3%

of the circulating

14Cwas attributed to the parent compound. The metabolites of 1-amino2,4-dibromoanthraquinoneinbloodwereprimarilyinthe

plasma fraction (blood:plasma ratioof approximately

0.5:1). The highest concentrations of 14C in tissues

15minutes after intravenous dosingwere in excretory

organs,lung, kidney,smallintestine,liver,adiposetissue,

andadrenalgland. Tissue:blood ratios(TBR)forthese

tissuesweregreaterthan or equalto 3:1. Onlythe liver

and kidney had TBRs significantly greater than 1:1 at

72 hours. The terminal half-life of 14C was approximately40hoursinthe liver andapproximately90hours

in the kidney. Adipose tissue contained primarily

unmetabolized1-amino-2,4-dibromoanthraquinone at

24 hours; liver, muscle, and skin contained mostly

metabolitesof1-amino-2,4-dibromoanthraquinone. The

elimination half -life of 1-amino -

2,4-dibromoanthraquinoneinadiposetissuewasapproximately 11 hours.

GENETIC TOXICOLOGY

1-Amino-2,4-dibromoanthraquinone (100 to

10,000:g/plate)wastestedforinductionofgenemutations in four strains of Salmonella typhimurium in a

preincubation protocol with and without

Aroclor 1254-induced male Sprague-Dawley rat or

Syrian hamster liver S9 (Table G1; Haworth et al.,

1983). 1-Amino-2,4-dibromoanthraquinone was
Results 79

positive in the absence of S9 in the frameshift

strains TA98 and TA1537; with S9, an equivocal

response was obtained in TA1537, and TA98 was

negative. In TA100, 1-amino-2,4-dibromoanthraquinone gave equivocal responses with and without S9, and all trials were negative with TA1535.

The equivocal calls were the results of positive or

weakly positive responses that were not duplicated

in a second trial. Precipitation of 1-amino2,4-dibromoanthraquinone occurred at concentrations of 100 :g/plate and above, and this may have

been a factor in the nonreproducibility of the

results.

1-Amino-2,4-dibromoanthraquinonewas tested in

two laboratories for induction of sister chromatid

exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells, with and

without Aroclor 1254-induced male SpragueDawley rat liver S9. In the sister chromatid

exchange test, one 

laboratory observed a significant increase in sister

chromatid exchanges only in the absence of S9,

while the second laboratory recorded positive

responses with and without S9 (Table G2; Loveday et al., 1990). This discrepancy cannot be

explained by a difference in dose levels employed

at the two laboratories because the positive responses with S9 were observed at 3, 10, 15, and

30 :g/mL at the second laboratory, whereas

negative trials resulted from testing doses up to

100 :g/mL at the first laboratory. In the chromosomal aberrations test, one laboratory observed a

weakly positive response only in the absence of S9

(Table G3). Another laboratory obtained a positive response in the first trial without S9 but did

not duplicate the positive response in the second

trial, and the overall call without S9 was concluded to be equivocal (Loveday et al., 1990).

Neither laboratory observed an increase in chromosomal aberrations with 1-amin o -

2,4-dibromoanthraquinone in the presence of S9.
80 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

 
PLATE 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; 140x
 

PLATE 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 (arrows) 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-dibromoanthraquinone in feed for 2 years. Note 

the disruption of the muscularis mucosa (M) layer at right with formation 

of irregular-shaped neoplastic glands (arrows), 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; 320x
 

PLATE 9

An hepatoblastoma in the liver of a female B6C3F1 mouse exposed to 

20,000 ppm l-amino-2,4-dibromoanthraquinone 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 B6C3F1 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 B6C3F1 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 CONCLUSIONS

Anthraquinonesrepresentthe largestgroupof naturallyoccurringquinones. Bothnatural andsynthetic

anthraquinoneshave been and continue to bewidely

usedas colorantsinfood,drugs,cosmetics,hairdyes,

andtextiles. Dantron(1,8-dihydroxyanthraquinone)

andemodin(1,3,8-trihydroxy-6-methylanthraquinone)

are also usedtherapeuticallyas cathartics and purgatives. Chrysophanol (1,8-dihydroxy3-methylanthraquinone)occursin cascara sagrada,

senna, and various species of Rumex and Rheum

(rhubarb).

Anthraquinoneand five substitutedanthraquinones

wereselected fortoxicologiccharacterizationfrom a

large group of amino-, alkyl-, nitro-, or halogencontaining anthraquinones. The basisforselection

centeredmainlyon four criteria: 1)lackof available

dataoncarcinogenicity, 2)magnitudeof production

andusepatterns,3) awareness of potential andactual

humanexposure,4) and representationof as broad a

spectrum of structural diversity within this class as

possible. 1-Amino-2,4-dibromoanthraquinonewas

selected froma groupof 36environmentallysignificant aryl bromides. Since all other substituted

anthraquinonechemicalsalreadyevaluatedforlongtermeffectsinducedcarcinogenicresponsesinlaboratoryanimals(NCI,1978a,1978b,1978c;IARC,1982,

1987; NTP, 1986a ), 1-amino-2,4-

dibromoanthraquinonewas predicted tobe carcinogenic in laboratory animals as well (Fung et al.,

1993).

1-Amino-2,4-dibromoanthraquinonewasstudiedfor

long-termtoxicityand carcinogenesis usinga "startstop" experimentaldesign. Oneofthe first chemicals

to be studied by the National Toxicology Program

(NTP) with a start-stop protocol, 1-amino2,4-dibromoanthraquinonewaspredictedtobe carcinogenic,sothe experimentaldesign wasselectedinan

attempt to gain some insight into the progression

and/orregressionof chemical-inducedlesions aswell

asto perhapsgainknowledge aboutpotentialmechanisms of action.

Because1-amino-2,4-dibromoanthraquinone caused

significantcarcinogenic responsesinmaleandfemale

rats and mice and in several organs and tissues, the

discussionoflesionsthatfollows hasbeengroupedby

organ.

Liver: 1-Amino-2,4-dibromoanthraquinone differs

fromother chemicalsstudied bythe NTP because it

induced greater than 90% incidences of multiple

benignandmalignanthepatocellularneoplasmsinrats

withfrequentmetastases(almost50%)ofthemalignant liver neoplasms. Although other chemicals

including 3,3N-dimethylbenzidinedihydrochloride

(NTP, 1991) and furan (NTP, 1993a) have caused

significant increases in benign and malignant liver

neoplasmsthat approacha100%incidence,metastases

occurred in only one or two instances in a group of

50 rats. Only in the 18-month study of methyl

carbamate(NTP,1988)hasa similarlyhighincidence

ofmalignant,metastatic liver neoplasmsoccurredin

male rats.

The liverlesionspresent after13weeksinthe25,000

and50,000ppmgroups ofratsincludedproliferative

bile duct lesions (cholangiofibrosis) and foci of

hepatocellular alteration. Based on morphological

features of this proliferative bile duct lesion and

resultsoftransplantationandstop-exposure studies,

cholangiofibrosis has been considered a

"premalignant" lesion which is autonomous and

progressive and not qualitatively different from

cholangiocarcinoma (Maronpot etal., 1991). While

thereissome disagreementonthebiologicalbehavior

of cholangiofibrosis,thishas generallybeenconsidered to be a preneoplastic lesion (Bannasch and

Massner, 1976; Ohshima et al., 1984).

Cholangiofibrosis has been described in toxicity

studies of methapyrilene (Ohshima et al., 1984),

aflatoxin (Wilson et al., 1985), and furan (NTP,

1993a) in rats. 

Exposure concentrations of 1-amino2,4-dibromoanthraquinone administered in the

2-year study were below those which produced

cholangiofibrosis at 13 weeks; however,manyof

the benign and malignant liver neoplasms in the

2-year study in rats were composed of a mixed

growth pattern of both hepatocytes and bile duct

formation. The incidences of foci
82 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

of hepatocellular alteration were increased with

1-amino-2,4-dibromoanthraquinone exposure at

13 weeks and all scheduled intervals examined

during the 2-year study. A detailed analysis of foci

of hepatocellular alteration in the liver of rats from

thisstudyhas been reported byHarada et al.(1989).

In addition to overall exposure-related increased

incidences of eosinophilic and clear cell foci of

alteration,there were increasesin size,number, and

volume fractionof atypicaleosinophilic, basophilic,

and clear cellfociinratsthat correlatedwithconcentration and d ur ation o f 1-amino2,4-dibromoanthraquinone exposure.

Although foci of hepatocellular alteration in rats are

believed to be precursors of liver neoplasms, their

biological nature and potential for progression to

neoplasms is uncertain (Popp and Goldsworthy,

1989;Squire,1989). Some ofthisuncertaintyresults

from a considerable variation in phenotypes of

hepatocellularfoci andthedifferentbiomarkersused

in their classification. Inmany studies, the conversion rateoffocito neoplasmshasbeenconsidered to

be extremelylow, and,in some instances, increases

in the incidences of basophilic foci have not been

associatedwithliverneoplasms(MacDonald et al.,

1988;Harada et al., 1989; Squire, 1989). Clear and

acidophilic cell foci have been suggested to be

important in the development of some chemicalinduced liver neoplasms (Bannasch et al., 1989;

Bannasch and Zerban, 1992). The atypical

eosinophilic foci that occurred in rats administered

1-amino-2,4-dibromoanthraquinone were rarely

observed in controls orin groups ofratsreceiving

other hepatocarcinogens (Harada et al., 1989).

Adenomasin the liversofratstreatedwith1-amino2,4-dibromoanthraquinone often contained cells

morphologically identical to those in the atypical

eosinophilic foci,suggestingthatsome ofthese foci

mayhave beenprecursorsforthehepaticneoplasms.

Liver effectsinmalemice administered 1-amino2,4-dibromoanthraquinonefor13weeks consistedof

pigmentationandhypertrophythatpersistedthroughoutthe 2-yearstudy. Although hypertrophydidnot

occurin female mice during this early period, pigmentation in the liver was present by 15 months.

Foci of hepatocellular alterationwerenot presentin

the 13-weekstudy, and after 2 years, the incidences

were only slightlyincreased inmice. After 2 years,

therewereincreasedincidencesofliverneoplasmsin

all groups of mice exposed to 1-amino2,4-dibromoanthraquinone in feed. This response

was more prominent in females that also had a

greater number of hepatocellular carcinomas and

more multiple liver neoplasms than male mice.

Unlike the highly metastatic liver neoplasms observed in rats, only a few neoplasms in mice had

detectable metastatic foci. The incidences of

hepatoblastomaswere alsoincreasedinthe exposed

groups of male and female mice. These distinctive

liver neoplasms rarelyoccur in control animals but

have been induced in mice administered

acetylaminofluorene (Nonoyama et al., 1988) or

N-nitrosodiethylamine (Ward et al., 1983).

Large Intestine: Adenomatous polyps(adenomas)

and carcinomas of the large intestine (distal colon

and rectum) in rats were generally observed after

15 months of exposure to 1-amino2,4-dibromoanthraquinone, although one adenomatous polyp (adenoma) was observed as early as 9

monthsin the 20,000 ppm group of male rats in the

"stop-exposure" study. Further, these lesions were

often grossly visible. Even when exposure was

stoppedafter 9months,the percentage of chemicalinducedrectalneoplasms was equaltoorgreaterthan

that observed with continuous exposure for 15

months. Inmanyrats,theseneoplasmsweremultiple

and malignant, based upon local invasion and/or

metastases. Neoplasms of the large intestine have

not been observed for other previously tested

anthraquinones. One other chemical studied bythe

NTP,bromodichloromethane(NTP,1987a),resulted

in similarlyhigh incidencesofbenign andmalignant

neoplasms of the large intestine.

Kidney: Accumulationofpigmentin the kidneywas

observed in both male and female rats by 13 weeks

and throughout the 2-year study. 1-Amino2,4-dibromoanthraquinone (ormetabolite) pigment

in the kidney of mice was not evident until after

the 15-month evaluation; there were no increased

incidencesofothernonneoplasticlesionsorneoplasms

of the kidney in mice. In the kidney of rats, several

changes in addition to pigment were present at 13

weeks. In male rats, increased accumulation of

hyaline droplets was observed in the cytoplasm of

the renaltubuleepithelium, yetno chemical-related

exacerbationofrenaltubuleepithelial regeneration

was observed at 13 weeks. There was a slight

enlargement(karyomegaly)ofsome nucleiinthe renal

tubule epithelium of male and female rats. At
Discussion and Conclusions 83

the 15-monthevaluation,thisslightnuclear enlargement was still evident, and the severity of

nephropathy(tubuleepithelialregeneration;transitional cell hyperplasia of the renal pelvis) was

increasedinexposedmaleandfemale rats. Accumulationofhyalinedropletswas not presentinexposed

malerats afterthe9-monthevaluation. Themorphological appearance of hyaline droplets and their

presenceonlyinmalesissuggestiveof accumulation

of "2:-globulin in the renal tubule epithelium, although the identity ofthe proteindroplets was not

determined. Theirabsence inthekidneytubulecells

of exposedmalerats afterthe 9-month evaluation is

consistentwiththenormallydecreasedproductionof

"2:-globulinby the liver beginning at 5 months of

age (Baetcke et al., 1991). Chemicalsthat cause a

hyaline droplet nephropathy syndrome are often

empirically associated with increasesin the incidences of benign and malignant renal tubule

neoplasms,linearfoci ofmineralizationofthe renal

medulla,andenhancednephropathyinmaleratsafter

2 years (Baetcke et al., 1991);however,other alternative mechanistic explanations exist that do not

show a dominant role for "2:-globulin. The key to

thisviewcenterson severalchemicalsthatincitethe

"hyalinedropletsyndrome," yetdonotinduce tubule

cellneoplasms ofthekidney(BarrettandHuff,1991;

Huff,1992, 1993; Melnick, 1992). Another strong

neoplastic response in the kidney of female rats

showsthatmechanisms other than those associated

with hyaline droplet nephropathy were involved in

the renaltubuleneoplasmresponse in rats administered1-amino-2,4-dibromoanthraquinone. Increased

incidencesofbenign andmalignantneoplasms ofthe

kidney occurred inmaleand female rats in the NTP

study of bromodichloromethane (NTP, 1987a).

Urinary Bladder: Chemical-related increased

incidencesof proliferative lesions (hyperplasia and

neoplasia) of the transitional cell epithelium of the

urinarybladderoccurredinmaleandfemale ratswith

a greater number of neoplasms observed in female

rats(45/146,31%)than in male rats (16/146, 11%).

In the stop-exposure groups,transitional cellhyperplasiawas present in four female rats at 9 months,

and, with the absence of continued chemical exposure, hyperplasia did not develop in male rats atthe

15-monthevaluation. Atransitional cell carcinoma

occurred in one female ratfromthe 15-monthexposure group. With continuous exposure to 1-amino2,4-dibromoanthraquinone, benign and malignant

neoplasms of the urinary bladder

developed by 15monthsin female rats and in both

male and female rats after 2 years of exposure. In

rats following chronic administration of

1,4,5,8-tetraaminoanthraquinone, a spectrum of

nonneoplastic lesions and neoplasms ofthe urinary

bladder was observed with similar morphologic

featuresincludingsquamousmetaplasia,squamous

cell carcinoma, and proliferation of fat (fatty

metaplasia)in thewallof the urinarybladder(NTP,

1986a). In that study, calculi were present in the

urinarybladder ofmostrats, yet there was a significant increase in the incidence of smooth muscle

neoplasms ofthe wall of the urinarybladder. The

hypothesis of cell proliferation and development of

urinary bladder neoplasms has been described

(Greenfield et al., 1984; Cohen et al., 1991). The

mechanismforformationofneoplasmsoftheurinary

bladder is uncertain. Increased cell proliferation

evidenced by transitional cell hyperplasia in the

urinarybladderdidnot occurbefore9monthsinrats.

Potential local irritant effects and associated neoplasmformationin the urinary bladder attributed to

calculusformation (Okumara et al., 1992)werenot

identified in thisstudy. Mostmice in that study had

calculi of the urinary bladder, yet did not have any

evidence of carcinogenic activity. No scientific

consensus existsthat endorsesthenotionthat calculi

or stones cause cancer; there may be some cocarcinogenicorpromotionactivity, yet eventhisdoes

not occur consistently (Huff, 1992, 1993).

Forestomach: In both rats and mice, there were

severalnonneoplasticproliferative andinflammatory

lesionsin the forestomach at the end of the 2-year

studies. These forestomach lesions were not observedin either species in the 13-week studies or in

rats at the 9- and 15-monthevaluations. In the stopexposure evaluation,rats exposed to 20,000 ppm

developednonneoplasticlesions ofthe forestomach

by 15 months. However, in the 9-month stopexposure group,forestomachlesionswereobserved

in a few males by the 15-month evaluation, but the

incidenceswere higher than those observedinmale

rats with the continuous 15-month exposure. In

female rats, forestomach lesions were present at

15 months with continuous exposure but were not

observed at 9 months or after 6 months of

nonexposure. Chemical-related lesions consistedof

hyperplasia,hyperkeratosisandassociatedinflammation, and focal erosionorulcerationofthe squamous

mucosa. The inflammatory and ulcerative lesions

were generallymore severe and more common in
84 1-Amino-2,4-dibromoanthraquinone, NTP TR 383

rats, butsignificant increasesin the incidences of

benign andmalignantforestomachneoplasmswere

limitedtomice. These dataprovide further evidence

that inflammation or ulceration does not always

resultin neoplasia (Berenblum, 1944; Huff, 1992,

1993;Melnicketal.,1993a,1993b). The absenceof

forestomachneoplasmsinratsmayhavebeenrelated

to lower exposure concentrations. Many of the

malignant forestomach neoplasms of mice

metastasized or invaded adjacent organs. 

Increasesinthe incidencesofforestomachneoplasms

have not been observed in mice or rats following

long-term administration of four other structurally

related anthraquinones. However,administration of

1-amino-2-methylanthraquinone(NCI, 1978b)torats

for 62 weeksfollowed by a 6-month nonexposure

periodwas associatedwithanincreasedincidence in

hyperplasia of the forestomach. Administration of

2-methyl-1-nitroanthraquinone (NCI, 1978c)torats

for 78 weeksfollowed by a 6-month nonexposure

period was also associated with an increased incidence of proliferative lesions ofthe forestomach.

Marked increases in the incidences of forestomach

neoplasms have been reported for some chemicals

that caused a sustainedproliferative response in the

squamousmucosathatwas evidentwithinthe first 2

to 13 weeks of chemical administration (NTP,

1987b). However, a number of other chemicals

causingforestomachneoplasms in rodentshave not

been associated with an early,sustainedincrease in

the incidence of hyperplasia (NTP, 1990a, 1990b,

1990c).

Lung: The incidences of alveolar/bronchiolar

adenoma andmultiplealveolar/bronchiolar adenoma

(malesonly) ofthe lungwere significantlyincreased

in mice in the 10,000 and 20,000 ppm groups.

Althoughtherewasnoevidencefor anincrease inthe

incidence of hyperplasia or for a progression of the

lung neoplasms to malignancy, the incidence of

adenoma in all four exposuregroups exceeded the

NTP historical control ranges. Administration of a

structurally related anthraquinone, C.I. Disperse

Blue 1 (1,4,5,8-tetraaminoanthraquinone)(NTP,

1986a), resulted in a marginal increase in the

incidenceof alveolar/bronchiolar adenoma in male

micewithno associatedincrease in the incidence of

alveolar/bronchiolar hyperplasia. Other chemicals

tested by the NTP have also caused increased

incidences of lung neoplasms without increased

incidencesof alveolar/bronchiolarhyperplasia(NTP,

1994),butmore commonly an increase in the incidence of alveolar/bronchiolar hyperplasiaorinflammation is also present with increased incidences of

lung neoplasms (NTP,1986b, 1989, 1990a, 1992).

Although the incidence of alveolar/bronchiolar

carcinoma was not increased in the lungs of mice

administered 1-amino-2,4-dibromoanthraquinone,a

number of chemicals have induced both alveolar/

bronchiolar adenomasandcarcinomas(NTP,1990b,

1990c, 1993b; Huff, 1994).

CONCLUSIONS

Under the conditions of these 2-yearfeed studies,

there was clear evidence of carcinogenic activity*

of 1-amino-2,4-dibromoanthraquinone inmaleand

femaleF344/Nratsbasedonincreasedincidences of

neoplasms in the liver, large intestine, kidney, and

urinary bladder. There was clear evidence of

carcinogenic activity of 1-ami n o -

2,4-dibromoanthraquinone in male and female

B6C3F1 mice based on increased incidences of

neoplasms in the liver, forestomach, and lung.

Exposure of male and female rats to 1-amino2,4-dibromoanthraquinonefor2yearswasassociated

withbasophilic focus (males only), clear cellfocus,

eosinophilic focus, and pigmentation in the liver;

renaltubulehyperplasia, renaltubulepigmentation,

and transitional cellhyperplasiainthekidney; transitional cell hyperplasia,squamousmetaplasia, and

stromal metaplasia (females only) in the urinary

bladder; squamous hyperplasia, hyperkeratosis,

ulceration, and inflammation of the forestomach

mucosa; and seminal vesicle atrophy. Exposure of

male and female mice to 1-amino2,4-dibromoanthraquinonefor2yearswasassociated

withcentrilobularhepatocellularhypertrophy(males

only), basophilic focus, clear cell focus (females

only),eosinophilic focus, and pigmentationin the

liver; pigmentation in the kidney; and hyperplasia,

basal cellhyperplasia,hyperkeratosis,andinflammation of the forestomach mucosa.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 13. A summary of the Technical Reports Review Subcommittee

 comments and the public discussion on this Technical Report appears on page 15.
85

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92 1-Amino-2,4-dibromoanthraquinone, NTP TR 383
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Appendix B: Tissues examined for histopathology in 

mice and rats in the NTP bioassays of ADBAQ 
9/19/02 RoC Background Document for 1-Amino-2,4-dibromoanthraquinone 

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51

Tissues examined for histopathology in mice 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, 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. 

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. 
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