1978 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

NO. 108 

13-WEEK SUBCHRONIC 

TOXICITY STUDIES OF 

DIRECT BLUE 6, DIRECT BLACK 38, 

AND DIRECT BROWN 95 DYES 

NCI-CG-TR-108 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 

13-WEEK SUBCHRONIC TOXICITY STUDIES
 

DIRECT BLUE 6, DIRECT BLACK 38, AND
 

DIRECT BROWN 95 DYES
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
 

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 78-1358
 

13-WEEK SUBCHRONIC TOXICITY STUDIES
 

DIRECT BLUE 6, DIRECT BLACK 38, AND
 

DIRECT BROWN 95 DYES
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD; This report presents the results of 13-week subchronic
 

toxicity studies of direct blue 6, direct black 38, and direct
 

brown 95 dyes conducted for the Carcinogenesis Testing Program,
 

Division of Cancer Cause and Prevention, National Cancer 

Institute (NCI), National Institutes of Health, Bethesda, 

Maryland. These subchronic studies were conducted as part of the 

bioassay protocol to establish concentrations for use in 2-year
 

bioassays of the test chemicals in rats and mice. In view of the
 

results obtained in the 13-week studies, the 2-year bioassays
 

will not be performed.
 

The Carcinogenesis Testing Program is designed to determine
 

whether selected chemicals have the capacity to produce cancer in
 

animals. Negative results, in which the test animals do not have
 

a greater incidence of cancer than control animals, do not
 

necessarily mean that the test chemical is not a carcinogen,
 

inasmuch as the experiments are conducted under a limited set of
 

circumstances. Positive results demonstrate that the test
 

chemical is carcinogenic for animals under the conditions of the
 

test and indicate that exposure to the chemical is a potential
 

risk to man. The actual determination of the risk to man from
 

animal carcinogens requires a wider analysis.
 

CONTRIBUTORS: The 13-week subchronic toxicity studies of direct
 

blue 6, direct black 38, and direct brown 95 dyes were conducted
 

by Battelle Memorial Institute, Columbus Laboratories, Columbus,
 

Ohio, under a subcontract to Tracer Jitco, Inc. , prime contractor
 

for the NCI Carcinogenesis Testing Program.
 

iii
 
The principal investigator for the studies of the direct dyes was
 

Dr. A. C. Peters*. Drs. Peters and J. F. Robens^ were
 

responsible for the selection of the doses administered. Drs.
 

Peters and G. S. Dill* prepared the reports of the methods and
 

results from the laboratory. Mr. M. Hughes^ was responsible for
 

the care of the animals. Histopathologic examinations were
 

performed by Dr. Dill, rat hepatic lesions were reviewed by Dr.
 

J. M. Ward^; the diagnoses included in this report represent
 

their interpretation.
 

Animal pathology tables were compiled at EG&G Mason Research
 

Institute . The statistical analyses were performed by Dr. J. R.
 

Joiner , using methods selected for the bioassay program by Dr.
 

J. J. Gart . Chemicals used in the studies were analyzed at
 

Midwest Research Institute under the direction of Dr. E.
 

Murrill"; stability tests were performed by Mr. J. R. Wagner" and
 

Dr. T. C. Carpenter", and tests for benzidine in the dyes were
 

performed by Ms. J. Huerner". Test diets were analyzed for dye
 

content at Battelle, Columbus Laboratories, by Mr. D. Emmerlingl.
 

Tests for benzidine in the urine were performed at Battelle,
 

Columbus Laboratories, by Dr. A. P. Leber* and tests for methemoglobin in the blood were performed by Ms. S. D. Guthrie*. The
 

results of the analyses were reviewed by Dr. S. S. Olin^.
 

This report was prepared at Tracer Jitco^ under the direction of
 

NCI. Those responsible for the report at Tracer Jitco were Dr.
 

L. A. Campbell, Director of the Bioassay Program; Dr. S. S. Olin,
 

Deputy Director for Science; Dr. J. F. Robens, toxicologist; Dr.
 

R. L. Schueler, pathologist; Dr. G. L. Miller and Mr. W. D.
 

Reichardt, bioscience writers; and Dr. E. W. Gunberg, technical
 

editor, assisted by Ms. Y. E. Presley and Ms. P. J. Graboske.
 

The statistical analysis was reviewed by members of the
 

Mathematical Statistics and Applied Mathematics Section of NCI^:
 

Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, and Dr.
 

Robert E. Tarone.
 

The following other scientists at NCI were responsible for evaluating the studies, interpreting the results, and reporting the
 

findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto, Jr., Dr. J.
 

iv
 
Fielding Douglas, Dr. Dawn G. Goodman, Dr. Richard A. Griesemer,
 

Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Jerrold M. Ward, and
 

Dr. Carrie E. Whitmire.
 

Ifiattelle Memorial Institute, Columbus Laboratories, 505 King
 

Avenue, Columbus, Ohio.
 

2

Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

^Carcinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

-"Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute, National
 

Institutes of Health, Bethesda, Maryland.
 

^Midwest Research Institute, 425 Volker Boulevard, Kansas City,
 

Missouri.
 

SUMMARY
 

Thirteen-week subchronic toxicity studies of direct blue 6,
 

direct black 38, and direct brown 95 dyes were conducted by
 

administering the test chemicals in feed to Fischer 344 rats and
 

B6C3F1 mice.
 

Groups of 10 rats and 10 mice of each sex were administered one
 

of the three dyes at one of five concentrations for 13 weeks and
 

then necropsied, beginning the second day after the end of the
 

dosing period. The concentrations used for the rats were 190,
 

375, 750, 1,500, and 3,000 ppm. The concentrations used for the
 

mice were 750, 1,500, 3,000, 6,000, and 12,500 ppm, -except for
 

the female mice administered direct brown 95 dye, which were
 

given concentrations of 375, 750, 1,500, 3,000, and 6,000 ppm.
 

Matched controls consisted of groups of 10 untreated rats and 10
 

untreated mice of each sex.
 

Mean body weights of the male and female rats administered the
 

two or three highest doses of any one of the test dyes were lower
 

than mean body weights of the corresponding controls, and the
 

depressions in mean body weight were dose related. Mean body
 

weights of the male and female mice administered the highest dose
 

of any one of the test dyes were slightly lower than mean body
 

weights of the corresponding controls; mean body weights of mice
 

administered lower doses were generally unaffected.
 

All male and female rats administered 3,000 ppm of any one of the
 

dyes or 1,500 ppm of direct brown 95 dye died before the end of
 

the studies. One male administered 1,500 ppm direct blue 6 dye,
 

six males administered 1,500 ppm direct black 38 dye, and two
 

males administered 750 ppm direct brown 95 dye also died by the
 

end of the studies. No deaths occurred in any other dosed group
 

or in any control group of rats. All male and female mice
 

administered the test dyes survived to the end of the studies,
 

except for one male whose death was attributed to bacterial
 

infection.
 

vii
 
Benzidine and monoacetyl benzidine were detected in the urine of
 

male and female rats and mice administered the test dyes, but
 

neither compound was detected in the urine of control rats and
 

mice. Determinations of methemoglobin in control and dosed rats
 

showed no differences.
 

In rats, neoplastic lesions occurred only in dosed groups and
 

consisted of hepatocellular carcinomas and neoplastic nodules of
 

the liver. The incidences of hepatocellular carcinomas in female
 

rats administered 3,000 ppm direct blue 6 dye (4/9) and male rats
 

administered 1,500 ppm direct black 38 dye (4/9) were significant
 

(P = 0.033) when related to the incidences of the tumors in the
 

corresponding controls (0/10); hepatocellular carcinomas were
 

also observed in two male rats administered 1,500 ppm direct blue
 

6 dye and in one female rat administered 1,500 ppm direct brown
 

95 dye. No control rats from any of the three studies developed
 

hepatocellular carcinomas.
 

When incidences of neoplastic nodules were combined with those of
 

hepatocellular carcinomas, the significance increased to P <
 

0.001 for male rats administered 1,500 ppm direct blue 6 dye, P =
 

0.001 for females administered 3,000 ppm direct blue 6 dye, P <
 

0.001 for males administered 1,500 ppm direct black 38 dye, and
 

P = 0.007 for females administered 1,500 ppm direct brown 95 dye.
 

No controls developed neoplastic nodules. Female rats administered direct black 38 dye developed no hepatocellular carcinomas,
 

but had an incidence of neoplastic nodules of 5/10, with a
 

significance of P = 0.016. Male, rats administered direct brown
 

95 dye developed neither hepatocellular carcinomas nor neoplastic
 

nodules, but as indicated below, had significant incidences of
 

preneoplastic lesions. The failure of groups of rats administered 3,000 ppm dye to develop tumors when other groups
 

administered 1,500 ppm did develop tumors may be due to earlier
 

deaths at the higher dose.
 

Preneoplastic hepatic lesions (basophilic foci) occurred only in
 

dosed rats and did not occur in the controls. The incidences of
 

the basophilic foci were significant (P _< 0.033) in male (4/9)
 

and female (7/9) rats administered 3,000 ppm direct blue 6 dye
 

and in male rats (7/8) administered 1,500 ppm direct brown 95
 

dye. Basophilic foci also occurred, at lower incidences, in
 

viii
 
males (1/10) administered 1,500 ppm direct blue 6 dye, in males
 

(3/9) administered 1,500 ppm direct black 38 dye, in females
 

(1/8) administered 3,000 ppm direct black 38 dye, in males
 

administered 750 ppm (3/10) or 3,000 ppm (2/9) direct brown 95
 

dye, and in females administered 1,500 ppm (3/8) or 3,000 ppm
 

(3/8) direct brown 95 dye. When incidences of foci of cellular
 

alteration, a possible preneoplastic lesion, were added to those
 

of basophilic foci, significance occurred in additional dosed
 

groups.
 

In mice, no neoplastic lesions occurred in the liver or other
 

tissues of groups administered the different dyes. However,
 

three mice administered 12,500 ppm direct black 38 dye and one
 

mouse administered 12,500 ppm direct brown 95 dye had foci of
 

cellular alteration, in which the cells were basophilic when
 

compared with surrounding normal cells.
 

It is concluded that under the conditions of these 13-week
 

subchronic toxicity studies, direct blue 6 and direct black 38
 

dyes were carcinogenic in male and female Fischer 344 rats and
 

direct brown 95 was carcinogenic in female rats; all three dyes
 

induced hepatocellular carcinomas and neoplastic nodules in the
 

liver. The test dyes were not carcinogenic for B6C3F1 mice in
 

the 13-week subchronic toxicity studies.
 

TABLE OF CONTENTS
 

I. Introduction	 1
 

II. Materials and Methods	 3
 

A. Chemicals	 3
 

B. Dietary Preparation	 4
 

C. Animals	 5
 

D. Animal Maintenance	 6
 

E. Two-Week Toxicity Tests	 7
 

F. Thirteen-Week Subchronic Toxicity Studies 10
 

G. Clinical and Pathologic Examinations	 10
 

H. Data Recording and Statistical Analyses 14
 

III. Results - Rats	 19
 

A. Body Weights and Clinical Signs (Rats)	 19
 

B. Benzidine and Methemaglobin Studies (Rats) 19
 

C. Survival (Rats)	 23
 

D. Pathology (Rats)	 27
 

E. Statistical Analyses of Results (Rats)	 31
 

IV. Results -Mice	 39
 

A. Body Weights and Clinical Signs (Mice)	 39
 

B. Benzidine Studies (Mice)	 39
 

C. Survival (Mice)	 43
 

D. Pathology (Mice)	 43
 

E. Statistical Analyses of Results (Mice)	 45
 

V. Discussion	 47
 

VI.	 Bibliography 55
 

APPENDIXES
 

Appendix A Lesions in Rats Administered Direct
 

Table Al Lesions Observed Only in Rats Administered
 

Dyes in the Diet 59
 

Direct Blue 6 in the Diet 61
 

XI
 
Page 

Table A2 Other Lesions in Rats Administered 

Direct Blue 6 in the Diet 66 

Table A3 Lesions Observed Only in Rats Administered 

Direct Black 38 in the Diet 68 

Table A4 Other Lesions in Rats Administered 

Direct Black 38 in the Diet 73 

Table A5 Lesions Observed Only in Rats Administered 

Direct Brown 95 in the Diet 75 

Table A6 Other Lesions in Rats Administered 

Direct Brown 95 in the Diet 80 

Appendix B Lesions in Mice Administered Direct 

Dyes in the Diet 83 

Table Bl Lesions Observed Only in Mice 

Administered Direct Blue 6 in the Diet 85 

Table B2 Other Lesions in Mice Administered 

Direct Blue 6 in the Diet 86 

Table B3 Lesions Observed Only in Mice 

Administered Direct Black 38 in the Diet... 87 

Table B4 Other Lesions in Mice Administered 

Direct Black 38 in the Diet 88 

Table B5 Lesions Observed Only in Male Mice 

Administered Direct Brown 95 in the Diet... 89 

Table B6 Lesions Observed Only in Female Mice 

Administered Direct Brown 95 in the Diet... 90 

Table B7 Other Lesions in Mice Adminsitered 

Direct Brown 95 in the Diet 91 

Appendix C Analyses of the Incidence of Tumors 

or Foci Alterations in the Liver of 

Rats Fed Direct Dyes in the Diet 93 

xii
 
Table Cl
 

Table C2
 

Table C3
 

Table C4
 

Table C5
 

Table C6
 

Appendix D
 

Table Dl
 

Table D2
 

Appendix E
 

Table 1
 

Table 2
 

Page
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Male Rats
 

Fed Direct Blue 6 in the Diet 95
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Female
 

Rats Fed Direct Blue 6 in the Diet 98
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Male
 

Rats Fed Direct Black 38 in the Diet 101
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Female
 

Rats Fed Direct Black 38 in the Diet 104
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Male
 

Rats Fed Direct Brown 95 in the Diet 106
 

Analyses of the Incidence of Tumors or
 

Foci Alterations in the Liver of Female
 

Rats Fed Direct Brown 95 in the Diet 108
 

Benzidine in the Urine of Rats and
 

Mice Fed Direct Dyes Ill
 

Benzidine Excretion (//g/24 hr) per Rat 118
 

Benzidine Excretion (Aig/24 hr) per Mouse... 119
 

Molecular Structures of Direct Blue 6,
 

Direct Black 38, and Direct Brown 95 121
 

TABLES
 

Thirteen-Week Subchronic Toxicity Studies
 

of Direct Blue 6, Direct Black 38, and
 

Direct Brown 95 Dyes Administered in
 

Feed to Rats 11
 

Thirteen-Week Subchronic Toxicity Studies
 

of Direct Blue 6, Direct Black 38, and
 

Direct Brown 95 Dyes Administered in
 

Feed to Mice 12
 

xiii
 
Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Figure 5
 

Figure 6
 

Figure 7
 

Figure 8
 

Figure 9
 

Figure 10
 

Figure 11
 

Figure 12
 

FIGURES
 

Growth Curves for Rats Administered 

Direct Blue 6 in the Diet 20 

Growth Curves for Rats Administered 

Direct Black 38 in the Diet 21 

Growth Curves for Rats Administered 

Direct Brown 95 in the Diet 22 

Survival Curves for Rats Administered 

Direct Blue 6 in the Diet 24 

Survival Curves for Rats Administered 

Direct Black 38 in the Diet 25 

Survival Curves for Rats Administered 

Direct Brown 95 in the Diet 26 

Hepatic Lesions Observed in Rats 

Administered Direct Blue 6 in the Diet 33 

Hepatic Lesions Observed in Rats 

Administered Direct Black 38 in the Diet... 34
 

Hepatic Lesions Observed in Rats
 

Administered Direct Brown 95 in the Diet... 35
 

Growth Curves for Mice Administered
 

Direct Blue 6 in the Diet 40
 

Growth Curves for Mice Administered
 

Direct Black 38 in the Diet 41
 

Growth Curves for Mice Administered
 

Direct Brown 95 in the Diet 42
 

xiv
 
I. INTRODUCTION
 

Direct blue 6 (CAS 2602-46-2; NCI C54579), direct black 38 (CAS
 

1937-37-7; NCI C54557), and direct brown 95 (CAS 16071-86-6; NCI
 

C54568) are azo dyes used on textiles such as cotton, silk, wool,
 

nylon, and acetate. All three dyes also have commercial use on
 

leather. In addition to use as textile dyes, direct blue 6 and
 

direct black 38 are used in aqueous printing inks and as
 

biological stains, and one or another of these dyes has been used
 

in plastics (direct black 38, direct brown 95), paper (direct
 

blue 6, direct brown 95), wood stains (direct black 38), and wood
 

flour (direct black 38) (Society of Dyers and Colourists, 1971).
 

Two of these dyes, direct blue 6 and direct black 38, have been
 

used in hair dyes (Comptroller General of the U.S., 1977).
 

The United States International Trade Commission (1977a) reports
 

that 70,753 pounds of direct black 38 and 8,205 pounds of direct
 

brown 95 were imported for use in the United States in 1976. In
 

the same year, U.S. manufacturers produced 3,759,000 pounds of
 

direct black 38, and 595,000 pounds of direct brown 95. Similar
 

data are not available for direct blue 6, although its production
 

is believed to be greater than 5,000 pounds annually (USITC,
 

1977a and 1977b).
 

These three dyes were selected for study in the Carcinogenesis
 
Testing Program because they are derived from benzidine, which is
 

known to be carcinogenic in animals and man (IARC, 1972), because
 

large quantities were used industrially, and because of the
 

potential for long-term human exposure both through industrial
 

use and through contact with products containing the dyes.
 
II. MATERIALS AND METHODS
 

A. Chemicals
 

The chemicals used were technical-grade factory-strength (unformulated) dyes, manufactured by GAF Corporation (New York, N.Y.).
 

Direct blue 6 (Phenamin Blue BB-FS) was obtained in one batch
 

(Lot No. 43762), direct black 38 (Phenamin Black E-FS) in one
 

batch (Lot No. 43761), and direct brown 95 (Fastusal Brown
 

LBRS-FS) in one batch (Lot No. 43763). The molecular structures
 

of these test dyes are given in Appendix E and show the
 

occurrence of the benzidine moiety in each structure. The
 

identity and purity of each chemical were determined by analyses
 

at Midwest Research Institute. According to the manufacturer,
 

the purities by dyestuff assay of direct blue 6, direct black 38,
 

and direct brown 95 were 66%, 86%, and 79%, respectively;
 

according to analyses performed at Midwest Research Institute,
 

the corresponding purities by titration of azo groups with
 

titanous chloride were 59.9 + 1.9%, 87.1 + 3.4%, and 72.2 + 7.0%.
 

Elemental analyses (for all elements except oxygen) were
 

reasonably consistent with the molecular formulas of direct blue
 

6 (C32H2oOi4N6Na4S4), direct black 38 (C34H25N907S2Na2), and
 

direct brown 95 (C3iH20N609SNa2*Cu), after correction for the
 

percent dye determined by titanous chloride titration, the water
 

content, and sodium chloride (estimated from analyses for Na and
 
Cl); direct blue 6 and direct brown 95 were somewhat high in C,
 

H, and Na. Water analyses (Karl Fischer) were 9.18 + 0.51%,
 

7.13+ 0.54%, and 4.99 + 0.22%, respectively, and NaCl
 

concentrations were estimated at 20.8%, 7.9%, and 14.9%. The
 

infrared spectra of direct black 38 and direct brown 95 were
 

consistent with those in the literature (Sadtler, 1960); the
 

infrared spectrum for direct blue 6 was not consistent with that
 

in the literature and could not be taken as assurance of
 

identity. Thin-layer chromatographic analyses using two
 

different solvent systems showed 8-15 minor or trace impurities.
 

No attempt was made to identify or quantitate these impurities.
 

High-pressure liquid chromatography showed several small impurity
 

peaks in each of the dyes, but no benzidine (detection limit,
 

0.004%). The methodology used would have detected total amounts
 

of benzidine — that is, both benzidine salts and free benzidine.
 

Mazola® corn oil (Best Foods, Division of CPC International,
 

Inc., Englewood Cliffs, N. J.) was added to the dyes as a dust
 

suppressant. The concentration of corn oil in the dye was 1.3%.
 

Bulk dyes containing the corn oil were stored at 5°C.
 

B. Dietary Preparation
 

A 1-week supply of each diet was formulated 1 or 2 days before
 

use by mixing Purina® Laboratory Chow® animal meal (Ralston
 
Purina Co., St. Louis, Mo.) and dye containing 1.3% corn oil.
 

Weighed amounts of animal meal were combined with weighed amounts
 

of dye containing the corn oil and mixed in a Patterson-Kelly
 

twin shell blender for 15 minutes to assure homogeneity.
 

Formulated diets were stored at 23°C until used. The control
 

diets contained corn oil in amounts equal to that in the highest
 

dose groups for each species; i.e., 39 ppm for rats and 163 ppm
 

for mice. Corn oil was present in the diets containing the dyes
 

at 3 to 39 ppm for rats and 5 to 163 ppm for mice.
 

Stability of diets formulated with 10% of the bulk dyes containing 1.3% corn oil was determined by analyses performed after
 

storage for 2 weeks at -20°, 5°, 25°, or 45°C. Spectrophotometric analysis of extracts of the diets showed that each of
 

the dyes was stable in feed for 2 weeks at all temperatures
 

tested. Analyses for benzidine were not performed.
 

As a quality control test on the accuracy of diet preparations,
 

the concentration of dye in one sample at each dose level was
 

determined for each dye during the studies and verified to be
 

within +_ 10% of the required concentration.
 

C. Animals
 

Fischer 344 rats and B6C3F1 mice of each sex were obtained from
 

Frederick Cancer Research Center, Frederick, Maryland, through
 
contracts with the Division of Cancer Treatment, National Cancer
 

Institute. On arrival at the laboratory, the rats were 4 weeks
 

of age and the mice were 4-5 weeks of age. All animals were
 

quarantined (rats for 12 days, mice for 13 days) prior to the
 

start of the studies. During the quarantine periods, all animals
 

of each species and sex were examined, and several were
 

necropsied to detect observable disease. For the study of each
 

chemical, rats and mice of each sex were randomized into dosed or
 

control groups from the quarantine pool by tables of random
 

numbers, and marked to assure individual identification.
 

D. Animal Maintenance
 

All animals were housed in temperature- and humidity-controlled
 

rooms. The temperature range was 21-23°C, and the relative
 

humidity was maintained at 40-60%. The air in each room was
 

filtered with high-efficiency particulate air (HEPA) filters and
 

changed 20-25 times per hour. Fluorescent light provided 12
 

hours of illumination per day. Tap water was available ad
 

libitum, and diets were replenished as necessary, usually at
 

2-day intervals. Fresh control and test diets were provided
 

every week.
 

Both rats and mice were housed five per cage in solid polycarbonate cages (Lab Products, Inc., Garfield, N.J.) suspended from
 
stainless steel racks. Rack shelves were covered with spun-


bonded polyester filters (Dupont 2024). Absorb-Dri® hardwood
 

chip bedding (Lab Products, Inc., Garfield, N.J.) was used for
 

all cages and was changed two times per week. All rat and mouse
 

cages were changed two times per week and were mechanically
 

washed at temperatures not less than 82°C using Exceed® detergent
 

(Economics Labs, Inc., Osborn Building, St. Paul, Minn.). All
 

feed hoppers were changed once per week. Automatic watering
 

systems provided water for both the rats and the mice.
 

All rats were housed in one room, and the mice were housed in
 

another room. No animals administered any other test compounds
 

were housed in these rooms. Neither cage positions within the
 

racks nor rack positions within the rooms were rotated.
 

E. Two-Week Toxicity Tests
 

Two-week toxicity tests were conducted with Fischer 344 rats and
 

B6C3F1 mice to estimate the toxicity of each of the test dyes; on
 

the basis of these tests various concentrations were selected for
 

use in 13-week studies. In the 2-week tests, the dyes were
 

administered in the feed at concentrations of 3,000, 6,000,
 

12,500, 25,000, and 50,000 ppm. Five males and five females of
 

each species were administered each dose, and five males and five
 

females of each species were given basal diets. After the
 
administration of the dyes for 2 weeks, all animals were killed
 

and necropsied.
 

Rats administered the dyes had severe dose-related decreases in
 

food consumption and dose-related depressions in mean body
 

weight. The effects appeared even at the lowest concentrations
 

administered. In rats administered direct blue 6 dye, one male
 

given 50,000 ppm died on day 6; in rats administered direct black
 

38 dye, all males given 50,000 ppm except one and all females
 

given 50,000 ppm except one died by day 9; and in rats
 

administered direct brown 95 dye, one male given 50,000 ppm died
 

on day 12 and all females given 12,500 ppm died by day 11. All
 

other animals survived to the end of the tests. Gross
 

observations of rats administered the different dyes included
 

thymic atrophy, splenic enlargement, and darkening of the spleen
 

and kidneys. In rats administered the direct black 38,
 

pigmentation of the liver also was noted. The pigmentation of
 

the spleen and kidneys was dose related; the thymic atrophy was
 

attributed to the low consumption of food. Methemoglobin was
 

measured in four animals from tests using each of the dyes and
 

was found to be elevated.
 

Mice administered the dyes had depressions in mean body weights
 

in all groups except the males administered direct blue 6 dye.
 

The effects were generally dose related and extended in most
 

8
 
cases to all but the lowest doses administered. In mice
 

administered direct blue 6 or direct brown 95 dyes, no deaths
 

occurred; in mice administered direct black 38 dye, three males
 

given 25,000 ppm died by day 10 and two females given 50,000 ppm
 

died by day 12. All other animals survived to the end of the
 

tests. Hunched appearance and lethargic body movement were noted
 

in mice at the higher concentrations. Gross observations of mice
 

administered the different dyes consisted primarily of
 

pigmentation of spleen and kidneys in mice at the higher doses of
 

the dyes, related directly to the dye. The brown-colored viscera
 

and blood of mice administered direct black 38 dye was attributed
 

to methemoglobin, although tests for concentration of methemoglobin in the blood were not performed for this species.
 

Concentrations for the 13-week subchronic toxicity studies were
 

selected mainly on the basis of the effects of the dyes on mean
 

body weight. Because of generally excessive weight losses in the
 

male and female rats at 6,000 ppm or higher, the concentrations
 

set for the rats were 190, 375, 750, 1,500, and 3,000 ppm;
 

similarly, because of generally excessive weight losses in the
 

male and female mice at 25,000 ppm or higher, the concentrations
 

set for the mice were 750, 1,500, 3,000, 6,000, and 12,500 ppm,
 

except for the females administered the direct brown 95 dye, for
 

which the highest concentration was set at 6,000 ppm.
 
F. Thirteen-Week Subchronic Toxicity Studies
 

The test groups, doses administered, and times on study of the
 

13-week subchronic toxicity studies are shown in tables 1 and 2.
 

These studies were conducted as a part of the bioassay protocol
 

to establish concentrations for use in the 2-year bioassays of
 

the test chemicals in both rats and mice.
 

G. Clinical and Pathologic Examinations
 

Inspections for mortality and morbidity were carried out twice
 

daily. Clinical observations were recorded daily. Body weights
 

of individual animals were determined weekly. Tests for
 

benzidine in the urine were performed at weeks 4 and 12 of the
 

studies for the rats and at weeks 3 and 11 for the mice. Tests
 

for methemoglobin of the rats were performed at the end of the
 

studies.
 

Moribund animals and those animals that survived to the end of
 

the studies were killed using C02 anesthesia and necropsied.
 

Necropsies were also performed on all animals found dead, except
 

one that was cannibalized. The tissues were preserved in 10%
 

buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin. The following tissues were examined
 

microscopically in all control rats and in rats administered
 

1,500 or 3,000 ppm of each dye and 750 ppm of direct brown 95
 

10
 
Table 1. Thirteen-Week Subchronic Toxicity Studies of
 

Direct Blue 6, Direct Black 38,and Direct Brown 95 Dyes
 

Administered in Feed to Rats
 

Sex and
 

Test
 

Group3
 

Male
 

Matched-Controld
 

190 ppm
 10 91
 2
 

375 ppm
 10 91
 2
 

750 ppm
 10 91
 2
 

1,500 ppm
 10 91
 2
 

3,000 ppm
 10 91
 2
 

Female
 

Matched-Controld
 

190 ppm
 10 91
 2
 

375 ppm
 10 91
 2
 

750 ppm
 10 91
 2
 

1,500 ppm
 10 91
 2
 

3,000 ppm
 10 91
 2
 

Initial Time
 

No. of Dosed
 

Animals^ (days)
 

10
 

10
 

on Study
 

Observed
 

(days)c
 

93
 

93
 

a

Each dye was mixed with animal meal to give the concentrations
 

indicated. Corn oil was also present in the various dosed diets,
 

with concentrations ranging from 3 to 39 ppm.
 

^Male and female rats were 6 weeks of age when placed on study.
 

c

Surviving animals were necropsied beginning the second day
 

after the end of the dosing period.
 

"Matched-control rats were fed animal meal containing 39 ppm
 

corn oil.
 

11
 
Table 2. Thirteen-Week Subchronic Toxicity Studies of
 

Direct Blue 6, Direct Black 38, and Direct Brown 95 Dyes
 

Administered in Feed to Mice
 

Sex and Initial Time on Study
 

Test No. of Dosed Observed
 

Group3 Animals" (days) (days)c
 

Male
 

Matched-Controld 10 93
 

750 ppm 10 91 2
 

1,500 ppm 10 91 2
 

3,000 ppm 10 91 2
 

6,000 ppm 10 91 2
 

12,500 ppm 10 91 2
 

Female
 

Matched-Controld 10 93
 

750 ppm 10 91 2
 

1,500 ppm 10 91 2
 

3,000 ppm 10 91 2
 

6,000 ppm 10 91 2
 

12,500 ppm 10 91 2
 

a

Each dye was mixed with animal meal to give the concentration
 

indicated. The concentrations given the female mice administered
 

diet containing direct brown 95 dye were 375 to 6,000 ppm instead
 

of the concentrations indicated. Corn oil was also present in the
 

various dosed diets, with concentrations ranging from 5 to 163
 

ppm.
 

"Male and female mice were 6-7 weeks of age when placed on study.
 

12
 
Table 2. Thirteen-Week Subchronic Toxicity Studies of
 

Direct Blue 6, Direct Black 38, and Direct Brown Dyes
 

Administered in Feed to Mice
 

c

Surviving animals were necropsied beginning the second day after
 

the end of the dosing period.
 

"Matched-control mice were fed animal meal containing 163 ppm
 

corn oil.
 

13
 
dye: skin, lung, bone marrow, spleen, mandibular lymph node,
 

mesenteric lymph node, thymus, heart, salivary gland, liver,
 

pancreas, stomach, small intestine, colon, kidney, bladder,
 

adrenals, thyroids, testes, and epididymis. Microscopic
 

examination was also performed on the above tissues (plus bile
 

duct) of control mice, males and females administered 12,500 ppm
 

of each dye, females administered 6,000 ppm of direct brown 95
 

dye, and a male mouse that was administered 750 ppm of direct
 

brown 95 dye and that died early. In addition, certain tissues
 

were examined in rats and mice administered lower doses, as
 

indicated in Appendix A, tables A1-A6 and Appendix B, tables
 

B1-B7.
 

A few tissues from some animals were not examined. Thus, the
 

number of animals from which particular organs or tissues were
 

examined microscopically varies, and does not necessarily
 

represent the number of animals that were placed on study in each
 

group.
 

H. Data Recording and Statistical Analyses
 

Pertinent data on this experiment have been recorded in an automatic data processing system, the Carcinogenesis Bioassay Data
 

System (Linhart et al. , 1974). The data elements include descriptive information on the chemicals, animals, experimental design,
 

14
 
clinical observations, survival, body weight, and individual
 

pathologic results, as recommended by the International Union
 

Against Cancer (Berenblum, 1969). Data tables were generated for
 

verification of data transcription and for statistical review.
 

Data on the incidences of neoplastic and nonneoplastic lesions
 

were analyzed using the statistical techniques described in this
 

section. Those analyses of the experimental results that bear on
 

the possibility of carcinogenicity are discussed in the
 

statistical narrative sections.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

given as the ratio of the number of animals bearing such lesions
 

at a specific anatomic site (numerator) to the number of animals
 

in which that site is examined (denominator). In most instances,
 

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic examination was required' to detect lesions prior to histologic sampling
 

(e.g., skin or mammary tumors), or when lesions could have
 

appeared at multiple sites (e.g., lymphomas), the denominators
 

consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

15
 
animals. As a part of these analyses, the one-tailed Fisher
 

exact test (Cox, 1970) was used to compare the tumor incidence of
 

a control group with that of a group of dosed animals at each
 

dose level. When results for a number of dosed groups (k) are
 

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

made. The Bonferroni inequality (Miller, 1966) requires that the
 

P value for any comparison be less than or equal to 0.05/k. In
 

cases where this correction was used, it is discussed in the
 

narrative section.. It is not, however, presented in the tables,
 

where the Fisher exact P values are shown.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared to its control was calculated
 

from the exact interval on the odds ratio (Gart, 1971). The
 

relative risk is defined as p^/pc where pt is the true binomial
 

probability of the incidence of a specific type of tumor in a
 

dosed group of animals and pc is the true probability of the
 

spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

a specific tumor in a dosed group and the proportion in a control
 

group corresponds to a relative risk of unity. Values in excess
 

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

16
 
The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

approximately 95% of a large number of identical experiments, the
 

true ratio of the risk in a dosed group of animals to that in a
 

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P < 0.025 one-tailed test when the control
 

incidence is not zero, P < 0.050 when the control incidence is
 

zero) has occurred. When the lower limit is less than unity, but
 

the upper limit is greater than unity, the lower limit indicates
 

the absence of a significant result while the upper limit
 

indicates that there is a theoretical possibility of the
 

induction of tumors by the test chemical, which could not be
 

detected under the conditions of this test.
 

17
 

III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of the male and female rats dosed with the two
 

or three highest doses of any one of the test dyes were lower
 

than mean body weights of the corresponding controls, and the
 

depressions in mean body weight were dose related (figures 1, 2,
 

and 3). No other clinical signs related to administration of the
 

dyes were reported.
 

B. Benzidine and Methemoglobin Studies (Rats)
 

Urine collected over a 24-hour period during weeks 4 and 12 of
 

the subchronic toxicity studies from male and female rats
 

receiving each of three respective test dyes contained benzidine
 

and monoacetyl benzidine, while specimens of urine taken from
 

corresponding controls contained neither compound. The benzidine
 

and monoacetyl benzidine were identified by thin-layer
 

chromatography and mass spectroscopy. Quantities excreted in the
 

urine were determined by combined extraction and spectrometric
 

procedures. In most tests, the amounts excreted were dose
 

related for each of the dyes administered. No consistent
 

differences in results were found between males and females.
 

Details of the methods and results are given in Appendix D.
 

19
 
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a 190 ppm 

• CONTROLS 

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A 3 000 ppm 

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Figure 1. Growth Curves for Rats Administered Direct Blue 6 in the Diet 

20 
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• CONTROLS 0­

36 43 50 57 

TIME ON STUDY (DAYS) 

Figure 3. Growth Curves for Rats Administered Direct Brown 95 in the Diet 

22 
Concentrations of methemoglobin (Evelyn and Malloy, 1938) were
 

measured because methemoglobin was elevated in selected rats at
 

the higher dose concentration in the 2-week toxicity test.
 

However, the dye concentrations administered in the 13-week
 

studies were much lower, and determinations of methemoglobin in
 

rats administered various doses of each of the dyes were not
 

different from those of control rats.
 

C. Survival (Rats)
 

Curves of survival of control rats and of rats dosed with each of
 

the test dyes are shown in figures 4, 5, and 6. All male and
 

female rats administered 3,000 ppm of any one of the dyes and all
 

male and female rats administered 1,500 ppm direct brown 95 dye
 

died before the termination of the studies. One male
 

administered 1,500 ppm direct blue 6 dye, six males administered
 

1,500 ppm direct black 38 dye, and two males administered 750 ppm
 

direct brown 95 dye also died before the termination of the
 

studies. No deaths occurred in any other dosed group or in any
 

control group. The mortality was dose related, and, based on
 

times and incidences of deaths, direct brown 95 dye was most
 

toxic, followed in order by direct black 38 dye, then direct blue
 

6 dye.
 

23
 
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D. Pathology (Rats)
 

Gross Lesions. Gross lesions that were related to administration
 

of any one of the dyes varied, depending on the dose and the
 

length of time the animals survived. Livers of the rats given
 

the highest doses and dying first (generally the males) were
 

pale, yellow, or tan; these animals had ascites, hydrothorax, and
 

edema of subcutaneous tissues and intestinal submucosa. Rats
 

surviving longer but not to the end of the studies had, in
 

addition, roughened surfaces on their livers. Rats given the
 

highest doses at which survival was complete had livers with more
 

severely roughened surfaces, due to random, multiple, pale, 2- to
 

3-mm spherical nodules that were scattered throughout the hepatic
 

parenchyma and that elevated the capsule.
 

Histopathologic Lesions. Histopathologic lesions observed in
 

control and dosed rats are summarized in Appendix A. Tables Al,
 

A3, and A5 list those lesions that were observed only in rats
 

administered dye in the diet; tables A2, A4, and A6 list other
 

lesions. The histopathologic lesions varied, depending largely
 

on the time of death. The terms "basophilic foci," "foci of
 

cellular alteration," "neoplastic nodule," "hepatocellular
 

carcinoma," and "cholangiofibrosis," applying to lesions of the
 

liver, are used as defined by Squire and Levitt (1975). The term
 

"basophilic foci" is used separately from the term "foci of
 

27
 
cellular alteration," because of the possible greater
 

significance of the basophilic lesion. The term "nodular
 

regeneration" refers to lobules containing hepatocytes that
 

appear (1) to be normal in arrangement, size, shape, and
 

tinctorial quality, but often lacking in central veins or portal
 

areas or both, (2) to be pushing against adjacent areas, and (3)
 

to be larger than normal lobules. They are delineated by focal
 

biliary hyperplasia and fibrosis.
 

The first animals to die during administration of each of the
 

dyes had varying degrees of biliary hyperplasia, lymphoid
 

depletion of the spleen and the thymus, and myeloid depletion of
 

the bone marrow. Animals that survived longer had more numerous
 

proliferative changes of the liver, including biliary hyperplasia, cholangiofibrosis, nodular regeneration, foci of cellular
 

alteration, neoplastic nodules, and hepatocellular carcinomas.
 

Some of the rats had histopathologic evidence of bacterial
 

septicemia just prior to death.
 

The most severely affected livers were usually of one of two
 

types: (1) a liver with severe oval cell (biliary, cholangiolar)
 

hyperplasia, multiple foci of cellular alteration, and nodules or
 

(2) a liver with cirrhosis and nodules. In the first type (oval
 

cell), hyperplasia started as a mild increase in periportal oval
 

cells and progressed to large numbers of these cells,
 

28
 
proliferating along tne sinusoids in such a fashion as to almost
 

obscure the hepatocytes throughout the lobule. The initial
 

proliferative hepatocellular lesions seen were multiple foci
 

consisting of 10 to 20 or more cells that were larger and much
 

more basophilic than normal hepatocytes. These cells also had
 

larger, more vesicular nuclei, and some mitotic figures were
 

seen. The basophilic foci appeared to progress to neoplastic
 

nodules with basophilic hepatocytes, compressing adjacent
 

parenchyma. The larger nodules with foci of prominent trabecular
 

formation and acini were diagnosed as hepatocellular carcinomas.
 

At least one carcinoma invaded the wall of a vein. None
 

metastasized. The other foci of cellular alteration of
 

hepatocytes included cells with clear cytoplasm, with cytoplasm
 

containing eosinophilic droplets, or with cytoplasm having an
 

eosinophilic, ground-glass appearance.
 

The second type of severely affected liver occurred in those rats
 

that survived to the end of the studies after receiving the
 

highest dose. These rats had cirrhosis (nodular regeneration and
 

biliary hyperplasia, focal) characterized by multifocal, roughly
 

spherical, nodular aggregations of hepatocytes; the hepatocytes
 

generally appeared normal, although the lobules sometimes lacked
 

central veins. Oval cells and connective tissue separated the
 

regenerative nodules from each other. These livers contained
 

29
 
neoplastic nodules composed of large hepatocytes with
 

eosinophilic cytoplasm. Hepatocellular carcinomas were diagnosed
 

when neoplastic nodules contained foci of basophilic hepatocytes
 

forming prominent trabeculae. Bizarre mitotic figures were
 

occasionally noted. Cholangiofibrosis was commonly seen in
 

livers of rats with neoplastic nodules and hepatocellular
 

carcinomas. The severity of the proliferative changes of the
 

liver decreased as the doses decreased; rats administered 190 or
 

375 ppm direct blue 6 dye and rats administered 190 ppm direct
 

black 38 dye had essentially normal livers.
 

Lesions of the spleen, thymus, and bone marrow were characterized
 

by a marked decrease in the number of mature lymphocytes in the
 

white pulp of the spleen and in the cortex of the thymus and of
 

the myeloid elements in the bone marrow.
 

Those rats receiving 1,500 ppm direct black 38 dye or 750 ppm
 

direct brown 95 dye 'and surviving to the end of the studies had
 

subacute glomerulonephropathy characterized by an eosinophilic
 

amorphous material in Bowman's space and in the lumen of adjacent
 

tubules. Some affected glomeruli had parietal epithelial cells
 

in Bowman's capsule. There was some thickening of glomerular
 

basement membranes. These kidney lesions were not observed in
 

the rats administered direct blue 6 dye.
 

30
 
All females administered 375 ppm direct brown 95 dye had a
 

degenerative change in pancreatic acinar epithelial cells.
 

Individual cells had separated from the basement membrane and
 

were rounded, with pyknotic nuclei.
 

Other lesions were considered incidental and not related to
 

administration of the test dyes.
 

Based on the histopathologic examination, it was concluded that
 

proliferative and neoplastic lesions were induced in the livers
 

of Fischer 344 rats by each of the three test dyes administered
 

for 13 weeks.
 

E. Statistical Analyses of Results (Rats)
 

Tables C1-C6 in Appendix C contain the statistical analyses of
 

liver tumors which, along with other morphology concerning
 

changes in liver cells, were observed in the studies of the three
 

chemicals. Since each group for each chemical contained a
 

maximum of 10 animals, the power of the Fisher exact test to
 

determine significance of results is low; for example, with 0/10
 

incidence of a lesion in the controls, a significant result of
 

P <_ 0.05 is not seen until the incidence of the lesion in a dosed
 

group is over 4/10 (40%), at which incidence P = 0.043. The
 

higher dosed groups developed neoplastic or nonneoplastic lesions
 

that did not appear in the controls or in lower dose groups. In
 

31
 
some instances, mostly those involving nonneoplastic morphology,
 

Fisher exact test results have P values lower than the 0.01 level
 

required for an overall 0.05 significance level, taking into
 

account the criterion for multiple comparisons of five dosed
 

groups with a single control.
 

In male rats, liver tumors were observed in the 1,500 ppm- (8/10,
 

80%; P < 0.001) and the 3,000 ppm- (1/9, 11%; P is not significant) dose groups fed direct blue 6 as well as in the 1,500 ppm­

(9/9, 100%; P < 0.001) dose group fed direct black 38 (see
 

figures 7, 8, and 9). No incidence of these tumors appeared in.
 

any of the three control groups or in any dosed group fed direct
 

brown 95. Foci of cellular alteration or basophilic foci were
 

observed in significant incidences (P < 0.01) in the 750 ppm- and
 

1,500 ppm-dose groups fed direct blue 6, in the 375 ppm- and 750
 

ppm-dose groups fed direct black 38, and in the 375 ppm- and
 

1,500 ppm-dose groups fed direct brown 95, when compared with
 

corresponding control groups. Incidences of these foci were also
 

observed in the 3,000 ppm-dose group fed direct blue 6, the 1,500
 

ppm-dose group fed direct black 38, and in the 750 ppm- and 3,000
 

ppm-dose groups fed direct brown 95. These observed incidences
 

are in contrast to the absence of such incidences in any of the
 

control and 190 ppm-dose groups. In some of the higher dosed
 

groups, occurrences of either neoplastic nodules or cell changes
 

32
 
9 

INCIDENCE INCIDENCE 

o

5? 

to o o n •a Z CD I^; 

o x A 

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30% 

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MALES 

KEY 

Hepatocellular 

I

Carcinomas and 

Neoplastic Nodules 

Basophilic Foci 

190ppm 375ppm 

100% 

90% 

80% 

70% 

60% 

50% 

40% 

30%' 

20%' 

10%­

0 ­

*ALL ANIMALS DEAD AT FIFTH WEEK 

FEMALES 

KEY 

Hepatocellular 

Carcinomas and INeoplastic Nodules 

Basophilic Foci 

190ppm 375 ppm 

750 ppm 1,500 ppm 3,000 ppm* 

750 ppm 1,500 ppm 3 000 ppm * 

*AU ANIMALS DEAD AT TWELFTH WEEK 

Figure 8. Hepatic Lesions Observed in Rats Administered Direct Black 38 in the Diet 

34 
100% 

MALES 90% 

80% 

70% 

KEY 

60% Hepatocellular

 

Carcinomas and 

50% I Neoplastic Nodules 

40% 

Basophihc Foci 

30%­

20%' 

10%' 

190ppm 375ppm 750 ppm 1,500 ppm* 3,000 ppm* 

*ALL ANIMALS DEAD AT FIFTH WEEK. 

100% 

FEMALES 90% 

80%­

70% 

KEY 

60% 

I

Hepatocellular 

Carcinomas and 

50% Neoplastic Nodules

 

40%-

Basophihc Foci 

30%' 

20% 

10%' 

0' 190ppm 375ppm 750 ppm 1,500 ppm 3,000 ppm* 

*ALL ANIMALS DEAD AT SIXTH WEEK 

Figure 9. Hepatic Lesions Observed in Rats Administered Direct Brown 95 in the Diet 

35 
were observed as early as week 4 on study. The data on control
 

male Fisher 344 rats, compiled to date from 2-year studies
 

performed by all laboratories in the Carcinogenesis Testing
 

Program, indicate an incidence of neoplastic nodules or
 

hepatocellular adenomas or carcinomas of 32/1,806 (1.8%). There
 

were no such tumors in the 220 male rats that died before week
 

78.
 

In female rats, liver tumors were observed in the 3,000 ppm-dose
 

group (7/9, 77%; P = 0.001) fed direct blue 6, in the 1,500
 

ppm-dose group (5/10, 50%; P = 0.01.6) fed direct black 38, and in
 

the 1,500 ppm-dose group (5/8, 63%; P = 0.007) fed direct brown
 

95, but in none of the controls or the three lower dosed groups
 

of each study in the females. Foci of cellular alteration or
 

basophilic foci occurred in significant incidences (P < 0.01) in
 

the 750 ppm-, 1,500 ppm-, and 3,000 ppm-dose groups fed direct
 

blue 6, in the 750 ppm- and 1,500 ppm-dose groups fed direct
 

black 38, and in the 1,500 ppm-dose group fed direct brown 95.
 

Some incidences of these foci were observed in the 375 ppm- and
 

3,000 ppm-dose groups fed direct black 38 and in the 375 ppm-,
 

750 ppm-, and 3,000 ppm-dose groups fed direct brown 95.
 

Historical records from 2-year studies indicate that in control
 

animals the incidence of neoplastic nodules or hepatocellular
 

adenomas or carcinomas was 55/1,765 (3.1%). There were two such
 

36
 
tumors in the 182 female rats that died prior to week 78 on
 

study.
 

In summary, the occurrence of lesions of the liver at
 

statistically significant levels in dosed rats when compared with
 

controls as well as the comparison of incidences of the lesions
 

in these present 13-week subchronic toxicity studies with those
 

in historical records indicate that the observed hepatocellular
 

carcinomas, neoplastic nodules, and related proliferative lesions
 

are associated with the administration of the test dyes.
 

37
 

IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of the male and female mice administered the
 

highest dose of any one of the test dyes were slightly lower than
 

mean body weights of the corresponding controls; mean body
 

weights of mice administered lower doses were generally
 

unaffected (figures 10, 11, and 12). No other clinical signs
 

related to administration of the dyes were reported.
 

B. Benzidine Studies (Mice)
 

Urine collected over a 24-hour period during weeks 3 and 11 of
 

the subchronic toxicity studies from male and female mice dosed
 

with any one of the test dyes was found to contain benzidine and
 

monoacetyl benzidine, while specimens of urine taken from
 

corresponding control groups contained neither compound. The
 

benzidine and monoacetyl benzidine were identified by thin-layer
 

chromatography and mass spectroscopy. Quantities excreted in the
 

urine were determined by combined extraction and spectrometric
 

procedures. In most tests, the amounts excreted were dose
 

related for each of the dyes administered. No consistent
 

differences in results were found between males and females.
 

Details of the methods and results are given in Appendix D.
 

39
 
48­

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• CONTROLS 

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0­

36 43 50 57 

TIME ON STUDY (DAYS) 

Figure 10. Growth Curves for Mice Administered Direct Blue 6 in the Diet 

40 
GH _ 32 ­

(2 

9

•

I

?

0 

A 

> 

O 20 

O 

ffi 

Z 16 

5

UJ 

 12- MALE MICE 

A 12,500 ppm 

0 6,000 ppm 

D 3,000 ppm 

O 1.500 ppm 

4- O 750 ppm 

8­

• CONTROLS 

0 

36 43 60 57 

TIME ON STUDY (DAYS] 

u 32­

io 

O 20­

m 1 

FEMALE MICE 

A 12,500 ppm 

0 6,000 ppm 

D 3,000 ppm 

O 1,600 ppm 

O 750 ppm 

• CONTROLS 

36 43 50 57 

TIME ON STUDY (DAYS) 

Figure 11. Growth Curves for Mice Administered Direct Black 38 in the Diet 

41 
•3 

S 

Q 

O 

m 

z 

UJ 

5 

2

hI 

<D

UJ 

a

0 o 

£

48­

44­

40­

36­

32­ 1 

28- 9 * ' r3 O 6 D D

 

24, ^ ^ A J

§ ^ A 

20-

1 ' I 1 A £ A A A i ^ ^ 

16­

12- MALE MICE 

A 12 500 ppm 

{> 6,000 ppm 8­ Q 3,000 ppm 

O 1,500 ppm 

4- Ct 750 ppm 

• CONTROLS 

0­

36 43 50 57 

TIME ON STUDY (DAYS) 

48 

44­

40­

36­

 32H 

 28­

 >H 

 20­

FEMALE MICE 

6 000 ppm 

3 000 ppm 

1 500 ppm 

750 ppm 

375 ppm 

CONTROLS 

36 43 50 57 

TIME ON STUDY (DAYS) 

Figure 12. Growth Curves for Mice Administered Direct Brown 95 in the Diet 

42 
C. Survival (Mice)
 

All male and female mice administered any one of the doses of the
 

dyes survived to the end of the subchronic toxicity studies,
 

except for one male administered 750 ppm direct brown 95 dye.
 

The death of this animal was attributed, however, to bacterial
 

infection, and was not related to administration of the dye.
 

D. Pathology (Mice)
 

Gross Lesions. Gross lesions observed in mice administered
 

direct blue 6 at doses of 6,000 or 12,500 ppm consisted of
 

bluish-black and slightly enlarged spleens. Those observed in
 

mice administered direct black 38 dye at 12,500 ppm consisted of
 

slightly darkened livers and of enlarged and darkened spleens;
 

those observed in the mice administered 6,000 ppm consisted of
 

slightly enlarged and darkened spleens. Similar lesions were not
 

reported in mice administered direct brown 95 dye.
 

Histopathologic Lesions. Histopathologic lesions observed in
 

control and dosed mice are summarized in Appendix B. Tables Bl,
 

B3, and B5 list those lesions that were observed only in mice
 

administered dye in the diet; tables B2, B4, B6, and B7 list
 

other lesions. The findings observed in animals administered any
 

one of the test dyes consisted mainly of hemosiderosis of the
 

kidney and pigmentation of the liver at the highest doses and of
 

43
 
hemosiderosis of the spleen at low as well as high doses. The
 

splenic hemosiderosis was characterized by an increase in the
 

golden brown, iron-positive pigment that is normally seen in
 

macrophages in the red pulp. The kidney hemosiderosis consisted
 

of a finely granular, iron-positive pigment in epithelial cells
 

of the proximal convoluted tubules; the pigment was difficult to
 

detect without use of the Prussian-blue reaction. The hepatic
 

pigment was yellow to yellowish-green and occasionally
 

iron-positive; it occurred in occasional macrophages lining the
 

sinusoids. Pigment of the thyroid, observed only in mice
 

administered the two highest doses of direct black 38 dye, was
 

finely granular, yellow, iron-negative, and located largely in
 

the follicular cells.
 

Biliary hyperplasia was observed in the mice administered the
 

highest doses of direct black 38 and direct brown 95 dyes, but
 

not in mice administered direct blue 6 dye. The hyperplasia
 

consisted of a modest increase in the number of biliary cells
 

adjacent to most portal areas. Other hepatic lesions were
 

observed in mice administered direct black 38 dye or direct brown
 

95 dye, but not in mice administered direct blue 6 dye. Hepatocellular degeneration, observed in 9/10-10/10 male and female
 

mice administered 3,000, 6,000, or 12,500 ppm direct black 38 dye
 

and in 10/10 male mice administered 12,500 ppm direct brown 95
 

44
 
dye, was characterized by pleomorphic nuclei, cytoplasmic
 

vacuolization, eosinophilic droplet formation, and hydropic
 

change, and, in the mice administered the direct black 38 dye,
 

also an increased mitotic index. Three mice administered the
 

highest dose of direct black 38 dye and one mouse administered
 

the highest dose of direct brown 95 dye had foci of cellular
 

alteration in their livers; in these instances, the hepatocytes
 

were distinctly basophilic when compared with surrounding normal
 

cells.
 

Based on the histopathologic examination, hemosiderosis of the
 

spleen and kidney and pigmentation of the liver were the
 

principal lesions occurring in B6C3F1 mice administered any one
 

of the test dyes. The hepatocellular degeneration found in large
 

numbers of mice given high doses of direct black 38 and direct
 

brown 95 dyes also was related to administration of dye. No
 

hepatic lesions occurred in mice administered direct blue 6 dye.
 

E. Statistical Analyses of Results (Mice)
 

No neoplasms occurred in the mice administered any one of the
 

test dyes.
 

45
 

V. DISCUSSION
 

These subchronic toxicity studies of direct blue 6 dye, direct
 

black 38 dye, and direct brown 95 dye were conducted as a part of
 

the bioassay protocol for testing for possible carcinogenicity.
 

Thirteen-week studies are conducted to establish respective doses
 

of test chemicals to use in 2-year studies with both rats and
 

mice.
 

In these feeding studies of the three dyes, mean body weights of
 

the male and female rats administered the two or three highest
 

concentrations of the test dyes were markedly lower than mean
 

body weights of the corresponding controls throughout the
 

studies, and the depressions in mean body weight were dose
 

related. Mean body weights of the male and female mice administered the highest dose of any one of the test dyes were slightly
 

lower than mean body weights of the corresponding controls; mean
 

body weights of mice administered lower doses were generally
 

unaffected.
 

All male and female rats administered 3,000 ppm of any one of the
 

dyes or 1,500 ppm direct brown 95 dye died before the end of the
 

studies. One male rat administered 1,500 ppm direct blue 6 dye,
 

six males administered 1,500 ppm direct black 38 dye, and two
 

males administered 750 ppm direct brown 95 dye also died by the
 

47
 
end of the subchronic toxicit-y studies. No deaths occurred in
 

any other dosed group or in any control group of rats. Mortality
 

in the rats was dose related, and based on times and incidences
 

of deaths, direct brown 95 dye was most toxic, followed in order
 

by direct black 38 dye, then direct blue 6 dye. All male and
 

female mice administered the test dyes survived to the end of the
 

studies, except for one male whose death was attributed to
 

bacterial infection.
 

In rats, neoplastic lesions occurred only in dosed groups and
 

consisted of hepatocellular carcinomas and neoplastic nodules of
 

the liver. The time to onset of the tumors was remarkably short.
 

The incidences of the hepatocellular carcinomas in female rats
 

administered 3,000 ppm direct blue 6 dye (4/9) and male rats
 

administered 1,500 ppm direct black 38 dye (4/9) were significant
 

(P = 0.033) when related to the incidences of the tumors in the
 

corresponding controls (0/10); hepatocellular carcinomas were
 

also observed in two male rats administered 1,500 ppm direct blue
 

6 dye and in one female rat administered 1,500 ppm direct brown
 

95 dye. No control rats from any of the three studies developed
 

hepatocellular carcinomas.
 

When incidences of neoplastic nodules were combined with those of
 

hepatocellular carcinomas, the significance increased to P <
 

0.001 for male rats administered 1,500 ppm direct blue 6 dye, P =
 

48
 
0.001 for females administered 3,000 ppm direct blue 6 dye, P <
 

0.001 for males administered 1,500 ppm direct black 38 dye, and
 

P = 0.007 for females administered 1,500 ppm direct brown 95 dye.
 

No controls developed neoplastic nodules. Female rats administered direct black 38 dye developed no hepatocellular carcinomas,
 

but had an incidence of neoplastic nodules of 5/10, with a significance of P = 0.016. Male rats administered direct brown 95 dye
 

developed neither hepatocellular carcinomas nor neoplastic
 

nodules, but as indicated below, had significant incidences of
 

preneoplastic lesions. The failure of groups of rats
 

administered 3,000 ppm dye to develop tumors when other groups
 

administered 1,500 ppm did develop tumors may be due to earlier
 

deaths at the higher dose.
 

Preneoplastic hepatic lesions (basophilic foci as described by
 

Squire and Levitt, 1975) occurred only in dosed rats and did not
 

occur in controls. The incidences of the basophilic foci were
 

significant (P <^ 0.033) in male (4/9) and female (7/9) rats
 

administered 3,000 ppm direct blue 6 dye and in male rats (7/8)
 

administered 1,500 ppm direct brown 95 dye. Basophilic foci also
 

occurred, at lower incidences, in males (1/10) administered 1,500
 

ppm direct blue 6 dye, in males (3/9) administered 1,500 ppm
 

direct black 38 dye, in females (1/8) administered 3,000 ppm
 

direct black 38 dye, in males administered 750 ppm (3/10) or
 

49
 
3,000 ppm (2/9) direct brown 95 dye, and in females administered
 

1,500 ppm (3/8) or 3,000 ppm (3/8) direct brown 95 dye. When
 

incidences of foci of cellular alteration, a possible
 

preneoplastic lesion, were added to those of basophilic foci,
 

significance occurred in additional dosed groups.
 

In mice, no neoplastic lesions occurred in the liver or other
 

tissues of groups administered the different dyes. The principal
 

nonneoplastic lesions found in mice consisted of hemosiderosis of
 

the kidney and pigmentation of the liver at doses of 6,000 or
 

12,500 ppm and of hemosiderosis of the spleen at low as well as
 

high doses. Other nonneoplastic lesions in the dosed mice
 

involved the liver. Both biliary hyperplasia and hepatocellular
 

degeneration occurred in mice given high doses of direct black 38
 

dye or direct brown 95 dye. In addition, three mice administered
 

12,500 ppm direct black 38 dye and one mouse administered 12,500
 

ppm direct brown 95 dye had foci of cellular alteration, in which
 

the cells were basophilic when compared with surrounding normal
 

cells. No mice administered direct blue 6 dye had these lesions
 

of the liver.
 

In previous work, Rinde and Troll (1975) reported that when azo
 

dyes direct blue 6, direct black 38, direct brown 95, or an
 

additional azo dye (direct red 28) were administered by gavage to
 

rhesus monkeys, benzidine appeared in the urine in yields that
 

50
 
approximated those of animals administered equivalent amounts of
 

free benzidine. In Wistar rats, the metabolic breakdown of
 

benzidine-derived azo dyes to free benzidine has been
 

demonstrated in incubation mixtures of such dyes with intestine
 

(Miyakawa et al., 1973). In the present studies, benzidine and
 

monoacetyl benzidine were detected in the urine of male and
 

female rats and mice administered the test dyes, but neither
 

compound was detected in the urine of control rats and mice.
 

The biliary (oval cell) lesions observed in Fischer 344 rats in
 

the present studies have been previously reported to be induced
 

in SHR, Wistar, Sprague-Dawley, and Buffalo rats by several
 

chemicals that cause hepatocellular carcinoma (Ito et al. , 1973)
 

and by benzidine itself in Sherman rats (Spitz et al., 1950).
 

The foci of cellular alteration, nodules, and carcinomas are
 

identical to those caused by benzidine in Sherman rats (Spitz et
 

al. , 1950). In addition, the administration of benzidine in the
 

diet of Wistar rats and hamsters has been reported to induce
 

cholangiomas and hepatocytic tumors (Boyland et al. , 1954;
 

Saffiotti et al. , 1967). Direct blue 6 dye and direct black 38
 

dye were reported not to induce tumors in female mice when the
 

dyes were administered by implantation in the bladder in wax
 

pellets (Niitsu, 1973); however, foci of alteration as well as
 

hepatocellular carcinomas have been described in both male and
 

51
 
female mice given benzidine (Frith and Dooley, 1976). The
 

failure of the dyes tested in the present studies to induce
 

tumors in mice may have been due, however, to the short period of
 

administration and/or observation. Papillomas and carcinomas of
 

the bladder were found to develop in 3/7 dogs administered
 

benzidine orally by capsule (Bonser et al., 1956; Spitz et al.,
 

1950), although no control dogs were tested at the same time.
 

Humans exposed to benzidine during its manufacture or industrial
 

use have a significantly high incidence of cancer of the bladder
 

(Case et al., 1954; Goldwater et al. , 1965; Hueper, 1969; Mancuso
 

and El-Attar, 1967; Scott, 1952; Uebelin and Pletscher, 1954).
 

The presence of benzidine in the urine of rats and mice and of
 

liver lesions in rats and mice identical to those caused by
 

benzidine alone suggests that the benzidine released from the
 

metabolism of the dyes may be responsible for the liver lesions.
 

The failure of male rats receiving direct brown 95 dye to develop
 

hepatocellular carcinomas or neoplastic nodules may be due to the
 

toxicity of the chemical, which resulted in deaths of all animals
 

in the highest two dose groups by week 5 of the study. Two-year
 

studies of these three dyes in mice were not conducted, since
 

benzidine was detected in the urine of mice in these studies, and
 

since there is evidence from prior studies that benzidine can
 

produce hepatocellular carcinomas in mice (Frith and Dooley,
 

1976).
 

52
 
It is concluded that under the conditions of these 13-week
 

subchronic toxicity studies, direct blue 6 and direct black 38
 

dyes were carcinogenic in male and female Fischer 344 rats and
 

direct brown 95 was carcinogenic in female rats; all three dyes
 

induced hepatocellular carcinomas and neoplastic nodules in the
 

liver. The test dyes were not carcinogenic for B6C3F1 mice in
 

the 13-week subchronic toxicity studies.
 

53
 

VI. BIBLIOGRAPHY
 

Berenblum, I. , ed. , Carcinogenicity Testing; _A Report on the
 

Panel on Carcinogenicity of the Cancer Research Commission
 

of the UICC, Vol. 2. International Union Against Cancer,
 

Geneva, 1969.
 

Bonser, G. M., Clayson, D. B. , and Jull, J. W. , The induction of
 

tumours of the subcutaneous tissues, liver and intestine in
 

the mouse by certain dye-stuffs and their intermediates.
 

Brit. J_._ Cancer _10:653-667, 1956.
 

Boyland, E. , Harris, J. , and Horning, E. S. , The induction of
 

carcinoma of the bladder in rats with acetamidofluorene.
 

Brit. J. Cancer 8(4):647-654, 1954.
 

Case, R. A. M. , Hosker, M. E. , McDonald, D. B. , and Pearson, J.
 

T. , Tumours of the urinary bladder in workmen engaged in the
 

manufacture and use of certain dyestuff intermediates in the
 

British chemical industry. Brit. J_._ industr. Med. 11; 75-79
 

and 94-95, 1954.
 

Comptroller General of the United States, Cancer and coal tar
 

hair dyes: an unregulated hazard to consumers. Report of the
 

Comptroller General of the United States HRD-78-22, General
 

Accounting Office, Washington, D. C., 1977.
 

Cox, D. R., Analysis of Binary Data, Methuen & Co., Ltd., London,
 

1970, pp. 48-52.
 

Evelyn, K. A. and Malloy, H. T. , Microdetermination of
 

oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single
 

sample of blood. J. Biol. Chem. 126:655-662, 1938.
 

Frith, C. H. and Dooley, K. , Hepatic cytologic and neoplastic
 

changes in mice given benzidine dihydrochloride. J. Natl.
 

Cancer Inst. 56:679-682, 1976.
 

Gart, J. J., The comparison of proportions: a review of
 

significance tests, comfidence limits and adjustments for
 

stratification. Rev. Int. Statist. Inst. 39:148-169, 1971.
 

Goldwater, L. J. , Rosso, A. J. , and Kleinfeld, M. , Bladder tumors
 

in a coal tar dye plant. Arch. Envir. Health 11:814, 1965.
 

55
 
Hueper, W. C., Cancers of the urinary system. In: Occupational
 

and Environmental Cancers of_ the Urinary System, Yale
 

University Press, New Haven, Conn., 1969, pp. 1-67.
 

International Agency for Research on Cancer, Benzidine. IARC
 

Monographs on the Evaluation of Carcinogenic Risk of
 

Chemicals to Man, Vol. l_, World Health Organization,
 

International Agency for Research on Cancer, Lyon, 1972, pp.
 

80-86.
 

Ito, M., Masuko, K. , Matsuyama, M., Suzuki, H. , Nagayo, T. , and
 

Aoki, K. Proliferation of bile ductular cells in the
 

spontaneously hypertensive rat fed N,N'-2,7-fluoranylenebisacetamide. J. Natl. Cancer Inst. 50:699-706, 1973.
 

Linhart, M. S., Cooper, J. , Martin, R. L., Page, N., and Peters,
 

J., Carcinogenesis bioassay data system. Comp. and Biomed.
 

Res. 7:230-248, 1974.
 

Mancuso, T. F. and El-Attar, A., Cohort study of workers exposed
 

to betanaphthylamine and benzidine. J. Occup. Med.
 

_9(6):277-285, 1967.
 

Miller, R. G., Jr., Simultaneous Statistical Inference,
 

McGraw-Hill Book Co., New York, 1966, pp. 6-10.
 

Miyakawa, M. , Harada, T. , and Yoshida, 0., Reduction of benzidine
 

dye, Direct Deep Black Ex, in the intestine of the rat and
 

mouse. Medicine and Biology 86(6):355-360, 1973.
 

Niitsu, K. , Studies on the metabolism and carcinogenicity of azo
 

dyes used for food colors and direct dyestuffs. (Part II)
 

Studies on the metabolism and carcinogenicity of direct
 

dyestuffs Blue BB and Black Ex. Tokyo Jikeikai Ika Daigaku
 

Zasshi 88(3);467-471, 1973.
 

Rinde, E. and Troll, W. , Colorimetric assay for aromatic amines.
 

Anal. Chem. 48(3).-542-544, 1976.
 

Rinde, E. and Troll, W. , Metabolic reduction of benzidine azo
 

dyes to benzidine in rhesus monkey. J. Natl. Cancer Inst.
 

55(1):181-182, 1975.
 

Sadtler Commercial Spectra, Dyes, Pigments, and Stains. IR Nos.
 

x 2579, x 3020, and x 2909, Standard Research Laboratories,
 

Philadelphia, 1960.
 

56
 
Saffiotti, U. , Cefis, F. , Montesano, R. , and Sellakumar, A. R. ,
 

Induction of bladder cancer in hamsters fed aromatic amines.
 

In: Bladder Cancer — A^ Symposium, Deichmann, W. B., ed. ,
 

Aesculapius Publishing Co., Birmingham, Ala., 1967, pp.
 

129-135.
 

Scott, T. S. , The incidence of bladder tumours in a dyestuffs
 

factory. Brit. J_._ industr. Med. jh!27-132.
 

Society of Dyers and Colourists, Colour Index, Vol. 2 and Vol.
 

4., Third edition, 1971.
 

Spitz, S., Maguigan, W. H. , and Dobriner, K. , The carcinogenic
 

action of benzidine. Cancer 3:789-804, 1950.
 

Squire, R. A. and Levitt, M. H. , Report of a workshop on
 

classification of specific hepatocellular lesions in rats.
 

Cancer Res. 35:3214-3215, 1975.
 

Uebelin, F. and Pletscher, A., Aetiologic und Prophylaxe 

geiwerblicher Tumoren in der FarbstoffIndustrie. 

Schweizerische Medizinische Wochenschrift 84(32);917-920, 

T95T; 

United States International Trade Commission, Imports of
 

Benzenoid Chemicals and Products, 1976. USITC Publication
 

828, United States International Trade Commission,
 

Washington, D.C., 1977a, pp. 54-55.
 

United States International Trade Commission, Synthetic Organic
 

Chemicals, United States Production and Sales, 1976. USITC
 

Publication 833, United States International Trade
 

Commission, Washington, D.C., 1977b, p. 57.
 

57
 

APPENDIX A
 

LESIONS IN RATS ADMINISTERED DIRECT DYES
 

IN THE DIET
 

59
 

Table Al. Lesions Observed Only in Rats Administered Direct Blue 6 

in the Diet 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female 

Liver 

Hepatocellular Carcinomas 0/10 0/10 0/10 0/10 0/10 0/10 2/10 0/10 0/9 4/9 

Neoplastic nodules 0/10 0/10 0/10 0/10 0/10 0/10 6/10 0/10 1/9 3/9 

Foci of cellular 

alteration 0/10 0/10 0/10 0/10 10/10 9/10 7/10 10/10 0/9 0/9 

Basophilic foci 0/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10 4/9 7/9 

Biliary hyperplasia 0/10 0/10 0/10 0/10 4/10 0/10 10/10 8/10 9/10 9/9 

Nodular 

regeneration 0/10 0/10 0/10 0/10 0/10 0/10 9/10 10/10 0/9 0/9 

Hepatocellular 

degeneration 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 2/9 0/9 

Cholangiofibrosis 0/10 0/10 0/10 0/10 0/10 0/10 8/10 0/10 0/9 0/9 

Portal fibrosis 0/10 0/10 0/10 0/10 0/10 0/10 5/10 3/10 0/9 2/9 
Table Al. Lesions Observed Only in Rats Administered Direct Blue 6
 

in the Diet
 

(continued)
 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm
 

ON
 

N3
 

Tissue/Lesion
 

Spleen
 

Lymphoid depletion
 

Lymphoid necrosis
 

Bacterial
 

septicemia
 

Thymus
 

Lymphoid depletion
 

Bone Marrow
 

Myeloid depletion
 

Kidney
 

Subactute glomerulonephritis
 

Bacterial
 

septicemia
 

Male Female Male Female Male Female Male Female Male Female
 

a a a a a a 1/9 0/10 9/9 5/7
 

a a a a a a 0/9 0/10 1/9 0/8
 

a a a a a a 0/9 0/10 1/9 0/8
 

a a a a a a 1/8 0/4 6/7 2/2
 

a a a a a a 1/10 0/10 10/10 8/9
 

a a a a a a 1/10 0/10 0/10 0/9
 

a a a a a a 0/10 0/10 1/10 0/9
 
Table Al. Lesions Observed Only in Rats Administered Direct Blue 6 

in the Diet 

(continued) 

Tissue/Lesion 

190 ppm

Male Female

 375 ppm

 Male Female

 750 ppm

 Male Female

 1,500 ppm 

 Male Female 

3,000 ppm 

Male Female 

Pancreas 

Edema a a a a a a 0/8 0/9 0/9 1/4 

Bacterial 

septicemia a a a a a a 0/8 0/9 0/9 1/4 

Acinar cell 

necrosis a a a a a a 0/8 0/9 1/8 1/4 

Acinar cell 

atrophy a a a a a a 1/10 0/9 0/8 1/4 

Bladder 

Edema a a a a a a 0/8 0/10 1/9 0/9 

Epithelial necrosis a a a a a a 0/8 0/10 0/9 1/7 

Heart 

Bacterial 

septicemia a a a a a a 0/10 0/10 1/10 0/9 
Table Al. Lesions Observed Only in Rats Administered Direct Blue 6 

in the Diet 

(continued) 

Tissue/Lesion 

190 ppm

Male Female

 375 ppm

 Male Female

 750 ppm

 Male Female

 1,500 ppm 

 Male Female 

3,000 ppm 

Male Female 

Lung 

Bacterial 

septicemia a a a a a a 0/10 0/10 1/10 2/9 

Pulmonary edema a a a a a a 0/10 0/10 1/10 0/9 

Salivary Gland 

Acinar cell 

necrosis a a a a a a 1/9 0/10 7/10 9/9 

Acinar cell 

atrophy a a a a a a 0/9 0/10 6/10 0/9 

Skin 

Subcutaneous edema a a a a a a 1/10 0/10 7/10 0/9 

Lymph Nodes 

Lymphoid depletion a a a a a a 0/10 0/10 1/9 8/9 

Large Intestinal 

Lymphoid Follicle 

Lymphoid necrosis a a a a a a 0/7 0/8 1/10 0/8 
Table Al. Lesions Observed Only in Rats Administered Direct Blue 6 

in the Diet 

(continued) 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female 

Testes 

Interstitial 

hemorrhage a a a 1/10 6/9 

Large Intestine 

Edema a a a a a a 0/7 0/8 3/9 1/6 

Colon 

Acute enteritis a a a a a a 0/7 0/8 1/10 0/8 

a

Not examined. 
Table A2. Other Lesions in Rats Administered
 

Direct Slue 6 in the Diet
 

Control Combined 

Groups Dose Groups3 

Tissue Lesion Male Female Male Female 

Lung Nodular lymphoid hyperplasia 9/10 10/10 9/20 9/19 

Lymphocytic perivasculitis 4/10 1/10 3/20 1/19 

Acute hemorrhage 0/10 0/10 3/20 2/19 

Acute congestion 0/10 0/10 1/20 0/19 

Liver Subacute hepatitis 1/10 1/10 0/49 2/49 

Focal granuloma 0/10 0/10 0/49 1/49 

Extramedullary hematopoiesis 0/10 0/10 3/49 2/49 

Kidney Tubular sclerosis, focal 1/10 0/10 0/20 0/18 

Tubular regeneration 8/10 0/10 4/20 0/18 

Interstitial nephritis 1/10 0/10 0/20 0/18 

Heart Subacute myocarditis 5/10 1/10 4/20 1/19 

Subendocardial hemorrhage 0/10 0/10 3/20 0/19 

Adrenals Acute congestion 0/10 0/10 2/10 0/18 

Acute hemorrhage 0/10 0/10 6/20 3/18 

Tracheobronchial 

Lymph Nodes Acute hemorrhage 0/10 0/10 3/20 0/19 

Submandibular 

Lymph Node Acute hemorrhage 0/10 0/10 1/20 1/19 

66
 
Table A2. Other Lesions in Rats Administered
 

Direct Blue 6 in the Diet
 

(continued)
 

Control Combined
 

Groups Dose Groups3
 

Tissue Lesion Male Female Male Female
 

Mesenteric
 

Lymph Node Acute hemorrhage 0/10 1/10 2/19 0/19
 

Mesenteric Fat Necrosis 0/10 0/10 5/19 1/19
 

Salivary Gland Lymphocytic sialoadenitis 0/10 0/10 1/20 0/19
 

Seminal Vesicle Spermatic granuloma 1/10 0/20
 

Testicle Degeneration and mineralization 0/10 1/20
 

Stomach Acute hemorrhage 0/10 0/10 4/19 1/18
 

Colon Nematodiasis 1/10 0/10 1/19 1/18
 

a

For lesions of the liver, the combined group consisted of all dosed
 

groups. In lesions of all other tissues, the combined dose groups
 

consisted of the 1,500 ppm dose and 3,00 ppm dose groups.
 

67
 
Table A3. Lesions Observed Only in Rats Administered Direct Black 38
 

in the Diet 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female 

Liver 

Hepatocellular carcinomas 0/10 0/10 0/10 0/9 0/10 0/10 4/9 0/10 0/9 0/8 

Neoplastic nodules 0/10 0/10 0/10 0/9 0/10 0/10 5/9 5/10 0/9 0/8 

Foci of cellular 

alteration 0/10 0/10 7/10 5/9 9/10 10/10 2/9 10/10 0/9 0/8 

Basophilic foci 0/10 0/10 0/10 0/9 0/10 0/10 3/9 0/10 0/9 1/8 

00

 Biliary hyperplasia 1/10 0/10 0/10 0/9 9/10 6/10 9/9 9/10 9/9 7/8 

Nodular regeneration 0/10 0/10 0/10 0/9 2/10 0/10 4/9 10/10 0/9 1/8 

Cholangiof ibrosis 0/10 0/10 0/10 0/9 1/10 0/10 3/9 0/10 0/9 0/8 

Portal fibrosis 0/10 0/10 0/10 0/9 1/10 0/10 4/9 9/10 0/9 1/8 

Bacterial septicemia 0/10 0/10 0/10 0/9 0/10 0/10 0/9 0/8 4/9 4/8 

Fatty metamorphosis 0/10 0/10 0/10 0/9 0/10 0/10 0/9 0/8 0/9 1/8 

Multifocal necrosis 0/10 0/10 0/10 0/9 0/10 0/10 0/9 0/8 1/9 4/8 
Table A3. Lesions Observed Only in Rats Administered Direct Black 38 

in the Diet 

(continued) 

Tissue/Lesion 

190 ppm

Male Female

 375 ppm

 Male Female

 750 

 Male 

ppm 

Female 

1,500 ppm 

Male Female 

3,000 ppm 

Male Female 

Spleen 

Lymphoid depletion a a a a 0/10 0/10 7/9 10/10 9/9 6/7 

Bacterial septicemia a a a a 0/10 0/10 0/9 0/10 5/9 3/7 

Acute congestion a a a a 0/10 0/10 0/9 0/10 1/9 1/7 

Hemosiderosis a a a a 0/10 0/10 0/9 0/10 2/9 3/7 

vO Thymus 

Lymphoid depletion a a a a a a 2/4 0/2 5/5 5/5 

Lymphoid necrosis a a a a a a 1/4 0/2 0/5 0/5 

Bone Marrow 

Myeloid depletion a a a a 0/10 0/10 3/9 7/10 9/9 8/8 

Kidney 

Subactute glomerulonephritis 2/10 4/9 10/10 0/9 0/8 
Table A3. Lesions Observed Only in Rats Administered Direct

in the Diet 

 Black 38 

(continued) 

Tissue/Lesion 

190 ppm 

Male Female

375 ppm

 Male Female

 750 ppm 

 Male Female

1,500 ppm

 Male Female

 3,000 ppm 

 Male Female 

Pancreas 

Necrosis of ancinar 

epithelium a a a a 1/10 0/9 0/9 0/8 1/8 0/8 

Atrophy of acinar 

epithelium a a a a 0/10 0/9 4/9 2/8 0/9 0/8 

Edema a a a a 0/10 0/9 2/9 0/8 1/8 0/8 

Lung 

Acute pneumonia a a a a a a 0/8 0/10 1/9 0/8 

Leucocytosis a a a a a a 1/9 0/10 1/9 0/8 

Bacterial septicemia a a a a a a 0/8 0/10 0/9 1/8 

Skin 

Subcutaneous edema 1/9 0/10 0/9 0/8 
Table A3. Lesions Observed Only in Rats Administered Direct Black 38
 

in the Diet
 

(continued)
 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm
 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female
 

Adrenals
 

Congestion a a a a a a 0/9 0/10 5/9 5/8
 

Hemorrhage a a a a a a 2/9 0/10 6/9 0/8
 

Acute cortical necrosis a a a a a a 0/9 0/10 0/9 2/8
 

Salivary Gland
 

Atrophy of acinar
 

epithelium a a a a 0/10 0/10 1/7 2/9 7/9 5/7
 

Acinar epithelial necrosis a a a a 0/10 0/10 3/7 0/9 0/9 0/7
 

Lymph Nodes
 

Lymphoid depletion a a a a a a 0/8 0/9 0/9 1/7
 

Acute hemorrhage a a a a a a 1/8 0/9 0/9 1/7
 

Mandibular lymph nodes a a a a a a 0/8 0/9 4/9 1/7
 
Table A3. Lesions Observed Only in Rats Administered Direct Black 38 

in the Diet 

(continued) 

Tissue/Lesion

190 ppm

 Male Female

 375 ppm

 Male Female

 750 ppm

 Male Female

 1,500 ppm

 Male Female

 3,000 ppm 

 Male Female 

Testes 

Seminiferous tubular 

degeneration a a a 1/9 3/9 

Acute interstitial 

hemorrhage a a a a a a 1/9 a 1/9 a 

Small Intestine 

Edema a a a a a a 1/9 0/10 0/9 0/8 

Colon 

Submuscosal edema a a a a a a 0/9 0/10 1/6 0/8 

Mesentery 

Fat necrosis a a a a a a 1/9 0/10 0/9 0/8 

a

Not examined. 
Table A4. Other Lesions in Rats Administered
 

Direct Black 38 in the Diet
 

Tissue
 

Lung
 

Kidney
 

Liver
 

Heart
 

Thyroid
 

Bladder
 

Stomach
 

Colon
 

Testes
 

Combined
 

Dose Groupsa
 

Male Female
 

0/17 0/18
 

0/17 1/18
 

1/17 0/18
 

0/27 1/18
 

0/28 0/28
 

4/28 2/28
 

1/28 0/28
 

1/28 0/28
 

2/48 0/47
 

1/27 1/28
 

0/27 2/28
 

0/17 1/18
 

1/17 0/18
 

1/18 0/18
 

2/18 0/18
 

0/18 0/18
 

1/18
 

Lesion
 

Nodular lymphoid hyperplasia
 

Lymphocytic perivasculitis
 

Acute hemorrhage
 

Proliferative interstitial
 

pneumonia
 

Focal subacute glomerulitis
 

Tubular regeneration
 

Focal interstitial nephritis
 

Focal tubular degeneration
 

Subacute hepatitis
 

Subacute myocarditis
 

Acute hemorrhage
 

Acute interstitial hemorrhage
 

Thyroglossal duct cyst
 

Acute submucosal hemorrhage
 

Acute hemorrhage
 

Nematodiasis
 

Atrophy
 

Male
 

8/10
 

0/10
 

0/10
 

0/10
 

1/10
 

8/10
 

0/10
 

0/10
 

0/10
 

2/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

1/10
 

1/10
 

Control
 

Groups
 

Female
 

9/10
 

5/10
 

1/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

0/10
 

73
 
Table A4. Other Lesions in Rats Administered
 

Direct Black 38 in the Diet
 

(continued)
 

Control Combined
 

Groups Dose Groups3
 

Tissue Lesion Male Female Male Female
 

Epididymis Spermatic granuloma 0/10 1/18
 

Spermatic cyst 0/10 1/18
 

a

ln lesions of the liver, the combined dose groups consisted of all dosed
 

groups. For tissues of the kidney and heart, the combined dose groups
 

consisted of the 750 ppm, 1,500 ppm, and 3,000 ppm dose groups. For
 

lesions of the remaining tissues given in the table, the combined dose
 

groups consisted of the 1,500 ppm and 3,000 ppm dose groups.
 

74
 
Table A5. Lesions Observed Only in Rats Administered Direct Brown 95
 

in the Diet 

190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female 

Liver 

Hepatocellular carcinomas 0/10 0/10 0/10 0/10 0/10 0/10 0/9 1/8 0/9 0/8 

Neoplastic nodules 0/10 0/10 0/10 0/10 0/10 0/10 0/8 4/8 0/9 0/8 

Foci of cellular 

alteration 0/10 0/10 10/10 3/10 5/10 4/10 0/8 3/8 0/9 0/8 

Basophilic foci 0/10 0/10 0/10 0/10 3/10 0/10 7/8 3/8 2/9 3/8 

m Biliary hyperplasia 0/10 0/10 2/10 0/10 5/10 0/10 8/9 8/8 9/9 8/8 

Nodular regeneration 0/10 0/10 0/10 0/10 6/10 6/10 0/9 5/8 0/9 0/8 

Portal fibrosis 0/10 0/10 0/10 0/10 8/10 4/10 0/9 5/8 0/9 0/8 

Hepatic necrosis 0/10 0/10 0/10 0/10 0/10 0/10 0/9 0/8 3/9 0/8 

Bacterial septicemia 0/10 0/10 0/10 0/10 0/10 0/10 0/9 0/8 0/9 3/10 

Spleen 

Lymphoid depletion 0/10 0/10 0/10 0/10 5/10 5/10 6/8 7/8 5/8 5/8 

Lymphoid necrosis 0/10 0/10 0/10 0/10 0/10 0/10 1/8 0/8 1/8 0/8 

Bacterial septicemia 0/10 0/10 0/10 0/10 0/10 0/10 0/8 0/8 0/8 3/8
 
Table A5. Lesions Observed Only in Rats Administered Direct Brown 95 

in the Diet 

(continued) 

Tissue/Lesion 

190 ppm 

Male Female

375 ppm

 Male Female

 750 ppm 

 Male Female

1,500 ppm

 Male Female

 3,000 ppm 

 Male Female 

Thymus 

Lymphoid depletion a a a a 2/8 0/10 4/4 2/2 7/7 6/6 

Bone Marrow 

Myeloid depletion 0/10 0/10 0/10 0/10 1/10 6/10 10/10 8/8 9/8 9/10 

Kidney 

Subacute glomerulonephritis 0/10 0/10 0/10 0/10 8/10 10/10 9/10 2/8 0/9 0/10 

Bacterial septicemia 0/10 0/10 0/10 0/10 0/10 0/10 6/10 0/8 1/9 0/10 

Pancreas 

Degeneration of 

individual acinar 

epithelial cells 0/10 0/10 0/10 9/10 0/8 0/10 0/6 0/6 0/7 0/5 

Acinar cell atrophy 0/10 0/10 0/10 0/10 4/8 3/10 0/6 3/6 0/7 0/5 

Edema 0/10 0/10 0/10 0/10 1/8 0/10 0/6 2/6 0/7 0/5 
Table A5. Lesions Observed Only in Rats Administered Direct Brown 95
 

(continued)
 

Tissue/Lesion
 

Salivary Gland
 

Acinar cell atrophy
 

Necrosis of acinar epithelium
 

Heart
 

Bacterial septicemia
 

Testes
 

Interstitial hemorrhage
 

Degeneration of
 

germinal epithelium
 

Large Intestine
 

Submucosal edema
 

Epithelial necrosis
 

Submucosal hemorrhage
 

190 ppm


Male Female


a a


a a


a a


a


a


a a


a a


a a


in the Diet
 

 375 ppm


 Male Female


 a a


 a a


 a a


 a


 a


 a a


 a a


 a a


 750
 

 Male
 

 0/9
 

 1/9
 

 1/10
 

 1/10
 

 1/10
 

 1/10
 

 0/10
 

 0/10
 

ppm
 

Female
 

0/10
 

0/10
 

0/10
 

0/9
 

0/9
 

0/9
 

1,500 ppm
 

Male Female
 

3,000 ppm
 

Male Female
 

2/10
 

5/10
 

5/9
 

2/9
 

0/9
 

a
 

2/7
 

2/7
 

3/7
 

3/7
 

0/7
 

1/5
 

0/5
 

0/5
 

5/5
 

1/5
 

1/8
 

3/9
 

2/9
 

1/7
 

0/9
 

0/9
 

9/10
 

1/10
 

0/10
 

1/7
 

0/7
 

0/7
 
Table A5. Lesions Observed Only in Rats Administered Direct Brown 95 

in the Diet 

(continued) 

Tissue/Lesion 

190 ppm

Male Female

 375 ppm

 Male Female

 750 ppm

 Male Female

 1,500 ppm

 Male Female

 3,000 ppm 

 Male Female 

Skin 

Subcutaneous edema a a a a 1/10 0/10 0/10 1/8 0/9 1/10 

Lung 

Bacterial septicemia a a a a 0/10 0/10 5/8 0/7 1/8 0/9 

Mandibular Lymph Nodes 

Bacterial septicemia a a a a 0/9 0/10 1/10 0/5 0/5 1/9 

Lymphoid necrosis a a a a 1/9 0/10 0/10 0/5 0/5 2/9 

Bronchial Lymph Nodes 

Bacterial septicemia a a a a 0/9 0/10 0/10 0/5 1/5 0/9 

Mesenteric Lymph Nodes 

Lymphoid necrosis a a a a 0/9 0/10 0/10 0/5 0/5 1/9 
Table A5. Lesions Observed Only in Rats Administered Direct Brown 95 

in the Diet 

(continued) 

Tissue/Lesion

190 ppm

 Male Female

 375 ppm

 Male Female

 750 ppm

 Male Female

 1,500 ppm

 Male Female

 3,000 ppm 

 Male Female 

Stomach 

Submucosal edema a a a a 0/10 0/9 0/8 2/7 0/9 0/9 

Bladder 

Submucosal edema a a a a 0/6 0/6 0/9 0/7 1/9 0/8 

a

Not examined. 
Table A6. Other Lesions in Rats Administered
 

Direct Brown 95 in the Diet
 

Tissue
 

Lung
 

Heart
 

Liver
 

Kidney
 

Spleen
 

Lesion
 

Lymphoid hyperplasia, nodular
 

Subacute focal pneumonia
 

Acute focal hemorrhage
 

Lymphocytic perivasculitis
 

Congestion
 

Subacute to chronic myocarditis
 

Subendocardial hemorrhage
 

Myocardial degeneration and
 

mineralization
 

Extramedullary hematopoiesis
 

Subacute hepatitis
 

Hemorrhage
 

Tubular regeneration
 

Focal tubular sclerosis
 

Focal glomerulitis
 

Tubular degeneration
 

Hemosiderosis
 

Extramedullary hematopoiesis
 

Congestion
 

Control
 

Groups
 

Male Female
 

10/10 7/10
 

2/10 0/10
 

1/10 0/10
 

4/10 0/10
 

0/10 0/10
 

1/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

8/10 1/10
 

0/10 1/10
 

0/10 0/10
 

0/10 0/10
 

1/10 0/10
 

0/10 0/10
 

0/10 0/10
 

Combined
 

Dose Groups3
 

Male Female
 

5/29 5/27
 

0/29 0/27
 

5/29 2/27
 

0/29 0/27
 

0/29 3/27
 

3/29 0/27
 

5/29 2/27
 

1/29 1/27
 

1/49 3/46
 

0/49 1/46
 

1/49 0/46
 

13/49 9/48
 

0/49 0/48
 

0/49 2/48
 

0/49 1/48
 

0/59 1/57
 

0/59 1/57
 

2/59 1/57
 

80
 
Table A6. Other Lesions in Rats Administered
 

Direct Brown 95 in the Diet
 

(continued)
 

Tissue
 

Brain
 

Thymus
 

Adrenals
 

Bladder
 

Mesentery
 

Mesenteric
 

Lymph Node
 

Bronchial
 

Lymph Node
 

Mandibular
 

Lymph Node
 

Stomach
 

Small
 

Intestine
 

Lesion
 

Neuronal necrosis and
 

mineralization
 

Hemorrhage
 

Focal acute hemorrhage
 

Acute hemorrhage
 

Acute cortical congestion
 

Epithelial necrosis
 

Hemorrhage
 

Fat necrosis
 

Acute peritonitis
 

Acute hemorrhage
 

Lymphodenitis
 

Acute hemorrhage
 

Acute hemorrhage
 

Epithelial necrosis
 

Acute focal hemorrhage
 

Congestion
 

Control
 

Groups
 

Male Female
 

0/10 1/10
 

0/10 0/10
 

0/10 1/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

Combined
 

Dose Groupsa
 

Male Female
 

0/29 0/28
 

1/29 0/28
 

0/29 0/24
 

7/29 5/28
 

3/29 7/28
 

0/29 2/28
 

0/29 3/28
 

1/29 2/27
 

0/29 1/27
 

1/29 0/27
 

0/29 1/27
 

5/29 1/28
 

6/29 3/28
 

0/26 1/29
 

4/26 1/29
 

1/29 0/26
 

81
 
Table A6. Other Lesions in Rats Administered
 

Direct Brown 95 in the Diet
 

(continued) 

Tissue Lesion 

Control 

Groups 

Male Female 

Combined 

Dose Groupsa 

Male Female 

Colon Nematodiasis 1/10 1/10 0/29 0/26 

Epididymis Interstitial hemorrhage 0/10 1/29 

a

For lesions of the liver, kidney, or spleen, the combined dose groups
 

consisted of all dosed groups. For lesions of the other tissues given
 

in the table, the combined dose groups consisted of 750 ppm, 1,300 ppm,
 

and 3,000 ppm dose groups.
 

82
 
APPENDIX B
 

LESIONS IN MICE ADMINISTERED DIRECT DYES
 

IN THE DIET
 

83
 

Table Bl. Lesions Observed Only in Mice Administered Direct Blue 6 

in the Diet 

Tissue/Lesion 

Liver 

750 ppm

Male Female

 1,500 ppm

 Male Female

 3,000 ppm

 Male Female

 6,000 ppm

 Male Female

 12,500 ppm 

 Male Female 

oo

Pigment deposition 

Spleen 

Hemosiderosis 

Kidney 

 Hemosiderosis 

a

a

a

 a

 a 

a

 a

3/10

 a

 a

 1/10

 a

 a a

 10/10 10/10

 a 

 0/10 0/9

 10/10 10/10

a a

 9/10 7/10 

 10/10 10/10 

 6/10 10/10 

a

Not examined. 
Tissue
 

Liver
 

Pancreas
 

Prostate
 

Stomach
 

Table B2. Other Lesions in Mice Administered
 

Direct Blue 6 in the Diet
 

Lesion
 

Focal dystrophic mineralization
 

Subacute hepatitis
 

Hepatic necrosis
 

Extramedullary hematopoiesis
 

Lymphocytic hepatitis
 

Acute hepatitis
 

Focal pancreatic necrosis
 

Interstitial prostatitis
 

Acute focal gastritis
 

Control
 

Groups
 

Male Female
 

1/10 0/10
 

0/10 2/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10 0/10
 

0/10
 

0/10 0/10
 

Combined
 

Dose Groups3
 

Male Female
 

0/20 0/19
 

3/20 5/19
 

1/20 0/19
 

0/20 5/19
 

1/20 0/19
 

1/20 0/19
 

1/10 0/10
 

1/10
 

0/10 1/10
 

a

For lesions of the liver, the combined dose groups consisted of the 6,000
 

ppm and the 12,500 ppm dose groups. For lesions of the other tissues given
 

in the table, only the 12,500 ppm dose groups were examined.
 

86
 
Table B3. Lesions Observed Only in Mice Administered Direct Black 38 

in the Diet 

750 ppm 1,500 ppm 3,000 ppm 6,000 ppm 12,500 ppm 

Tissue/Lesion Male Female Male Female Male Female Male Female Male Female 

Liver 

Diffuse hepatocellular 

degeneration a 0/10 0/10 9/10 10/10 10/10 9/10 10/10 10/10 

Foci of cellular 

alteration a 0/10 0/10 0/10 0/10 1/10 0/10 2/10 1/10 

Biliary hyperplasia a a 0/10 0/10 0/10 0/10 0/10 0/10 9/10 10/10 

Pigment deposition a a 0/10 0/10 1/10 1/10 10/10 10/10 10/10 10/10 

Spleen 

Hemosiderosis 1/10 1/10 4/10 9/10 10/10 10/10 10/10 10/10 10/10 10/10 

Kidney 

Hemosiderosis a a a a 0/10 0/10 10/10 10/10 10/10 10/10 

Thyroid 

Pigment deposition a a a a 0/10 0/8 8/10 10/10 8/9 9/10 

a

Not examined. 
Table B4. Other Lesions in Mice Administered 

Direct Black 38 in the Diet 

Control Combined 

Groups Dose Groups8 

Tissue Lesion Male Female Male Female 

Liver Subacute hepatitis 1/10 2/10 5/40 12/40 

Focal hepatocellular necrosis 0/10 0/10 0/40 1/40 

Acute hepatitis 0/10 0/10 1/40 4/40 

Focal granuloma 0/10 0/10 0/40 1/40 

Stomach Acute gastritia 0/10 0/10 3/10 0/10 

Cerebellum Focal lipodystrophy 0/10 0/10 0/10 1/10 

Lung Lymphoid hyperplasia 1/10 0/10 0/10 0/10 

Kidney Tubular regeneration 1/10 0/10 1/30 0/30 

Amyloidosis, focal 0/10 0/10 0/30 1/30 

Lymphocytic interstitial 

nephritis 0/10 0/10 2/30 0/30 

a

For lesions of the liver, the combined dose groups consisted of the 1,500
 

ppm, 3,000 ppm, 6,000 ppm, and 12,500 ppm dose groups. For lesions of the
 

kidney, the combined dose groups consisted of the 3,000 ppm, 6,000 ppm, and
 

12,500 ppm dose groups. For lesions of the remaining tissues given in the
 

table, only the 12,500 ppm dose groups were examined.
 

88
 
Table B5. Lesions Observed Only in Male Mice Administered Direct Brown 95
 

Tissue/Lesion
 

Liver
 

Pigment deposition
 

Biliary hyperplasia
 

Hepatocellular
 

degeneration
 

Foci of cellular alteration
 

Spleen
 

Hemosiderosis
 

Kidney
 

Hemosiderosis
 

a

Not examined.
 

750 ppm
 

0/1
 

0/1
 

0/1
 

0/1
 

7/10
 

0/1
 

in the Diet
 

1,500 ppm 3,000 ppm
 

a 1/10
 

a 0/10
 

a 0/10
 

a 0/10
 

10/10 10/10
 

a 0/10
 

6,000 ppm 12,500 ppm 

10/10 10/10 

0/10 3/10 

0/10 10/10 

0/10 1/10 

10/10 10/10 

10/10 10/10 
Table B6. Lesions Observed Only in Female Mice Administered Direct Brown 95
 

in the Diet
 

Tissue/Lesion 375 ppm 750 ppm 1,500 ppm 3,000 ppm 6,000 ppm 

Liver 

Pigment deposition a a 0/10 10/10 7/10 

Biliary hyperplasia a a 0/10 0/10 0/10 

Hepatocellular 

degeneration a a 0/10 0/10 0/10 

Foci of cellular alteration a a 0/10 0/10 0/10 

Spleen 

Hemosiderosis 5/9 10/10 10/10 10/10 10/10 

Kidney 

Hemosiderosis a a 0/10 0/10 9/10 

a

Not examined.
 
Table B7. Other Lesions in Mice Administered
 

Direct Brown 95 in the Diet
 

Tissue Lesion 

Control 

Groups 

Male Female 

Combined 

Dose Groups3 

Male Female 

Liver Subacute hepatitis 0/10 1/10 2/31 13/30 

Acute hepatitis 0/10 0/10 2/31 1/30 

Extramedullary hematopoiesis 0/10 0/10 1/31 1/30 

Hepatocellular necrosis 0/10 0/10 1/31 1/30 

Hepatocellular cytoplasmic 

vacuolization 0/10 0/10 1/31 1/30 

Focal granuloma 0/10 0/10 1/31 1/30 

Heart Myocardial degeneration 0/10 0/10 1/11 0/10 

Thymus Lymphoid depletion 0/10 0/10 1/11 0/10 

Salivary 

Gland Multiple abscesses 0/10 0/10 1/11 0/10 

Lymphocytic sialoadenitis 0/10 0/10 0/11 1/10 

Bone Marrow Granulocytic hyperplasia 0/10 0/10 1/11 0/10 

Submandibular 

Lymph Node Acute hemorrhage 0/10 0/10 0/11 1/10 

Kidney Lymphocytic interstitial 

nephritis 0/10 0/10 1/31 0/30 

Parathyroid Cyst 0/10 0/10 0/11 0/10 

91
 
Table B7. Other Lesions in Mice Administered
 

Direct Brown 95 in the Diet
 

:ontinued)
 

Control Combined
 

Groups Dose Groups3
 

Tissue Lesion Male Female Male Female
 

Testes Degeneration of germinal
 

epithelium 0/10 1/11
 

Pancreas Lymphocytic pancreatitis 0/10 0/10 2/11 0/10
 

a

For lesions of the liver or kidney, the combined dose groups consisted
 

of the 3,000 ppm, 6,000 ppm, and 12,500 ppm dose groups of the males and
 

the 1,500 ppm, 3,000 ppm, and 6,000 ppm dose groups of the females. For
 

lesions of other tissues given in the table, only the 12,500 ppm dose group
 

of males and the 6,000 ppm dose group of females were exmained.
 

92
 
APPENDIX C
 

ANALYSES OF THE INCIDENCES OF TUMORS OR FOCI
 

ALTERATIONS IN THE LIVER OF RATS FED DIRECT DYES
 

IN THE DIET
 

93
 

Table Cl. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Blue 6 in the Diet3
 

Matched 190 ppm 375 ppm 750 ppm 1,500 ppm
 

Compound Control Dose Dose Dose Dose
 

Liver; Hepatocellular
 

Carcinomab 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 2/10 (20)
 

P Values0 N.S.
 

Relative Riskd Infinite
 

Lower Limit 0.330
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — —
 4


vo
 

Liver: Hepatocellular Carcinoma
 

or Neoplastic Noduleb 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 8/10 (80)
 

P Values0 P < 0.001
 

Relative Riskd Infinite
 

Lower Limit 2.747
 

Upper Limit Infinite
 

Weeks to First Observed Tumor —
 4


3,000 ppm
 

Dose
 

0/9 (0)
 

—
 

—
 

1/9 (11)
 

N.S.
 

Infinite
 

0.064
 

Infinite
 

4 
Table Cl. Analyses of the Incidence of Tumors or Foci Alterations in the Liver 

of Male Rats Fed Direct Blue 6 in the Diet3 

(continued) 

Compound

Matched

 Control

 190 ppm 

 Dose 

375 ppm

Dose

 750 ppm 

 Dose 

1,500 ppm 

Dose 

3,000 ppm 

Dose 

Liver: Basophilic Focib 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (10) 1/10 (10)e 4/9 (44) 

P Values0 

— N.S. P = 0.033 

Realtive Risk^ 

Lower Limit 

Upper Limit 

Infinite 

0.058 

Infinite 

Infinite 

1.183 

Infinite 

Weeks to First Observed Lesion — — — — 4 4 

vo

°"

 Liver: Foci of Cellular Alteration 

 or Basophilic Focib 0/10 (0) 0/10 (0) 0/10 (0) 10/10 (100) 8/10 (80)f 4/9 (44) 

P Values0 P < 0.001 P < 0.001 P = 0.033 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

3.968 

Infinite 

Infinite 

2.747 

Infinite 

Infinite 

1.183 

Infinite 

Weeks to First Observed Lesion 13 4 4 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm. 

^Number of lesion-bearing animals/number of animals examined at site (percent). 
Table Cl. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Blue 6 in the Diet3
 

(continued)
 

c

Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test
 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not
 

significant (N.S.) is indicated.
 

95% confidence interval of the relative risk between each dosed group and the control group.
 

e

This animal was also reported to have a liver tumor.
 

of these animals were also reported to have liver tumors.
 

VO
 
Table C2. Analyses of the Incidence of Tumors or Foci Alterations in the Liver 

of Female Rats Fed Direct Blue 6 in the Diet3 

Compound

Matched

 Control

 190 ppm

 Dose

 375 ppm

 Dose

 750 ppm

 Dose

 1,500 ppm 

 Dose 

3,000 ppm 

Dose 

Liver: Hepatocellular 

Carcinomab 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 4/9 (44) 

P Values0 P = 0.033 

Relative Riskd 

Lower

Upper

 Limit 

 Limit 

Infinite 

1.183 

Infinite 

Weeks to First Observed Tumor — — — — — 5 

oo Liver: Hepatocellular Carcinoma 

or Neoplastic Noduleb 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 7/9 (77) 

P Values0 P = 0.001 

Relative Riskd 

Lower

Upper

 Limit 

 Limit 

Infinite 

2.590 

Infinite 

Weeks to First Observed Tumor — — — — — 5 
Table C2. Analyses of the Incidence of Tumors or Foci Alterations in the Liver 

of Female Rats Fed Direct Blue 6 in the Dieta 

( continued) 

Compound

Matched

 Control

 190 ppm 

 Dose 

375 ppm

Dose

 750 ppm 

 Dose 

1,500 ppm 

Dose 

3,000 ppm 

Dose 

Liver: Basophilic Focib

 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 7/9 (77)e 

P Valuesc 

— — P = 0.001 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

2.590 

Infinite 

Weeks to First Observed Lesion — — — — — 5 

VO 

Liver: Foci of Cellular Alteration 

or Basophilic Focib 0/10 (0) 0/10 (0) 0/10 (0) 9/10 (90) 10/10 (100) 7/9 (77)e 

P Values0 P < 0.001 P < 0.001 P = 0.001 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

3.265 

Infinite 

Infinite 

3.968 

Infinite 

Infinite 

2.590 

Infinite 

Weeks to First Observed Lesion — — 13 13 5 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm. 

^Number of lesion-bearing animals/number of animals examined at site (percent). 
Table C2. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Female Rats Fed Direct Blue 6 in the Diet3
 

(continued)
 

c

Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test
 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not
 

significant (N.S.) is indicated.
 

95% confidence interval of the relative risk between each dosed group and the control group.
 

e

Five of these animals were also reported to have liver tumors.
 

o
 

o
 
Table C3. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Black 38 in the Dieta
 

Matched 190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm
 

Compound Control Dose Dose Dose Dose Dose
 

Liver: Hepatocellular
 

Carcinomab 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 4/9 (44) 0/9 (0)
 

P Valuesc
 P = 0.033
 —
 d
 

Relative Risk Infinite
 

Lower Limit 1.183
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — — — — 5
 —
 

Liver: Hepatocellular Carcinoma
 

or Neoplastic Noduleb 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 9/9 (100) 0/9 (0)
 

P Values^ P < 0.001
 —
 d
 

Relative Risk Infinite
 

Lower Limit 3.895
 

Upper Limit Infinite
 

Weeks to First Observed Tumor — — — — 4
 —
 
Table C3. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Black 38 in the Diet3
 

(continued)
 

Matched 190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm
 

Compound Control Dose Dose Dose Dose Dose
 

e
 Liver: Basophilic Focib

 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 3/9 (33) 0/9 (0)
 

P Values0 N.S.
 — — —
 

Relative Riskd Infinite
 

Lower Limit 0.759
 

Upper Limit Infinite
 

Weeks to First Observed Lesion — — 5
 — — —
 

Liver: Foci of Cellular Alteration
 

or Basophilic Focib 0/10 (0) 0/10 (0) 7/10 (70) 9/10 (90) 5/9 (55)e 0/9 (0)
 

P Values0 P = 0.002 P < 0.001 P = 0.011
 —
 

Relative Riskd Infinite Infinite Infinite
 

Lower Limit 2.291 3.265 1.628
 

Upper Limit Infinite Infinite Infinite
 

Weeks to First Observed Lesion — — 13 13 5
 —
 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm.
 

"Number of lesion-bearing animals/number of animals examined at site (percent),
 
Table C3. Analyses of the Incidence of Tumors or Foci Alterations in the Liver 

of Male Rats Fed Direct Black 38 in the Dieta 

(continued) 

c

Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not 

significant (N.S.) is indicated. 

"The 95% confidence interval of the relative risk be.iv> ^en each dosed group and the control group. 

e

All of these animals were also reported to have liver tumors. 

o 

CO 
Table C4. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Female Rats Fed Direct Black 38 in the Diet3
 

Compound
 

Liver: Neoplastic Nodule^
 

P Values0
 

Relative Riskd
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Basophilic Focib
 

P Values0
 

Relative Riskd
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Lesion
 

Matched 190 ppm


Control Dose


0/10 (0) 0/10 (0)


0/10 (0) 0/10 (0)


—
 

—
 

 375 ppm 750 ppm 1,500 ppm 3,000 ppm 

 Dose Dose Dose Dose 

 0/10 (0) 0/10 (0) 5/10 (50) 0/8 (0) 

P = 0.016 — 

Infinite 

1.454 

Infinite 

13 — 

 0/9 (0) 0/10 (0) 0/10 (0) 1/8 (13) 

N.S. 

Infinite 

0.072 

Infinite 

5 
Table C4. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Female Rats Fed Direct Black 38 in the Dieta
 

(continued)
 

Matched 190 ppm 375 ppm 750 ppm 1,500 ppm 3,000 ppm
 

Compound Control Dose Dose Dose Dose Dose
 

Liver: Foci of Cellular Alteration
 

or Basophilic Focib 0/10 (0) 0/10 (0) 5/9 (56) 10/10 (100) 10/10 (100) 1/8 (13)
 

P Values0 P = 0.011 P < 0.001 P < 0.001 N.S.
 

Relative Riskd Infinite Infinite Infinite Infinite
 

Lower Limit 1.628 3.968 3.968 0.072
 

Upper Limit Infinite Infinite Infinite Infinite
 

o
 Weeks to First Observed Lesion — 13 13 13
 Ul
 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm.
 

^Number of lesion-bearing animals/number of animals examined at site (percent).
 

°Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test
 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not
 

significant (N.S.) is indicated.
 

"The 95% confidence interval of the relative risk between each dosed group and the control group.
 

5 
Table C5. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Brown 95 in the Diet3
 

Compound

Matched

 Control

 190 ppm 

 Dose 

375 ppm 

Dose 

750 ppm 

Dose 

Liver: Basophilic Focib 0/10 (0) 0/10 (0) 0/10 (0) 3/10 (30) 

P Values0 

— N.S. 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

0.681 

Infinite 

Weeks to First Observed Lesion — — — 4 

Liver: Foci of Cellular 

Alteration or Basophilic Focib 0/10 (0) 0/10 (0) 10/10 (100) 5/10 (50) 

P Values0 P < 0.001 P = 0.016 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

3.968 

Infinite 

Infinite 

1.454 

Infinite 

Weeks to First Observed Lesion — — 14 4 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm.
 

"Number of lesion-bearing animals/number of animals examined at site (percent).
 

1,500 ppm
 

Dose
 

7/8 (88)
 

P < 0.001
 

Infinite
 

3.007
 

Infinite
 

4
 

7/8 (88)
 

P < 0.001
 

Infinite
 

3.007
 

Infinite
 

4
 

3,000 ppm
 

Dose
 

2/9 (22)
 

N.S.
 

Infinite
 

0.368
 

Infinite
 

4
 

2/9 (22)
 

N.S.
 

Infinite
 

0.368
 

Infinite
 

4
 
Table C5. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Male Rats Fed Direct Brown 95 in the Diet3
 

(continued)
 

c

Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test
 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not
 

significant (N.S.) is indicated.
 

95% confidence interval of the relative risk between each dosed group and the control group.
 

o
 

•-J
 
Table C6. Analyses of the Incidence of Tumors or Foci Alterations in the Liver 

of Female Rats Fed Direct Brown 95 in the Diet3 

Compound

Matched

 Control

 190 ppm 

 Dose 

375 ppm

Dose

 750 ppm

 Dose

 1,500 ppm 

 Dose 

3,000 ppm 

Dose 

Liver: Hepatocellular 

Carcinomab 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 1/8 (13) 0/8 (0) 

P Values0 N.S. — 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

0.072 

Infinite 

Weeks to First Observed Tumor — — 5 — 

00 Liver: Hepatocellular Carcinoma 

or Neoplastic Noduleb 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 5/8 (63) 0/8 (0) 

P Values0 P = 0.007 — 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

1.851 

Infinite 

Weeks to First Observed Tumor — 5 — 
Table C6. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Female Rats Fed Direct Brown 95 in the Dieta
 

(continued) 

Compound

Matched

 Control

 190 ppm 

 Dose 

375 ppm 

Dose 

750 ppm 

Dose 

Liver: Basophilic Focib 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 

P Valuesc — — 

Relative Riskd 

Lower Limit 

Upper Limit 

Weeks to First Observed Lesion — — — — 

Liver: Foci of Cellular Alterati

or Basophilic Focib

on 

 0/10 (0) 0/10 (0) 3/10 (30) 3/10 (30) 

P Values0 N.S. N.S. 

Relative Riskd 

Lower Limit 

Upper Limit 

Infinite 

0.681 

Infinite 

Infinite 

0.681 

Infinite 

Weeks to First Observed Lesion — — 13 13 

a

Dosed groups received 190, 375, 750, 1,500, or 3,000 ppm.
 

"Number of lesion—bearing animals/number of animals examined at site (percent),
 

1,500 ppm
 

Dose
 

3/8 (38)e
 

N.S.
 

Infinite
 

0.858
 

Infinite
 

5
 

6/8 (75)f
 

P = 0.002
 

Infinite
 

2.397
 

Infinite
 

5
 

3,000 ppm
 

Dose
 

3/8 (38)
 

N.S.
 

Infinite
 

0.858
 

Infinite
 

5
 

3/8 (38)
 

N.S.
 

Infinite
 

0.858
 

Infinite
 

5
 
Table C6. Analyses of the Incidence of Tumors or Foci Alterations in the Liver
 

of Female Rats Fed Direct Brown 95 in the Dieta
 

(continued)
 

c

Beneath the incidence of lesions in a dosed group is the probability level for the Fisher exact test
 

for the comparison of that dosed group with the matched-control group when P < 0.05; otherwise, not
 

significant (N. S.) is indicated.
 

95% confidence interval of the relative risk between each dosed group and the control group.
 

e

Two of these animals were also reported to have liver tumors.
 

*Four of these animals were also reported to have liver tumors.
 
APPENDIX D
 

BENZIDINE IN THE URINE OF RATS AND MICE FED
 

DIRECT DYES
 

111
 

APPENDIX D
 

Benzidine in the Urine of Rats and Mice Fed Direct Dyes
 

The purpose of this study was to determine whether mice and rats
 

fed diets containing direct blue 6, direct black 38, or direct
 

brown 95 metabolically reduced these compounds to benzidine.
 

These azo dyes are benzidine derivatives and have been shown to
 

be reduced to benzidine in rhesus monkeys (Rinde and Troll,
 

1975).
 

Materials and Methods
 

Urine was collected from three rats of each sex and three mice of
 

each sex at each of three dose levels of each of the three dyes
 

and from associated untreated controls during weeks 4 and 12
 

(rats) and weeks 3 and 11 (mice) of the 13-week subchronic
 

toxicity studies. The intent was to collect urine from the three
 

highest dose groups; however, lower dose groups, as indicated in
 

table Dl, had to be used because of mortality in the higher dose
 

groups. Animals were placed for 24 hours in metabolism cages.
 

The collection vessels for these cages each contained 2.0 ml of
 

0.1 N_ HCl added as a preservative. Animals were fasted during
 

urine collection, but water was available ad libitum. Volumes of
 

urine collected were recorded, and samples were stored frozen at
 

-20°C in the dark until analysis.
 

113
 
The benzidine assay procedure was a modification of the method of
 

Rinde and Troll (1975, 1976). To 5.0 ml of urine was added 0.5
 

ml of sodium citrate buffer, l.OM, pH 5.0. To each tube 2 ml of
 

chloroform was added, and the mixture was shaken by hand for 1
 

minute. After complete phase separation, 1.7 ml of the organic
 

layer was transfered to another test tube containing 2.0 ml of
 

0.01 _N HCl. This mixture was shaken for 1 minute by hand. A
 

1.6-ml aliquot of the aqueous layer was transfered to another
 

test tube containing 0.5 ml of 1.0 M citrate buffer, pH 5.0. A
 

0.2-ml volume of chloroform was added to each test tube. The
 

mixture was shaken, and the upper (aqueous) layer was withdrawn
 

and discarded.
 /
 

A 100-/J1 portion of the chloroform layer was then spotted onto
 

silica gel thin-layer chromatography (tic) plates (Quantum
 

Industries, LQ6DF plates). The plates were developed in
 

chloroform-ethanol (9:1). After drying in a fume hood in the
 

dark, each plate was sprayed with Fluram reagent (1.0 mg/ml
 

fluorescamine in glacial acetic acid). Yellow spots were scraped
 

from the plates, and the yellow color eluted with methanol (1.0
 

ml).
 

Each sample was centrifuged, transfered to cuvettes, and read at
 

415 nanometers, using methanol as blank. Benzidine standards
 

(Nanogen, Watsonville, California) were routinely used as a
 

114
 
reference for Rf values and for determining and validating
 

standard curves.
 

Mass spectral analyses were run on pooled samples, both from rats
 

and mice. For these samples, spots on tic plates were visualized
 

by ultraviolet light (rather than spray reagent), scraped, and
 

extracted in chloroform. Each sample was then subjected to
 

direct probe mass spectral analysis, on a Finnigan 4000 Mass
 

Spectrometer, using the chemical ionization technique.
 

Results and Discussion
 

A benzidine standard curve (absorbance vs. figs of benzidine
 

spotted) was constructed by spotting benzidine (0.2 - 8.0 ^g) in
 

duplicate onto tic plates and proceeding as described in the
 

previous section.
 

All urine samples from dosed animals and all standards contained
 

sufficient benzidine so that Fluram spraying produced a yellow
 

spot detectable to the naked eye at Rf 0.82. Most samples from
 

dosed animals also produced a second spot on the tic plates at Rf
 

0.68. None of the samples from untreated-control rats or mice
 

contained substances which produced color on tic plates following
 

Fluram spraying.
 

Tables Dl and D2 present the 24-hour excretion of benzidine for
 

115
 
rats and mice, respectively. Each value represents a mean of
 

three samples, except where indicated, with one rat or three mice
 

per sample. Standard deviations are presented in parentheses.
 

There are large standard deviations associated with many of the
 

dosed groups. For the lowest dose groups, the lower limits of
 

sensitivity for the quantitation of benzidine were approached.
 

The tic spot with Rf 0.68 was not quantitated for lack of
 

standard.
 

The data in tables Dl and D2 clearly demonstrate that benzidine
 

is excreted in the urine by rats and mice dosed with direct blue
 

6, direct black 38, or direct brown 95. The quantity of
 

benzidine excreted generally increases with increasing dietary
 

concentration of the direct dye, although the values for rats at
 

high doses are unexpectedly low at 12 weeks. Mice dosed with
 

direct black 38 excreted somewhat more benzidine than mice dosed
 

with the other dyes, but no difference among the dyes was
 

apparent in rats. There are no consistent differences between
 

males and females in either rats or mice.
 

These data cannot be explained by possible residual benzidine in
 

the dyes. No benzidine was detected in any of the dyes, with a
 

detection limit of 0.004%. Even if it is assumed that the dyes
 

did contain 0.004% benzidine and that all of it is excreted in
 

the urine (Rinde and Troll, 1975, report finding 1.45% of an oral
 

116
 
dose of benzidine in the urine in rhesus monkeys), the levels
 

found in this study in most cases exceed the maximum possible
 

from residual benzidine in the dyes. In addition, the portion of
 

benzidine excreted as the monoacetyl derivative was not quantitated and would increase the values reported in tables 1 and 2.
 

The identities of the Rf 0.82 and 0.68 tic spots were confirmed
 

by chemical ionization mass spectrometry. From mice and from
 

rats, the chemical at Rf 0.82, reported above as benzidine, was
 

verified as benzidine (parent and base peak at m/e 185,
 

corresponding to protonated benzidine). The chemical at Rf 0.68
 

was identified as monoacetylbenzidine (base peak at m/e 227,
 

corresponding to the protonated form; correct fragmentation
 

pattern); Rinde and Troll (1975) identified this derivative as a
 

second metabolite of these same three dyes in rhesus monkeys.
 

117
 
Table Dl. Benzidine Excretion (0g/24 hr) Per Rata
 

Dye Weeks on Diet 

Dietary 4 12 

Concentration, ppm Male Female Male Female 

Direct Blue 6 

3,000 or l,50Qb 5.8 (0.9)c

 8.0 (6.7) 0.77 (0.65) 0.55 (0.29) 

750 1.4 (0.8) 0.94 (0.27) 0.32 (0.10) 0.29 (0.18) 

190 0.85 (0.18) 0.62 (0.17) 0.44 (0.41) 0.16 (0.10) 

Direct Black 38 

1,500 3. 6 (4.8) 16. 8 (n=2) 0.16 (0.03) 0. 31 (0.16) 

750 1. 7 (n=2) 2. 1 (0.06) 0.46 (0.09) 1. 4 (0.35) 

190 0.55 (0.3D 0.44 (0.13) 0.49 (0.39) 0.43 (0.32) 

Direct Brown 95 

750 4. 2 (1. 3) 3. 7 (2.9) 0.44 (0.12) 1. 1 (n=l) 

375 

190 

1. 

0. 

0 

80 

(0.77) 

(n=2) 

4. 

0. 

2 

66 

(1. 

(0. 

3) — 

24) 0.29 (0.11) 

5. 

0. 

8 

27 

(9.7) 

(0.05) 

a

Samples from untreated controls taken at weeks 4 and 12 showed
 

no benzidine when spotted on tic plates.
 

^Female rats at week 4 were from the 3,000-ppm group, male rats at
 

week 4 and both males and females at week 12 were from the 1,500-ppm
 

group.
 

c

Numbers in parentheses are standard deviations. If fewer than three
 

samples were averaged, the number of samples is given in parentheses
 

instead.
 

118
 
Table D2. Benzidine Excretion (Mg/24 hr) Per Mousea
 

Dye Weeks on Diet 

Dietary 11 

Concentration, ppm Male Female Male Female 

Direct Blue 6 

12,500 5.2 (0.85)b 5.1 (n=2) 2.4 (1.3) 5.5 (1.0) 

3,000 0.97 (0.32) 1.1 (0.35) 1.7 (0.62) 3.1 (0.94) 

750 0.55 (0.65) 0.31 (0.023) 1.1 (0.72) 0.52 (0.17) 

Direct Black 38 

12,500 12.8 (2.8) 6.08 (1.8) 14.4 (2.7) 8.6 (1.0) 

3,000 3.5 (2.1) 7.3 (n=2) 7.3 (2.2) 7.4 (1.7) 

750 3.6 (3.4) 3.0 (2.7) 2.8 (3.2) 2.0 (1.8) 

Direct Brown 95 

12,500 9.4 (n=2) 7.5 (0.90) 

3,000 4.7 (0.93) 1.1 (0.20) 

750 0.39 (0.09) 0.49 (0.23) 

6,000 3.5 (1.8) 3.2 (0.59) 

1,500 2.8 (0.85) 0.35 (0.12) 

375 0.56 (0.19) 0.19 (0.12) 

a

Samples from untreated controls taken at weeks 3 and 11 showed no
 

benzidine when spotted on tic plates.
 

^Numbers in parentheses are standard deviations. If fewer than three
 

samples were averaged, the number of samples is given in parentheses
 

instead.
 

119
 

APPENDIX E
 

MOLECULAR STRUCTURES OF DIRECT BLUE 6,
 

DIRECT BLACK 38, AND DIRECT BROWN 95
 

121
 

APPENDIX E 

MOLECULAR STRUCTURES OF DIRECT BLUE 6, 

DIRECT BLACK 38, AND DIRECT BROWN 95 

NH2 OH OH NH2 

Na03S S03Na 

DIRECT BLUE 6 C

32H20°14N6Na4S4 

NH2

NaO,S

0 

DIRECT BLACK 38 

 OH 

N=N 

v

S03Na 

C

34H25N9°7S2Na2 

NaOOC 

HO N=N 

DIRECT BROWN 95 

S03Na 

c

31H2N6°9SNa2 -Cu complex 

Copper complexed at * 

123 

Review of the Bioassay of Direct Blue 6, Direct Black 38,
 and Direct Brown 95 Dyes*for Carcinogenicity by the
 

Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

March 6, 1978
 

The Clearinghouse on Environmental Carcinogens was
 

established in May, 1976, in compliance with DHEW Committee
 

Regulations and the Provisions of the Federal Advisory
 

Committee Act. The purpose of the Clearinghouse is to
 

advise the Director of the National Cancer Institute (NCI)
 

on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be
 

exposed. The members of the Clearinghouse have been drawn
 

from academia, industry, organized labor, public interest
 

groups, State health officials, and quasi-public health
 

and research organizations. Members have been selected on
 

the basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in laboratory
 

animal sciences, chemistry, biochemistry, biostatistics,
 toxicology, pathology, and epidemiology. Representatives
 

of various Governmental agencies participate as ad hoc
 

members. The Data Evaluation/Risk Assessment Subgroup of
 

the Clearinghouse is charged with the responsibility of
 

providing a peer review of reports prepared on NCI-sponsored
 

bioassays of chemicals studied for carcinogenicity. It is
 

In this context that the below critique is given on the
 

bioassay of Direct Blue 6, Direct Black 38, and Direct
 

Brown 95 dyes for carcinogenicity.
 

The primary reviewer agreed with the conclusions given
 

in the report that Direct Blue 6 and Direct Black 38 were
 

carcinogenic in both sexes of the Fischer 3^4 rat and Direct
 

Brown 95 was carcinogenic In the female rat, under the
 

conditions of test. He described the experimental design
 

employed during the 13-week subchronic study and noted that
 

the technical grade dyes contained unidentified impurities.
 Despite the fact that only 10 animals of each sex were used
 

in each treatment group, given the demonstrated carcino­ genicity of the tested materials, it did not compromise the
 

study. Determinations of methemoglobin and urinary benzidine
 

levels and the well-conducted pathology supported the con­ fidence that could be given to the study. The primary
 

reviewer concluded that the tested materials posed a potential
 

carcinogenic risk to humans.
 

125
 
The secondary reviewer expressed concern regarding
 

the high percent of impurities contained in the tested
 

materials. He agreed with the primary reviewer, however,
 that the dyes were strong hepatic carcinogens in the rat.
 

With respect to the impurities, a Program staff member
 

noted that each batch of the tested material was analyzed
 

for free benzidine and benzidine salt. Since none was
 

found, it was concluded that the urinary benzidine in the
 

treated animals was a metabolic product of the dyes. A
 

Subgroup member pointed out that the majority of the
 

impurity was probably sodium chloride, based on analyses
 

given in the report. The secondary reviewer agreed that
 

the significance of the impurity was diminished if, in
 

fact, most of it was salt.
 

A Program staff member said that there were no plans
 

to initiate a 2-year chronic study on the dye materials,
 

since all of them already have been shown to be carcinogenic
 

A Subgroup member commented that he would expect a chronic
 

study to last no more than six or nine months based on
 

the demonstrated carcinogenicity of the dyes in the 13-week
 

test. He added that a smaller number than the standard 50
 

animals per test group could be used and suggested that
 

parallel studies be conducted using dyes of different
 

purities. Given the Program's mission and the fact that
 

benzidine was likely the carcinogenic metabolite, it was
 

felt that an additional study was not necessary.
 

A staff pathologist showed slides of the liver tumors
 

induced in the treated rats. He described the histological
 

changes and indicated the nomenclature applied to the
 

pathology. Although some tumors appeared to invade or
 

push into the wall of veins, no metastases were observed.
 

He attributed the lack of metastases to the short study
 

period.
 

A motion was made that the report on the bioassay of
 

the direct dyes be accepted as written. The motion was
 

seconded and approved unanimously.
 

Members present were
 

Gerald N. Wogan (Chairman), Massachusetts Institute of
 

Technology
 

Arnold Brown, Mayo Clinic
 

Lawrence Garfinkel, American Cancer Society
 

E. Cuyler Hammond, American Cancer Society
 

Joseph Highland, Environmental Defense Fund
 

126
 
Henry Pitot, University of Wisconsin Medical Center
 

George Roush, Jr., Monsanto Company
 

Sheldon Samuels, Industrial Union Department, AFL-CIO
 

Michael Shimkin, University of California at San Diego
 

John Weisburger, American Health Foundation
 

Sidney Wolfe, Health Research Group
 

Subsequent to this review, changes may have been made
 

in the bioassay report either as a result of the review
 

or other reasons. Thus, certain comments and criticisms
 

reflected in the review may no longer be appropriate.
 

127
 

DREW Publication No. (NIH) 78-1358