1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 134 

BIOASSAY OF 

C. I. VAT YELLOW 4 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 128-66-5 

NCI-CG-TR-134 

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

Public Health Service 

National Institutes of Health 

BIO AS SAY OF
 

C. I. VAT YELLOW 4
 

FOR POSSIBLE CARCINOGENICITY
 

Careinogenesis 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) 79-1389
 
ii

 
BIOASSAY OF
 

C. I. VAT YELLOW 4
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD; This report presents the results of the bioassay of
 

C.I. vat yellow 4 conducted for the Carcinogenesis Testing
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

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

Maryland. This is one of a series of experiments 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 chemicals found to be carcinogenic to animals requires
 

a wider analysis.
 

CONTRIBUTORS; This bioassay of C. I. vat yellow 4 was conducted
 

at the NCI Frederick Cancer Research Center (FCRC) (1),
 

Frederick, Maryland, operated for NCI (2) by Litton Bionetics,
 

Inc. (3), Kensington, Maryland.
 

The manager of the bioassay at FCRC was Dr. B. Ulland, the
 

toxicologist was Dr. E. Gordon, and Drs. R. Cardy and D. Creasia
 

compiled the data. Ms. S. Toms was responsible for management of
 

data, Mr. D. Cameron for management of histopathology, Mr. L.
 

Callahan for management of the computer branch, and Mr. R. Cypher
 

for management of the facilities. Mr. A. Butler performed
 

computer services. Histopathologic evaluations were performed by
 

Dr. C. E. Gilmore (4), and the diagnoses included in this report
 

represent his interpretation.
 

Animal pathology tables and survival tables were compiled at EG&G
 

Mason Research Institute (5). Statistical analyses were
 

performed by Dr. J. R. Joiner (6) and Ms. P. L. Yong (6), using
 

methods selected for the bioassay program by Dr. J. J. Gart (7).
 

The chemicals used in this bioassay were analyzed at FCRC by Dr.
 

ill
 
W. Zielinsky (1). The results of these analyses were reviewed by
 

Dr. C. W. Jameson (6).
 

This report was prepared at Tracor Jitco (6) under the direction
 

of NCI. Those responsible for the report at Tracor 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,
 

Ms. M. S. King, 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 following scientists at NCI (2) were responsible for
 

evaluating the bioassay, interpreting the results, and reporting
 

the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cue to, Jr., Dr.
 

J. Fielding Douglas, Dr. Thomas E. Hamm, Dr. William V. Hartwell,
 

Dr. Richard A. Griesemer, Dr. Morton H. Levitt, Dr. Harry A.
 

Milman, Dr. Thomas W. Orme, Dr. Sherman F. Stinson, Dr. Jerrold
 

M. Ward, and Dr. Carrie E. Whitmire.
 

(1) Frederick Cancer Research Center, P.O. Box B, Frederick,
 

Maryland.
 

(2) Careinogenesis Testing Program, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

(3) Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington,
 

Maryland.
 

(4) Experimental Pathology Laboratories, Inc., P.O. Box 474,
 

Herndon, Virginia.
 

(5) EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

(6) Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,
 

Maryland.
 

(7) 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.
 

IV
 
SUMMARY

 

A bioassay of C. I. vat yellow 4, a commercial formulation 

containing dibenzo(b,def)chrysene-7,l4-dione, for possible 

carcinogenicity was conducted by administering the test chemical 

in feed to Fischer 344 rats and B6C3F1 mice. 

Groups of 50 rats of each sex and 50 mice of each sex were 

administered C. I. vat yellow 4 in the diet at one of two doses, 

either 3,500 or 7,000 ppm for the rats, either 25,000 or 50,000 

ppm for the male mice, and either 12,500 or 25,000 ppm for the 

female mice. The rats were administered the test chemical for 

104 weeks; the mice, for 106 weeks. Matched controls consisted 

of 20 untreated rats and 20 untreated mice of each sex. All 

surviving animals were killed at the end of the period of 

administration of the test chemical. 

Mean body weights of the dosed rats were lower than those of 

corresponding controls throughout the bioassay, but the 

differences in weights were slight for the males. Mean body 

weights of the dosed mice were not affected by the test 

chemical. Survival of the rats and mice was not affected 

adversely by the chemical, and sufficient numbers of dosed and 

control rats and mice of each sex were at risk for the 

development of late-appearing tumors. 

In the male and female rats and the female mice, no tumors 

occurred at incidences that were significantly higher in dosed 

groups than in control groups. 

In the male mice, lymphomas occurred at incidences that were dose 

related (P = 0.002), and, in a direct comparison, the incidence 

of the tumor in the high-dose group was significantly higher (P = 

0.019) than that in the control group (controls 3/20, or 15%; 

low-dose 7/47, or 15%; high-dose 22/50, or 44%). The incidence 

of lymphomas and leukemias in historical-control maie B6C3F1 mice 

at this laboratory was 38/323 (12%). 

It is concluded that under the conditions of this bioassay, the 

formulated product containing C. I. vat yellow 4 was not 

carcinogenic for male or female Fischer 344 rats or for female 

B6C3F1 mice, but was carcinogenic for male B6C3F1 mice, causing 

an increased incidence of lymphomas. 
vi

 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. An imals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies 7
 

F. Chronic Studies 10
 

G. Clinical and Pathologic Examinations 13
 

H. Data Recording and Statistical Analyses 14
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 23
 

D. Statistical Analyses of Results (Rats) 25
 

IV. Results - Mice 27
 

A. Body Weights and Clinical Signs (Mice) 27
 

B. Survival (Mice) 27
 

C. Pathology (Mice) 29
 

D. Statistical Analyses of Results (Mice) 32
 

V. Discussion 37
 

VI. Bibliography 41
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in Rats
 

Administered C. I. Vat Yellow 4 in the Diet... 43
 

Table Al Summary of the Incidence of Neoplasms in Male
 

Rats Administered C. I. Vat Yellow 4 in the
 

Diet 45
 

vii
 
Table A2 Summary of the Incidence of Neoplasms in Female 

Rats Administered C. I. Vat Yellow 4 in the 

Diet 49 

Appendix B Summary of the Incidence of Neoplasms in Mice 

Administered C. I. Vat Yellow 4 in the 

Diet 53 

Table Bl Summary of the Incidence of Neoplasms in Male 

Mice Administered C. I. Vat Yellow 4 in the 

Diet 55 

Table B2 Summary of the Incidence of Neoplasms in Female 

Mice Administered C. I. Vat Yellow 4 in the 

Diet 59 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered C. I. Vat Yellow 4 

in the Diet 63 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered C. I. Vat 

Yellow 4 in the Diet 65 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered 

C. I. Vat Yellow 4 in the Diet 68 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered C. I. Vat Yellow 4 

in the Diet 73 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered C. I. Vat 

Yellow 4 in the Diet 75 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered C. I. Vat 

Yellow 4 in the Diet 78 

Appendix E Analyses of the Incidence of Primary Tumors in 

Rats Administered C. I. Vat Yellow 4 in 

the Diet 81 

Vlii 
Table El
 

Table E2
 

Appendix F
 

Table Fl
 

Table F2
 

Table 1
 

Table 2
 

Table 3
 

Figure 1
 

Figure 2
 

Figure 3
 

Figure 4
 

Figure 5
 

Analyses of the Incidence of Primary Tumors in 

Male Rats Administered C. I. Vat Yellow 4 in 

the Diet 83 

Analyses of the Incidence of Primary Tumors in 

Female Rats Administered C. I. Vat Yellow 4 in 

the Diet 90 

Analyses of the Incidence of Primary Tumors in 

Mice Administered C. I. Vat Yellow 4 in 

the Diet 95 

Analyses of the Incidence of Primary Tumors in 

Male Mice Administered C. I. Vat Yellow 4 in 

the Diet 97 

Analyses of the Incidence of Primary Tumors in 

Female Mice Administered C. I. Vat Yellow 4 in 

the Diet 101 

TABLES 

C. I. Vat Yellow 4 Subchronic Feeding Studies 

in Rats and Mice 9 

C. I. Vat Yellow 4 Chronic Feeding Studies 

in Rats 11 

C. I. Vat Yellow 4 Chronic Feeding Studies 

in Mice 12 

FIGURES 

Growth Curves for Rats Administered C. I. Vat 

Yellow 4 in the Diet 22 

Survival Curves for Rats Administered C. I. Vat 

Yellow 4 in the Diet 24 

Growth Curves for Mice Administered C. I. Vat 

Yellow 4 in the Diet 28 

Survival Curves for Mice Administered C. I. Vat 

Yellow 4 in the Diet 30 

Life Table for Male Mice Administered C. I. Vat 

Yellow 4 in the Diet: Incidence of Lymphoma .. 34
 

ix
 

I. INTRODUCTION
 

C. I. vat yellow 4 (CAS 128-66-5; NCI C03565) is the common name
 

adopted in the Colour Index (Society of Dyers and Colourists,
 

1971) for a commercial formulation that consists in part of
 

dibenzo(b,def)chrysene-7,14-dione, the color-imparting component
 

(Owens and Ward, 1974). This chemical has an anthraquinoid
 

structure. In the dye process, the keto groups are reduced to
 

the hydroxyl level of oxidation, which allows the dye to
 

penetrate the fiber of the material being dyed. The dye is then
 

fixed by oxidation back to the keto state, reforming the original
 

insoluble dyestuff. C. I. vat yellow 4 is used on cellulose
 

fibers, on some cellulose synthetics such as cellulose acetate
 

and cellulose polyester, and on wool and silk. It is also used
 

as a pigment for paper.
 

C. I. vat yellow 4 is used by the armed services as a smokescreen
 

and as a signaling agent (Owens and Ward, 1974). Smoke dyes were
 

effectively used to mask the movement of troops in World War II
 

and in Korea, where in some instances men were exposed to
 

chemical smokescreens for several months (Puro, 1964). Although
 

there are no production data on C. I. vat yellow 4, the vat dyes
 
as a class achieved a production volume of 1,254,000 pounds in
 

1976 (USITC, 1977).
 

This chemical was selected for study in the Carcinogenesis
 

Testing Program because these uses indicated that instances of
 

significant long-term human exposure may exist.
 
II. MATERIALS AND METHODS
 

A. Chemical
 

The commercial formulated material used in the bioassay was 

obtained in a single batch from the American Aniline Products, 

Inc., Philadelphia, Pennsylvania. This material was manufactured 

by the American Hoechst Corporation in Somerville, New Jersey, as 

a formulated product. The specifications from the manufacturer 

required 18.2% of the color-imparting component, 30.8% sorbitol, 

5.5% dispersant (Lomar TWC), 2.7% glycerine, and 42.8% water. 

The test material was stored at 5 C until used. 

B. Dietary Preparation 

Test diets containing C.I. vat yellow 4 were prepared every 1 to 

1-1/2 weeks in 6- to 12-kg batches at appropriate doses. A known 

weight of the formulated chemical was first mixed with an equal 

weight of autoe laved, powdered Wayne Sterilizable Lab Meal 

containing 4% fat (Allied Mills, Inc., Chicago, 111.), using a 

mortar and pestle. The mixing was continued with second and 
third additions of feed, and final mixing was performed with the 

remaining quantity of feed for a minimum of 15 minutes in a 

Patterson-Kelly twin-shell blender. The diets were routinely 

stored at 7 C until used. 

No homogeneity or stability studies of the C.I. vat yellow 4 feed 

mixtures were performed. 

C. Animals 

Male and female Fischer 344 rats and B6C3F1 mice were obtained
 

through a National Cancer Institute contract from the Frederick
 

Cancer Research Center Animal Farm (Frederick, Md.) as 4-week-old
 

weanlings, all within 3 days of the same age. The animals were
 

housed within the test facility for 2 weeks and then were
 

assigned four rats to a cage and five mice to a cage by a
 

procedure that gave the same average weights per cage for each
 

cage of animals of a given species and sex. For use in the
 

chronic study, the male rats were required to weigh 90 to 105 g,
 

averaging at least 100 g; the female rats, 80 to 95 g, averaging
 

at least 90 g; the male mice, 18 to 22 g, averaging at least 19.5
 

g; and the female mice, 17 to 21 g, averaging at least 18.5 g.
 

Individual animals were identified by ear punch.
 
D. Animal Maintenance
 

The animals were housed in polycarbonate cages (Lab Products,
 

Inc., Garfield, N. J.), 19 x 10-1/2 x 8 inches for the rats and
 

11-1/2 x 7-1/2 x 5 inches for the mice. The cages were suspended
 

from aluminum racks (Scientific Cages, Inc., Bryan, Tex.) and
 

were covered by nonwoven polyester-fiber 12-mil-thick filter
 

paper (Hoeltge, Inc., Cincinnati, Ohio). The bedding used was
 

Absorb-dri

©)

 hardwood chips (Northeastern Products, Inc., 

Warrenburg, N. Y.). The feed supplied was Wayne Sterilizable 

Lab Meal, which was steam autoclaved for 20 minutes before mixing 

with the test compound. The feed was provided ad_ libitum in 

suspended stainless steel hoppers and replenished at least three 

times per week. Water, acidified to pH 2.5, was supplied ad_ 

libitum from glass bottles. Sipper tubes (Lab Products, Inc.) 

were suspended through the tops of the cages. 

The contaminated bedding was disposed of through an enclosed 

vacuum line that led to a holding tank from which the bedding was 

fed periodically into an incinerator. The cages were sanitized 

twice per week and the feed hoppers twice per month at 82 to 

88 c in a tunnel-type cagewasher (Industrial Washing Machine 

Corp., Mataway, N. J.), using the detergents Clout® (Pharmacal 

Research Laboratories, Greenwich, Conn.) or Oxford D'Chlor 
(Oxford Chemicals, Atlanta, Ga.). The water bottles were 

sanitized at 82 to 88 C in a tunnel-type bottle washer 

(Consolidated Equipment Supply Co., Mercersburg, Pa.) three times 

per week, using a Calgen Commercial Division detergent (St. 

Louis, Mo.). The racks for the cages were sanitized at or above 

82 C in a rack washer (Consolidated Equipment Supply Co.) once 

per month, using the Calgen Commercial Division detergent, and 

the filter paper was changed at the same time. 

The air in the animal rooms was maintained at a temperature of 22 

to 24 C and a relative humidity of 45 to 55%. Nonrec irculated 

air was passed through a filter of 65% efficiency and a bag 

filter of 95% efficiency at the intake and through "Z"-type 

roughing filter of 30% efficiency and a bag system of 90 to 95% 

efficiency at the exhaust (American Air Filters, Louisville, Ky.; 

Mine Safety Appliances, Pittsburgh, Pa.). The rate of movement 

allowed 15 changes of room air per hour. The air pressure was 

maintained negative to a clean hallway and positive to a return 

hallway. Fluorescent lighting was provided on an automatic 

12-hour-per-day cycle. 

Both control and dosed rats were housed in the same room as rats 

on feeding studies of the following chemicals: 
(CAS 298-00-0) methyl parathion 

(CAS 148-18-5) sodium diethyldithiocarbamate 

Both control and dosed mice were housed in the same room as mice 

on feeding studies of the following chemicals: 

(CAS 103-33-3) azobenzene 

(CAS 20941-65-5) ethyl tellurac 

(CAS 298-00-0) methyl parathion 

(CAS 88-06-2) 2,4,6-trichlorophenol 

(CAS 72-56-0) p,p'-ethyl-DDD 

(CAS 85-44-9) phthalic anhydride 

(CAS 51-03-6) piperonyl butoxide 

E. Subchronic Studies 

Subchronic feeding studies were conducted to estimate the maximum 

tolerated doses (MTD's) of C. I. vat yellow 4, on the basis of 

which two concentrations (hereinafter referred to as "low" and 

"high" doses) were selected for administration in the chronic 

studies. Groups of five rats of each sex and five mice of each 

sex were administered feed containing C. I. vat yellow 4 at one 

of several doses, and groups of five control animals of each 

species and sex were administered basal diet only. The period of 

administration of the test chemical was 7 weeks, followed by 1 

week of additional observation. Each animal was weighed twice 

per week. Table 1 shows the survival of animals in each dosed 

group at the end of the course of administration and the week on 
study when the last animal died; the table also shows the mean 

body weights of dosed animals at week 7, expressed as percentages 

of mean body weights of controls. 

At the end of the subchronic studies, all animals were killed 

using CO and necropsied. On clinical and histopathologic 

examination oi: the rats, slight to moderate centrilobular 

cytoplasmic vacuolation was noted in the liver of one male, and 

trace amounts of the vacuolation were noted in four females at 

50,000 ppm. Tissues examined in rats at 6,200 ppm were 

essentially normal in both males and females. In mice, tissues 

examined in males at 50,000 and in females at 25,000 ppm were 

essentially normal. 

Ten percent depression in body weight was taken as the major 

criterion for estimation of MTD's. The doses that were required 

to produce this response were determined by the following 

procedure: first, least squares regressions of mean body weights 

versus days on study were used to estimate mean body weights of 

each of the dosed groups at day 49. Next, probits of the percent 

weights of dosed groups at day 49 relative to weights of 

corresponding control groups were plotted against the logarithms 

of the doses, and least squares regressions fitted to the data 
Table 1. C. I. Vat Yellow 4 Subchronic Feeding
 

Studies in Rats and Mice
 

Male Female 

Week on Week on 

Study Mean Weight Study Mean Weight 

when Last at Week 7 when Last at Week 7 

Dose Surviv­ Animal as % of Surviv- Animal as % of 

(ppm) al (a) Died Control al (a) Died Control 

Rats
 

6,200 5/5 84 5/5 91
 

12,500 5/5 87 5/5 85
 

25,000 5/5 88 5/5 87
 

50,000 5/5 77 5/5 83
 

Mice
 

6,200 5/5 115 5/5 105
 

12,500 5/5 116 5/5 111
 

25,000 5/5 114 5/5 104
 

50,000 5/5 110 3/5 7 120
 

(a) Number surviving/number in group.
 
were used to estimate the doses required to induce 10% depression 

in weight. 

Based on the data thus obtained, the low and high doses for 

chronic studies using rats were set at 3,500 and 7,000 ppm. The 

low dose for the chronic studies using male mice was set at 

25,000 ppm, and the high dose was set at 50,000 ppm, the maximum 

amount recommended in Guidelines for Carcinogen Bioassay in Small 

Rodents (Sontag et al., 1976) in the Careinogenesis Testing 

Program. The low and high doses for the chronic studies using 

female mice were set at 12,500 and 25,000 ppm. 

F. Chronic Studies
 

The test groups, doses administered, and durations of the chronic
 

feeding studies are shown in tables 2 and 3.
 

G. Clinical and Pathological Examinations
 

All animals were observed twice daily for deaths. Observations 

on sick, tumor-bearing, or moribund animals were recorded daily. 

Clinical examination and palpation for masses were performed each 

month, and the animals were weighed at least once per month. 

10 
Table 2. C. I. Vat Yellow 4 Chronic Studies in Rats
 

C. I. Vat 

Sex and Initial Yellow 4 (b) 

Test No. of in Diet (c) Time on Study 

Group An ima Is (a ) (ppm) (weeks) 

Male 

Matched-Control 20 0 104 

Low-Dose 50 3,500 104 

High-Dose 50 7,000 104
 

Female
 

Matched-Control 20 0 104
 

Low-Dose 50 3,500 104
 

High -Dose 50 7,000 104
 

(a) All animals were approximately 6 weeks of age when placed on
 

study.
 

(b) Commercially formulated product (see "Introduction" and
 

"Chemical" sections, above).
 

(c) Test and control diets were provided ad libitum.
 

11
 
Table 3. C. I. Vat Yellow 4 Chronic Studies in Mice
 

C. I. Vat 

Sex and Initial Yellow 4 (b) 

Test No. of in Diet (c) Time on Study 

Group Animals (a) (ppm) (weeks) 

Male 

Matched-Control 20 0 106 

Low-Dose 50 25,000 106 

High-Dose 50 50,000 106 

Female
 

Matched-Control 20 0 106
 

Low-Dose 50 12,500 106
 

High -Dose 50 25,000 106
 

(a) All animals were approximately 6 weeks of age when placed on
 

study.
 

(b) Commercially formulated product (see "Introduction" and
 

"Chemical" sections, above).
 

(c) Test and control diets were provided ad libitum.
 

12
 
Moribund animals and animals that survived to the end of the
 

bioassay were killed using C0_ and necropsied. Necropsies were
 

also performed on all animals found dead, unless precluded by
 

autolysis or severe cannibalization.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross
 

lesions. The tissues were preserved in 10% buffered formalin,
 

embedded in paraffin, sectioned, and stained with hematoxylin and
 

eosin. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone marrow (femur), spleen,
 

lymph nodes (mesenteric and submandibular), thymus, heart,
 

salivary glands (parotid, sublingual, and submaxillary), liver,
 

pancreas, esophagus, stomach (glandular and nonglandular), small
 

and large intestines, kidney, urinary bladder, pituitary,
 

adrenal, thyroid, parathyroid, mammary gland, testis, prostate,
 

uterus, ovary, brain (cerebrum and cerebellum), and all tissue
 

masses. Peripheral blood smears also were made for all animals,
 

whenever possible.
 

A few tissues from some animals were not examined, particularly
 

from those animals that may have died early, been missing, or
 

been in advanced states of cannibalization or autolysis. Thus,
 

the number of animals from which particular organs or tissues
 

13
 
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 Careinogenesis Bioassay Data
 

System (Linhart et al., 1974). The data elements include
 

descriptive information on the chemicals, animals, experimental
 

design, 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.
 

These data were analyzed using the appropriate 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
 

section.
 

Probabilities of survival were estimated by the product-limit
 

14
 
procedure of Kaplan and Meier (1958) and are presented in this 

report in the form of graphs. Animals were statistically 

censored as of the time that they died of other than natural 

causes or were found to be missing; animals dying from natural 

causes were not statistically censored. Statistical analyses for 

a possible dose-related effect on survival used the method of Cox 

(1972) for testing two groups for equality and Tarone's (1975) 

extensions of Cox's methods for testing for a dose-related 

trend. One-tailed P values have been reported for all tests 

except the departure from linearity test, which is only reported 

when its two-tailed P value is less than 0.05. 

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 

15 
determine whether animals receiving the test chemical developed a 

significantly higher proportion of tumors than did the control 

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 Cochran-Armitage test for linear trend in proportions, with 

continuity correction (Artnitage, 1971), was also used. Under the 

assumption of a linear trend, this test determines if the slope 

of the dose-response curve is different from zero at the onetailed 0.05 level of significance. Unless otherwise noted, the 

direction of the significant trend is a positive dose relationship. This method also provides a two-tailed test of departure 

from linear trend. 

A time-adjusted analysis was applied when numerous early deaths 

16 
resulted from causes that were not associated with the formation 

of tumors. In this analysis, deaths that occurred before the 

first tumor was observed were excluded by basing the statistical 

tests on animals that survived at least 52 weeks, unless a tumor 

was found at the anatomic site of interest before week 52. When 

such an early tumor was found, comparisons were based exclusively 

on animals that survived at least as long as the animal in which 

the first tumor was found. Once this reduced set of data was 

obtained, the standard procedures for analyses of the incidence 

of tumors (Fisher exact tests, Cochran-Armitage tests, etc.) were 

followed. 

When appropriate, life-table methods were used to analyze the 

incidence of tumors. Curves of the proportions surviving without 

an observed tumor were computed as in Saffiotti et al. (1972). 

The week during which an animal died naturally or was sacrificed 

was entered as the time point of tumor observation. Cox's 

methods of comparing these curves were used for two groups; 

Tarone's extension to testing for linear trend was used for three 

groups. The statistical tests for the incidence of tumors which 

used life-table methods were one-tailed and, unless otherwise 

noted, in the direction of a positive dose relationship. 

Significant departures from linearity (P less than 0.05, twotailed test) were also noted. 

17 
The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

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

relative risk is defined as p./p where p is the true
 

binomial probability of the incidence of a specific type of tumor
 

in a dosed group of animals and p 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.
 

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 less than 0.025 one-tailed test when the
 

control incidence is not zero, P less than 0.050 when the control
 

incidence is zero) has occurred. When the lower limit is less
 

18
 
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.
 

19
 
20

 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

Mean body weights of dosed male rats were only slightly depressed
 

throughout the bioassay when compared with those of matched
 

controls; mean body weights of females were more depressed
 

throughout the bioassay than those of the males (figure 1). No
 

differences were observed in the effects of the high and low
 

doses. Some fluctuation in the growth curves may be due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation. Clinical signs including
 

wasting, arched back, rough hair coats, and alopecia occurred at
 

low incidences in dosed groups and may have been related to
 

administration of the test chemical. Cornea 1 opacity and tissue
 

masses occurred at comparable incidences in dosed and control
 

groups.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered C. I. vat yellow 4
 

21
 
n n 

a 6 o g A o 

8 § § A O 

_DO 

tO 

X 300­ <a 

uj

5 

Q 

O to 

ui 

5 

MALE RATS 

D MATCHED CONTROL 10011

 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

2 

I — 6

A 

o AA 

Q e e e 

O 

00 ana g g e 

4 

UJ 

s 

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered C.I. Vat Yellow 4 in the Diet 

22 
in the diet at the doses of this bioassay, together with those of
 

the matched controls, are shown in figure 2. In male rats, the
 

result of the Tarone test for dose-related trend in mortality is
 

not significant. In females, the result of the Tarone test is
 

significant (P = 0.039), but in the negative direction, because
 

the dosed groups lived longer than the control animals.
 

In male rats, 39/50 (78%) of the high-dose group, 37/50 (74%) of
 

the low-dose group, and 19/20 (95%) of the control group lived to
 

the end of the study. In females, 45/50 (90%) of the high-dose
 

group, 40/50 (80%) of the low-dose group, and 14/20 (70%) of the
 

control group lived to the end of the study.
 

Sufficient numbers of rats of each sex were at risk for the
 

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

Appendix A, tables Al and A2; findings on nonneoplastic lesions
 

are summarized in Appendix C, tables Cl and C2.
 

23
 
a -- o 

4 6

"° 

A-0-^ * '"A-^OT Mi 1 

6 

Cfl 

O ncn 

m 

0 

cc 

FEMALE RATS 

0.20 • 

0.10­

D

O

' 

1

 MATCHED CONTROL 

 LOW DOSE 

 HIGH DOSE 

onn ­

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered C.I. Vat Yellow 4 in the Diet 

24 

ABILIT

Y

O

8

 C

i 

CD 

O 

CC 

MALE RATS 

0.20 • D MATCHED CONTROL 

O LOW DOSE 

0.10 /1 HIGH DOSE • 

n no • 

45 60 75 

TIME ON STUDY (WEEKS) 

ftwtiu 1 

\c£\ i 0.90 , - \j "-Qi /V • • • Tft 

i

"K \0

<t 

<ft 

Li. 

O „,-„ BILIT

Y 3 C 

3 C 
The incidence of proliferative lesions, with the exception of
 

interstitial-cell tumors of the testes in male rats, was low and
 

approximately equal in control and dosed groups of animals. As
 

expected from findings in previous studies using the Fischer 344
 

strain of rat, most males had interstitial-cell tumors involving
 

one or both testicles.
 

Occasionally, there were inflammatory and degenerative lesions
 

such as chronic nephritis (especially in males), pneumonia, and
 

cysts of the pituitary and ovaries. Incidences of these changes
 

also appeared to be approximately equal in dosed and control
 

animals.
 

Based on the pathologic examination, there is no evidence that C.
 

I. vat yellow 4 was carcinogenic or contributed to the incidence
 

of nonneoplastic lesions in Fischer 344 rats under the conditions
 

of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

25
 
least two animals of one group and at an incidence of at least 5%
 

in one or more than one group.
 

In each sex, the results of the Cochran-Armitage test for
 

positive dose-related trend in proportions and those of the
 

Fisher exact test comparing the incidences in the dosed groups
 

with those in the control group in the positive direction are not
 

significant.
 

Significant results in the negative direction are observed in the
 

incidences of pheochromocytomas of the adrenal and C-cell tumors
 

of the thyroid in male rats and in the incidence of hematopoietic
 

tumors in female rats where the incidences in the control group
 

exceed those in the dosed groups.
 

In each of the 95% confidence intervals of relative risk, shown
 

in the tables, the value of one or less than one is included;
 

this indicates the absence of significant positive results. It
 

should also be noted that each of the intervals (except that for
 

the incidences of adrenal pheochromocytoma in low-dose male rats
 

and hematopoietic tumors in high-dose female rats) has an upper
 

limit greater than one, indicating the theoretical possibility of
 

the induction of tumors by C. I. vat yellow 4, which could not be
 

detected under the conditions of this test.
 

26
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of dosed male mice were unaffected by
 

administration of the test chemical; mean body weights of the
 

dosed females also were unaffected, except for a dose-related
 

depression during the last 16 weeks of weight measurement (figure
 

3). Fluctuation in the growth curves may have been due to
 

mortality; as the size of a group diminishes, the mean body
 

weight may be subject to variation. Clinical signs included
 

tissue masses, which occurred at comparable incidences in dosed
 

and control groups; corneal opacity occurred in two low-dose
 

females. Wasting, arched back, and paralysis each occurred in
 

one or two mice, but none of the clinical signs could be clearly
 

related to administration of the test chemical.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered C. I. vat yellow 4
 

in the diet at the doses of this bioassay, together with those of
 

27
 
60­

6 B

H n

2 

(9 40­ e 8 

g 

UJ 

o 

O 

m 

Z 

ui 

S 

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 50 60 70 80 90 100 110 

TIME ON STUDY (WEEKS) 

B ^ 2 o 8 

8 8 

O 

tO 

ui 

5 

S 

n9 

o .8 

o 

CD 

20; 

FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 50 60 70 80 90 100 110 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered C.I. Vat Yellow 4 in the Diet 

28 
the matched controls, are shown in figure 4. In each sex, the
 

result of the Tarone test for dose-related trend in mortality is
 

not significant.
 

In male mice, 32/50 (64%) of each dosed group and 16/20 (80%) of
 

the control group were still alive at the end of the bioassay.
 

In females, 39/50 (78%) of the high-dose group, 35/50 (72%) of
 

the low-dose group, and 14/20 (70%) of the control group were
 

alive at the end of the bioassay.
 

Sufficient numbers of mice of each sex were at risk for the
 

development of late-appearing tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

Appendix B, tables Bl and B2; findings on nonneoplastic lesions
 

are summarized in Appendix D, tables Dl and D2.
 

There was increased incidence of hepatocellular carcinomas in the
 

dosed male and female mice when compared with respective
 

controls. The incidences of these neoplasms were as follows:
 

29
 
W 6­ 'A­ ___*.„ _ 

"*kl 

*"& 

^kl 

-1 0.70 ­

^ 0.60 ­

</} 

^\$>-A 0 

*& 

BILIT

Y 3 C 

, 8 

m 

0 

EC 

MALE Ml(:E 

0.20­ D MATCHED CONTROL — 

O LOW DOSE 

0.10­ /\ HIGH DOSE 

onn46 60 75 

TIME ON STUDY (WEEKS) 

-,UJn*S--l 

n 

Ht ^"&e 

[ 1 ^A 

I

8 

IVAIAU

 1

n (0 

o 0.50 • 

t 

_J 

m 

O 

cc 

FEMALE MICE 

0.20- D MATCHED CONTROL

 

O LOW DOSE 

0.10 - Li HIGH DOSE 

0.00 • 

60 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered C.I. Vat Yellow 4 in the Diet 

30 
Controls Low-Dose High-Dose 

Males 3/20 (15%) 22/47 (47%) 21/50 (42%) 

Females 2/19 (11%) 6/48 (13%) 9/50 (18%) 

Metastases of the tumor were found in one control male, three 

low-dose males, and two high-dose males. 

The hepatocellular carcinomas had a wide morphologic spectrum, 

varying from well to very poorly differentiated. Some were small 

expanding and invasive nodules usually with increased basophilic 

staining; others were large masses involving large portions of 

the liver. These varied from a trabecular pattern to solid 

masses of large anaplastic cells. All had increased mitoses. 

Although the incidence of the hepatocellular carcinomas is higher 

in dosed than in control male mice, the tumor is known to occur 

frequently in this strain of mouse. In addition, there is no 

evidence of a dose relationship in the incidence of the tumor in 

this study. 

There was an increased incidence of malignant lymphoma, which 

frequently occurred in multiple sites in high-dose male mice. 

The incidences of lymphomas in the mice were as follows: 

31 
Controls Low-Dose High-Dose
 

Males 3/20 (15%) 7/47 (15%) 22/50 (44%)
 

Females 6/19 (32%) 12/48 (25%) 17/50 (34%)
 

Other proliferative or neoplastic lesions were of a type,
 

incidence, and distribution commonly found in aged B6C3F1 mice
 

and therefore were believed to be unrelated to administration of
 

the test compound.
 

In addition to the proliferative lesions, there were inflammatory
 

and degenerative changes in some mice in each of the groups.
 

These included focal mineralization of the brain, focal
 

hepatitis, focal hepatic necrosis, testicular atrophy, and cystic
 

uterine endometrium. The incidence of these changes appeared to
 

be approximately equal in control and dosed groups.
 

Based on the pathologic examination, administration of C.I. vat
 

yellow 4 may be associated with the increased incidence of
 

lymphomas in male B6C3F1 mice.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

32
 
of the incidences of those primary tumors that occurred in at 

least two animals of one group and at an incidence of at least 5% 

in one or more than one group. 

In male mice, the result of the Cochran-Armitage test for 

positive dose-related trend in the incidence of lymphomas is 

significant (P = 0.002) and the Fisher exact test shows that the 

incidence in the high-dose group is significantly higher (P * 

0.019) than that in the control group. When the life-table 

method is used on the incidence of lymphoma, as shown in figure 

5, the result of the Tarone test shows a P value of 0.004. The 

statistical evaluation suggests that the incidence of lymphomas 

in male mice is associated with the administration of C. I. vat 

yellow 4. 

Although the result of the Cochran-Armitage test for the 

incidence of hepatocellular carcinomas in male mice is not 

significant at the 0.05 level (the trend statistic has P = 0.07 

with the departure statistic of P = 0.056), the Fisher exact 

comparisons of the incidences in the low- and high-dose groups 

with that in the control group show P values of 0.012 and 0.027, 

respectively. The latter is above the 0.025 level for 

significance when the Bonferroni inequality criterion is used for 

multiple comparison. Historical records at this laboratory 

33 
 =J ..r.r.r.r.r.r. ir.r..".-..-.."..- r.r.r.r.r.r.r. — n.o­ - — -1 

£c " A---: ^~ 6» 1

1 0.90­ . A 

^o,,­

J I 

>

> 

(O 

O 060­ A 

3 

TM a4°" m 

O 

ec 

MALE Ml( :E 

0.20­ P MATCHED CONTROL 

O LOW DOSE 

0.10­ Li HIGH DOSE 

45 60 75 

TIME ON STUDY (WEEKS) 

Figure 5. Life Table for Male Mice Administered C.I. Vat Yellow 4 in the Diet: 

Incidence of Lymphoma 

34 
indicate an overall incidence of hepatocellular carcinomas in
 

control male B6C3F1 mice of 93/323 (29%); two separate control
 

groups out of 11 groups had incidences of these tumors as high as
 

10/20 (50%) and 8/17 (47%) as compared with 22/47 (47%) in the
 

low-dose group and 21/50 (42%) in the high-dose group of the
 

present study. In view of these historical incidences, the
 

occurrences of hepatocellular carcinoma in the dosed male mice of
 

the present test do not convincingly establish a dose association.
 

The results of the Cochran-Armitage test and of the Fisher exact
 

test are not significant for the incidences of any tumors in the
 

female mice.
 

35
 
36

 
DISCUSSION
 

Depressions in mean body weight occurred throughout the bioassay
 

in the rats administered C. I. vat yellow 4, a commercial
 

formulation of the color-imparting chemical, dibenzo(b,def)­

chrysene-7,14-dione. However, the depression was slight for the
 

dosed male rats, and the mean body weights of the mice were not
 

appreciably affected by the test chemical except for the last 16
 

weeks of the bioassay in the female mice. Clinical signs
 

observed at low incidences in the dosed rats were wasting, arched
 

back, rough hair coats, and alopecia; these signs may have been
 

related to the chemical. Survival of the rats and mice was not
 

affected by the C. I. vat yellow 4 except for an increased
 

survival in the dosed female rats compared with the controls; in
 

every group 82% or more of the animals were alive at week 90 of
 

the bioassay. Thus, sufficient numbers of dosed and control rats
 

and mice of each sex were at risk for the development of
 

late-appearing tumors.
 

No tumors occurred in male or female rats or female mice at
 

incidences that were significantly higher in dosed groups than in
 

control groups.
 

37
 
Hepatocellular carcinomas occurred in the low-dose male mice at
 

an incidence that was higher (P = 0.012) than that in the control
 

group (controls 3/20, low-dose 22/47, high-dose 21/50). However,
 

the incidence of this tumor in the high-dose group was not
 

significant when the Bonferroni criterion was applied; the
 

incidences in the dosed groups were not significantly dose
 

related; and the incidence in the control group (3/20, or 15%)
 

was lower than that observed for hepatocellular carcinomas or
 

adenomas in historical-control male B6C3F1 mice at this
 

laboratory (93/323, or 29%). Thus, the occurrence of hepatocellular carcinomas in the dosed male B6C3F1 mice of the present
 

bioassay cannot be clearly related to administration of the test
 

chemical.
 

Lymphomas occurred in the male mice at incidences that were dose
 

related (P = 0.002), and, in a direct comparison, the incidence
 

of the tumor in the high-dose group was significantly higher (P =
 

0.019) than that in the control group (controls 3/20, or 15%;
 

low-dose 7/47, or 15%; high-dose 22/50, or 44%). When the
 

life-table method is used on the incidence of lymphoma, a P value
 

of 0.004 is obtained with the Tarone test. The incidence of
 

lymphomas and leukemias in historical-control male B6C3F1 mice at
 

this laboratory was 38/323 (12%).
 

38
 
No oral LD values have been reported for C. I. vat yellow 4 

in rats or mice, and no chronic feeding studies with the test 

chemical have previously been carried out. No tumors appeared in 

strain P. D. mice in 12-month studies when 1.2 ml of a 0.5% 

solution of the chemical was injected subcutaneously or when a 

0.4% solution in benzene was administered 115 times to the skin 

(Kleinenberg, 1939). Dibenzo(b,def)chrysene, the parent 

hydrocarbon analog of the color-imparting component of vat yellow 

4, is a potent carcinogen (Haddow and Ron., 1947; Dipple, 1976) 

and may occur in small amounts as a contaminant in vat yellow 4 

(Owens and Ward, 1974). No evidence has been reported for the 

metabolic reduction of dibenzo(b,def)chrysene-7,14-dione to 

dibenzo(b,def)chrysene. 

It is concluded that under the conditions of this bioassay, the 

formulated product containing C. I. vat yellow 4 was not 

carcinogenic for male or female Fischer 344 rats or for female 

B6C3F1 mice, but was carcinogenic for male B6C3F1 mice, causing 

an increased incidence of lymphomas. 

39 
40

 
VI. BIBLIOGRAPHY
 

Armitage, P., Statistical Methods in Medical Research, John Wiley
 

& Sons, Inc., New York, 1971, pp. 362-365.
 

Berenblum, I., ed., Carcinogenicity Testing: A Report p_f the
 

Panel on Careinogenic ity c^f the Cancer Research Commission of the
 

UICC, Vol. 2_. International Union Against Cancer, Geneva, 1969.
 

Cox, D. R., Regression models and life tables. J_. R. Statist.
 

Soc. B 34;187-220, 1972.
 

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

1970, pp. 48-52.
 

Dipple, A., Polynuclear aromatic hydrocarbons. In: Chemical
 

Carcinogens, Searle, C. E., ed., American Chemical Society,
 

Washington, D. C., 1976, pp.245-314.
 

Gart, J. J., The comparison of proportions: a review of significance tests, confidence limits and adjustments for stratification. Rev. Int. Stat Inst. 39:148-169, 1971.
 

Haddow, A. and Kon, G. A. R., Chemistry of carcinogenic compounds.
 

Brit. Med. J. 4:314-325, 1947.
 

Kaplan, E. L. and Meier, P., Nonparametric estimation from
 

incomplete observations. J_^ Am. Statist. Assoc. 5^:457-481, 1958
 

Kleinenberg, H. E., Investigations on the blastomogenic effect of
 

3,4,8,9-dibenzpyrene and some of its derivatives. Archives des
 

sciences biologiques 56(3);40-47, 1939.
 

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

Peters, J. A., Carcinogenesis bioassay data system. Comp. and
 

Biomed. Res. 7:230-248, 1947.
 

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


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

Owens, E. J., and Ward, D. M., A Review o_f the Toxicology o_f
 

Colored Chemical Smokes and Colored Smoke Dyes, National
 

Technical Information Service, U. S. Department of Commerce,
 

Springfield, Va., 1974, pp. 1-71.
 

41
 
Puro, T. E., Chemical warfare. In: Kirk-Othmer Encyclopedia p_f
 

Chemical Technology, Vol. 4_, Interscience Publishers, Inc., New
 

York, 1964, pp. 900-907.
 

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

Kaufman, D. G., Respiratory tract carcinogenesis in hamsters
 

induced by different numbers of administrations of benzo (a)
 

pyrene and ferric oxide. Cancer Res. 32:1073-1081, 1972.
 

Society of Dyers and Colourists, Vat Dyes. In: Colour Index,
 

Vol. 3, Dean House, Piccadilly, England, 1971, pp. 3719-3721.
 

Sontag, J. M., Page, N. P., and Saffiotti, U., Guidelines for
 

Carcinogen Bioassay i_n Small Rodents, DHEW Publication No. (NIH)
 

76-801, U.S. Government Printing Office, Washington, D.C. 1976.
 

Tarone, R. E., Tests for trend in life table analysis.
 

Biometrika 62(3);679-682. 1975.
 

United States International Trade Commission, Dyes. In:
 

Synthetic Organic Chemicals - United States Production and Sales,
 

1976, USITC Publication 833, U. S. Government Printing Office^
 

Washington, D. C., 1977, pp. 79-80.
 

42
 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED C. I. VAT YELLOW 4 IN THE DIET
 

43
 
44

 
TABLE A1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

MATCHED
 

CONTROL LOW DOSE
 

ANIMALS INITIALLY IN STUDY 20 50
 

ANIMALS NECROPSIED 20 50
 

ANIHALS EXAMINED HISTOPATHOLOGICALLY 20 50
 

INTEGUMENTARY SYSTEM 

*SKIN

SEBACEOUS ADENOCARCINOMA 

LIP01A 

 (20) (50) 

2 (UX) 

*SUBCUT TISSUE

FIBROMA 

FIBROSARCOMA 

LIPOMA 

HSMANGIOSARCOMA

 (20) 

1 (5%) 

(50) 

1

2

 (2X) 

 (4X) 

RESPIRATORY SYSTEM
 

*LUNG (20) (50)
 

ALVEOLAR/BPONCHIOLAR ADENOMA 1 (5X) 3 (6X)
 

ALVEOLAR/BRONCHIOLAR CABCIHOHA 5 (10X)
 

HEMANGIOSARCOMA
 

OSTEOSARCONA 1 (2X)
 

OSTEOSARCOMA, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

*KULTIPLE ORGANS (20) (50)
 

MALIGNANT LYMPHOMA, HOS 3 (6X)
 

^DIFFERENTIATED LEOKEHIA 1 (5X) 1 (2X)
 

*SPLEEN (20) (50)
 

ONDIFFERENTIATED LEUKEHIA 2 (10X) 2 (H%)
 

fTHYMUS (12) (18)
 

MALIGNANT LYMPHOHA, HOS
 

CIRCULATORY SYSTEM
 

NONE
 

# NUMBER OF ANIMALS WITH TISSUE EXAHIHED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

HIGH DOSE
 

50
 

50
 

50
 

(50) 

1 (2X) 

(50) 

1 (2X) 

1 <2X)
 

1 (2X)
 

(50)
 

1 (2X>
 

3 (6X)
 

1 (2X)
 

(50)
 

1 (2X)
 

1 (2X)
 

(50)
 

1 (2X)
 

(24)
 

1 (UX)
 

45
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIV E SYSTE M 

flIV1?P. (20) (50) (50) 

HEPATOCFLLULA R ADENOM A 1 (2X) 3 (6X) 

•COLON (18) (US) (50) 

ADENOCARCINOMA , NOS 1 (2%) 

#CECU M (18) (48) (50) 

ADENOCAPCINOMA , NOS, INVASIV! 1 (2*) 

LIPOM A 1 (2X) 

URINAR Y SYSTE M 

NONE 

ENDOCRIN E SYSTEH 

• PITUITAR Y (19) (U9) 

ADENOMA , NO S 3 (16%) 4 (9X) 6 (12X) 

ADENOCARCINONA , NOS 2 (UX) 

• ADRENA L (20) (50) (50) 

CORTICAL CARCIKOHA 1 (2X) 

PHEOCHROMOCYTOBA 4 (20%) 1 (2X) 5 (10X) 

tTHYROID	 (19) (50) (50) 

FOLLICOLAR-CELL ADENOMA 

FOLLICULAR-CELL CARCIBOHA 1 <2X) 

C-CELL ADENOM A 3 (16*) 1 (2X) 

C-CELL CARCINOHA 1 (5X) 1 (2X) 

• PANCREATIC ISLETS (19) (50)
 

ISLET-CELL ADENOMA 3 (6X)
 

REPRODUCTIVF SYSTEH
 

*BAMMARY GLAND (20) (50) (50)

 

CYSTADENOHA, NOS 1 (2X)
 

FIBROADENOMA 1 (5X)
 

•PROSTATE	 (19) (49) (49) 

1.12X1, 

# NUMBE R OF ANIMAL S WITH TISSUE EHHI5ED MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECBOPSIBD 

46 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

fTESTI S (20) (50) (50) 

INTERSTITIAL-CEL L TUMO R 16 (80*) HH (88*) 39 (78*) 

*EPIDIDYMI S (20 ) (50) (50) 

LIPOK A 1 (2*) 

NERVOU S SYSTE M 

*ERAI N (20) (U9 ) (50) 

GLIOMA , NOS 1 (2*) 

SPECIA L SENS E ORGAN S 

NON E 

MUSCULOSKELSTA L SYSTEM 

*EONE/LOWE R EXTREMIT Y (20) (50) (50) 

OSTEOSARCOM A 1 (2«) 

BOE Y CAVITIE S 

*F2RITON1?U M (20 ) (50) (50) 

MESOTHELIOHA , NOS 1 (2%) — —• ——_ _ — —— _ _ _ _ ___ H _ — — _ » — — __... — _._.«_«..__«_..__«_•.___ »*»— —— _ — ——*•-, — — — — _— • — _ _ «.«, — •*—— —— 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMAL S INITIALLY IN STUDY 20 50 50
 

NATURA L DEATHS 1 10 9
 

MORIBUND SACRIFICE 3 2
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINA L SACRIFICE 19 37 39
 

ANIMAL "ISSING
 

^_II!£!HDES_A.UTOLYZED_ ANIMALS
 

* NU1BF P OF ANIMAL S WIT H TISSUE EXAMINE D HICROSCOPICALLT 

* NUM8 rP OF ANIMAL S NECROPSIED 

47 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIMARY TUMORS*


TOTAL PRIMARY TUMORS


TOTAL ANIMALS WITH BENIGN TDMOBS


TOTAL PENIGN TUMORS


TOTAL ANIMALS WITH MALIGNANT TUMORS


TOTAL MALIGNANT TUMORS


TOTAL ANIMALS WITH SECONDARY TUMORS*


TOTAL SECONDARY TUMORS


TOTAL ANIMAL S WITH TUMORS UNCERTAINEENIGN OR MALIGNANT


TOTAL UNCERTAIN TUMORS


TOTAL ANIMAL S WITH TUMORS UNCERTAINPSIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

MATCHED
 

CONTROL
 

 18


 33


 18


 28


 4


 5


LOW DOSE HIGH DOSE 

 50 47 

 82 75 

 46 43 

 64 58 

 17 13 

 18 16 

1 1 

1 1 

1 

1 

* PPIMAPY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: METASTATIC TUMOES OP TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

48
 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

ANI1ALS INITIALL Y IN STUD Y

ANIMAL S NECROPSIE D

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY

INTFGUMENTAR Y SYSTEM 

*SKIN


SEBACEOUS ADENOCARCINOMA


KERATOACANTHOMA


*SUBCUT TISSUE


SEBACEOUS ADENOCARCINOMA


FIBHOSARCOMA


LIPOMA


RESPIRATORY SYSTEM
 

#LUNG


ALVEOLAVBRONCHIOLAR ADENOMA


HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS


MALIGNANT LYMPHOMA, NOS


UNDIFFERENTIATED LEUKEMIA


#SPLEEN


MALIGNANT LYMPHOMA, NOS


UNDIFFERSNTIATED LEUKEMIA


CISCULATOBY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

NOSE
 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 20 50 50 

 20 5" 50 

 20 50 50 

 (20) (50) (50) 

1 (2%) 

1 (2*) 

 (20) (50) (50) 

1 (2X) 

2 (U%) 1 (2%) 

1 (5%) 

 (20) (50) (50) 

1 (5%) « (8%) 

 (20) (50) (50) 

1 (5*) 1 (2X) 

1 (5%) 3 (6X) 

 (20) (U8) (50) 

1 (5%) 

 .1 (5%) 2 (4X) 

# NUMBEF OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

49
 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

URINAR Y SYSTE M 

NONE 

ENDOCRIN E SYSTEM 

*PITUITAR Y (19 ) (50) (50) 

ADENOMA , NOS 2 (11X ) 12 (24*) 6 (12*) 

ADENOCARCINOMA , NO S 1 (5*) 

*ADRENA L (20) (U9) (50) 

PHEOCHPOMOCYTOMA 1 (2X) 

tTHYROT D (20) (50) (50) 

C-CELL ADENOM A 2 (10*) 1 (2*) 1 (2*) 

C-CELL CARCINOM A 1 (2X) 3 (6*) 

REPRODUCTIV E SYSTEH 

*MAMMAR Y GLAN D (20) (50) (50) 

ADENOMA , NOS 1 (2*) 

CYSTADENOMA , NOS 1 (5*) 2 (U*) 1 (2X) 

FIBROADENOH A 1 (5X) 8 (16X) 6 (12«) 

*UTERUS/* 1NDOMETBIUM (20) (U9 ) (50) 

ADENOCARCINOMA , NOS 1 (5*) 

NFFVODS SYSTEM
 

fBRAIN (20) (50) (50)
 

GLIOMA, NOS 1 (2S) 1 (2*)
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

# NUMBE R OF ANIMAL S HITH TISSUE EXAMINE D HICBOSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

50 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMA L DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

NATUPAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINA L SACRIFICE 45
 

ANIMAL MISSING
 

9 INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS HITH PRIMARY TOMORS* 9 26 23
 

TOTAL PRIMARY TUMORS 14 34 27
 

TOTAL ANIMALS WITH BENIGN TUMORS 7 22 16
 

TOTAL BENIGN TDMORS 8 24 20
 

TOTAL ANIMALS WITH MALIGNANT TOMORS 5 8
 

TOTAL MALIGNANT TUMORS 6 10
 

TOTAL ANIMALS WITH SECONDARY TOMORS*
 

TOTAL SECONDARY TUHORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OP MALIGNANT
 

TOTAL UNCERTAIN TOMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUHORS
 

# SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAH
 

51
 
52

 
APPENDIX R
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED C. I. VAT YELLOW 4 IN THE DIET
 

53
 
54

 
TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

50 

ANIMAL S MISSING 2 

ANIMAL S NECROPSIE D 2 0 47 50 

ANIMAL S EXAMINE D HISTOPATHOLOGIC ALL Y 2 0 47 50 

ANIMAL S INITIALL Y IN STUDY 2 0 50 

INTEGUMENTAR Y SYSTEM 

*SKI N (20) («7) (50) 

SQUAMOU S CELL CARCINOM A 1 (2%) 

*SU3CUT TISSUE (20) (47 ) (50) 

ADENOCABCINOHA , NOS 1 (2%) 

KERATOACANTHOM A 1 (5%) 

FIBPOM A 1 (2%) 

FIBROSARCOK A 2 (4%) 

LIPOM A 2 (10% ) 1 (2%) 4 (8%) 

HEMANGIOM A 1 (2%) 

HSMANGIOSARC0.1 A 2 (4% ) 

RESPIRATOR Y SYSTE M 

*LUN G (20) (47 ) (50) 

HEPATOCELLULA R CARCINOMA , METAST 1 (5%) 3 (6%) 1 (2X> 

ALVEOLAP/BRONCHIOLA R ADENOM A 2 (4X) 4 (8%) 

ALVEOLAP/BRONCHIOLA R CARCINOM A 4 (20% ) 12 (26*) 11 (22%) 

HF1ATOPOIETIC SYSTE M 

* MULTIPL E ORGAN S

MALIGNAN T LYMPHOMA , NOS

MALIG.LYKPHOMA , HISTIOCYTIC TYPE 

 (20) 

2 (10% ) 

(U7 ) 

U (9S) 

(50) 

16

2

 (32%) 

 (4%) 

*S'JBCUT TISSUE

MALIGNAN T LYMPHOMA, NOS 

 (20) (47) 

1 (2«) 

(50) 

1 (2%) 

#SPLEE N

SARCOMA , NOS 

HEKiNGIOSiRCQN A _ _ 

 (19 ) 

__..

(45) 

1_12%L . _

(50) 

1

 ­1

 (2%) 

IIH 

# NUMBER OF ANIHALS WITH TISSUE * NUMBER OF ANIMALS NECROPSIED 

 EXAMINED MICROSCOPICALLY 

55
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MALIGNAN T LYMPHOMA , NOS 1 (2X) 1 (2%) 

#MESFNTERI C L. NOD E (18) (46) (49) 

MALIGNAN T LYMPHOMA , NOS 1 (6%) 1 (2%) 1 (2%) 

fLIVFR (20) (47) (50)
 

MALIGNANT LYMPHOMA, NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

#LIVER (20) (H7) (50)
 

HFPATOCELLULAR CARCINOMA 3 (15%) 22 (47X) 21 (42%)
 

HSMANGIOSARCOMA 2 (4X)
 

*SMALL INTESTINE (19) (46) (49)
 

ADENOCARCINOBA, NOS 3 (7%) 1 (2%)
 

SARCOMA, NOS 1 (2%)
 

URINAR Y SYSTEM
 

#KIDNFY (20) (47) (50)
 

HFPATOCELLUtAR CARCINOMA, HETAST 1 (5%)
 

TUBULAR-CELL ADENOMA 1 (5%)
 

ENDOCRINE SYSTEM
 

#ADRENA L (19) (46) (46)
 

CORTICAL CARCINOMA 1 (5%)
 

PHEOCHROMOCYTOHA 1 (2%)
 

#THYROID (20) (45) (48)
 

ADENOCARCI.NOMA, NOS 1 (2X)
 

FOLIICULAR-CELL CARCINOMA 1 (2%)
 

•PANCREATIC	 ISLETS (19) (46) (47)
 

ISLET-CFLL ADENOMA 1 (5X) 1 (2%)
 

REPRODUCTIVE SYSTEM
 

NONE
 

t NUMBER OF ANIMALS WITH TISSOE EXAMINED MICROSCOP1CALL?
 

* NUMBER OF ANIMALS NECROPSIED
 

56
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NERVOUS SYSTEM 

*BRAI N

GLIOMA , NOS

 (20) (U7 ) (50) 

1 (2%) 

SPECIAL SENS E ORGAN S 

*FYE/LACRIMA L GLAN D

ADENOMA , NOS

 (20) (U7 )

 2 (UX )

 (50) 

1 (2%) 

MUSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*PERITONEU«

LIPOMA

 (20) (47) (50) 

1 (2%) 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SOMHARY 

ANIMALS INITIALLY IN STUDY

NATUPAL DEATH8

MORIBUND SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE

ANIMA L MISSING

 20

 4

 16

 50

 16

 32

 2 

 50 

 17 

1 

 32 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

57
 
TABLE 81. MALE MICE: NEOPLASMS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMO R SUMMAR Y 

TOTAL ANIMALS WITH PRIMARY TUMORS* 12 42 48 

TOTAL PRIMAR Y TUMORS 16 55 78

 

TOTAL ANIMAL S WITH BENIGN TUMORS 4 7 10 

TOTAL BENIG N TUMORS 5 8 11 

TOTAL ANIMAL S WITH MALIGNAN T TUMORS 10 38 44 

TOTAL MALIGNAN T TUMORS 11 47 67 

TOTAL ANIMALS WITH SECONDARY TUMORSf 1 3 1
 

TOTAL SECONDARY TUMORS 2 3 1
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OP MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRISARY OP. METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* FFI1ARY TUMORS: ALL TUHORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TUMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

58
 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAIS INITIALLY IN STDDY 20 50 50
 

ANIKALS HISSING 1 1
 

ANIMALS NECROPSIED 19 US 50
 

ANIMALS EXAMINED HISTOPATHOLOGIC ALIY 19 U8 50
 

INTEGUMENTARY SYSTEM
 

*SU3CUT TISSUE (19) (US) (50)
 

FIBROSARCOMA 1 (2%) 2 (UK)
 

LIPOMA 1 (5%) 1 (2%) 1 (2«)
 

RESPIRATORY SYSTEM
 

#LUN G (19) (U8) (50) 

ALVSOLAR/BRONCHIOLA R ADENOH A 2 («%) 1 (2«) 

ALVEOLAR/BRONCHIOLA R CARCINOM A 2 (11*) 3 (6X) 2 (4%) 

FIBROSAPCOMA 1 (2%) 

OSTEON A 1 (5%) 

HEMATOPOICTI C SYSTEM 

*MULTIPL ? ORGAN S (19 ) (48) (50) 

MALIGNAN T LYMPHOMA , NOS 3 (16% ) 8 (17*) 9 (18*) 

MALIG.LYMPHOHA , HISTIOCYTIC TYPE 1 (5%) 2 (H%) 2 (4X) 

* P rRITONEA L CAVIT Y (19) (U8 ) (50) 

MALIGNAN T LYMPHOMA , NOS 1 (2X) 

*SIJ3CUT TISSUE (19) (U8 ) (50) 

MALIGNAN T LYMPHOBA , NOS 1 (2%) 

#SPLFE N (19) (47) (50) 

HEMJNGIOSARCOM A 1 (2S) 

1ALIG. LYMPHOMA , HISTIOCYTIC TYPE 1 (2*) 2 (HI) 

tMESENTERI C L. NODE (19) (48) (50) 

MALIGNAN T LYMPKOMA , NOS 1 (2%) 

*LIVPR (19) (48) (50) 

1_12*L_ 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

59 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

#SMAL L INTESTINE

MALIGNAN T LYHPHOMA , NOS

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

 (19) 

1 (5%) 

1 (5%) 

(<*5) (12) 

1 (2*) 

CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

fSALIVAKY GLAND 

HHMANGIOMA 

(18) (47) (50) 

1 (2*) 

(•LIVER 

HEPATOCELLOLAR CARCINOMA 

HEMANGIOSARCOMA 

tSMALL INTESTINE

ADENOCARCINOHA, NOS

(19) 

2 (11%) 

 (19)

(U8) 

6 (13*) 

1 (2*) 

 (U5)

 1 (2%) 

(50) 

9 (18%) 

 (<*2) 

UPINAPY SYSTEM 

NONE 

ENCOCRIN E SYSTEM 

• PITUITAR Y

ADENOMA, NOS

 (19)

 1 (5%)

 (<*3)

 1 (2%)

 (<»8) 

1 (2%) 

iADRENA L

COPTTCAL ADENOM A

#THYPOIt >

FOLLICULAR-CELL ADENOMA

 (19 )

 (19)

 (U8)

 1 (2%) 

 (U7 )

 1 (2%)

 (1*9) 

 (U9) 

1 (2%) 

REPRODUCTIV E SYSTEM 

#UTEPUS

LFIOMYOSARCOMA

 (19) (48) (1*8) 

1 (2*) 

NERVOUS SYSTEM 

NONE ^ 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED

* NUMBER OF ANIMALS NECROPSIED 

 MICROSCOPICALLY 

60
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) _. ._ ____..___—_.__.._____. . w ____•_ W«_»W_VH.A«___»_V_.«V __ «——— _— —•*—.•——•—————..— — — —«•• 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

__—_~__~__~___«__ _ .._______.._•_•_*_«._•_______________________________•-»_•••»•»••«••• 

SPECIA L SENSE ORGAN S 

*EYE/LACRIMA L GLAN D (19) (t8) (50) 

ADENOMA , NOS 1 (5%) 2 (IX) 2 (<»*) 

MUSCULOSKELFTAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

NATURAL DEATH8 H 14 11
 

10RIBOND SACRIFICE 1
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACBIFICE 1« 35 39
 

ANIMAL MISSING 1 1
 

»_ INC LODE S^AUTOLY,Z ED SHI jj A.J.S
 

* SOMBER OF ANIMAL S WITH TISSUE EXAMINED HICROSCOPICALLY
 

* NUMBER OF ANIMAL S HECROPSIED
 

61
 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUHORS* 13 29 29

 

TOTA L PRIMAR Y TUMORS m 33 39

 

TOTAL ANIMAL S WITH BENIG N TDMORS 3 8 5 

TOTA L BENIG N TUMORS H 8 7 

T OTAL ANIMAL S WITH MALIGNAN T TUMORS 10 21 28 

TOTA L MALIGNAN T TDMORS 10 25 32 

TOTAL ANIMALS WITH SECONDARY TUMORS*
 

TOTAL SECONDARY TUMORS
 

TOTAL ANIMAL S WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOTAL UNCERTAIN TUMORS
 

TOTAL ANIMALS WITH TUMORS UNCERTAINPRTMAEY OH METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* PFIBAEY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

* SECONDARY TDMORS: METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

62
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED C. I. VAT YELLOW 4 IN THE DIET
 

63
 
64

 
TABLE C1. 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

_ _ _• — _______„____ _ » ._*______.».•—« ­. — ­. — — — _— — ————— — — — —— — •._..— — •*»—w— <— •— •— — — •*«*•• —•. 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMALS NECROPSIED 20 50 50 

ANIMALS EXAMINED HISTOPATHOLOGICALLY 20 50 50 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (50)
 

EPIDERMAL INCLUSION CYST 1 (2*)
 

*SUBCUT TISSUE (20) (50) (50)
 

CYST, NOS 1 (5%)
 

RESPIRATORY SYSTEM
 

#LUNG (20) (50) (50)
 

ATELECTASIS 2 (10%) l» (8%)
 

INFLAMMATION, NOS 1 (2X)
 

ALVEOLAR MACROPHAGES 1 (5%)
 

HEWATOPOIETIC SYSTEM
 

tSPLEEN (20) (50) (50)
 

FIBROSIS 1 (2%) 1 (2X)
 

HEMATOPOIESIS 1 (5«) 3 (6%) 3 (6X)
 

CIRCULATORY SYSTEM
 

•MYOCARDIUM	 (20) (50) (50)
 

INFLAMMATION, FOCAL 1 (555) 3 (6X)
 

INFLAMMATION, CHRONIC 1 (5%)
 

FIBROSIS 2 (10%) 1 (2%)
 

DIGESTIVE SYSTEM
 

tLIVER	 (20) (50) (50)
 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED BICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

65
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HEPATOCYTOMEGAL Y 

HYPEEPLASIA, NODULAR 

REGENERATIV E NODDLE 

ttLIVFR/HFPATOCYTES 

CYTOPLASMIC VACUOLIZATIO N 

*BILE DUCT 

HYPFRPLASIA , NOS 

#PANCREA S 

INFLAMMATION , NO S 

PERIARTERITI S 

ATROPHY , NO S 

CSTOMAC H 

INFLAMMATION , NO S 

fCOLO N 

POLYPOID HYPERPLASI A 

URINAR Y SYSTE M 

tKIONE Y 

INFLAMMATION , CHRONIC 

INFLAMMATION , GR ANDLOMATOU S 

INFARCT , HEALE D 

#KIDNSY/CORTE X 

CYST, NOS 

ENDOCRIN E SYSTE M 

• PITUITAR Y 

CYST, NOS 

ANGIECTASI S 

IADRENA L 

ANGIFCTASI S 

#ADPFNA L MEDULL A 

ANGIECTASI S 

tTHYROI D 

CYSTIC_£OiHC.LJS 

MATCHED 

CONTROL 

(20) 

(20) 

3 (15%) 

(19) 

2 (11%) 

(20) 

(18) 

(20) 

17 (85%) 

1 (5%) 

(20) 

(19) 

2 (11%) 

2 (11% ) 

(20) 

(20) 

(19) 

HIGH DOSE 

2 <<»») 

(50) 

H (8%) 

(50) 

« (8X) 

(50) 

1 (2)1) 

1 (2%) 

13 (26%) 

(50) 

1 (2%) 

(50) 

1 (2%) 

(50) 

43 (86%) 

1 (2%) 

(50) 

(49) 

3 (6%) 

1 (2%) 

(50) 

1 (2*) 

(50) 

(50) 

2_iaSL_ 

LOW DOSE 

2 (<t%) 

1 (2*) 

1 (2%) 

(50) 

6 (12%) 

(50) 

« (8%) 

(U9) 

3 (6%) 

6 (12%) 

(50) 

(U8 ) 

(50) <•<* (88%) 

(50) 

1 (2%) 

(US) 

3 (7%) 

U (9%) 

(50) 

3 (6%) 

(50) 

1 (2%) 

(50) 

1.J281 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

66
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

REPRODUCTIVE SYSTEH
 

*MAMMABY GLAND (20) (50) (50)
 

CYST, NOS 1 (2*)
 

f PROSTATE (19) 

INFLAMMATION, NOS 2 (11*) 5 (10*) 3 (6*) 

HYPERPLASIA, CYSTIC 1 (2*) 

*TFSTIS (20) (50) (50)
 

ATROPHY, NOS 2 (4*)
 

NERVOUS SYSTEM
 

IEEAIN (20) (49) (50)
 

INFLAMMATION, FOCAL 1 (2*)
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SYSTEM
 

NONE
 

BODY CAVITIES
 

NONE
 

ALL OTHER SYSTEMS
 

NONE
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LSSION REPORTED 1
 

AUTO/NECROPSY/HISTO PERF 1 1
 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

67
 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED C.I. VAT YELLOW 4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUDY

ANIMAL S NECROPSIE D

ANIMAL S EXAMINE D HISTOPA THOLOGICALL Y

 20

 20

 20

 50

 50

 50

 50 

 50 

 50 

INTEGUMENTAR Y SYSTFM 

NON E 

RESPIRATOR Y SYSTE M 

#LUN G

INFLAMMATION ,

INFLAMMATION ,

 SUPPUPATIV E

 GRANOLOMATOU S

 (20) (50)

 1 (2%) 

1 (2%) 

 (50) 

HEMA^OPOIETI C SYSTEM 

*BONE BARRO W

HYPOPLASIA , HEMATOPOIETIC

 (20)

 1 (5X) 

 (50) (50) 

frSPLEEN

PIGMENTATION , NOS

HYPERPLASIA , LYNPHOI D

HEMATOPOIESI S

 (20)

 1

 7

 (5%)

 (35%)

 (U8 )

 2 (tX) 

 19 (40S)

 (50) 

1 (2X ) 

 16 (32%) 

CIRCULATOR Y SYSTE M 

#HEART

THROMBUS, MURAL

 (20) (50) (50) 

2 (1%) 

#«YOCARDIUM

INFLAMMATION, FOCAL

 (20) (50)

 2 (4%)

 (50) 

1 (2*) 

DIGESTIVE SYSTEM 

#SALIVARY GLAND (20) (50) (U9) 

* NUMBE R OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

68
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

•IIVFR (20) C»9)

 

NECROSIS , FOCA L 

HEPATOCYTOMEGAL Y 1 (5%)

 

HYPERPLASIA , NODULA R 1 (2%)

 

#LIVER/HEPATOCYTE S (20) (U9 ) 

CYTOPLASMI C VACUOLI Z ATIO N 4 (20% ) 3 (6*) 

*BTL F DUCT (20) (50)

 

HYPERPLASIA , NO S 1 (5% ) 

• PANCREA S (19) (48) 

ECTOFI A 

ATROPHY , NO S 6 (13*) 

• STOMAC H (20) (U9 ) 

INFLAMMATION , NO S 1 (2«) 

INFLAMMATION , FOCAL 1 (5%) 

HYPERPLASIA , EPITHELIA L 1 (2X) 

HYPFRKERATOSI S 1 (2X) 

URINAP Y SYSTE M 

• KIDNE Y (20) (49) 

INFLAMMATION , CHRONIC U (20% ) 6 (12*) 

• KIDNEY/CORTE X (20) (H9 ) 

CYST, NOS 

ENL'OCRIN E SYSTE M 

• PITUITAR Y (19) (50) 

CYST , NOS 2 (11% ) 3 (6*) 

ANGIECTASI S 5 ( 26 %) 2 (<**) 

• ADRENA L (20) («9)

ANGIECTASI S 1 (5% ) 2 C*S) 

• THYROI D (20) (50) 

CYSTIC FOLLICLES 

HYPERPLASIA. , C-CELL 3 11521. 6 i'2SL 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECFOPSIED
 

HIGH DOSE 

(50) 

1 (2*) 

(50) 

5 (105S) 

(50) 

(50) 

1 <2X) 

13 (26*) 

(50) 

(50) 

10 (20*) 

(50) 

1 (2X) 

(50) 

5 (10*) 

14 (28*) 

(50) 

2 («»*) 

(50) 

1 (2*) 

69
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

REPRODUCTIV E SYSTE M 

*MAMMAF Y GLAN D (20) (50) (50) 

HYPFRPLASIA , CYSTIC 2 (10%) 1 (2%) 2 (4%) 

tUTERU S (20) (49) (50) 

POLYPOI D HYPERPLASI A U (20% ) 8 (16%) U (8%) 

#UTEFtJS/FNDOHETHIU H (20) (49) (50) 

CYST , NOS 2 (10%) 1 (2%) 1 (2X) 

#OVHF Y (20) (U9 ) (50) 

CYST, NOS 1 (5%) 3 (6%) 6 (12%) 

NERVOU S SYSTE M 

#ERAI N (20) (50) (50) 

HEMORRHAG E 1 (5%) 1 (2%) 

SPECIAL SENSE ORGAN S 

NON E 

MUSCULOSKELETA L SYSTEM 

NON E 

BOD Y CAVITIES 

NON E 

A L L OTHER SYSTEMS 

NON E 

SPECIA L MOFPHOLOGY SUMMAR Y 

NO_LESION_EEiOjeTED 1. 2 § 

* NUMBE R OF ANIMAL S WITH TISSDE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIED 

70 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW OOSE HIGH DOSE 

AUTO/NECROPSY/HISTO PERF 

* NUNBER OF ANIMALS WITH TISSUE EX*HINED MICROSCOPICALLY 

* NUHBER OF ANIHAL S NECROPSIED 

71 
72

 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED C. I. VAT YELLOW 4 IN THE DIET
 

73
 
74

 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED C.I. VAT YELLOW 4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

ANIMAL S KISSIN G 2 

ANIMAL S NTCROPSIE D 20 <*7 50 

ANIMAL S EXAMINE D HISTOPATHOLOGIC ALL Y 20 H7 50 

INTEGUMENTAR Y SYSTEM 

*SKIN (20) («7) (50)
 

SPIDERMAL INCLUSION CYST 1 (2*)
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN (19) (US) (50)
 

HYPERPLASIA, LYMPHOID 1 (5*) 5 (11*) 5 (10*)
 

HEMATOPOIESIS 2 (11*) 10 (22*) 3 (6*)
 

tMESENTERIC L. NODE (18) (46) («9)
 

HYPERPIASIA, LYHPHOID 3 (6*)
 

CIRCULATORY SYSTEM
 

#HEART (20) («7) (50)
 

MINERALIZATION 1 (2*)
 

#MYOCARDIUM . (20) (l»7) (50)
 

INFLAMMATION, FOCAL 1 (5*)
 

DIGESTIVE SYSTEM
 

fLIVEH (20) (47) (50)
 

INFLAMMATION, FOCAL 1 (2*)
 

# NUMBER OF ANIMALS WITH TISSOE EXAHIHED MICROSCOPICALLY
 * NUMBER OF ANIMALS NECROPSIED
 

75
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

#STOMACH
 

INFLAMMATION, FOCAL
 

*COLON
 

PARASITISM
 

URINAF Y SYSTEM
 

*KIDNEY
 

INFLAMMATION, INTERSTITIAL
 

INFLAMMATION, CHRONIC
 

INFAPCT, HEALED
 

LYMPHOCYTOSIS
 

ttKIDNFY/CORTEX
 

CYST, NOS
 

ENLOCP.INE SYSTEM
 

* THYROID
 

ATROPHY, NOS
 

#Pf,NCREATIC ISLETS
 

HYPFFPLASIA, NOS
 

REPRODUCTIVE SYSTEM
 

*SEMINAL VESICLE
 

CAST, NOS
 

•TESTIS
 

ATROPHY^.NOS
 

(19) (1*7) (50) 

2 (<*X) 2 (4X) 

(19) (46) (46) 

1 (2X) 

(20) (47) (50) 

1 (5%) 1 (2X) 

1 (5X) 

1 (2X) 

1 (5%) 1 (2X) 

(20) ((47) (50) 

1 (2X) 

(20) (15) (48) 

1 (5X) 1 (2X) 

(19) (46) (47) 

3 (16X) 5 (11X) 1 (2X) 

(20) (50) 

1 (2X) 

(19) (50) 

# NUMBFR OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

76
 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NERVOU S SYSTEM 

# BP.A IN

(1 IN REALIZATIO N

 (20)

 5 (25%)

 (U7 )

 12 (26%)

 (50) 

7 (14X) 

SPECIA L

NON E 

 SENS E ORGAN S 

MUSCULOSKELETA L

NONE 

 SYSTEPI 

BODY CAVITIES 

NON F 

A LL OTHER

NON E 

 SYSTEM S 

SPECIAL MORPHOLOG Y SUMMAR Y 

NO LESION EEPOETED

ANIMA L MISSING/NO NECROPS Y

AOTOLYSIS/N O NECROPSY

# NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S NECHOPSIE D 

 EXAMINE D

 <t

 MICROSCOPICALL Y 

1

 2 

1 

2 

77 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED C.I. VAT YELLOW4 IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

50 

ANIMAL S MISSIN G 1 1 

ANIMAL S NFCROPSIE D 19 48 50 

ANIMAL S EXAMINE D HISTOPATHOtOGICALLY 19 48 50 

ANIMAL S INITIALL Y IN STUDY 20 50 

INTFGUMENTARY SYSTEM
 

*SU3CUT TISSUE (19) (48) (50)
 

CYST, NOS 2 (4S)
 

RESPIRATORY SYSTEM
 

#LUNG (19) (48) (50)
 

HEMORRHAGE 1 (5%) 1 (2%) 1
 

HEMATOPOIFTIC SYSTEM
 

#SPLEE N (19) (47 ) (50) 

HYPFRPLASIA , LYMPHOI D 3 (6X) 2 (4X) 

HEMATOPOIESI S 7 (37%) 17 (36%) 12 (24% ) 

tKANDIBUlA R L. NODE (19) (U8) (50) 

HYPERPLASIA , LYHPHOI D 1 (2%) 

"MESENTFFI C L. NODE (19) (48) (50) 

INFLAMMATION , GRANULOMATOD S 1 (2%) 

HYPERPLASIA , LYMPHOI D 1 (2% ) 

fTHYM S (11) (23) (37) 

HYPERPLASIA , LYMPHOI D 1 (3*) 

CISCULATORY SYSTEM
 

• MYOCARDIUM (19) («8) (50)
 

INFLAMMATION, FOCAL 1 (5«)
 

DIGESTIVE SYSTEM
 

*LIVER (19) (48) (50)
 

» NUMBER OF ANIMALS WITH TISSUE EXAMINE D MICROSCOPICALLY
 

* NUMBER OF ANIMAL S NECROPSIED
 

78 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

INFLAMMATION , GRANULOMftTOUS

HYPERPLASIA , NODULA R

LYMPHOCYTOSI S

*PANCRFAS

CYSTIC DUCTS

#STOMACH


INFLAMMATION, FOCAL


URINAF Y SYSTEM
 

#KIDNSY


INFLAMMATION, CHRONIC


INFAPCT, NOS


METAPLASIA, OSSEOUS


LYMPHOCYTOSIS


#UBTNARY BLADDER


LYMPHOCYTOSIS


ENCOCRINT SYSTEM
 

#THYROID


CYSTIC FOLLICLES


REPRODUCTIVE SYSTEM
 

tUTEPUS


POLYPOID HYPERPLASIA


#UT?RUS/ENDOHETRIUH


CYST, NOS


tOVARY


CYST, NOS


NERVOUS SYSTEM
 

#BRAI N

MINFRALIZATIO N

SPECIA L SENS E ORGAN S 

NONE 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

1 (2X) 

1 (5*) 2 (4X) 5 (10*) 

1 (5%) 

 (19) (43) (45) 

1 (5*) 3 (7*) 

 (19) (U8) (49) 

2 (4X) 3 (6X) 

 (19) (48) (50) 

1 (2*) 

1 (2X) 

1 (2X) 

1 (5X) 3 (6X) 

 (17) (46) (44) 

2 (5X) 

 (19) (47) (49) 

1 (2«) 1 (2%) 

 (19) (48) (48) 

1 (2*) 

 (19) (48) (48) 

7 (37*) 14 (29%) 7 (15*) 

 (17) (48) (46) 

1 (6*) 10 (21*) 7 (15*) 

 (19 ) (48) (50) 

2 (11*) 7 (15*) 10 (20*) 

# NUMBER OF ANIMAL S WITH TISSUE EXABIHBD HICROSCOPICALLT
 

* NUMBER OF ANIMALS NECHOPSIED
 

79
 
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) _ _ — _________ _ _ —— _ _ —_ _ _ .— _ _ _ •._ _ _ _ _ _ _ ______ _ _ _ _ _______ _ _ _ __-«._ ____________________ _ 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKELETS L SYSTEM 

NON E 

BOD Y CAVITIF S 

NON^ 1 

A L L OTHE R SYSTEM S 

*1ULTIPLS ORGANS (19) (48) (50)
 

PIPROSI? 1 (2%)
 

SPECIAL MORPHOLOGY SUMMARY
 

NO LESION REPORTED U 5
 

»NT«A L MISSING/NO NECROPSY 1 1
 

AOTOLYSTS/NO NECROPSY 1
 

# NUMBER OF ANIMAL S WITH TISSUF EXAMINED MICROSCOPICALLY
 

* N'IMBER OF ANIMALS NECROPSIED
 

80
 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS IN
 

RATS ADMINISTERED C. I. VAT YELLOW & IN THE DIET
 

81
 
82

 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered C. I. Vat Yellow 4 in the Diet (a) 

Topography; Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar 

Carcinoma (b) 

0/20 (0) 5/50 (10) 0/50 (0) 

P Values (c,d) N.S. N.S. 

Departure from Linear Trend (e) P = 0.010 

CO 

CO 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Infinite 

0.525 

Infinite 

104 

— 

— 

— 

— 

Lung: Alveolar/Bronchiolar Carcinoma 

or Adenoma (b) 1/20 (5) 8/50 (16) 1/50 (2) 

P Values (c,d) N.S. N.S. N.S. 

Departure from Linear Trend (e) P = 0.019 

Relative Risk (f) 

Lower Limit 

Upper Limit 

3.200 

0.482 

138.771 

0.400 

0.005 

30.802 

Weeks to First Observed Tumor 104 101 104 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered C. I. Vat Yellow 4 in the Diet (a) 

(continued) 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Hematopoietic System:

or Leukemia (b)

 Lymphoma 

 3/20 (15) 6/50 (12) 4/50 (8) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.800 

0.195 

4.615 

0.533 

0.102 

3.410 

oo 

-F* 

Weeks to First Observed Tumor 104 89 59 

All Sites: Hemangiosarcoma (b) 1/20 (5) 0/50 (0) 3/50 (6) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.000 

0.000 

7.475 

1.200 

0.106 

61.724 

Weeks to First Observed Tumor 104 104 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered C. I. Vat Yellow 4 in the Diet (a) 

(continued) 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Adenoma (b) 0/20 (0) 1/50 (2) 3/50 (6) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.022 

Infinite 

Infinite 

0.250 

Infinite 

Weeks to First Observed Tumor 104 101 

oo 

Pituitary: Adenoma, NOS (b) 3/19 (16) 4/45 (9) 6/49 (12) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.563 

0.108 

3.578 

0.776 

0.191 

4.463 

Weeks to First Observed Tumor 104 91 96 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats 

Administered C. I. Vat Yellow 4 in the Diet (a) 

(continued) 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Pituitary: Adenoma or 

Adenocarcinoma, NOS (b) 3/19 (16) 4/45 (9) 8/49 (16) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.563 

0.108 

3.578 

1.034 

0.288 

5.620 

00 Weeks to First Observed Tumor 104 91 96 

Adrenal: Pheochromocytoma (b) 4/20 (20) 1/50 (2) 5/50 (10) 

P Values (c,d) N.S. P = 0.021 (N) N.S. 

Departure from Linear Trend (e) P = 0.015 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.100 

0.002 

0.944 

0.500 

0.124 

2.322 

Weeks to First Observed Tumor 104 104 91 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Matched Low
 

Topography; Morphology Control Dose
 

Thyroid: Follicular-cell
 

Adenoma or Carcinoma (b) 0/19 (0) 3/50 (6)
 

P Values (c,d) N.S. N.S.
 

Relative Risk (f) Infinite
 

Lower Limit 0.238
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 104
 

oo
 

Thyroid: C-cell Adenoma or
 

Carcinoma (b) 4/19 (21) 2/50 (4)
 

P Values (c,d) P = 0.034 (N) P = 0.045 (N)
 

Relative Risk (f) 0.190
 

Lower Limit 0.019
 

Upper Limit 1.230
 

Weeks to First Observed Tumor 104 104
 

High
 

Dose
 

1/50 (2)
 

N.S.
 

Infinite
 

0.021
 

Infinite
 

104
 

2/50 (4)
 

P = 0.045 (N)
 

0.190
 

0.019
 

1.230
 

104
 
__ 

Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Topography; Morphology 

Pancreatic Islets: Is letcell Adenoma (b) 

P Values (c,d) 

Departure from Linear Trend (e) 

Relative Risk (f) 

00 

00 Weeks to

Testis:

P Values

Relative

Lower Limit 

Upper Limit 

 First Observed Tumor 

 Interstitial-cell Tumor

 (c,d) 

 Risk (f) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor
 

Hatched Low High 

Control Dose Doae 

0/19 (0) 3/49 (6) 0/50 (0) 

N.S. U.S. — 

P » 0.047 

Infinite 

0.243 — Infinite — 

104 — — 

 (b) 16/20 (80) 44/50 (88) 39/50 (78) 

U.S. N.S. N.S. 

1.100 0.975 

0.884 0.775 

1.458 1.382 

104 83
 91 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

(a) Dosed groups received 3,500 or 7,000 ppm.
 

(b) Number of tumor-bearing animals/number of animals examined at site
 

(percent).
 

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors 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 less than 0.05;
 

otherwise, not signficant (N.S.) is indicated.
 

oo
 (d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

Matched Low
 

Topography: Morphology


Lung: Alveolar/Bronchiolar Adenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


VO
 

o
 

Hematopoietic System: Lymphoma
 

or Leukemia (b)


P Values(c, d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


 Control Dose 

 1/20 (5) 0/50 (0) 

 N.S. N.S. 

0.000 

0.000 

7.475 

 104 — 

 4/20 (20) 5/50 (10) 

P = 0.011 (N) N.S. 

0.500 

0.124 

2.322 

 95 93

High
 

Dose
 

4/50 (8)
 

N.S.
 

1.600
 

0.175
 

77.169
 

104
 

1/50 (2)
 

P = 0.021 (N)
 

0.100
 

0.002
 

0.944
 

 104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Pituitary: Adenoma, NOS (b) 2/19 (11) 12/50 (24) 6/50 (12) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.280 

0.587 

19.837 

1.140 

0.232 

10.985 

Weeks to First Observed Tumor 103 96 98 

Pituitary: Adenoma or 

Adenocarcinoma, NOS (b) 3/19 (16) 12/50 (24) 6/50 (12) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 1.520 0.760 

Lower Limit 0.481 0.187 

Upper Limit 7.762 4.377 

Weeks to First Observed Tumor 91 96 98
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Thyroid: C-cell Carcinoma (b) 0/20 (0) 1/50 (2) 3/50 (6)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.022 0.250
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor — 101 104
 

VO
 

NJ Thyroid: C-cell Carcinoma
 

or Adenoma (b) 2/20 (10) 2/50 (4) 4/50 (8)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 0.400 0.800
 

Lower Limit 0.032 0.128
 

Upper Limit 5.277 8.436
 

Weeks to First Observed Tumor 91 101 104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Topography: Morphology


Mammary Gland: Adenoma or
 

Cystadenoma, NOS (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 vo
 

U>
 

Mammary Gland: Fibroadenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 1/20 (5)
 

 N.S.
 

 91
 

 1/20 (5)
 

 N.S.
 

104
 

Low
 

Dose
 

3/50 (6)
 

N.S.
 

1.200
 

0.106
 

61.724
 

102
 

8/50 (16)
 

N.S.
 

3.200
 

0.482
 

138.771
 

83
 

High
 

Dose
 

1/50 (2)
 

N.S.
 

0.400
 

0.005
 

30.802
 

104
 

6/50 (12)
 

N.S.
 

2.400
 

0.325
 

108.021
 

104
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

(a) Dosed groups received 3,500 or 7,000 ppm.
 

(b) Number of tumor-bearing animals/number of animals examined at site (percent).
 

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors 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 less than 0.05;
 

otherwise, not signficant (N.S.) is indicated.
 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 

vo
 

**	 (e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TURMORS IN
 

MICE ADMINISTERED C. I. VAT YELLOW 4 IN THE DIET
 

95
 
6

 9
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Integumentary System: Lipoma of

the Subcutaneous Tissue (b)

 the 

 2/20 (10) 1/47 (2) 4/50 (8) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.213 

0.004 

3.909 

0.800 

0.128 

8.436 

Weeks to First Observed Tumor 106 97 105 

Lung: Alveolar /Bronchiolar 

Carcinoma (b) 4/26 (20) 12/47 (26) 11/50 (22) 

P Values (c,d) U.S. U.S. U.S. 

Relative Risk (f) 1.277 1.100 

Lower Limit 0.457 0.384 

Upper Limit 4.926 4.321 

Weeks to First Observed Tumor 100 85 87
 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Administered C. I. Vat Yellow 4 in the Diet (a) 

(continued) 

Topography; Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Lung: Alveolar/Bronchiolar

or Adenoma (b)

 Carcinoma 

 4/20 (20) 14/47 (30) 15/50 (30) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.489 

0.554 

5.616 

1.500 

0.566 

5.627 

VO 

00 

Weeks to First Observed Tumor 100 85 82 

Hematopoietic System: Lymphoma (b) 3/20 (15) 7/47 (15) 22/50 (44) 

P Values (c,d) P = 0.002 N.S. P = 0.019 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.993 

0.261 

5.532 

2.933 

1.040 

13.947 

Weeks to First Observed Tumor 85 87 53 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice 

Administered C. I. Vat Yellow 4 in the Diet (a) 

(continued) 

Topography: Morphology

Matched 

 Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma (b) 3/20 (15) 22/47 (47) 21/50 (42) 

P Values (c,d) N.S. P = 0.012 P = 0.027 

Relative Risk (f) 

Lowe r Limi t 

Upper Limit 

3.121 

1.110 

14.730 

2.800 

0.986 

13.384 

Weeks to First Observed Tumor 80 27 83 

\o 

VO 

Small

NOS

 Intestine:

 (b)

 Adenoc arc inoma, 

 0/19 (0) 3/46 (7) 1/49 (2) 

P Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.259 

Infinite 

Infinite 

0.021 

Infinite 

Weeks to First Observed Tumor 106 106 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

(a) Dosed groups received 25,000 or 50,000 ppm.
 

(b) Number of tumor-bearing animals/number of animals examined at site (percent).
 

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors 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 less than 0.05;
 

otherwise, not signficant (N.S.) is indicated.
 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 H1
 

o
 

0

 (e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

Topography: Morphology


Lung: Alveolar/Bronchiolar
 

Carcinoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


Lung: Alveolar/Bronchiolar Carcinoma
 

or Adenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

 Control Dose Dose 

 2/19 (11) 3/48 (6) 2/50 (4) 

 N.S. U.S. N.S. 

0.594 0.380 

0.076 0.030 

6.774 5.009 

 106 106 106 

 2/19 (11) 5/48 (10) 3/50 (6) 

 N.S. N.S. N.S. 

0.990 0.570 

0.184 0.073 

9.880 6.511 

106 106 106 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Heraatopoietic System:
 

Lymphoma (b) 6/19 (32) 12/48 (25) 17/50 (34)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 0.792 1.077
 

Lower Limit 0.336 0.499
 

Upper Limit 2.267 2.920
 

o Weeks to First Observed Tumor 77 62 26
 N3
 

Liver: Hepatocellular Carcinoma (b) 2/19 (11) 6/48 (13) 9/50 (18)
 

P Values (c,d) N.S. N.S. N.S.
 

Relative Risk (f) 1.188 1.710
 

Lower Limit 0.242 0.407
 

Upper Limit 11.426 15.426
 

Weeks to First Observed Tumor 106 87 93
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered C. I. Vat Yellow 4 in the Diet (a)
 

(continued)
 

(a) Dosed groups received 12,500 or 25,000 ppm.
 

(b) Number of tumor-bearing animals/number of animals examined at site
 

(percent).
 

(c) Beneath the incidence of tumors in the control group is the probability level for the Cochran-


Armitage test when P less than 0.05; otherwise, not significant (N.S.) is indicated. Beneath
 

the incidence of tumors 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 less than 0.05;
 

otherwise, not signficant (N.S.) is indicated.
 

(d) A negative trend (N) indicates a lower incidence in a dosed group than in a control group.
 

(e) The probability level for departure from linear trend is given when P less than 0.05 for any
 

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
104

 
Review of the Bioassay of C.I. Vat Yellow 4* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup of the
 

Clearinghouse on Environmental Carcinogens
 

August 31, 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 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
 

C.I. Vat Yellow 4 for carcinogenicity.
 

Although the primary reviewer noted the increased incidence of
 

lymphomas and hepatomas among treated high dose male mice, he said that
 

the evidence was insufficient to designate C.I. Vat Yellow 4 to be a
 

carcinogen. He commented on the large disparity between the dose levels
 

administered to rats (3,500 and 7,000 ppm) compared to mice (male:
 

25,000 and 50,000 ppm; female: 12,500 and 25,000 ppm). Except for a
 

question regarding the disparity in dosages, the primary reviewer said
 

that the study was adequate. He said that the lack of a dose-response
 

relationship for hepatomas may be due to the fact that both dose levels
 

are on the upper slope of the curve. He opined that the absence of a
 

response among treated female mice may be a result of the lower dose
 

levels administered. The primary reviewer suggested that the report
 

over emphasized statistics and that not enough attention was given to
 

pharmacological considerations. He recommended that both the increased
 

incidence of hepatomas and lymphomas, among treated male mice, recieve
 

mention in the report's summary conclusion. He proposed that the
 

conclusion be reworded as follows, "Increased incidences of lymphomas
 

and of hepatomas were observed in male mice receiving the higher dose,
 

s^&gesting carcinogenic activity and requiring consideration for further
 

tests." He concluded by stating that the human risk posed by C.I. Vat
 

Yellow 4, if any, is very low.
 

105
 
A program staff member said that the staff had rejected the
 

significance of the liver tumors in the high dose male mice based on the
 

Bonferroni criterion. In addition, the historical control data did not
 

support the significance of the liver tumors. In regard to the lymphomas,
 

he said that their significance was sustained by both the Bonferroni
 

criterion and historical control data.
 

The primary reviewer said that the basic disagreement was not with
 

the interpretation of the data but rather with how it should be stated.
 

A vote on a proposal that the conclusion be reworded, as suggested, was
 

defeated. Subsequently, a recommendation was approved unanimously that
 

the report on the bioassay of C.I. Vat Yellow 4 be accepted as written.
 

Members present were:
 

Arnold Brown (Chairman), University of Wisconsin Medical School
 

Joseph Highland, Environmental Defense Fund
 

Michael Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

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.
 

Jf.U.5. GOVERNMEN T PRINTIN G OFFICE : 1 9 7 8-?. 8 1 - 2 I 7 / 3 2 7 0
 

106
 

DHEW Publication No. (NIH) 79-1389