National Cancer Institute

CARCINOGENESIS

Technical Report Series

No. 71

1978

BIOASSAY OF

L-TRYPTOPHAN

FOR POSSIBLE CARCINOGENICITY

CAS No. 73-22-3

NCI-CG-TR-71

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

Public Health Service

National Institutes of Health


BIOASSAY OF

L-TRYPTOPHAN

FOR POSSIBLE CARCINOGENICITY

Carcinogenesis Testing Program

Division of Cancer Cause and Prevention

National Cancer Institute

National Institutes of Health

Bethesda, Maryland 20014

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

Public Health Service

National Institutes of Health

DHEW Publication No. (NIH) 78-1321

BIOASSAY OF

L-TRYPTOPHAN

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

L-tryptophan 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 animal carcinogens requires a wider analysis.

CONTRIBUTORS; The bioassay of L-tryptophan was conducted by

Southern Research Institute, Birmingham, Alabama, initially under

direct contract to NCI and currently under a subcontract to

Tracer Jitco, Inc., prime contractor for the NCI Carcinogenesis

Testing Program.

The experimental design and doses were determined by Drs. D. P.

Griswold^, J. D. Prejean^, E. K. Weisburger^, and J. H.

Weisburger^»3. Ms. J. Belzer* and Mr. I. Brown^ were responsible

for the care and feeding of the laboratory animals. Data management and retrieval were performed by Ms. C. A. Dominick .

Histopathologic examinations were performed by Drs. S. D.

Kosanke* and J. C. Peckham^-, and the diagnoses included in this

report represent their interpretation.

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

Mason Research Institute . The statistical analyses were performed by Dr. J. R. Joiner-*, using methods selected for the

bioassay program by Dr. J. J. Gart . Chemicals used in this

bioassay were analyzed under the direction of Dr. E. Murrill^,

and the analytical results were reviewed by Dr. C. W. Jameson^.

The structural formula was supplied by NCI2.

This report was prepared at Tracor Jitco-> under the direction of

NCI. Those responsible for the report at Tracor Jitco were

Dr. Marshall Steinberg, Director of the Bioassay Program; Dr. L.

A. Campbell, Deputy Director for Science; Drs. J. F. Robens and

C. H. Williams, toxicologists; Dr. G. L. Miller, Ms. L. A. Waitz,

and Mr. W. D. Reichardt, bioscience writers; and Dr. E. W.

Gunberg, technical editor, assisted by Ms. Y. E. Presley.

The statistical analysis was reviewed by members of the

Mathematical Statistics and Applied Mathematics Section of NCI":

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

Robert E. Tarone.

The following other scientists at NCI2 were responsible for

evaluating the bioassay experiment, interpreting the results, and

reporting the findings: Dr. Kenneth C. Chu, Dr. Cipriano Cueto,

Jr., Dr. J. Fielding Douglas, Dr. Dawn G. Goodman, Dr. Richard A.

Griesemer, Dr. Harry A. Milman, Dr. Thomas W. Orme, Dr. Robert A.

Squire", and Dr. Jerrold M. Ward.

•'•Southern Research Institute, 2000 Ninth Avenue South,

Birmingham, Alabama.

2Carcinogenesis Testing Program, Division of Cancer Cause and

Prevention, National Cancer Institute, National Institutes of

Health, Bethesda, Maryland.

oJNow with the Naylor Dana Institute for Disease Prevention,

American Health Foundation, Hammond House Road, Valhalla,

New York.

iv
^EG&G Mason Research Institute, 1530 East Jefferson Street,

Rockville, Maryland.

5Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville,

Maryland.

"Mathematical Statistics and Applied Mathematics Section,

Biometry Branch, Field Studies and Statistics, Division of

Cancer Cause and Prevention, National Cancer Institute, National

Institutes of Health, Bethesda, Maryland.

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

Missouri.

^Now with the Division of Comparative Medicine, Johns Hopkins

University, School of Medicine, Traylor Building, Baltimore,

Maryland.

V

SUMMARY

A bioassay of the amino acid L-tryptophan for possible carcinogenicity was conducted by administering the test chemical in feed

to Fischer 344 rats and B6C3F1 mice.

Groups of 35 rats and 35 mice of each sex were administered

L-tryptophan at one of two doses, either 25,000 or 50,000 ppm, 5

days per week for 78 weeks, and then observed for 26 or 27 weeks.

Matched controls consisted of groups of 15 rats or 15 mice of

each sex. All surviving rats and mice were killed at 104 or 105

weeks.

L-Tryptophan had little toxic effect on the rats; mean body

weight loss was minimal and survival of dosed groups of both

sexes was high. In the mice, mean body weights of dosed animals

were lower than those of controls throughout most of the

bioassay, particularly in the females. Sufficient numbers of

rats were at risk to termination of the study for development of

late-appearing tumors, and sufficient numbers of mice were at

risk beyond 52 weeks of the study for development of tumors.

No neoplasms occurred in a statistically significant incidence

among dosed rats when compared with controls.

In both male and female mice, neoplasms of the hematopoietic

system occurred at higher incidences in the low-dose groups than

in the matched-control groups (males: controls 0/12, low-dose

9/34, high-dose 2/33; females: controls 2/13, low-dose 6/33,

high-dose 1/35). These incidences, however, are not

statistically significant, using the Bonferroni correction, and

therefore, no tumors are considered to be related to the

administration of the test chemical.

It is concluded that under the conditions of this bioassay,

L-tryptophan was not carcinogenic for Fischer 344 rats or B6C3F1

mice.

vii

TABLE OF CONTENTS

Page

I. Introduction 1

II. Materials and Methods 3

A. Chemical 3

B. Dietary Preparation 4

C. Animals 4

D. Animal Maintenance 5

E. Subchronic Studies 7

F. Designs of Chronic Studies 8

G. Clinical and Pathologic Examinations 8

H. Data Recording and Statistical Analyses... 12

III. Results -Rats 17

A. Body Weights and Clinical Signs (Rats) 17

B. Survival (Rats) 17

C. Pathology (Rats) 20

D. Statistical Analyses of Results (Rats) 21

IV. Results - Mice 23

A. Body Weights and Clinical Signs (Mice) 23

B. Survival (Mice) 23

C. Pathology (Mice) 26

D. Statistical Analyses of Results (Mice) 30

V. Discussion 33

VI. Bibliography 35

APPENDIXES

Appendix A Summary of the Incidence of Neoplasms in

Rats Fed L-Tryptophan in the Diet 39

Table Al Summary of the Incidence of Neoplasms in

Male Rats Fed L-Tryptophan in the Diet 41

Table A2 Summary of the Incidence of Neoplasms in

Female Rats Fed L-Tryptophan in the Diet 45

ix
Page

Appendix B Summary of the Incidence of Neoplasms in

Mice Fed L-Tryptophan in th Diet 49

/

Table Bl Summary of the Incidence of Neoplasms in

Male Mice Fed L-Tryptophan in the Diet 51

Table B2 Summary of the Incidence of Neoplasms in

Female Mice Fed L-Tryptophan in the Diet 54

Appendix C Summary of the Incidence of Nonneoplastic

Lesions in Rats Fed L-Tryptophan in the Diet 57

Table Cl Summary of the Incidence of Nonneoplastic

Lesions in Male Rats Fed L-Tryptophan in the Diet.. 59

Table C2 Summary of the Incidence of Nonneoplastic

Lesions in Female Rats Fed L-Tryptophan

in the Diet 63

Appendix D Summary of the Incidence of Nonneoplastic

Lesions in Mice Fed L-Tryptophan in the Diet 67

Table Dl Summary of the Incidence of Nonneoplastic

Lesions in Male Mice Fed L-Tryptophan in the Diet.. 69

Table D2 Summary of the Incidence of Nonneoplastic

Lesions in Female Mice Fed L-Tryptophan

in the Diet 72

Appendix E Analyses of the Incidence of Primary Tumors

in Rats Fed L-Tryptophan in the Diet 77

Table El Analyses of the Incidence of Primary Tumors

in Male Rats Fed L-Tryptophan in the Diet 79

Table E2 Analyses of the Incidence of Primary Tumors

in Female Rats Fed L-Tryptophan in the Diet 84

Appendix F Analyses of the Incidence of Primary Tumors

in Mice Fed L-Tryptophan in the Diet 89

Table Fl Analyses of the Incidence of Primary Tumors

in Male Mice Fed L-Tryptophan in the Diet 91

Table F2 Analyses of the Incidence of Primary Tumors

in Female Mice Fed L-Tryptophan in the Diet........ 95

x
Page

TABLES

Table 1 Design of L-Tryptophan Chronic Feeding

Studies in Rats 9

Table 2 Design of L-Tryptophan Chronic Feeding

Studies in Mice 10

FIGURES

Figure 1 Growth Curves for Rats Fed L-Tryptophan

in the Diet 18

Figure 2 Survival Curves for Rats Fed L-Tryptophan

in the Diet 19

Figure 3 Growth Curves for Mice Fed L-Tryptophan

in the Diet 24

Figure 4 Survival Curves for Mice Fed L-Tryptophan

in the Diet 25

xi

I. INTRODUCTION

L-Tryptophan (CAS 73-22-3; NCI C01729) is an essential amino acid

for humans, and a precursor of the neurohormones serotonin

(5-hydroxytryptamine) and melatonin (N-acetyl-5-methoxytryptamine), and the B vitamin nicotinic acid (Orten and Neuhaus,

1975). It is found in small concentrations in casein, and in

many foods (Stecher, 1968; Food and Agriculture Organization,

1970).

In the 1950's, there were two reports that the dietary

administration of DL-tryptophan to rats modified the carcinogenic

effects of 2-acetylaminofluorene. The combined administration of

these compounds resulted in the production of bladder tumors

which were not found in animals dosed with 2-acetylaminofluorene

alone (Dunning et al., 1950; Boyland et al., 1954). In

subsequent studies in which the tryptophan metabolites indole,

indican, or 3-hydroxyanthranilic acid were administered by

subcutaneous injection to mice, malignant tumors of the reticuloendothelial system, and leukemia were observed, but there was no

evidence of bladder tumors (Ehrhart and Stich, 1957, and 1958;

Ehrhart et al., 1959). Tryptophan metabolites were also tested

in mice by bladder implantation techniques, and among those that

were positive were o-aminophenol derivatives such as 3-hydroxykynurenine, 3-hydroxyanthranilic acid, and 2-amino-3-hydro1
xyacetophenone (Allen et al., 1957; Bryan et al., 1964), although

L-tryptophan itself was negative (Boyland et al., 1964).

Finally, DL-tryptophan induced bladder hyperplasia but no cancer

in dogs when fed in high doses for 1 year (Radomski et al., 1969,

1970, and 1977) which led these authors to the conclusion that

tryptophan metabolites may act as co-carcinogens.

L-tryptophan was selected for study in the Carcinogenesis Testing

Program because the evidence available at the time of selection

suggested that tryptophan or a metabolite was involved in the

etiology of bladder cancer.

2
II. MATERIALS AND METHODS

A. Chemical

L-TRYPTOPHAN

L-Tryptophan (L-a-amino-/J-indolepropionic acid) was obtained in a

single batch (Lot No. C-8-30-72) for the chronic studies from

Carroll Products, Wood River Junction, Rhode Island. The

identity and purity of this batch was confirmed in analyses at

Midwest Research Institute. No impurities were found by thinlayer chromatography. The melting point was 275-284°C

(literature: 278°C, Dictionary of Organic Compounds, 1965).

Elemental analyses (C, H, N) were consistent with C^H^2N2^2»

 the

molecular formula of tryptophan. Nuclear magnetic resonance,

infrared, and ultraviolet spectra were in agreement with the

structure and matched the spectra given in the literature.

The chemical was stored in the original container at 5°C.

3
B. Dietary Preparation

Test diets were prepared every 2 weeks by mixing a known amount

of sifted L-tryptophan with a small amount of Wayne® Lab Blox

animal meal (Allied Mills, Inc., Chicago, 111.) in a portable

mixer, then adding this mixture to the required amount of animal

meal and mixing in a twin-shell blender for 10 minutes. Tests of

the concentration or stability of the chemical in feed were not

performed.

The prepared diets were stored at room temperature in sealed

plastic containers.

C. Animals

For the subchronic studies, male Sprague-Dawley rats were

obtained from Charles River Breeding Laboratories, Inc.,

Wilmington, Massachusetts, and male Swiss mice were obtained from

Purina Laboratories, St. Louis, Missouri. All animals were 30

days of age on arrival at the laboratory. They were quarantined

for 7 days and then placed on study.

For the chronic studies, Fischer 344 rats and B6C3F1 mice were

obtained from Charles River Laboratories under a contract with

the Division of Cancer Treatment, National Cancer Institute. On

arrival at the laboratory, male and female rats were 30 days of

4
age, male mice 31 days of age, and female mice 38 days of age.

All animals were quarantined for 12 days. Animals with no

visible signs of disease were assigned to control or dosed groups

and earmarked for individual identification.

D. Animal Maintenance

All animals were housed in temperature- and humidity-controlled

rooms. The temperature range was 20-24°C, and the relative

humidity was 40-60%. The room air was changed 15 times per hour

and passed through both intake and exhaust fiberglass roughing

filters. In addition to natural light, illumination was provided

by fluorescent light for 9 hours per day. Food and water were

supplied daily and were available ad libitum.

All animals were housed five per cage in solid-bottom stainless

steel cages (Hahn Roofing and Sheet Metal Co., Birmingham, Ala.).

The rat cages were provided with Iso-Dri® hardwood chip bedding

(Carworth, Edison, N.J.), and cage tops were covered with

disposable filter bonnets; mouse cages were provided with

Sterolit® clay bedding (Englehard Mineral and Chemical Co., New

York, N.Y.) and cage tops were covered with filter bonnets

beginning at week 86. Bedding was replaced once per week; cages,

water bottles, and feeders were sanitized at 82°C once per week;

and racks were cleaned once per week.
The rats and mice were housed in separate rooms. Control animals

were housed with respective dosed animals. Animals administered

L-tryptophan were maintained in the same rooms as animals of the

same species being dosed with the following chemicals:

»

RATS

Feed Studies

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide

(acetohexamide) (CAS 968-81-0)

anthranilic acid (CAS 118-92-3)

l-butyl-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide

(chlorpropamide) (CAS 94-20-2)

5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine

(pyrimethamine) (CAS 58-14-0)

2,6-diamino-3-(phenylazo)pyridine hydrochloride (phenazopyridine

hydrochloride) (CAS 136-40-3)

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)

N-(p-toluenesulfonyl)-N'-hexamethyleniminourea

(tolazamide) (CAS 1156-19-0)

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)

4,4'-thiodianiline (CAS 139-65-1)

ethionamide (CAS 536-33-4)

MICE

Feed Studies

4-acetyl-N-((cyclohexylamino)carbonyl)benzenesulfonamide

(acetohexamide) (CAS 968-81-0)

anthranilic'acid (CAS 118-92-3)

l-butyl-3-(p-tolylsulfonyl)urea (tolbutamide) (CAS 64-77-7)

4-chloro-N-((propylamino)carbonyl)benzenesulfonamide

(chlorpropamide) (CAS 94-20-2)

5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine

(pyrimethamine) (CAS 58-14-0)

2,6-diamino-3-(phenylazo)pyridine hydrochloride (phenazopyridine

hydrochloride) (CAS 136-40-3)

N-9H-fluoren-2-ylacetamide (CAS 53-96-3)
N-(p-toluenesulfonyl)-N'-hexamethyleniminourea

(tolazamide) (CAS 1156-19-0)

1-phenethylbiguanide hydrochloride (phenformin) (CAS 114-86-3)

pyrazinecarboxamide (pyrazinamide) (CAS 98-96-4)

4,4'-sulfonyldianiline (dapsone) (CAS 80-08-0)

4,4'-thiodianiline (CAS 139-65-1)

ethionamide (CAS 536-33-4)

Gavage Studies

cholesterol (p-(bis(2-chloroethyl)amino)phenyl)acetate

(phenesterin) (CAS 3546-10-9)

estradiol bis((p-(bis(2-chloroethyl)amino)phenyl)acetate)

(estradiol mustard) (CAS 22966-79-6)

Intraperitoneal Injection Studies

4'-(9-acridinylamino)methansulfon-m-aniside monohydrochloride

(MAAM) (NSC 141549)

acronycine (CAS 7008-42-6)

5-azacytidine (CAS 320-67-2)

beta-2'-deoxy-6-thioguanosine monohydrate (beta-TGdR)

(CAS 789-61-7)

1,4-butanediol dimethanesulfonate (busulfan) (CAS 55-98-1)

emetine dihydrochloride tetrahydrate (CAS 316-42-7)

3,3'-iminobis-l-propanol dimethanesulfonate (ester)

hydrochloride [1PD] (CAS 3458-22-8)

(+;)-4,4'-(l-methyl-l,2-ethanediyl)bis-2,6-piperazinedione

(ICRF-159) (CAS 21416-87-5)

N,3-bis(2-chloroethyl)tetrahydro-2H-l,3,2-oxazaphosphorin-2-

amine-2-oxide (isophosphamide) (CAS 3778-73-2)

N-(2-thloroethyl)-N-(l-methyl-2-phenoxyethyl)benzylamine

hydrochloride (phenoxybenzamine hydrochloride) (CAS 63-92-3)

N-(l-methylethyl)-4-((2-methylhydrazino)methyl)benzamide

monohydrochloride (procarbazine) (CAS 366-70-1)

tris(l-aziridinyl)phosphine sulfide (thio-TEPA) (CAS 52-24-4)

2,4,6-tris(dimethylamino)-s-triazine (CAS 645-05-6)

E. Subchronic Studies

Subchronic feeding studies were conducted to estimate the maximum

tolerated doses of L-tryptophan, on the basis of which two
different concentrations (hereinafter referred to as "low doses"

and "high doses") were determined for administration in the

chronic studies. In the subchronic studies, the chemical was

administered in feed at concentrations of 1,000, 5,000, 10,000,

25,000, or 50,000 ppm to both male Sprague-Dawley rats and male

Swiss mice. Dosed animals received the test diets 7 days per

week for 45 days and then were observed for an additional 45

days. Five animals of each species were dosed at each

concentration, and 19 rats and 20 mice were used as untreated

controls.

There were no deaths at any dose among either the rats or the

mice, and final body weights of the dosed animals were within 15%

of the controls. The low and high doses for the chronic studies

using rats or mice were set at 25,000 and 50,000 ppm to avoid

exceeding the limit of 5% of the test compound in feed.

F. Designs of Chronic Studies

The designs of the chronic studies are shown in tables 1 and 2.

G. Clinical and Pathologic Examinations

All animals were observed twice daily for signs of toxicity, and

animals that were moribund were killed and necropsied. Rats and

mice were weighed individually every 2 weeks through week 86, and
Table 1. Design of L-Tryptophan Chronic Feeding Studies in Rats

Sex and

Test

Group

Male

Ma tched-Control

Low-Dose

High-Dose

Female

Ma tched-Control

Low-Dose

High-Dose

Initial

No. of

Animals3

15

35

35

15

35

35

L-Tryptophan

in Dietb

(ppm)

0

25,000

50,000

0

25,000

50,000

Time on Study

Dosed Observed

(weeks ) (weeks )

105

78 26-27

78 26

105

78 26-27

78 26-27

a

All animals were 42 days of age when placed on study.

^The dosed animals were fed test diets 5 days per week and

control diets 2 days per week.

9
Table 2. Design of L-Tryptophan Chronic Feeding Studies in Mice

Sex and

Test

Group

Male

Matched-Control

Low-Dose

High-Dose

Female

Matched-Control

Low-Dose

High-Dose

Initial

No. of

Animalsa

15

35

35

15

35

35

L-Tryptophan

in Dietb

(ppm)

0

25,000

50,000

0

25,000

50,000

Time

Dosed

(weeks )

78

78

78

78

on Study

Observed

(weeks )

104

26

26

104

26

26

aMale mice were 43 days of age and female mice were 50 days of

age when placed on study.

10
once every month for the remainder of the study. Palpation for

masses was carried out at each weighing.

The pathologic evaluation consisted of gross and microscopic

examination of major tissues, major organs, and all gross lesions

from killed animals and from animals found dead. The following

tissues were examined microscopically: skin, muscle, lungs and

bronchi, trachea, bone marrow, spleen, lymph nodes, thymus,

heart, salivary gland, liver, gallbladder and bile duct (mice),

pancreas, esophagus, stomach, small intestine, large intestine,

kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, mammary gland, prostate or uterus, testis or ovary, brain,

and sensory organs. Peripheral blood smears were prepared from

each animal whenever possible. Occasionally, additional tissues

were also examined microscopically. The different tissues were

preserved in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Special staining

techniques were utilized when indicated for more definitive

diagnosis.

A few tissues from some animals were not examined, particularly

from those animals that died early. Also, some animals were

missing, cannibalized, or judged to be in such an advanced state

of autolysis as to preclude histopathologic evaluation. Thus,

the number of animals from which particular organs or tissues

11
were examined microscopically varies, and does not necessarily

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

group.

H. Data Recording and Statistical Analyses

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

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

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 statistical techniques

described in this section. Those analyses of the experimental

results that bear on the possibility of carcinogenicity are

discussed in the statistical narrative sections.

Probabilities of survival were estimated by the product-limit

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

12
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

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

13
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 (Armitage, 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

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

14
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 < 0.05, two-tailed test)

were also noted.

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 pt/pc where pt is the true binomial

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

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

spontaneous incidence of the same type of tumor in a control

15
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 < 0.025 one-tailed test when the control

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

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

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

the absence of a significant result while the upper limit

indicates that there is a theoretical possibility of the

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

detected under the conditions of this test.

16
III. RESULTS - RATS

A. Body Weights and Clinical Signs (Rats)

Mean body weights of the low- and high-dose male rats were lower

than those of the male matched controls, while body weights of

the female rats were similar to those of the female matched

controls. Fluctuation in the growth curve may be due to

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

weight may be subject to wide variation. No other chemicalrelated signs of toxicity in the dosed animals were recorded

(figure 1).

To control respiratory disease, rats received oxytetracycline in

the drinking water at 0.6 mg/ml during weeks 39 to 43 and at 0.3

mg/ml during weeks 43 to 44.

B. Survival (Rats)

The Kaplan and Meier curves estimating the probabilities of

survival for male and female rats fed L-tryptophan in the diet at

the doses of this bioassay, togethe-r with those of the matched

controls, are shown in figure 2.

In each sex, the Tarone test result for positive dose-related

trend in mortality is not significant. In male rats, 28/35 (80%)

of the high-dose group, 27/35 (77%) of the low-dose group, and

17
II

o

Q

O

m

UJ

5

IX

Hi 250-

g

Q

O 200-

m

Figure 1. Growth Curves for Rats Fed L-Tryptophan in the Diet

18
19

Figure 2. Survival Curves for Rats Fed L-Tryptophan in the Diet
8/15 (53%) of the matched controls lived to the end of the study.

In females, 31/35 (89%) of the high-dose group, 26/35 (74%) of

the low-dose group, and 11/15 (73%) of the matched controls 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.

A variety of neoplasms occurred in both the matched-control and

dosed groups. Some types of neoplasms occurred only in rats of

dosed groups, or with a greater frequency in dosed groups when

compared with controls. Fibromas of the subcutaneous tissue

occurred in 4/34 low-dose and 3/34 high-dose male rats, but in

0/15 controls. These lesions, however, are not uncommon in this

strain of rat independent of any treatment.

In addition to the neoplastic lesions, a number of degenerative,

proliferative,. and inflammatory changes were also encountered in

animals of the control and dosed groups. These nonneoplastic

lesions are commonly seen in aged rats.

In the judgment of the pathologists, L-tryptophan was not carcino20
genie when fed to 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 were observed in

at least two animals in one group and with 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 and of the Fisher exact test for direct

comparison of incidences between the matched-control group and

each of the dosed groups in the positive direction are not

significant.

In male rats, the Cochran-Armitage test results indicate a linear

trend in the negative direction in the incidences of leukemia

(P = 0.014), C—cell adenomas or carcinomas of the thyroid (P =

0.004), and interstitial-cell tumors of the testis (P = 0.016).

The probability level of the Fisher exact test of the incidence

of C-cell adenomas or carcinomas of the thyroid in male rats is

0.007, reflecting the higher incidence in the control group

(5/14, 36%) than in the high-dose group (1/32, 3%). These

significant results in the negative direction cannot be explained

by differential survival. In female rats, the Cochran-Armitage

21
test results of the incidence of fibroadenomas in the mammary

gland also indicate a significant trend (P = 0.027) in the

negative direction.

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

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

indicating the absence of positive significant results. It

should also be noted that most of the intervals have upper limits

greater than one, indicating the theoretical possibility of the

induction of tumors by L-tryptophan, which could not be detected

under the conditions of this test.

22
IV. RESULTS - MICE

A. Body Weights and Clinical Signs (Mice)

Mean body weights of both low- and high-dose mice of each sex

were lower than those of the matched controls, becoming

increasingly lower from week 20 through week 78 of administration

of the chemical (figure 3). The body weights of the dosed groups

approached those of the controls after L-tryptophan feeding was

discontinued, especially in the males. Fluctuation in the growth

curve may be due to mortality; as the size of a group diminishes,

the mean body weight may be subject to wide variation. There

were no other clinical signs of chemical-related toxicity.

To control respiratory disease, mice received oxytetracycline in

the drinking water at 0.6 mg/ml during week 66 and at 0.3 mg/ml

during week 67. Propylene glycol was vaporized in the mouse room

during weeks 66 to 76 to decrease the transmission of microorganisms that may have caused the respiratory disease.

B. Survival (Mice)

The Kaplan and Meier curves estimating the probabilities of

survival for male and female mice fed L-tryptophan in the diet at

the doses of this bioassay, together with those of the matched

controls, are shown in figure 4.

23
Figure 3. Growth Curves for Mice Fed L-Tryptophan in the Diet

24
25

Figure 4. Survival Curves for Mice Fed L-Trytophan in the Diet
In each sex, the Tarone test result for positive dose-related

trend in mortality is not significant. In male mice, 21/33 (64%)

of the high-dose group, 12/35 (34%) of the low-dose group, and

3/15 (20%) of the matched controls were alive at the last week

(104) of the study. In females, 13/35 (37%) of the high-dose

group, 22/35 (63%) of the low-dose group, and 2/15 (13%) of the

matched-control group lived to the end of the study. More than

50% of the mice in each group of either sex studied lived beyond

week 75 on study, providing sufficient numbers of animals 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.

With the exception of the lymphatic tumors and a few hematopoietic tumors, the neoplasms listed in Appendix B appeared with

approximately equal frequency in control and dosed mice or

appeared in insignificant numbers.- These lesions are not

uncommon in the B6C3F1 strain of mouse independent of any

treatment.

The incidence of hematopoietic neoplasms was higher in the dosed

than in the matched-control groups, with the highest incidence in

26
the low-dose groups. The incidences of these lesions were as

follows:

MALES

Number of Mice Necropsied

Brain: Malignant lymphoma,

histiocytic type

Multiple organs, lymphoreticular:

Malignant lymphoma, histiocytic or

mixed type

Spleen: Malignant lymphoma,

lymphocytic type

Mandibular lymph node: Malignant

lymphoma, lymphocytic type

Mesenteric lymph node: Malignant

lymphoma, lymphocytic or histiocytic

type

Liver: Malignant lymphoma, histiocytic

type

Total incidence of mice with tumors (%)

Matched

Control

(12)

0

0

0

0

0

2

0/12(0)

Low

Dose

(34)

4

1

0

1

2

!_

9/34(26)

High

Dose

(33)

0

1

1

0

0

0

2/33(6)

27
Matched Low High

Control Dose Dose

Number of Mice Necropsied

Brain: Malignant lymphoma,

histiocytic type

Multiple organs, lymphoreticular and

hematopoietic: Malignant lymphoma,

lymphocytic or histiocytic type

lymphocytic leukemia

Mesenteric lymph node: Malignant

lymphoma, histiocytic type

Payer's patches: Malignant lymphoma,

histiocytic type

Total incidence of mice with tumors (%)

(13)

0

0

0

1

_1

2/13(15)

(33)

0

5

1

0

_0

6/33(18)

(35)

1

0

0

0

j)

1/35(3)

Malignant lymphomas consisted of three cell types. (1) The

lymphocytic type was comprised of cells having a small, darkly

basophilic to large, lightly basophilic vesicular nucleus and a

rim of eosinophilic cytoplasm. (2) The histiocytic type was

comprised of cells with a large, open-faced vesicular nucleus and

a distinct eosinophilic nucleolus. (3) The mixed type was a

combination of the lymphocytic and histiocytic types of cells.

The malignant lymphomas were observed to be either generalized,

involving several organs, or solitary, involving only one organ.

The generalized lymphomas always involved the spleen, liver, and

one or more lymph nodes. The solitary lymphomas involved the

28

FEMALES
spleen, liver, mandibular lymph nodes, mesenteric lymph nodes,

Peyer's patches or brain. The brain lesions usually involved the

meninges and choroid plexuses with variable degrees of

perivascular cuffing.

Neoplastic cells having a small, darkly basophilic nucleus and

minimal cytoplasm were the predominant type of cell in

lymphocytic leukemia. The lymphocytic infiltration within the

liver was diffuse when compared with the more solid arrangement

of the cells during lymphoma. A large area of hemorrhage within

the brain was another feature of the leukemia.

A mast-cell sarcoma involving the wall of the stomach, liver, and

mesentery was observed in a low-dose female. The neoplastic mast

cells had a large, basophilic nucleus and an abundant cytoplasm

engorged with basophilic granules.

In addition to the neoplastic lesions, a number of degenerative,

proliferative, and inflammatory changes were also encountered in

animals of the control and dosed groups (Appendix D). These

nonneoplastic lesions are commonly seen in aged mice; however,

the suppurative lesions involving the trachea and lungs were

associated with early deaths. The decreased life spans,

especially prominent in the low-dose males, high-dose females,

29
and both control groups, may have resulted in a reduced incidence

of tumors in several of these groups.

The incidence of lymphoreticular and other neoplasms of the

hematopoietic system was higher in both male and female low-dose

groups of mice fed L-tryptophan than in either the matchedcontrol or high-dose mice of either sex.

In the judgment of the pathologists, L-tryptophan may be

associated with the increased incidence of lymphoreticular

neoplasms in low-dose male and female B6C3F1 mice under the

conditions of this bioassay.

D. Statistical Analyses of Results (Mice)

Tables Fl and F2 in Appendix F contain the statistical analyses

of the incidences of those primary tumors that were observed in

at least two animals in one group and with an incidence of at

least 5% in one or more than one group.

In male mice, when the incidence of lymphomas in the low-dose

group is compared with that in the control group, there is a

higher proportion in the low-dose group of male mice (P = 0.048),

but this probability level is above that of 0.025 required for

significance using the Bonferroni inequality for multiple

comparisons. The incidences of this tumor in the high-dose group

30
of male mice and in both dosed groups of female mice are not

statistically significant when the Fisher exact test is applied.

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

positive dose-related trend and of the Fisher exact test for

direct comparison of the incidence in the matched-control group

with the incidences in each of the dosed groups are not

significant for any of the tumors.

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

for the occurrence of lymphoma in low-dose male mice, the value

of one is included; this indicates the absence of significant

positive results. It should also be noted that each of the

intervals has an upper limit greater than one, indicating the

theoretical possibility of the induction of tumors by

L-tryptophan, which could not be detected under the conditions of

this test.

31

V. DISCUSSION

There was little evidence that the administration of L-tryptophan

was toxic to rats. Differences in mean body weight among dosed

and control groups were minimal, and survival of the dosed groups

of both sexes was high. Sufficient numbers of rats were at risk

for development of late-appearing tumors.

In the mice, however, the administration of L-tryptophan resulted

in lowered mean body weights throughout most of the study,

particularly in the females. Survival rates were lower in

low-dose males, high-dose females, and corresponding matched

controls than in high-dose males and low-dose females. More than

50% of the mice in each group lived to week 52 or longer; thus,

sufficient numbers of mice were at risk for development of tumors

appearing up to that time.

No neoplasms occurred in a statistically significant incidence

among the dosed rats when compared with the control rats.

In both male and female mice, neoplasms of the hematopoietic

systems (all sites) occurred at higher incidences in the low-dose

groups than in the matched-control groups (males: controls 0/12,

low-dose 9/34, high-dose 2/33; females: controls 2/13, low-dose

6/33, high-dose 1/35); the incidence in females was not

significant, and in males the probability of P = 0.048 was not

33
significant using the Bonferroni correction. Because of the lack

of statistical significance and because of the known variability

of the incidence of these tumors in B6C3F1 mice, they are not

considered to be related to administration of the chemical.

In previous studies, malignant reticuloendothelial tumors and

leukemia were reported in mice administered various metabolites

of tryptophan by oral or subcutaneous routes (Ehrhart and Stich,

1957 and 1958; Ehrhart et al., 1959; Rauschenbach et al., 1963;

Rauschenbach et al. , 1966). In addition, numerous studies have

been conducted to determine the effect of tryptophan on the

carcinogenicity of other compounds. Dunning et al. (1950) and

Kawachi et al. (1968) found that tryptophan increased or extended

the carcinogenicity of known carcinogens, while Okajima et al.

(1971), Oyasu et al. (1972), and Evarts and Brown (1977)

demonstrated that tryptophan decreased the incidence of liver

tumors induced by known carcinogens.

It is concluded that under the conditions of this bioassay,

L-tryptophan was not carcinogenic for Fischer 344 rats or B6C3F1

mice.

34
VI. BIBLIOGRAPHY

Allen, M. J., Boyland, E., Dukes, C. E., Horning, E. S., and

Watson, J. G., Cancer of the urinary bladder induced in mice

with metabolites of aromatic amines and trytophan. Brit. J.

Cancer 11;212-228, 1957.

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

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

Berenblum, I., ed., Carcinogenicity Testing: A Report of £he

Panel on Carcinogenicity of the Cancer Research Commission

of the UICC, Vol. _2, International Union Against Cancer,

Geneva, 1969.

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

carcinoma of the bladder in rats with acetamidofluorene.

Brit. J. Cancer 8:647-654. 1954.

Bryan, G. T., Brown, R. R., and Price, J. M., Mouse bladder

Carcinogenicity of certain tryptophan metabolites and other

aromatic nitrogen compounds suspended in cholesterol.

Cancer Res. 24:596-602, 1964.

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

Soc. B 34 (2):187-220, 1972.

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

1970, pp. 48-52.

Dictionary of Organic Compounds, Vol. 5, Oxford University Press,

New York, 1965, p. 3211.

Dunning, W. F., Curtis, M. R. , and Maun, M. E. , The effect of

added dietary tryptophane on the occurrence of 2-acetylaminofluorene-induced liver and bladder cancer in rats. Cancer

Res. 10(7):454-459, 1950.

Ehrhart, H. , Georgii, A., and Stanislawksi, K. Ueber die

leukamogene Wirkung von 3-Hydroxy-anthranilsaure bei

RFH-Mausen. Klinische Wochenschrift 37(20);1054-1059, 1959.

Ehrhart, H. and Stich, W., Leukamieerzeugung bei Mausen durch

Injektion wassriger Indicanlosung. Klinische Wochenschrift

36(22):1056-1061, 1958.

35
Ehrhart, H. and Stich, W., Die Indol-Leukatnie bei der weissen

Maus. Klinische Wochenschrift 35(10);504-511. 1957.

Evarts, R. P. and Brown, C. A., Effect of L-tryptophan on

diethylnitrosamine and 3'-methyl-4-N-dimethylaminoazobenzene

hepatocarcinogenesis. Fd. Cosmet. Toxicol. 15;431-435, 1977.

Food and Agriculture Organization of the United Nations,

Amino-Acid Content of Foods and Biological Data on Proteins,

No. 24, Food and Agriculture Organization, Rome, 1970.

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

significance tests, confidence limits and adjustments for

stratification. Rev. Int. Stat. Inst. 39 (2):148-169, 1971.

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

incomplete observations. J. Am. Statist. Assoc. 53;457-481,

1958.

Kawachi, T., Hirata, Y. , and Sugimura, T., Enhancement of

N-nitrosodiethylamine hepatocarcinogenesis by L-tryptophan

in rats. GANN 59:523-525, 1968.

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, 1974.

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

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

Okajima, E., Hiramatsu, T., Motomiya, Y., Iriya, K. , Ijuin, M.,

and Ito, N., Effect of DL-tryptophan on tumorigenesis in the

urinary bladder and liver of rats treated with N-nitrosodibutylamine. GANN 62:163-169, 1971.

Or ten, J. M. and Neuhaus, 0. W., Basic principles of

biochemistry. Human Biochemistry, The C. V. Mosby Co., St.

Louis, 1975, pp. 332-336.

Oyasu, R., Kitajima, T., Hopp, M., and Sumie, H. , Enhancement of

urinary bladder tumorigenesis in hamsters by coadministration of 2-acetylaminofluorene and indole. Cancer Res.

^2: 2027-2033, 1972.

36
Radomski, J. L. , Radomski, T. , and MacDonald, W. E.,

Cocarcinogenic interaction between D,L-tryptophan and

4-aminobiphenyl or 2-naphthylamine in dogs. J. Natl. Cancer

Inst. 58(6);1831-1834. 1977.

Radomski, J. L. , Deichmann, W. B., and Brill, E. , Tryptophan as a

co-carcinogen in bladder cancer. Proc. Am. Assoc. Cancer

Res. 10:71, 1969.

Radomski, J. L. , Deichmann, W. B., and Brill, E. , Transitional

cell hyperplasia in the bladders of dogs fed trytophan.

Proc. Am. Assoc. Cancer Res. 11;65, 1970.

Rauschenbach, M. 0., Zharova, E. I., Ivanova, W. D., Kuznezova,

L. E. , Miasitcheva, N. W., and Sergeev, A. V., The

investigation of the mechanisms of leucomogenic action of

the tryptophan metabolites. Ninth International Cancer

Congress, Abstracts, Cancer Institute, Tokyo, 1966, p. 194.

Rauschenbach, M. 0., Jarova, E. I., and Protasova, T. C.,

Blastomogenic properties of certain metabolites of

tryptophane. Acta-Unio Internationalis Contra Canerum

^: 660-662, 1963.

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.

Stecher, P. G. , ed., The Merck Index, 8th ed., Merck & Co., Inc.

Rahway, N. J., 1968, pp. 216 and 1086.

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

Biometrika 62 (3):679-682, 1975.

37

APPENDIX A

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

RATS FED L-TRYPTOPHAN IN THE DIET

39

TABLE A1.

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS

FED L-TRYPTOPHAN IN THE DIET

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

MATCHED

CONTROL

15

15

ANIMALS EXAMINED HISTOPATHOL06ICALLY 15

INTEGUMENTARY SYSTEM

*SKIN

SQUAHOUS CELL PAPILLOHA

SQUAMOUS CELI CARCINOMA

*SUBCUT TISSDE

FIBROMA

LI POM A

NEUROFIBROSARCONA

RESPIRATORY SYSTEM

tLUNG

ALVTOLAR/BRONCHIOLAR ADENOMA

ALVEOLAR/BRONCHIOLAR CARCINOMA

NEUROFIPROSARCOMA, MET^STATIC

HEMATOPOIETIC SYSTEM

*NULTIPLE ORGANS

LEUKEMIA, NOS

ONDIFFERENTIATED LEUKEMIA

LYMPHOCYTIC LEUKEMIA

tMANDIBULAR L. NODE

NEUROFIBROSARCOMA, METASTATIC

CIRCULATORY SYSTEM

fMYOCARDIUM

FIBROMA

(15)

1 (7%)

(15)

(15)

1 (7%)

(15)

U (21%)

(2)

(15)

. __ L.I7J1 _

LOW DOSE

35

3U

3«

<3t)

1 (3«)

(34)

4 (12«)

1 (3%)

(34)

1 (3%)

(34)

2 (6X)

1 (3*)

(4)

(34)

HIGH DOSE

35

34

34

(34)

(34)

3

1

1

(34)

1

1

(34)

1

(9)

1

(34)

(9X)

(3%)

(3%)

(3*)

(3%)

(3X)

(11*)

41

I NUHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIFD
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

DIGESTIVE SYSTEH

iCOLON

ADENOHATOU S POLYP, NOS

MOCINOUS ADENOCARCINOHA

URINAR Y SYSTEH

NON E

ENDOCRIN E SYSTEM

#PITUITARY

CHROHOPHOBE ADENOM A

CRANIOPHARYNGIOM A

#ADRENA L

PHEOCHROMOCYTOM A

GANGLIONEUROM A

#THYROI D

F3LLICULAR-CELL CARCINOM A

C-CELL ADENOMA

C-CELL CARCINOM A

MUCINOU S ADENOCARCINOM A

tPANCREATIC ISLETS

ISLET-CELL ADENOM A

ISLET-CELL CARCINOM A

REPRODUCTIV E SYSTEM

*MAMMAR Y GLAN D

FIBROADENOH A

#TESTIS

INTERSTITIAL-CELL TUMO R

NERVOU S SYSTE M

NON E

MATCHED

CONTROL

(15)

**

(11)

1 (9%)

(15)

(1«)

4 (29%)

1 (7%)

(15)

(15)

2 (13X)

(15)

13 (87%)

LOW DOSE

(34)

(30)

2 (7X)

1 (3X)

(34)

1 (3X)

(33)

2 (6«)

2 (6%)

U (12«)

1 (3«)

(33)

2 (6%)

(3U)

(34)

31 (91%)

HIGH DOSE

(34)

1 (3% )

1 (3%)

(30)

5 (17% )

(34)

1 (3% )

(32)

1 (3%)

(34)

1 (3% )

(34)

(33)

21 (64% )

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

42
SPECIAL SENSE ORGAN S

+HARDERIA N GLAN D

ADENOHA , NOS

MOSCULOSKELETA L SYSTEM

NON E

BODY CAVITIES

*PERITONSUN

LIPOBA

BESOTHELIOBA BENIGN

*PLEURA

ALVEOLAR/BRONCHIOLA R CA, HETASTA

ALL OTHER SYSTEMS

NONE

ANIMA L DISPOSITION SUMMAR Y

ANIMALS INITIALLY IN STUDY

NATURA L DEATHS

MORIBUN D SACRIFICE

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED

TERMINAL SACRIFICE

ANIMA L MISSING

9_iaCfc2DES_ADTOiY2ID_MIHM.S

MATCHED

CONTROL

(15)

(15)

(15)

15

2

5

8

LOW DOSE

(34)

(34)

2 (6*)

(34)

35

3

5

27

HIGH DOSE

(34)

1 (3*)

(34)

1 (3«)

1 (3%)

(34)

1 (3X)

35

4

3

28

t NUMBER OF ANIMALS HITH TISSUE EXAMINED BICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

43

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED)

l\(

TUMOR SUMMARY

TOTAL ANIMALS WITH PRIMARY TDHORS*

TOTAL PRIMARY TUMORS

TOTAL ANIMALS WITH BENIGN TUHORS

TOTAL BENIGN TUMORS

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

TOTAL ANIMALS WITH SECONDARY TUMORS

TOTAL SECONDARY TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

BENIGN OR MALIGNANT

TOTAL UNCERTAIN TUHORS

TOTAL ANIMALS WITH TUHORS UNCERTAIN

PRIMARY OR HETASTATIC

TOTAL UNCERTAIN TUHORS

HATCHED

JONTROL

1i»

28

14

22

U

6

1

_

-

LOW DOSE

32

58

32

46

10

11

1

1

HIGH DOSE

29

U1

26

36

5

5

2

3

* PRIHARY TOHORS: ALL TOBOBS EXCEPT SECONDARY TtJHORS

* SECONDARY TOHORS: HETASTATIC TnHORS 3R TOHORS INVASIVE INTO AN ADJACENT ORGAN

44
TABLE A2.

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS

FED L-TRYPTOPHAN IN THE DIET

ANIMALS INITIALLY IN STODY

ANIMALS NECROPSIED

ANIBALS EXAMINED HISTOPATHOLOGICALLY

INTEGUBENTARY SYSTEM

*SOBCOT TISSUE

LIPOMA

RESPIRATORY SYSTEM

CLUNG

ADENOCARCINOBA, NOS, METASTATIC

ALVEOLAR/BBONCHIOLAR ADENOMA

HEMATOPOIETIC SYSTEM

*MULTIPLE ORGANS

UNDIPFERENTIATED LEUKEMIA

fBEDIASTINAL L.NODE

ADENOCARCINOMA, NOS, BETASTATIC

CIRCULATORY SYSTEB

NONE

DIGESTIVE SYSTEM

NONE

URINARY SYSTEB

NONE

MATCHED

CONTROL

15

14

1U

(1l»)

(1"0

1 (7%)

(1K)

1 (If,)

(3)

LOW DOSE

35

35

35

(35)

1 (3%)

(35)

1 (3X)

(35)

(5)

1 (20%)

HIGH DOSE

35

3U

3U

(3U)

(3U)

(3U)

(2)

45

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIFD
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

MATCHED

CONTROL

ENDOCRINE SYSTEM

IPITUITARY (12)

CHROHOPHOBE ADENOMA 1* (33X)

CHROMOPHOBE CARCINOM A

#ADRENA L (1H)

CORTICAL ADENOM A

PHEOCHROMOCYTOMA 1 (7X)

PHEOCHROMOCYTOMA, MALIGNAN T

#THYBOID (1<» )

FOLLICOLAR-CELL CARCINOMA

C-CELL ADENOM A 1 (7X)

REPRODUCTIVE SYSTEM

*MAHMAR Y GLAN D (1U )

ADENOMA , NOS

ADENOCARCINOMA , NOS

FIBROftDENOM A 4 (29%)

*UTERUS (13)

ADEHOCARCINOMA , NOS

SARCOMA . NOS

ENDOMETRIA L STROMA L POLYP 3 (23%)

NERVOU S SYSTE M

NONE

SPECIAL SENSE ORGANS

NON E

MDSCULOSKELETA L SYSTEM

NONE

BODY CAVITIES

*PERITONSUM (1<4 )

ADENOCARCINOMA., ' NOS. METASTATIC

LOW

(27)

12

1

(35)

(32)

1

2

(35)

7

(35)

1

1

3

(35)

1

DOSE

(HH%)

(«)

(3X)

(6%)

(20%)

(3%)

(3X)

(•)%)

J3X) .

HIGH DOSE

(32)

9 (28*)

2 (6%)

(3t)

1 (3X)

1 (3%)

(33)

1 (3«)

(3«)

2 (6«)

1 (3X )

2 (6% )

(33)

1 (3S)

3 (9X)

(34)

46

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED)

p

(

ALL OTHER SYSTEMS

NONE

ANIMAL DISPOSITION SDMMARY

ANIMRLS INITIALLY IN STUDY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED

TERMINAL SACRIFICE

ANIMAL MISSING

3 INCLUDES AUTOLYZED ANIMALS

TUMOR SUMMARY

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

TOTAL ANIMALS WITH SECONDARY TUMORS

TOTAL SECONDARY TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

BENIGN OR MALIGNANT

TOTAL UNCERTAIN TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

PRIMARY OR METASTATIC

TOTAL UNCERTAIN TUMORS

/IATCHED

CONTROL

15

3

1

11

11

15

11

1U

1

1

*

_

_

LOW DOSE

35

2

7

26

2H

29

21

25

U

H

1

3

HIGH DOSE

35

1

3

31

19

23

15

18

5

5

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TOMORS

* SECONDARY TDMORS: MBTASTATIC TOBORS OR TOMORS INVASIVE INTO AN ADJACENT ORGAN

47

APPENDIX B

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

MICE FED L-TRYPTOPHAN IN THE DIET

49

TABLE B1.

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE

FED L-TRYPTOPHAN IN THE DIET

ANIHALS INITIALLY IN STUDY

ANIMALS NECBOPSIED

ANIHALS EXAHINE D HISTOPATHOLOGICALLY

INTEGUMENTAR Y SYSTEM

*SOBCUT TISSUE

FIBROSARCOMA

RESPIRATOR Y SYSTEM

*LUNG

ALVEOLAR/BRONCHIOLAR ADENOMA

ALVBOLAR/BRONCHIOLAH CARCINOM A

HEMATOPOIETIC SYSTEM

*BRAIN

MALIG.LYHPHONA , HISTIOCYTIC TYPE

*MULTIPLE ORGAN S

MALI3.LYMPHOMA , HISTIOCYTIC TYPE

MALIGNAN T LYMPHOMA , MIXE D TYPE

ISPLEEN

MALIG. LYMPHOMA , LYBPHOCYTIC TYPE

tMANDIBOLA R L. NODE

MALIG.LYHPHOMA , LYMPHOCYTIC TYPE

#MESENTBHIC L. NODE

BALIG. LYMPHOMA, LYMPHOCYTIC TYPE

BALIS. LYHPHOHA , HISTIOCYTIC TYPE

*LIVER

MALIG.LYHPHOMA , HISTIOCYTIC TYPE

CIRCULATORY SYSTEM

NONE

MATCHED

CONTROL

15

12

12

(12)

(12)

(11)

(12)

i

(11)

CO

(»)

(12)

LOW DOSE

35

3H

3i»

(3<»)

1 (3*)

(33)

3 (9%)

1 (3X)

(32)

i» (13«)

(34)

1 (3*)

(32)

(HO

1 (7«)

(10)

1 (7X)

1 (7*)

(3«)

1 (3*)

HIGH DOSE

335

33

33

(33)

1 (3*)

(32)

2 (6*)

(32)

(33)

1 (3X)

(32)

1 (3«)

(3)

(3)

(33)

t RUBBER OF ANIMALS KITH TISSUE EXABINED MICROSCOPICALLY

* NOBBER OF AMIHALS NECROPSIED

» 35 MilHALS 8BRE IMITIALLY IN THE STUDY, EOT TWO AIHALS WEBB FOUND TO BE

FEMALES IN A HALE GROUP.

51
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

DIGESTIVE SYSTEM

fLIVER

HEPATOCELLDLAR ADENOMA

HEPATOCEILOLAR CARCINOMA

DRINARY SYSTEM

NONE

ENDOCRINE SYSTEM

tTHYROID

FOLLICULAR-CELL ADENOMA

REPRODUCTIVE SYSTEM

NONE

NERVOUS SYSTEM

*TRIGEMINAL GANGLION

NEURILEMOMA, MALIGNANT

SPECIAL SENSE ORGANS

NONE

MUSCULOSKELETAl SYSTEM

NONE

BODY CAVITIES

NONE

ALL OTHER SYSTEMS

NONE

MATCHED

CONTROL

(12)

1 (8X)

(11)

(12)

LOW DOSE

(31)

4 (12%)

1 (3*)

(31)

1 (3«)

(3<»)

2 (6%)

HIGH DOSE

(33)

5 (15X)

2 (6%)

(28)

(33)

52

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED)

"

ANIMAL DISPOSITION SUMMARY

ANIMALS INITIALLY IN STUDY

NATURAL DEATH9

MORIBUND SACRIFICE

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED

TERMINAL SACRIFICE

ANIMAL MISSING

ANIMAL DELITED (WRONG SEX)

3 INCLUDES AUTOLYZED ANIMALS

TUMOR SUMMARY

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN TUMORS

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

TOTAL ANIMALS WITH SECONDARY TUMORS

TOTAL SECONDARY TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

BENIGN OR MALIGNANT

TOTAL UNCERTAIN TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

PRIMARY OR METASTATIC

TOTAL UNCERTAIN TUMORS

MATCHED

CONTROL

15

5

5

2

3

1

1

1

1

*

_

_

LOW DOSE

35

9

13

1

12

15

22

7

8

12

1U

HIGH DOSE

35

8

5

20

2

12

12

7

7

5

5

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS

t SECONDARY TUMORS: BETASTATIC TUMORS 3R TUMORS INVASIVE INTO AN ADJACENT ORGAN

53
TABLE B2.

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE

FED L-TRYPTOPHAN IN THE DIET

r

(

ANIMAL S INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY

INTEGUMENTAR Y SYSTEM

*SUBCUT TISSUE

SARCOMA, NOS

RESPIBATORY SYSTEM

tLUN G

ALVEOLAR/BRONCHIOLA H ADENOMA

HEMATOPOIETIC SYSTEM

tBRAIN

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

*MDLTIPLE ORGANS

MALIG.LYMPHOMA , LYMPHOCYTIC TYPE

MALI3.LYMPHOMA , HISTIOCYTIC TYPE

MAST-CELL SARCOMA

LYHPHOCYTIC LEUKEMIA

tBESENTERIC L. NODE

MALIG.LYMPHOMA , HISTIOCYTIC TYPE

IPEYERS PATCH

HALIG.LYMPHOMA , HISTIOCYTIC TYPE

CIRCULATORY SYSTEM

NONE

DIGESTIVE SYSTEM

fLIVER

HfPATQCELLOLAR UDEHOHA

HATCHED

CONTROL

15

13

13

(13)

(13)

1 (8X)

(12)

(13)

(<»)

1 (25X)

(13)

1 <8X)

(13)

LOW DOSE

35

33

33

(33)

1 (3X)

(32)

(33)

(33)

1 (3X)

4 (12X)

1 (3X)

1 (3X)

(17)

(33)

(32)

._ __1_I34L

HIGH DOSE

35

35

35

(35)

(35)

1 (3%)

(35)

1 (3X)

(35)

(15)

(35)

(35)

._ __«

* NDMBER OF ANIHALS HITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

54
HEPATOCELLULAR CARCINOBA

URINAR Y SYSTEM

NON E

ENDOCRIN E SYSTEM

tPITUITARY

CHROMOPHOB E ADENOMA

REPRODUCTIV E SYSTEM

#UTERUS

ENDONETRIAL STROBAL POLYP

NERVOUS SYSTEM

*TRIGEBINA L GANGLIO N

NEURILEMOMA, MALIGNANT

SPECIAL SENSE ORGANS

NONE

MOSCULOSKELETAL SYSTEM

NON E

BODY CAVITIES

*PERITONEUM

SARCOMA, NOS

ALL OTHEH SYSTEMS

NQS?

MATCHED

CONTROL

(5)

1 (20*)

(13)

(13)

(13)

LOW DOSE

(22)

1 (5%)

(32)

1 (3%)

(33)

1 (3*)

(33)

1 (3*)

HIGH DOSE

1 (3%)

(28)

(3U)

(35)

(35)

I NDHBER OF ANIMALS WITH TISSUE EXAMINED HICROSCOPICALLT

* NUMBER OF ANIMALS NECROPSIED

55

TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED)

M

C

ANIMAL DISPOSITION SOBHARY

ANIMALS INITIALLY IN STODY

NATURAL DEATHS

MORIBUND SACRIFICE

SCHEDULED SACRIFICE

ACCIDENTALLY KILLED

TERMINAL SACRIFICE

ANIMAL MISSING

3 INCLUDES AUTOLYZED ANIMALS

TUMOR SUMMARY

TOTAL ANIMALS WITH PRIMARY TUMORS*

TOTAL PRIMARY TUMORS

TOTAL ANIMALS WITH BENIGN TUMORS

TOTAL BENIGN T'JMORS

TOTAL ANIMALS WITH MALIGNANT TUMORS

TOTAL MALIGNANT TUMORS

TOTAL ANIMALS WITH SECONDARY TUMORS

TOTAL SECONDARY TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

BENIGN OR MALIGNANT

TOTAL UNCERTAIN TUMORS

TOTAL ANIMALS WITH TUMORS UNCERTAIN

PRIMARY OR METASTATIC

TOTAL UNCERTAIN TUMORS

IATCHED

ONTROL

15

3

10

2

3

H

2

2

2

2

#

-

_

LOW DOSE

35

7

6

22

11

13

3

3

3

10

HIGH DOSE

35

6

16

13

3

3

1

1

2

2

* PRIMARY TUMORS: ALL TUHORS EXCEPT SECONDARY TUMORS

* SECONDARY TUMORS: METASTATIC TDMOR3 OR TUMORS INVASIVE INTO AN ADJACENT ORGAN

56
APPENDIX C

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS

IN RATS FED L-TRYPTOPHAN IN THE DIET

57

TABLE C1.

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE

RATS FED L-TRYPTOPHAN IN THE DIET

M

0

ANIMALS INITIALLY IN STUDY

ANIHALS NECROPSIED

(.ANIMALS EXAMINED HISTOPATHOLOGICALLY

INTEGUHENTARY SYSTEH

*SKIN

EPIDERMAL INCLUSION CYST

INFLAMMATION, FOCAL

INFLAMMATION, NECROTIZING

*SUBCUT TISSUE

HEMORRHAGE

HEMORRHAGIC CYST

HEMORRHAGE, CHRONIC

RESPIRATORY SYSTEM

*TRACHEA

INFLAMMATION, SUPPURATIVE

PLASMA-CELL INFILTRATE

#LUNGPNEUMONIA INTERSTITIAL CHRONIC

BBONCHOPNEUMONIA CHRONIC SUPPURA

HYPERPLASIA, ALVEOLAR EPITHELIUM

METAPLASIA, SQUAMOUS

tLUNG/ALVEOLI

EMPHYSEMA, NOS

H*MATOPOIETIC SYSTEM

#BONE MARROW

ATROPHY, NOS

*SPLEEN

-HYPERPLASIAi LYMPHOID_

ATCHED

ONTROL

15

15

15

(15)

1 (7*)

(15)

(I'M

5 (36%)

(15)

1 (7%)

(15)

(15)

2 (13*)

(15)

LOW DOSE

35

34

34

(34)

1 (3%)

(3<»)

1 (3X)

(33)

8 (24«)

(34)

1 (3%)

1 (3*)

1 (3%)

(34)

1 (3%)

(34)

7 (21X)

(34)

HIGH DOSE

35

34

34

(34)

2 (6%)

1 (3X)

1 (3%)

(34)

1 (3*)

1 (3%)

(34)

8 (24%) •

1 (3%)

(34)

1 (3X)

(34)

(31)

11 (35%)

(34)

.__ __!-J3*j.__

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

59
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

HBMATOPOIESIS

#MANDIBULA R L. NODE

HYPERPLASIA , PLASM A CELL

#MESENTERIC L. NODE

HYPERPLASIA , LYBPHOI D

CIRCULATORY SYSTEM

#HYOCARDIO H

CALCIFICATION, DYSTROPHIC

DIGESTIVE SYSTEM

fLIVER

INFLAMMATION , NECROTIZIN G

HYPERPLASIA , NODULA R

HEMAT3POIESI S

#LIVER/CENTRILOBULAR

NECROSIS, COAGOLATIVE

#PANCREAS

ATROPHY , NOS

tPANCREATIC ACINUS

ATROPHY , NOS

ATROPHY , FOCAL

*PEYERS PATCH

HYPERPLASIA , LYMPHOID

URINAB Y SYSTEM

IKIDNE Y

INFLAMMATION , CHRONIC

ENDOCRINE SYSTEM

tTHYHOID

-HIElRPLASIAi.C^ELL

MATCHED

CONTROL

2 (13%)

(2)

( 2)

(15)

1 (7%)

(15)

1 (7%)

2 (13%)

(15)

1 (7%)

(15)

(15)

(15)

(15)

11 (93%)

(11*)

LOW DOSE

2 (6%)

(1)

1 (25%)

W

1 (25%)

(3U)

(34)

2 (6%)

1 (3%)

(3H)

(33)

1 (3%)

(33)

2 (6%)

(3«)

1 (3%)

(3U)

30 (88%)

(33)

. 2_16JL

HIGH DOSE

(9)

(9)

(31)

(3t)

1 (3%)

1 (3%)

(31)

(31)

(31)

1 (3%)

1 (3%)

(31)

(31)

31 (91%)

(32)

1 1IS)__

I NDHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

60
REPRODUCTIVE SYSTEM

*NAM(JARY GLAND

CYST, NOS

*PREPUTIAL GLAND

INFLAMMATION, CHRONIC SUPPDRATIV

HYPERKERATCSIS

*PROSTATE

INFLAMMATION, SOPPORATIVE

INFLAMMATION, CHRONIC SOPPUHATIV

tTESTIS

ATROPHY, NOS

NERVOUS SYSTEM

tBRAIN

HEMORRHAGE

MALACIA

SPECIAL SENSE ORGANS

*EYE

PUS

* EYE/CORNEA

INFLAMMATION, CHRONIC

*EYE/CRYSTALLINE LENS

HINEFALIZATION

HUSCTJLOSKELETAL SYSTEM

NONE

BODY CAVITIES

*PERITONEUH

NECROSIS. FAT

MATCHED

CONTROL

(15)

(15)

(15)

(15)

(15)

3 (20*)

1 (7%)

(15)

1 (7*)

(15)

(15)

(15)

LOW DOSE

(34)

1 (3%)

(34)

1 (3X)

1 (3*)

(33)

1 (3X)

(34)

(34)

(34)

(34)

(34)

1 (3X)

(34)

1_13X)

HIGH DOSE

(34)

1 (3%)

(34)

(34)

1 (3X)

2 (6X>

(33)

2 (6X)

(34)

(34)

(34)

1 (3X)

(34)

1 (3X)

(34)

61

TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

t SOMBER OF ANIMALS WITH TISSOE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECHOPSIED
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

ALL OTHER SYSTEMS

NONE

SPECIAL HOPPHOLOGT SUHHAR Y

AUTOLYSIS/NO NECROPSY

MATCHED

CONTROL

LOW DOSE

" 1

HIGH DOSE

1

* NUHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

62
1

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

INTEGUMENTARY SYSTEM

NONE

RESPIRATORY SYSTEM

*NASAL CAVITY

INFLAMMATION, CHRONIC SUPPURATIV

tTRACHEA

INFLAMMATION, SOPPURATIVE

tLUNG

B30NCHOPNEOMONIA SUPPORATIVE

BRONCHOPNEUMONIA CHRONIC SDPPORA

HEHATOPOIBTIC SYSTEM

tBONE MARROW

ATROPHY, NOS

ISPLEEN

NECROSIS, COAGULATIVF

HEMAT3POIESIS

CIHCDLATORY SYSTEM

#MYOCARDIUM

INFLAMMATION, INTERSTITIAL

DIGESTIVE SYSTEM

#LIVBR

HEMORRHAGE

MATCHED

CONTROL

15

1ft

1ft

(1ft)

1 (1%)

(1ft)

ft (29%)

(1ft)

1 (7%)

(12)

7 (58%)

(1ft)

(1ft)

(1ft)

. 1 1Z*±._

LOW DOSE

35

35

35

(35)

(3ft)

8 (2ft*)

(35)

1 (3%)

(31)

19 (61%)

(35)

1 (3%)

ft (11%)

(35)

1 (3%)

(35)

HIGH DOSE

35

3ft

3ft

(3ft)

(3ft)

10 (29%)

(3ft)

1 (3%)

(33)

23 (70%)

(34)

1 (3%)

(3ft)

(3ft)

63

TABLE C2.

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS

FED L-TRYPTOPHAN IN THE DIET

t NUMBER OP ANIMALS WITH TISSUE EXAMINED MICBOSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

CYTOPLASMIC VACUOLI ZATION

FOCAL CEILULAR CHANGE

HYPERPLASIA, NODULAR

HEMATOPOIESIS

#PANCREATIC ACINUS

ATROPHY, NOS

ATROPHY, FOCAL

URINABY SYSTEM

#KIDNEY

MINERALIZATION

PYELONEPHRITIS SOPPURATIVE

INFLAMMATION, CHRONIC

NECROSIS, MEDOLLARY

ENDOCRINE SYSTEM

NONE

REPRODUCTIVE SYSTEM

*MAMSARY GLANE

CYST, NOS

fOTEKUS

DECIDUAL ALTERATION, NOS

ICERVIX UTERI

CYST, NOS

tUTEROS/ENDOMETRIUM

INFLAHHATION, StJPPORATIVE

HYPERPLASIA, CYSTIC

#OVARY/OVIDUCT

INFLAMMATION, SOPPORATIVE

tOVARY

CYST, NOS

INFLAMMiTIONi.SOPPORATIVE

MATCHED

CONTROL

(It)

(1«)

10 (71%)

(1i»)

6 (43%)

(13)

(13)

1 (8*)

(13)

3 (23%)

1 (8%)

(13)

(13)

9 (69*)

LOW DOSE

1 (3%)

1 (3%)

(35)

1 (3%)

(35)

1 (3«)

1 (3%)

19 (5U«)

1 (3X)

(35)

1 (3*)

(35)

(35)

(35)

7 (20X)

« (11*)

(35)

(32)

10 (HU%)

HIGH DOSE

1 (3X)

1 (3%)

(31)

1 (3X)

1 (3%)

(3t)

24 (71%)

(3t)

1 (3%)

(33)

1 (3%)

(33)

(33)

1 (12%)

3 (9%)

(33)

1 (3%)

(33)

11 (33%)

_1 -QS1_.

* NUHBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

64
INFLAMMATION , CHRONIC SUPPORATIV

NERVOU S SYSTE M

*SPINA L COR D

DEGENERATION , NOS

SPECIAL SENSE ORGANS

*EYE

ATROPHY, NOS

*EYE/CRYSTALLINE LENS

MINERALIZATIO N

MOSCOLOSKELETA1 SYSTEM

NONE

BODY CAVITIES

^ABDOMINA L CAVIT Y

STEATITIS

ALL OTHER SYSTEMS

NONE

SPECIAL MORPHOLOGY SUMMAR Y

NO LESION REPORTED

AOTOLYSIS/NO UECHOPSY

MATCHED

CONTROL

(14)

(14)

(1l»)

(14)

1

1

LOW DOSE

2 (6X)

(35)

(35)

2 (6*)

(35)

2 (6*)

(35)

HIGH DOSE

(30)

1 (3%)

(31)

(34)

(34)

1 OX)

1

65

TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED)

f NtJHBEF OF ANIMAL S WITH TISSOE EXAHINE D HICROSCOPICALLT

* HUHBER OF ANIHALS HECBOPSIED

APPENDIX D

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS

IN MICE FED L-TRYPTOPHAN IN THE DIET

67

TABLE D1.

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE

FED L-TRYPTOPHAN IN THE DIET

MATCHED LOW DOSE

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIMALS EXAMINED HISTOPATHOLOGICALLY

INTEGUMENTARY SYSTEM

*SKIN

INFLAMMATION, CHRONIC

*SUBCOT TISSUE

HEMORRHAGE

RESPIRATORY SYSTEM

fTRACHEA

INFLAMMATION, SUPPURATIVE

*LUNG/BRONCHUS

HYPERPLASIA, LYMPHOID

*LUNG

BHONCHOPNEUMONIA SUPPUHATIVE

BRONCHOPNEUNONIA CHRONIC SUPPURA

HYPERPLASIA, ALVEOLAE EPITHELIUM

HYPERPLASIA, PLASMA CELL

HEMATOPOIETIC SYSTEM

#BONE BARROW

ATROPHY, NOS

#SPLE1=!N

HEMATOPOIESTS

tMANDIBULAR L. NODE

HYPERPLASIA, LYMPHOID

KNESENTERIC L. NODE

CONTROL

15

12

12

(12)

1 (8«)

(12)

(12)

2 (17%)

(12)

(12)

3 (25%)

1 (8%)

(12)

1 (8%)

(11)

CO

(4)

HYPERPLAS!£t_LYMPHOID

35

3U

34

(34)

(34)

(3U)

5 (15%)

(33)

(33)

9 (27%)

1 (3%)

1 (3*)

(3«)

2 (6*)

(32)

1 (3«)

(14)

1 (7*)

(1»)

HIGH DOSE

a35

33

33

(33)

(33)

1 (3*)

(33)

1 (3*)

(32)

1 <3X)

(32)

4 (13%)

1 (3%)

2 (6%)

1 (3«)

(33)

(32)

1 (3X)

(3)

(3)

i_im

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NF.CROPSIED

a 35 ANIMALS WERE INITIALLY IN THE STUDY, BUT TWO AIBALS WERE FOUND TO BE

FEMALES IN A MALE GROUP.

69
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

HEMATOPOIESIS

CIRCULATORY SYSTEM

#MYOCARDIUM

INFLAMMATION, INTERSTITIAL

INFLAMMATION, SUPPURATIVE

INFLAMMATION, CHRONIC SUPPORATIV

FIBROSIS, DIFFUSE

NECROSIS, DIFFUSE

DIGESTIVE SYSTEM

*LIVER

THROMBOSIS, NOS

INFLAMMATION, NECROTIZING

INFLAMMATION, CHRONIC

FIBROSIS

NECROSIS, COAGULATIVE

CYTOPLASMIC VACOOLIZ ATION

HYPERPLASIA, NODULAR

ANGIECTASIS

HYPERPLASIA, RETICULUM CELL

HfiMATOPOIESIS

*LIVER/CENTRILOBULAR

NECROSIS, COAGULATIVE

tPANCFEAS

INFLAMMATION, NECROTIZING

URINARY SYSTEM

*KIDNEY

CYST, NOS

INFLAMMATION, CHRONIC

FIBROSIS, FOCAL

CALCIFICATION, FOCAL

ATROPHY, NOS

ATROPHY, FCCAL

ENDOCBINE SYSTEM

NONE

MATCHED

CONTROL

(12)

1 (8X)

1 (8X)

(12)

1 (8X)

1 (8X)

1 (8%)

2 (17%)

2 (17%)

(12)

(12)

(12)

1 (8%)

1 (8%)

1 (8%)

LOW DOSE

1 (7X)

(32)

1 (3X)

1 (3X)

<3«)

1 (3X)

1 (3%)

1 (3X)

1 (3%)

1 (315)

1 (3X)

<3<»)

1 (315)

(3i»)

1 (3*)

(34)

1 (3X)

1 (3X)

1 (3%)

HIGH DOSE

(32)

3 (9%)

2 (6X)

2 (6%)

(33)

3 (9%)

2 (6%)

1 (3%)

(33)

(32)

(32)

t NUMBER OF ANIMALS HITH TISSUE EXAMINED BICROSCOPICALIY

* NUMBER OF ANIMALS NECROPSIED

70
REPRODUCTIV E SYSTEM

*SENINA L VESICLE

CYST, NOS

NERVOU S SYSTE M

NON E

SPECIAL SENSE ORGANS

NON E

MUSCULOSKELETAl SYSTEM

NONE

BODY CAVITIES

*PERITONEUM

INFLAMMATION , CHRONIC

NECROSIS, FAT

ALL OTHER SYSTEMS

NON E

SPECIAL MORPHOLOGY SUMMAR Y

NO LESION REPORTED

NO NECROPSY PERFORME D

AUTO/NECROPSY/HISTO PERF

AUTOLYSIS/NO NECROPSY

MATCHED

CONTROL

(12)

1 (8X)

(12)

2

2

1

LOW DOSE

(34)

(3U)

1 (3«)

2 (6S)

8

1

1

HIGH DOSE

(33)

(33)

10

1

71

TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

t NUMBER OF ANIHALS WITH TISSUE EXAMINED BICROSCOPICALLY

* NUHBER OF ANIBALS NECROPSIED
TABLE 02.

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE

MICE FED L-TRYPTOPHAN IN THE DIET

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIHALS EXAMINED HISTOPATHOLOGICALL'

INTEGUMENTARY SYSTEM

NONE

RESPIRATORY SYSTEM

*NASAL TURBINATE

INFLAMMATION, CHRONIC SOPPURATI'

tTRACHEA

INFLAMMATION, SOPPURATIVE

ILUNG/BRONCHUS

INFLAMMATION, SOPPURATIVE

ILUNG

BRONCaOPNEDHONIA SOPPORATIVE

PNEONONIA INTERSTITIAL CHRONIC

BHONCHOPNEUMONIA CHRONIC SUPPUR,

HBMOSIDEROSIS

HYPERPLASIA, ALVEOLAR EPITHELIDI

HYPERPLASIA, PLASMA CELL

HYPERPLASIA, LYMPHOID

HEMATOPOIETIC SYSTEM

IBONE HARROW

ATROPHY, NOS

ISPLEEN

INFLAMMATION, CHROHIC FOCAL

ANGIECTASIS

HIP.£BEIASI*t iai£gi,oa CELL

MATCHED

CONTROL

15

13

I 13

(13)

V 1 (8X)

(13)

1 (8X)

(13)

1 (8%)

(13)

2 (15%)

H 8 (62%)

!i

(12)

(13)

LOW DOSE

35

33

33

(33)

(33)

3 (9«)

(32)

(32)

5 (16*)

1 (3*)

1 (3X)

1 (3«)

(31)

(32)

2_JiSl

HIGH DOSE

35

35

35

(35)

(35)

4 (11X)

(35)

(35)

1<» (HOX)

1 (3X)

1 (3X)

1 (3*)

3 (9X)

1 (3X)

(35)

2 (6X)

(35)

1 (3X)

2 (6X)

t NUMBER OF ANIHALS WITH TISSDE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

72
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

HEMATOPOIESIS

tMANDIBULAR L. NODE

HYPERPLASIA, PLASMA CELL

HYPERPLASIA, RETICULUM CELL

HYPERPLASIA, LYMPHOID

*MEDIASTINAL L.NODE

THROHBOSIS, NOS

INFLAMMATION, NECROTIZING

HYPERPLASIA, PLASHA CELL

HYPERPLASIA, RETICOLOM CELL

JHESENTERIC L. NODE

THROMBOSIS, NOS

CONGESTION, NOS

INFLAMMATION, NECROTIZING

ATROPHY, NOS

#THYMUS

HYPERPLASIA, LYMPHOID

CIRCULATORY SYSTEM

tMYOCARDIUM

MINERALIZATION

DIGESTIVE SYSTEM

#LIVER

THROMBOSIS, NOS

INFLAMMATION, SUPPURATIVE

ANGIECTASIS

#PANCREAS

CYST, NOS

*RECTUM

PROLAPSE

ULCER, NOS

UBINARY SYSTEM

NONE _

MATCHED

CONTROL

1 (8%)

(<»)

(«)

(")

(13)

(13)

1 (8%)

(13)

(13)

(13)

LOW DOSE

3 (9%)

O

7

)

1 (6X)

1 (6%)

(17)

1 (6%)

1 (6X)

1 (6%)

(17)

2 (12%)

1 (6%)

2 (12X)

1 (6X)

(31)

1 (3X)

(32)

(32)

(33)

1 (3X)

(33)

1 (3X)

1 (3X)

HIGI

7

(15)

1

(15)

2

(15)

(35)

(35)

(35)

1

1

2

(35)

(35)

HDOSE

(20%)

(7%)

(13%)

<3X)

(3%)

(6X)

f NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECROPSIED

73
TABLE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

ENDOCRINE SYSTfM

((THYROID

CYSTIC FOLLICLES

REPRODUCTIVE SYSTEM

*UTEH US/EN DO KETRIUI"!

INFLAMMATION, SUPPURATIVF

INFLAMMATION, CHRONIC

INFLAMMATION, CHRONIC SUPPUHATIV

HYPERPLASIA, CYSTIC

f OVARY/OVIDUCT

INFLAMMATION, CHRONIC

#OVARY

CYST, NOS

INFLAMMATION, SUPPURATIVE

INFLAMMATION, CHRONIC SUPPORATIV

NERVOUS SYSTEM

tBRAIN

HEMORRHAGE

SPECIAL SENSE ORGANS

*MIDDLE EAR

INFLAMMATION, SUPPURATIVE

INFLAMMATION, CHRONIC SUPPURATIV

MUSCULOSKELETAL SYSTEM

NONE

BODY CAVITIES

NONE

MATCHED

CONTROL

(13)

1 (8%)

(13)

2 (15%)

11 (85%)

(13)

(13)

(12)

(13)

1 (8%)

1 (8X)

LOW DOSE

(30)

(32)

2 (63)

3 (9%)

2U (7^%)

(32)

(32)

5 (16%)

2 (6X)

(33)

1 (3%)

(33)

HIGH DOSE

(31)

1 (3*)

(34)

2 (f.%)

1 (15)

19 (56%)

(31*)

1 (3%)

(3U)

5 (15%)

1 (3%)

2 (6%)

(35)

(35)

It NUMBE R O F ANIMAL S WIT H TISSUE EXAMINE D MICROSCOPICALL Y

* NUMBE R OF ANIMAL S NECROPSIED

74
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

MATCHED LOW DOSE HIGH DOSE

CONTROL

ALL OTHER SYSTEMS

*HOLTIPLE ORGANS

HYPERPLASIA, PLASMA CELL

HYPERPLASIA, LYHPHOID

SPECIAL MORPHOLOGY SUMMARY

NO LESION REPORTED

NO NECROPSY PERFORMED

AUTOLYSIS/NO NECROPSY

(13)

1

1

(33)

1 (3%)

2 (6X)

2

1

1

(35)

U

75

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY

* NUMBER OF ANIMALS NECPOPSIED

APPENDIX E

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

IN RATS FED L-TRYPTOPHAN IN THE DIET

77

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

Fed L-Tryptophan in the Diet3

Topography: Morphology

Subcutaneous Tissue: Fibroma^

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Hematopoietic System: Leukemia*5

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

0/15 (0)

N.S.

—

4/15 (27)

P = 0.014(N)

82

Low

Dose

4/34 (12)

N.S.

Infinite

0.437

Infinite

105

3/34 (9)

N.S.

0.331

0.058

1.754

95

High

Dose

3/34 (9)

N.S.

Infinite

0.281

Infinite

78

1/34 (3)

P = 0.026(N)

0.110

0.002

1.013

84
Table El. Analyses of the Incidence of Primary Tumors in Male Rats

Fed L-Tryptophan in the Dieta

(continued)

Topography: Morphology

Pituitary: Chromophobe Adenoma*3

P Valuesc>

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Thyroid: Follicular-cell Carcinoma*3

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

1/11 (9)

N.S.

101

0/14 (0)

N.S.

—

Low

Dose

2/30 (7)

N.S.

0.733

0.045

41.816

105

2/33 (6)

N.S.

Infinite

0.134

Infinite

105

High

Dose

5/30 (17)

N.S.

1.833

0.250

83.411

96

0/32 (0)

N.S.

__

—

—

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

Fed L-Tryptophan in the Diet3

(continued)

Topography: Morphology

Thyroid: C-cell Carcinoma*5

P Valuesc>

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Thyroid: C-cell Adenoma or

Carcinoma*5

P Values0 >d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

1/14 (7)

N.S.

91

5/14 (36)

P = 0.004(N)

91

Low

Dose

4/33 (12)

N.S.

1.697

0.195

80.844

100

6/33 (18)

N.S.

0.509

0.167

1.823

100

High

Dose

0/32 (0)

N.S.

0.000

0.000

8.096

—

1/32 (3)

P = 0.007(N)

0.088

0.002

0.697

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

Fed L-Tryptophan in the Diet3

(continued)

Topography: Morphology

Pancreatic Islets: Islet-cell

Adenoma"

P Values0 >d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Pancreatic Islets: Islet-cell

Adenoma or Carcinoma^

P Valuesc'

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

0/15 (0)

N.S.

—

0/15 (0)

N.S.

—

Low

Dose

2/33 (6)

N.S.

Infinite

0.142

Infinite

105

2/33 (6>

N.S.

Infinite

0.142

Infinite

105

High

Dose

0/34 (0)

N.S.

__

—

—

—

1/34 (3)

N.S.

Infinite

0.025

Infinite

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

Fed L-Tryptophan in the Diet3

(continued)

Topography; Morphology

Testis: Interstitial-cell Tumorb

P Values0 »d

Relative Risk^

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

13/15 (87)

P = 0.016(N)

82

Low

Dose

31/34 (91)

N.S.

1.052

0.878

1.352

91

High

Dose

21/33 (64)

N.S.

0.734

0.595

1.145

100

aDosed groups received 25,000 or 50,000 ppm.

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

GBeneath the incidence of tumors in the control group is the probability level for the CochranArmitage test when P < 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 < 0.05; otherwise,

not significant (N.S.) is indicated.

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

eThe probability level for departure from linear trend is given when P < 0.05 for any comparison.

^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

Fed L-Tryptophan in the Diet3

Topography: Morphology

Pituitary: Chromophobe Adenoma"

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Pituitary: Chromophobe Carcinoma*5

P Valuesc»

d

Relative Risk^

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

4/12 (33)

N.S.

103

0/12 (0)

N.S.

—

Low

Dose

12/27 (44)

N.S.

1.333

0.543

4.670

75

1/27 (4)

N.S.

Infinite

0.025

Infinite

86

High

Dose

9/32 (28)

N.S.

0.844

0.313

3.218

92

2/32 (6)

N.S.

Infinite

0.120

Infinite

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

Fed L-Tryptophan in the Diet3

(continued)

Topography: Morphology

Pituitary: Chromophobe Adenoma

or Carcinoma^

P Values0 >d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Thyroid: Follicular-cell

Carcinoma*3

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

4/12 (33)

N.S.

103

0/14 (0)

N.S.

—

Low

Dose

13/27 (48)

N.S.

1.444

0.601

4.964

75

1/32 (3)

N.S.

Infinite

0.025

Infinite

105

High

Dose

11/32 (34)

N.S.

1.031

0.408

3.779

92

0/33 (0)

N.S.

__

—

—

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

Fed L—Tryptophan in the Diet3

(continued)

Topography: Morphology

Thyroid: C-cell Adenomab

P Values0 »

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Mammary Gland: Adenoma, NOS

(not otherwise specified)*3

P Values0 »

d

Relative Risk^

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched Low

Control Dose

1/14 (7) 2/32 (6)

N.S. N.S.

0.875

0.051

49.995

105 105

0/14 (0) 0/35 (0)

N.S. N.S.

__

—

—

— —

High

Dose

1/33 (3)

N.S.

0.424

0.006

32.328

104

2/34 (6)

N.S.

Infinite

0.130

Infinite

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

Fed L-Tryptophan in the Dieta

(continued)

Topography: Morphology

Mammary Gland: Fibroadenomab

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Mammary Gland: Adenoma or

Fibroadenoma"

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

4/14 (29)

P = 0.027(N)

96

4/14 (29)

N.S.

—

Low

Dose

7/35 (20)

N.S.

0.700

0.224

2.873

91

7/35 (20)

N.S.

0.700

0.224

2.886

—

High

Dose

2/34 (6)

N.S.

0.206

0.022

1.289

92

4/34 (12)

N.S.

0.412

0.094

1.957

92
00

00

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

Fed L-Tryptophan in the Diet3

(continued)

Topography; Morphology

Uterus: Endometrial Stromal

Polypb

P Values0 »d

Relative Risk^

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

3/13 (23)

N.S.

105

Low

Dose

3/35 (9)

N.S.

0.371

0.060

2.516

105

High

Dose

3/33 (9)

N.S.

0.394

0.064

2.664

104

aDosed groups received 25,000 or 50,000 ppm.

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

GBeneath the incidence of tumors in the control group is the probability level for the CochranArmitage test when P < 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 < 0.05; otherwise,

not significant (N.S.) is indicated.

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

e

The probability level for departure from linear trend is given when P < 0.05 for any comparison.

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

group.
APPENDIX F

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

IN MICE FED L-TRYPTOPHAN IN THE DIET

89

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

Fed L-Tryptophan in the Diet3

Topography: Morphology

Lung: Alveolar /Bronchiolar

Adenoma^

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Lung: Alveolar/Bronchiolar

Adenoma or Carcinoma*3

P Values0 »d

Relative Risk^

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

0/12 (0)

N.S.

__

0/12 (0)

N.S.

—

Low

Dose

3/33 (9)

N.S.

Infinite

0.237

Infinite

74

4/33 (12)

N.S.

Infinite

0.367

Infinite

74

High

Dose

2/32 (6)

N.S.

Infinite

0.120

Infinite

102

2/32 (6)

N.S.

Infinite

0.120

Infinite

102
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice

Fed L-Tryptophan in the Diet3

(continued)

Topography: Morphology

Hematopoietic System: Lymphoma^

P Valuesc»

d

Departure from Linear Trend6

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Trigeminal Ganglion:

Malignant Neurilenoma"

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

0/12 (0)

N.S.

P = 0.005

—

0/12 (0)

N.S.

—

Low

Dose

9/34 (26)

P = 0.048

Infinite

1.016

Infinite

62

2/34 (6)

N.S.

Infinite

0.113

Infinite

67

High

Dose

2/33 (6)

N.S.

Infinite

0.117

Infinite

67

0/33 (0)

N.S.

__

—

—

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

Fed L-Tryptophan in the Diet3

(continued)

Topography: Morphology

Liver: Hepatocellular Adenoma^

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Liver: Hepatocellular Carcinoma^5

P Values0 »d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

1/12 (8)

N.S.

104

0/12 (0)

N.S.

—

Low

Dose

4/34 (12)

N.S.

1.412

0.165

67.331

94

1/34 (3)

N.S.

Infinite

0.020

Infinite

100

High

Dose

5/33 (15)

N.S.

1.818

0.143

83.089

102

2/33 (6)

N.S.

Infinite

0.117

Infinite

104
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice

Fed L-Tryptophan in the Dieta

(continued)

Topography; Morphology

Liver: Hepatocellular

Adenoma or Carcinoma**

P Values0 »

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

1/12 (8)

N.S.

104

Low

Dose

5/34 (15)

N.S.

1.765

0.236

80.745

94

High

Dose

7/33 (21)

N.S.

2.545

0.396

110.503

102

aDosed groups received 25,000 or 50,000 ppm.

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

cBeneath the incidence of tumors in the control group is the probability level for the CochranArmitage test when P < 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 < 0.05; otherwise,

not significant (N.S.) is indicated.

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

eThe probability level for departure from linear trend is given when P < 0.05 for any comparison.

^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

Fed L-Tryptophan in the Diet3

Topography: Morphology

Hematopoietic System:

Lymphoma or Leukemia"

P Valuesc»

d

Relative Riskf

Lower Limit

Upper Limit

Weeks to First Observed Tumor

Matched

Control

2/13 (15)

N.S.

92

Low

Dose

6/33 (18)

N.S.

1.182

0.256

11.065

67

High

Dose

1/35 (3)

N.S.

0.186

0.003

3.348

61

aDosed groups received 25,000 or 50,000 ppm.

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

cBeneath the incidence of tumors in the control group is the probability level for the CochranArmitage test when P < 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 < 0.05; otherwise,

not significant (N.S.) is indicated.

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

eThe probability level for departure from linear trend is given when P < 0.05 for any comparison.

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

group.

Review of the Bioassay of L-Tryptophan for Carcinogenicity

by the Data Evaluation/Risk Assessment Subgroup

of the Clearinghouse on Environmental Carcinogens

March 7, 1978

The Clearinghouse on Environmental Carcinogens was

established in May, 1976, in compliance with DREW 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 L-Tryptophan for carcinogenicity.

The primary reviewer agreed with the conclusion in the

report that, under the conditions of test, L-Tryptophan

was not carcinogenic in rats or mice. After a brief

description of the experimental design and conditions of

test, he said that the study was adequate to support this

conclusion.

As the secondary reviewer, Dr. Kuschner noted that others

have reported tumors in mice administered L-Tryptophan. He

questioned the use of rodents as an appropriate species for

studying the carcinogenicity of L-Tryptophan and suggested

that the dog would be a more relevant experimental model.

A motion was made that the report on the bioassay of

L-Tryptophan be accepted as written. The motion was

seconded and approved unanimously.

Members present were:

Gerald N. Wogan (Chairman), Massachusetts Institute of

Technology g?
Arnold Brown, Mayo Clinic

E. Cuyler Hammond, American Cancer Society

Joseph Highland, Environmental Defense Fund

Henry Pitot, University of Wisconsin Medical Center

George Roush, Jr., Monsanto Company

Michael Shimkin, University of California at San Diego

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.

98

DHEW Publication No. (NIH) 78-1321