National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 26 

1978 

BIOASSAY OF 

NITROFEN 

FOR POSSIBLE CARCINOGENICITY
 

CAS No. 1836-75-5 

NCI-CG-TR-26 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

NITROFEN
 

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
 

DHEWJPublication No. (NIH) 78-826
 

REPORT ON THE ΒIOASSAY OF NITROFEN
 

FOR POSSIBLE CARCINOGENICITY
 

CARCINOGENESIS PROGRAM, DIVISION OF CANCER CAUSE AND PREVENTION
 

NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
 

CONTRIBUTORS: This report presents the results of the bioassay of
 

nitrofen conducted for the Carcinogen Bioassay and Program Resources
 

Branch, Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI), National Institutes of Health,
 

Bethesda, Maryland· This bioassay was conducted by Hazleton Laboratories America, Inc., Vienna, Virginia, initially under direct contract to the NCI and currently under a subcontract to Tracor Jitco,
 

Inc., prime contractor for the NCI Carcinogenesis Bioassay Program·
 

The experimental design was determined by the NCI Project Officers, Dr. J· Η. Weisburger (1,2) and Dr. E. K. Weisburger (1). The
 

principal investigators for the contract were Dr. Μ· Β· Powers (3),
 

Dr. R. W. Voelker (3), Dr. W. A. Olson (3,4) and Dr. W. M. Weatherholtz (3). Chemical analysis was performed by Dr. C. L. Guyton
 

(3,5); the technical supervisor of animal treatment and observation
 

was Ms. K. J. Petrovics (3).
 

Histopathology was performed by Dr. R· H. Habermann (3) and reviewed by Dr· R. W· Voelker (3) at the Hazleton Laboratories America,
 

Inc., and the diagnoses included in this report represent the interpretation of these pathologists. Pathologists from NCI (1) and
 

Tracor Jitco (6) have reviewed selected slides and concur with the
 

overall histopathologic evaluation of the study·
 

Compilation of individual animal survival, pathology, and
 

summary tables was performed by EG&G Mason Research Institute (7);
 

the statistical analysis was performed by Dr· J· R· Joiner (6) and
 

Mr. W. W. Belew (8), using methods selected for the Bioassay Program
 

by Dr. J. J. Gart (9).
 

This report was prepared at METREK, a Division of The MITRE
 

Corporation (8) under the direction of the NCI· Those responsible
 

for this report at METREK are the project coordinator, Dr. L. W.
 

Thomas (8), the task leader, Dr. M. R. Kornreich (8), and the senior
 

biologist, Ms. P. Walker (8). The final report was reviewed by
 

members of the participating organizations.
 

The following scientists at the National Cancer Institute were
 

responsible for evaluating the bioassay experiment, interpreting the
 

results, and reporting the findings: Dr. K. C. Chu (1), Dr. C. C.
 

iii
 
Cueto, Jr. (1), Dr. J. F. Douglas (l), Dr. D. G. Goodman (1), Dr.
 

R. A. Griesemer (1), Dr. R. A. Squire (1), and Dr. J. Μ. Ward (1).
 

1.	 Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health,
 

Bethesda, Maryland.
 

2.	 Now with the Naylor Dana Institute for Disease Prevention,
 

American Health Foundation, Hammon House Road, Valhalla,
 

New York.
 

3.	 Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike,
 

Vienna, Virginia.
 

4.	 Now with the Center for Regulatory Services, 2347 Paddock Lane,
 

Reston, Virginia.
 

5.	 Now with Rhodia, Inc., 23 Belmont Drive, Somerset, New Jersey.
 

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

Maryland.
 

7.	 EG&G Mason Research Institute, 1530 East Jefferson Street,
 

Rockville, Maryland.
 

8.	 The MITRE Corporation, METREK Division, 1820 Dolley Madison
 

Boulevard, McLean, Virginia.
 

9.	 Mathematical Statistics and Applied Mathematics Section, Field
 

Studies and Statistics Branch, Division of Cancer Cause and
 

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

IV
 
SUMMARY
 

A bioassay of technical-grade nitrofen for possible carcinogenicity was conducted using Osborne-Mendel rats and B6C3F1 mice.
 

Nitrofen was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species·
 

The time-weighted average high and low dietary concentrations of
 

nitrofen were 3656 and 2300 ppm for male rats, 2600 and 1300 ppm for
 

female rats, and 4696 and 2348 ppm for both male and female mice,
 

respectively. After a 78-week treatment period, observation of the
 

low dose and control male and all female rats continued for an additional 32 weeks; observation of the high dose male rats continued
 

for an additional 4 weeks. All mice were observed for an additional
 

12 weeks after the 78-week treatment period.
 

For each species, 20 animals of each sex were placed on test as
 

controls. No nitrofen was added to their diet.
 

The incidence of carcinomas of the pancreas had a statistically
 

significant positive association with concentration of nitrofen in
 

the diet of female rats. The incidence of this tumor in high dose
 

female rats was significant when compared to controls. Poor survival
 

related to chemical toxicity precluded the evaluation of the carcinogenicity of nitrofen in male rats.
 

In mice of both sexes, the incidence of hepatocellular carcinoma
 

at both high and low dose levels was highly significant when compared
 

to the controls. The incidence of hemangiosarcoma of the liver had a
 

statistically significant relationship with nitrofen concentration in
 

the diet for mice of both sexes, and the incidence in high dose male
 

mice was significant when compared to controls.
 

The results of this study indicate that orally administered
 

technical-grade nitrofen is a liver carcinogen in B6C3F1 mice of both
 

sexes. Nitrofen is also carcinogenic to female Osborne-Mendel rats.
 

TABLE OF CONTENTS
 

Page
 

I. INTRODUCTION 1
 

II. MATERIALS AND METHODS 3
 

A. Chemicals 3
 

B· Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 4
 

E. Selection of Initial Concentrations 6
 

F. Experimental Design 7
 

G. Clinical and Histopathologic Examinations 10
 

H· Data Recording and Statistical Analyses 12
 

III. CHRONIC TESTING RESULTS: RATS 17
 

A· Body Weights and Clinical Observations 17
 

B. Survival 19
 

C. Pathology 21
 

D. Statistical Analyses of Results 2 3
 

IV. CHRONIC TESTING RESULTS: MICE 35
 

A. Body Weights and Clinical Observations 35
 

B. Survival 35
 

C. Pathology 38
 

D. Statistical Analyses of Results 39
 

V. DISCUSSION 4 6
 

VI. BIBLIOGRAPHY 4 8
 

APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN A-l
 

RATS TREATED WITH NITROFEN
 

APPENDIX Β SUMMARY OF THE INCIDENCE OF NEOPLASMS IN B-l
 

MICE TREATED WITH NITROFEN
 

APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC C-l
 

LESIONS IN RATS TREATED WITH NITROFEN
 

APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC D-l
 

LESIONS IN MICE TREATED WITH NITROFEN
 

vii
 
LIST OF ILLUSTRATIONS 

Figure Number Page 

1 GROWTH CURVES FOR NITROFEN CHRONIC STUDY

RATS 

 18 

2 SURVIVAL COMPARISONS OF NITROFEN CHRONIC

STUDY RATS 

2 0 

3 GROWTH CURVES FOR NITROFEN CHRONIC STUDY

MICE 

 36 

4 SURVIVAL COMPARISONS OF NITROFEN CHRONIC

STUDY MICE 

3 7 

LIST OF TABLES 

Table Number Page 

1 DESIGN SUMMARY FOR OSBORNE-MENDEL RATS-­

NITROFEN FEEDING EXPERIMENT 

8 

2 DESIGN SUMMARY FOR B6C3F1 MICE--NITROFEN

FEEDING EXPERIMENT 

9 

3 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

AT SPECIFIC SITES IN MALE RATS TREATED WITH 

NITROFEN 

2 4 

4 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

AT SPECIFIC SITES IN FEMALE RATS TREATED WITH 

NITROFEN 

 2 7 

5 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

AT SPECIFIC SITES IN MALE MICE TREATED WITH 

NITROFEN 

 40 

6 ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS

AT SPECIFIC SITES IN FEMALE MICE TREATED WITH 

NITROFEN 

4 3 

Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

MALE RATS TREATED WITH NITROFEN 

 A-3 

viii
 
LIST OF TABLES (Concluded) 

Table Number Page 

A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

FEMALE RATS TREATED WITH NITROFEN 

 A-7 

Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

MALE MICE TREATED WITH NITROFEN 

 B-3 

B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN

FEMALE MICE TREATED WITH NITROFEN 

 B-6 

CI SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN MALE RATS TREATED WITH NITROFEN 

 C-3 

C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN FEMALE RATS TREATED WITH 

NITROFEN 

 C-8 

Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN MALE MICE TREATED WITH NITROFEN 

 D-3 

D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC

LESIONS IN FEMALE MICE TREATED WITH 

NITROFEN 

 D-6 

ix
 

I. INTRODUCTION
 

Nitrofen (NCI No. C00420), a substituted diphenyl ether, is one
 

of several agricultural pesticides selected for bioassay by the
 

National Cancer Institute because of a lack of adequate chronic
 

toxicity data·
 

The Chemical Abstracts Service (CAS) Ninth Collective Index
 

(1977) name for this compound is 2,4-dichloro-l-(4-nitrophenoxy)­

benzene· It is also known as 2,4-dichlorophenyl-p-nitrophenyl
 

ether, nitrophene, Tok E-25, and Nip·
 

Nitrofen is a selective contact herbicide used for pre- and post-


emergence control of annual grasses and broadleaf weeds on a variety
 

of food crops (Weed Science Society of America, 1974)·
 

Postemergence treatment is restricted to certain highly tolerant
 

crops and involves spraying the crops with 4 to 6 pounds of active
 

ingredient per acre in a water carrier. For preemergence treatment,
 

the spray is applied at a similar rate directly to the soil (Weed
 

Science Society of America, 1974).
 

Although specific production figures are unavailable, the listing
 

of nitrofen in the 1975 Directory of Chemical Producers, U.S.A.
 

(Stanford Research Institute, 1975) implies an annual commercial
 

production in excess of 1000 pounds or $1000 in value.
 

Occupational exposure to nitrofen, primarily through inhalation
 

and dermal contact, may occur among workers at pesticide production
 

*
 

The CAS registry number is 1836-75-5.
 
facilities and among agricultural workers engaged in treatment of
 

crops with the chemical. The major route of exposure for the general
 

population, however, is ingestion due to possible persistence of
 

residual quantities of nitrofen on food crops·
 

Adverse effects noted in agricultural workers following excessive
 

exposure to nitrofen over prolonged periods of time include reduction
 

in hemoglobin and leukocyte counts, inhibition of serum cholinesterase
 

and abnormalities in erythrocyte catalase and serum transaminase
 

levels (Doroshenko, 1975). In addition, dermal contact with the
 

concentrated emulsion (Tok E-25) may cause skin irritation (Weed
 

Science Society of America, 1974).
 

2
 
II. MATERIALS AND METHODS
 

A· Chemicals
 

Nitrofen, 2,4-dichloro-l-(4-nitrophenoxy) benzene, was purchased
 

from Rohm and Haas Chemical Company by Hazleton Laboratories America,
 

Inc., Vienna, Virginia, where the chemical analysis was performed·
 

The manufacturer's analysis indicated a purity of approximately
 

87 percent. Gas-liquid chromatography (GLC), utilizing the internal
 

standard assay, suggested a purity of greater than 80 percent. The
 

observed melting point (58° to 68°C) suggested the presence of
 

significant impurities, because of its wide range and variance from
 

that reported as an FDA standard (71° to 72°C). GLC total area
 

analysis indicated the presence of at least five impurities.
 

The material was analyzed by GLC total area analysis after
 

having been stored for one year. Five impurities were again detected
 

and, although the change in area suggested a different distribution
 

of these substances, no significant change in purity of the compound
 

over the 12-month period was indicated. The nature of the impurities, as suggested by the manufacturer, include xylene, dichlorophenol,
 

p-chloronitrobenzene, and chloronitrodiphenyl ethers.
 

Throughout this report the term nitrofen is used to represent
 

this technical-grade material.
 

B· Dietary Preparation
 

The basal laboratory diet for both control and dosed animals
 

consisted of 2 percent Duke's® corn oil (S. F. Sauer Company)
 
by weight added to Wayne Lab-Blox meal (Allied Mills, Inc·)· Fresh
 

mixtures of nitrofen in corn oil were prepared each week and stored
 

in the dark. The mixtures of nitrofen in corn oil were incorporated
 

into the appropriate amount of the basal laboratory diet in a twin-


shell blender fitted with an accelerator bar.
 

C· Animals
 

Two animal species, rats and mice, were used in the carcinogenicity bioassay. The Osborne-Mendel rat was selected on the basis of a
 

comparative study of the tumorigenic responsiveness to carbon tetrachloride of five different strains of rats (Reuber and Glover, 1970).
 

The B6C3F1 mouse was selected because it has been used by the NCI for
 

carcinogenesis bioassays and has proved satisfactory in this capacity.
 

Rats and mice of both sexes were obtained through contracts with
 

the Division of Cancer Treatment, National Cancer Institute. The
 

Osborne-Mendel rats were procured from the Battelle Memorial Institute,
 

Columbus, Ohio, and the B6C3F1 mice were obtained from the Charles
 

River Breeding Laboratories, Inc., Wilmington, Massachusetts. Upon
 

receipt, animals were quarantined for at least 10 days, observed for
 

visible signs of disease or parasites, and assigned to the various
 

treatment and control groups.
 

D· Animal Maintenance
 

All animals were housed by species in temperature- and humidity-


controlled rooms· The temperature range was 20° to 24°C and the
 

relative humidity was maintained between 45 and 55 percent. The air
 

4
 
conditioning system in the laboratory provided filtered air at a
 

rate of 12 to 15 complete changes of room air per hour. Fluorescent
 

lighting was provided on a 12-hour-daily cycle. The rats were individually housed in suspended galvanized-steel wire-mesh cages with
 

perforated floors. Mice were housed by sex in groups of 10 in solid-


bottom polypropylene cages equipped with filter tops. Sanitized
 

cages with fresh bedding (Sanichips , Shurfire) were provided once
 

each week for mice. Rats received sanitized cages with no bedding
 

with the same frequency. Food hoppers were changed and heat-


sterilized once a week for the first 10 weeks and once a month thereafter. Fresh heat-sterilized glass water bottles were provided three
 

times a week. Food and water were available ad libitum.
 

The nitrofen-treated and control rats were housed in the same
 

room with rats treated with trifluralin (1582-09-8), dioxathion (78­

34-2), dicofol (115-32-2), endosulfan (115-29-7), and mexacarbate
 

(315-18-4). All mice used in the nitrofen study, including controls,
 

were housed in the same room with mice treated with trifluralin (1582­

09-8), dioxathion (78-34-2), sulfallate (95-06-7), p,p'-DDT (50-29-3),
 

methoxychlor (72-43-5), p,p'-DDE (72-55-9), p,p'-TDE (72-54-8), dicofol (115-32-2), pentachloronitrobenzene (82-68-8), clonitralid (1420­

04-8), endosulfan (115-29-7), chlorobenzilate (510-15-6), mexacarbate
 

(315-18-4), amitrole (61-82-5), acetylaminofluorene (53-96-3), and
 

safrole (94-59-7).
 

*
 

CAS registry numbers are given in parentheses.
 

5 
Ε. Selection of Initial Concentrations
 

In order to establish the maximum tolerated doses of nitrofen for
 

administration to treated animals in the chronic studies, subchronic
 

toxicity tests were conducted with both rats and mice. Animals of
 

each species were distributed among six groups, each consisting
 

of five males and five females· Nitrofen was premixed with a small
 

amount of corn oil. The mixture was then incorporated into the basal
 

laboratory diet and fed ad libitum to five of the six rat groups at
 

concentrations of 1000, 1780, 3160, 5620, and 10,000 ppm and to five
 

of the six mouse groups at concentrations of 1780, 3160, 5620, 10,000
 

and 17,800 ppm. The sixth group of each species served as a control
 

group, receiving only the basal laboratory diet. The dosed dietary
 

preparations were administered for a period of 6 weeks, followed by
 

a 2-week observation period during which all animals were fed the
 

basal diet of corn oil and laboratory chow.
 

A dosage inducing no mortality and resulting in a retardation
 

in body weight gain (a retardation of approximately 20 percent) was
 

to be selected as the initial high dose. When weight gain criteria
 

were not applicable, mortality data alone were utilized.
 

In the male and female rats, no deaths were observed at any
 

concentration. In males, body weight gain retardation, expressed as
 

a percentage of the weight gain of the controls, was 10 and 25 percent at concentrations of 3160 and 5620 ppm, respectively. In
 

females, body weight gain retardation was 17 and 26 percent at
 

6
 
concentrations of 1780 and 3160 ppm, respectively· The initial high
 

doses selected for the rat chronic bioassay were 4600 ppm for males
 

and 2600 ppm for females.
 

In mice, retardation in body weight gain, although not clearly
 

dose-related, was observed at concentrations of 3160 ppm and above.
 

At the 3160 ppm concentration, body weight gain reduction was 12 percent for male mice and 8 percent for female mice· At 5620 ppm, body
 

weight gain reduction was 37 percent for male mice and 40 percent for
 

female mice. One male died at 5620 ppm· Mortality increased with
 

concentration in both sexes· The initial high dose selected for the
 

chronic study was 3550 ppm for both male and female mice.
 

F· Experimental Design
 

The experimental design parameters for the chronic study (species, sex, group size, concentrations administered, duration of
 

treated and untreated observation periods, and the time-weighted
 

average concentrations) are summarized in Tables 1 and 2.
 

At the initiation of the study the high dose, low dose, and control rats were all approximately 7 weeks old. The high and low concentrations of nitrofen initially utilized for male rats were 4600
 

and 2300 ppm, respectively. For female rats the initial high and low
 

concentrations were 2600 and 1300 ppm, respectively. During week 46
 

the concentration administered to the high dose male rats was decreased to 2300 ppm as intolerance to the higher dosage was observed.
 

All high and low dose rats were treated for 78 weeks. The low dose
 
MALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSEC
 

FEMALE
 

CONTROL
 

LOW DOSE
 

HIGH DOSE
 

TABLE 1 

DESIGN SUMMARY FOR OSBORNE-MENDEL RATS 

NITROFEN FEEDING ΕΧΡΪ&ΙΜΕΝΤ 

INITIAL 

GROUP 

SIZE 

NITROFEN 

CONCENTRATION3 

OBSERVATION PERIOD 

TREATED 

(WEEKS) 

UNTREATED 

(WEEKS) 

TIME-WEIGHTED 

AVERAGE 

CONCENTRATION 

20 0 110 0 

50 2300 

0 

78 

32 

2300 

50 4600 

2300 

0 

45 

33 

5 

3627 

20 0 110 0 

50 1300 

0 

78 

32 

1300 

50 2600 

0 

78 

32 

2600 

Concentrations in parts per million. 

Σ(concentration X weeks received)
 Time-weighted average concentration = -=rr ; :—: \
 

2J \weeks receiving treatment;
 

"These animals were terminated in week 83.
 
TABLE 2
 

DESIGN SUMMARY FOR B6F3F1 MICE
 

NITROFEN FEEDING EXPERIMENT
 

INITIAL OBSERVATION PERIOD TIME-WEIGHTED 

GROUP NITROFEN TREATED UNTREATED AVERAGE 

SIZE CONCENTRATION3 (WEEKS) (WEEKS) CONCENTRATION 

MALE 

CONTROL 20 0 90 0 

LOW DOSE 50	 1775 6 2348
 

2000 15
 

2500 57
 

0	 12
 

HIGH DOSE 50	 3550 6 4696
 

4000 15
 

5000 57
 

0	 12
 

FEMALE
 

CONTROL 20 0	 90 0
 

LOW DOSE 50	 1775 6 2348
 

2000 15
 

2500 57
 

0	 12
 

HIGH DOSE 50	 3550 6 4696
 

4000 15
 

5000 57
 

0	 12
 

Concentrations in parts per million·
 

b	 Σ(concentration X weeks received)
 Time-weighted average concentration =
 Σ(weeks receiving treatment)
 

9
 
and control males were observed for an additional 32 weeks during
 

which they were maintained on the basal laboratory diet and corn oil
 

mixture. The high dose males were observed for an additional 4 weeks
 

after treatment, during which they were maintained on the basal laboratory diet and corn oil mixture. All surviving high dose male rats
 

were sacrificed during week 83 of the study. High and low dose female
 

rats were treated for 78 weeks and then received the basal diet and
 

corn oil for an additional 32-week observation period.
 

At the initiation of the study all mice were approximately
 

weeks old. The high and low doses initially administered to the male
 

and female mice were 3550 and 1775 ppm, respectively. During week 7
 

the high and low dosages administered to the male and female mice
 

were increased to 4000 and 2000 ppm, respectively, as the animals had
 

apparently tolerated the previous dosages. Dosages were increased
 

again during week 22, to 5000 ppm for the high dose male and female
 

mice, and to 2500 ppm for the low dose male and female mice. The
 

treated mice were maintained on these nitrofen concentrations for 57
 

weeks, followed by a 12-week observation period during which the
 

animals received the basal diet and corn oil· Control mice received
 

the basal diet and corn oil for the entire study.
 

Both rat and mouse control groups were maintained and observed
 

in the same manner as the treated animals.
 

G. Clinical and Histopathologic Examinations
 

Animals were weighed immediately prior to initiation of the
 

experiment. From the first day, all animals were inspected daily
 

10
 
for mortality. Body weights, food consumption, and data concerning
 

appearance, behavior, signs of toxic effects, and incidence, size,
 

and location of tissue masses were recorded at weekly intervals for
 

the first 10 weeks and at monthly intervals thereafter. The presence
 

of tissue masses was determined by observation and palpation of each
 

animal.
 

A necropsy was performed on each animal regardless of whether it
 

died, was killed when moribund, or was sacrificed at the end of the
 

bioassay. The animals were euthanized by exsanguination under sodium
 

pentobarbital anesthesia, and were immediately necropsied. The histopathologic examination consisted of gross and microscopic examination
 

of major tissues, organs, or gross lesions taken from sacrificed animals and, whenever possible, from animals found dead.
 

Slides were prepared from the following tissues: skin, subcutaneous tissue, lungs and bronchi, trachea, bone marrow, spleen, lymph
 

nodes, thymus, heart, salivary gland, liver, gallbladder (mice) and
 

bile duct, pancreas, esophagus, stomach, small intestine, large intestine, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, pancreatic islets, testis, prostate, brain, muscle, uterus,
 

mammary gland, and ovary.
 

Tissues for which slides were prepared were preserved in 10 percent buffered formalin, embedded in paraffin, sectioned, and stained
 

with hematoxylin and eosin prior to microscopic examination. An
 

11
 
occasional section was subjected to special staining techniques for
 

more definitive diagnosis·
 

A few tissues were not examined for some animals, particularly
 

for those that died early· Also, some animals were missing, cannibalized, or judged to be in such an advanced state of autolysis as to
 

preclude histopathologic interpretation. Thus, the number of animals
 

for which particular organs, tissues, or lesions were examined microscopically varies and does not necessarily represent the number of
 

animals that were placed on experiment 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
 

12
 
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 used Tarone's (1975) extensions of Cox's methods for
 

testing 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 was 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, pp. 48-52) was used to compare the tumor incidence of a control
 

13
 
group to that of a group of treated animals at each dose level. When
 

results for a number of treated 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, pp. 6-10) 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, pp. 362-365), was also used.
 

Under the assumption of a linear trend, this test determined if the
 

slope of the dose-response curve is different from zero at the one-


tailed 0.05 level of significance. Unless otherwise noted, the direction of the significant trend was 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 the first tumor was
 

14
 
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 animals died naturally or were 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 to its control was calculated from
 

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

is defined as ρ / where ρ is the true binomial probability of the
 

t c c
 

incidence of a specific type of tumor in a treated group of animals
 

and ρ 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 treated group
 

and the proportion in a control group corresponds to a relative risk
 

15
 
of unity· Values in excess of unity represent the condition of a
 

larger proportion in the treated 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
 

percent of a large number of identical experiments, the true ratio
 

of the risk in a treated 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 (a P < 0.025
 

one-tailed test when the control incidence is not zero, Ρ < 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. CHRONIC TESTING RESULTS: RATS
 

A· Body Weights and Clinical Observations
 

As indicated in Figure 1, a dose-related depression of body
 

weight gain was observed in males and females throughout the 78-week
 

treatment period. Growth curves for rats surviving beyond the
 

treatment period tend to converge.
 

All animals exhibited generally normal appearance and behavior
 

during the first 10 weeks of the study with the exception of intermittent observations of hunched appearance, abdominal urine stains, and
 

labored respiration in a few treated rats. Beginning in week 14, a
 

hunched appearance was observed in a gradually increasing number of
 

treated rats and by week 78, at cessation of treatment, 75 percent of
 

the low dose and 95 percent of the high dose rats appeared hunched.
 

Urine stains and a slight decrease in body weight gain were also
 

evident» particularly in the high dose groups. A bloody-appearing
 

vaginal discharge was intermittently observed in one to five females
 

in each of the treatment groups during the second year of the study
 

and was consistently noted in these animals during the the last
 

3 months.
 

Respiratory signs, characterized by labored respiration, wheezing, and/or nasal discharge were observed at a low to moderate
 

incidence in all groups during the second year of the study. The
 

incidence increased as the animals aged. At termination of the
 

study in week 110, most of the surviving treated and control rats
 

17
 
TIME ON TEST (WEEKS) 

FIGURE 1 

GROWTH CURVES FOR NITROFEN CHRONIC STUDY RATS 

18 
appeared hunched and were showing respiratory signs. Other signs
 

often associated with aging that were noted at comparable rates in
 

all groups included sores on the body and/or extremities, localized
 

alopecia, reddened or squinted eyes, rough or stained fur, bloating,
 

and palpable nodules or tissue masses. Isolated, apparently spontaneous symptoms observed in one or two rats included incoordination,
 

ataxia, pale appearance, and hyperactivity.
 

. Survival
 

The estimated probabilities of survival for male and female rats
 

in the control and nitrofen-treated groups are shown in Figure 2.
 

For male rats the Tarone test for positive association between
 

increased dosage and accelerated mortality was significant (P < 0.001),
 

The departure from linear trend was also significant (P < 0.001),
 

principally because of the accelerated mortality in the high dose
 

group. Fifty percent of the high dose males were dead by week 45;
 

the 15 males still surviving by week 83 were then sacrificed.
 

Sixty percent of the low dose and 45 percent of the control
 

group survived until the end of the study. As such, there were
 

adequate numbers of low dose and control males, but inadequate numbers of high dose males, at risk to perform a meaningful statistical
 

analysis of the incidence of late-developing tumors.
 

For female rats the Tarone test indicated a significant (P =
 

0.032) positive association between increased dosage and accelerated
 

19
 
 τ τ τ

 ι ι ι 45 60 75 90 

TIME ON TEST (WEEKS) 

FIGURE 2 

SURVIVAL COMPARISONS OF NITROFEN CHRONIC STUDY RATS 

20 
mortality· Survival was adequate in all groups as 56 percent of the
 

high dose, 74 percent of the low dose, and 80 percent of the control
 

female rats survived until the end of the study.
 

C. Pathology
 

Histopathologic findings on neoplasms in rats are tabulated in
 

Appendix A (Tables Al and A2); findings on nonneoplastic lesions are
 

tabulated in Appendix C (Tables C1 and C2).
 

Long-term dietary intake of nitrofen was associated with an
 

increased incidence of carcinoma of the pancreas in female rats·
 

This unusual neoplasm occurred in 2/50 (4 percent) low dose and 7/50
 

(14 percent) high dose females. These were highly invasive neoplasms
 

characterized microscopically by the proliferation of anaplastic
 

epithelial cells forming glands, often sequestered in fibrous tissue,
 

and ducts and papillary structures that were lined by one or more
 

cell layers· Marked desmoplasia, ischemic necrosis, inflammation,
 

and hemorrhage were often associated with these tumors. Most appeared
 

to be ductal carcinomas, but in some areas, were so highly anaplastic
 

and poorly differentiated that little pattern was observed. Poorly
 

formed acini occasionally were recognized, consisting of polygonal,
 

highly basophilic cells with abundant cytoplasm and hyperchromatic
 

nuclei, but zymogen granules were not observed. Peritoneal spread and
 

invasion of abdominal viscera were common, and all metastasized to the
 

lung.
 

21
 
An equivocal increased incidence of neoplasms affecting the
 

reproductive system of female rats was observed including the following: in the vagina, a squamous-cell carcinoma in 1/50 (2 percent)
 

high dose females; in the uterus, a squamous-cell carcinoma in 1/50
 

(2 percent) low dose females; and uterine adenocarcinomas in 1/50 (2
 

percent) low dose and 1/49 (2 percent) high dose females· In the
 

ovary, a granulosa-cell carcinoma appeared in 1/20 (5 percent)
 

control and 1/50 (2 percent) low dose females, cystadenocarcinoma in
 

1/50 (2 percent) low dose females, and granulosa-cell tumors in 4/49
 

(8 percent) high dose females· Although these types of neoplasms
 

occurred in small numbers, the vaginal and uterine carcinomas represent unusual forms of neoplasia in this strain· There were 3/50
 

(6 percent) histiocytic malignant lymphomas of multiple organs and
 

1/49 (2 percent) lymphocytic malignant lymphoma of the uterus in
 

high dose females· A variety of other neoplasms were seen in all
 

groups but appeared unrelated to treatment·
 

In male rats, a life-shortening effect related to intake of
 

nitrofen was observed, particularly in high dose males· The high
 

dose male group was terminated in week 83 because only 15 animals
 

remained in the study. The principal toxic effect of nitrofen in the
 

high dose male group was massive hemorrhage involving the genitalia
 

and pelvic cavity· Massive centrilobular necrosis, a sequela of
 

hypoxia due to acute hemorrhage, was frequently recognized in the
 

livers of high dose animals. In low dose males, a high incidence
 

22
 
of chronic pneumonia, probably exacerbated by stress, was observed.
 

Although a carcinogenic effect was not demonstrated in male rats,
 

the possible masking effects of toxicity with early mortality should
 

not be dismissed·
 

Increased incidences of malignant tumors of the pancreas and
 

of the reproductive system provided evidence of carcinogenicity
 

in female rats. The absence of histopathologic evidence of carcinogenicity in male rats could be the result of abbreviated life spans
 

due to compound-related toxicity·
 

D· Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

rats are summarized in Tables 3 and 4. The analysis for every type
 

of tumor that was observed in more than 5 percent of any of the
 

nitrofen-dosed groups of either sex is included.
 

Two control groups were used for statistical analyses: the
 

control group originally assigned to nitrofen in the experimental
 

design (designated in this section as the "matched" control group)
 

and a pooled control group which combined the controls from the
 

studies of nitrofen, chlorobenzilate, endosulfan, and mexacarbate.
 

Each chlorobenzilate control group had 50 rats, each of the other
 

groups had 20. The control rats used for the pool were of the same
 

strain, were housed in the same room, were tested concurrently for
 

over a year, and were diagnosed by the same pathologists.
 

23
 
TABLE 3
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE RATS TREATED WITH NITROFENa
 

TOPOGRAPHY :MORPHOLOGY
 

Subcutaneous Tissue: Fibroma or Fibrosarcoma
 

 ValuesC
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma
 

 Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: Follicular-Cell Adenoma
 

 Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW
 DOSE
 

3/50(0.06)
 

N.S.
 

0.934
 

0.161
 

3.880
 

0.400
 

0.060
 

2.801
 

89
 

9/46(0.20)
 

N.S.
 

1.630
 

0.647
 

3.868
 

1.957
 

0.462
 

17.603
 

99
 

7/50(0.14)
 

N.S.
 

2.520
 

0.760
 

8.549
 

2.800
 

0.402
 

123>408
 

90
 

HIGH
 

DOSE
 

0/50(0.00)
 

 - 0.021**(Ν)
 

0.000
 

0.000
 

1.128
 

0.000
 

0.000
 

0.659
 

1/44(0.02)
 

N.S.
 

0.189
 

0.004
 

1.212
 

0.227
 

0.004
 

4.167
 

0/47(0.00)
 

N.S.
 

0.000
 

0.000
 

1.440
 

0.000
 

0.000
 

7.942
 

POOLED
 

CONTROL
 

7/109(0.06)
 

N.S.
 

12/100(0.12)
 

N.S.
 

 = 0.025
 

6/108(0.06)
 

N.S.
 

 = 0.006
 

MATCHED
 

CONTROL
 

3/20(0.15)
 

 - 0.012(Ν)
 

107
 

2/20(0.10)
 

N.S.
 

 - 0.031
 

71
 

1/20(0.05)
 

N.S.
 

 - 0.019
 

103
 

84 
TOPOGRAPHY:MORPHOLOGY
 

Thyroid: Follicular-Cell Adenoma or Carcinoma
 

 Valuesc
 

Departure from Linear Trend
 

Relative Risk (Fooled Control)d
 

Lower Limit
 

upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Thyroid: C-Cell Adenomab
 

 Values0
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)*
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Mammary Gland: Adenocarcinoma, NOS
 

 Values0
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 3 

(GONTISUED) 

POOLED MATCHED LOW HIGH 

CONTROL CONTROL DOSE DOSE 

9/108(0.08) 2/20(0.10) 8/50(0.16) 0/47(0.00) 

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

 * 0.035*(Ν) 

 ­ 0.010 - 0.042 

1.920 0.000 

0.681 0.000 

5.226 0.877 

1.600 0.000 

0.364 0.000 

14.699 1.429 

103 90 

0/108(0.00) 0/20(0.00) 1/50(0.02) 2/47(0.04) 

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

Infinite Infinite 

0.114 0.671 

Infinite Infinite 

Infinite Infinite 

0.022 0.130 

Infinite Infinite 

112 58 

3/109(0.03) 1/20(0.05) 2/50(0.04) 0/50(0.00) 

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

1.453 0.000 

0.124 0.000 

12.215 3.635 

0.800 0.000 

— — _ 0.045 0.000 46.273 7.475 

Ill 83 
TABLE 3
 

(CONCLUDED)
 

POOLED MATCHED LOW HIGH 

TOPOGRAPHY:MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Mammary Gland: Fibroadenoma 1/109(0.01) 0/20(0.00) 2/50(0.04) 0/50(0.00) 

 Values0 

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

Relative Risk (Pooled Control)d 4.360 0.000 

Lower Limit 0.231 0.000 

Upper Limit 252.025 40.652 

Relative Risk (Matched Control)d Infinite 

Lower Limit 0.123 —._ 

Upper Limit Infinite 

Weeks to First Observed Tumor 111 

T)osed groups received time-weighted average doses of 2300 and 3627 ppm in feed.
 

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

C

Beneath the incidence of each of the controls is the probability level for the Cochran-Armitage test for dose-related trend in proportions when it is
 

below 0.05, otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath the dose group
 

incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group with the pooled control group (*) and
 

the matched control group (**) when either is below 0.05, otherwise N.S. - not significant.
 

(N) Less incidence in the dose group (s) than in a control group results in a negative indication.
 

Relative Risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence interval for that
 

relative risk.
 
TABLE 4
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE RATS TREATED WITH NITROFEIT
 

TOPOGRAPH?:MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

 Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Hatched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Lymphoma1*
 

 Valuesc
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenomab
 

 ValuesC
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

LOW
 

DOSE
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

1.331
 

0.000
 

0.000
 

1.345
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

41.028
 

15/49(0.31)
 

N.S.
 

0.885
 

0.496
 

1.469
 

1.837
 

0.618
 

9.084
 

103
 

HIGH
 

DOSE
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

1.381
 

0.000
 

0.000
 

i.345
 

4/50(0.08)
 

 - 0.033*
 

8.800
 

0.698
 

424.104
 

Infinite
 

0.386
 

Infinite
 

88
 

18/48(0.38)
 

N.S.
 

1.085
 

0.645
 

1.717
 

2.250
 

0.783
 

10.870
 

105
 

POOLED
 

CONTROL
 

6/110(0.05)
 

 - 0.029(Ν)
 

—
 

1/110(0.01)
 

 - 0.016
 

—
 

—
 

—
 

37/107(0.35)
 

N.S.
 

...
 

MATCHED
 

CONTROL
 

2/20(0.10)
 

 - 0.024(N)
 

 - 0.042
 

- Μ —
 

Ill
 

0/20(0.00)
 

 - 0.039
 

3/18(0.17)
 

N.S.
 

_
 

98
 
TABLE 4 

(CONTINUED) 

POOLED MATCHED LOW TOPOGRAPHY: MORPHOLOGY CONTROL CONTROL DOSE 

Thyroid: Follicular-Cell Adenomab 4/108(0.04) 1/19(0.05) 2/49(0.04) 

 Values0 

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

Relative Risk (Pooled Control)d 1.102 

Lower Limit 0.103 

Upper Limit 7.363 

Relative Risk (Matched Control)d 0.776 

Lower Limit 0.044 

Upper Limit 44.838 

Weeks to First Observed Tumor Ill 103 

Thyroid: Follicular-Cell Adenoma or Carcinomab 5/108(0.05) 2/19(0.11) 3/49(0.06) 

 Values0 N.S. N.S. N.S. 

Relative Risk (Pooled Control)d 1.322 

Lower Limit 0.211 

Upper Limit 6.470 

Relative Risk (Matched Control ) d 0.582 

Lower Limit 0.074 

Upper Limit 6.640 

Weeks to First Observed Tumor Ill 103 

Thyroid: C-Cell Adenoma or Carcinoma1* 6/108(0.06) 0/19(0.00) 3/49(0.06) 

 Values0 N.S. N.S. N.S. 

Relative Risk (Pooled Control)d 1.102 

Lower Limit 0.184 

Upper Limit 4.897 

Relative Risk (Matched Control)d Infinite 

Lower Limit 0.243 

Upper Limit Infinite 

Weeks to First Observed Tumor — — ___ 112 

HIGH
 

DOSE
 

4/50(0.08)
 

N.S.
 

2.160
 

0.418
 

11.071
 

1.520
 

0.168
 

73.309
 

112
 

4/50(0.08)
 

N.S.
 

1.728
 

0.357
 

7.632
 

0.760
 

0.122
 

8.007
 

112
 

2/50(0.04)
 

N.S.
 

0.720
 

0.073
 

3.836
 

Infinite
 

0.117
 

Infinite
 

112
 
TABLE 4 

(CONTINUED) 

POOLED MATCHED 

TOPOGRAPHY :MORPHOLOGY CONTROL CONTROL 

Pancreas: Carcinoma, NOS 0/110(0.00) 0/20(0.00) 

 Values0 P< 0.001 ­ 0.021 

Relative Risk (Pooled Control)d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control) 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

Mammary Gland: Adenocarcinoma, NOS 3/110(0.03) 1/20(0.05) 

 Values0 

 ­ 0.094 N.S. 

Relative Risk (Pooled Control)d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control ) d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 98 

Mammary Gland: Fibroadenoma 32/110(0.29) 7/20(0.35) 

 Values0 N.S. N.S. 

Relative Risk (Pooled Control)d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor Ill 

LOW
 

DOSE
 

2/50(0.04)
 

N.S.
 

Infinite
 

0.640
 

Infinite
 

Infinite
 

0.123
 

Infinite
 

94
 

3/50(0.06)
 

N.S.
 

2.200
 

0.303
 15,792
 

1.200
 

0.105
 

61.724
 

14
 

10/50(0.20)
 

N.S.
 

0.688
 

0.324
 

1.300
 

0.571
 

0.240
 

1.558
 

89
 

HIGH
 

DOSE
 

7/50(0.14)
 

P< 0.001*
 

Infinite
 

4.206
 

Infinite
 

Infinite
 

0.809
 

Infinite
 

70
 

4/50(0.08)
 

N.S.
 

2.933
 

0.509
 

19.228
 

1.600
 

0.175
 

77.169
 

94
 

12/50(0.24)
 

N.S.
 

0.825
 

0.419
 

1.483
 

0.686
 

0.305
 

1.806
 

112
 
TABLE 4 

(CONTINUED) 

POOLED MATCHED 

TOPOGRAPHY :MORPHOLOGY CONTROL CONTROL 

Mammary Gland: Fibroma or Fibroadenoma 32/110(0.29) 7/20(0.35) 

 Values0 

N.S. N.S. 

Relative Risk (Pooled Control)d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)4 _ 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor Ill 

Uterus: Endometrial Stromal Polyp 8/109(0.07) 2/20(0.10) 

 Values0 

N.S. N.S. 

Relative Risk (Pooled Control)0 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor Ill 

Ovary: Granulosa-Cell Tumor 1/109(0.01) 0/20(0.00) 

 Values0 

 ­ 0.015 - 0.038 

Relative Risk (Pooled Control)d 

Lower Limit 

Upper Limit 

Relative Risk (Matched Control)d 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 

LOW
 DOSE
 

11/50(0.22)
 

N.S.
 

0.756
 

0.371
 

1.392
 

0.629
 

0.272
 

1.683
 

89
 

3/50(0.06)
 

N.S.
 

0.834
 

0.147
 

3.222
 

0.612
 

0.077
 

6.860
 

107
 

0/50(0.00)
 

N.S.
 

0.000
 

0.000
 

40,652
 

HIGH
 

DOSE
 

12/50(0.24)
 

N.S.
 

0.825
 

0.427
 

1.493
 

0.686
 

0.305
 

1.806
 

112
 

1/49(0.02)
 

N.S.
 

0.284
 

0.006
 

1.977
 

0.208
 

0.004
 

3.754
 

112
 

4/49(0.08)
 

 - 0.032*
 

8.898
 

0.905
 

428.730
 

Infinite
 

0.394
 

Infinite
 

112
 
TABLE 4
 

(CONCLUDED)
 

POOLED MATCHED LOW HIGH 

TOPOGRAPHY:MORPHOLOGY CONTROL CONTROL DOSE DOSE 

Ovary: Granulosa-Cell Tumor or Carcinoma0 2/109(0.02) 1/20(0.05) 1/50(0.02) 4/49(0.08) 

 Values0 

 = 0.050 N.S. N.S. N.S. 

Relative Risk (Pooled Control) 1.090 4.449 

Lower Limit 0.019 0.658 

Upper Limit 20.319 47.561 

Relative Risk (Matched Control)d 0.400 1.633 

Lower Limit 0.005 0.179 

Upper Limit 30.802 78.704 

Weeks to First Observed Tumor 99 112 112 

Dosed groups received time-weighted average doses of 1300 and 2600 ppm in feed.
 

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

°Beneath the incidence of each of the controls is the probability level for the Cochran-Armitage test for dose-related trend in proportions when it is
 

below 0.Q5, otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath the dose group
 

incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group with the pooled control group (*) and
 

the matched control group (**) when either is below 0.05, otherwise N.S. - not significant.
 

(N) Less incidence in the dose group(s) than in a control group results in a negative indication.
 

Relative Risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence intercal for that
 

relative risk.
 
The incidence of carcinomas of the pancreas was high in female
 

rats· The Cochran-Armitage test indicated a significant positive
 

association between increased dosage and elevated tumor incidence
 

when comparing either the pooled (P < 0,001) or the matched (P =
 

0.021) control. The Fisher exact test confirmed these results with
 

a statistically significant comparison (P < 0·001) of the high dose
 

group to the pooled control· The lower limit of the 95 percent confidence interval on the relative risk of the high dose to the pooled
 

control was greater than the value one·
 

In female rats the Cochran-Armitage test showed a significant
 

positive association between increased dosage and an elevated incidence of lymphomas whether compared to the matched control (P = 0.039)
 

or to the pooled control ( β 0.016). The Fisher exact test uid not
 

confirm this increased incidence of lymphomas as the probability level
 

(P = 0.033) of the comparison of the high dose to the pooled control
 

was not significant under the Bonferroni criterion.
 

The incidence of granulosa-cell tumors of the ovary was also
 

noted in female rats· The Cochran-Armitage test was significant
 

in comparisons involving both the pooled (P s 0·015) and the matched
 

(P = 0.038) controls. The Fisher exact tests did not support these
 

findings, however, when the Bonferroni criterion was applied. When
 

the incidences of ovarian carcinomas were combined with the incidences of ovarian granulosa-cell tumors, again the Fisher exact tests
 

32
 
did not indicate that the relationship between treatment and incidence
 

was statistically significant·
 

Based upon these results, the statistical conclusion is that in
 

female Osborne-Mendel rats the incidences of carcinomas of the pancreas were associated with the administration of nitrofen at the dose
 

levels used in this experiment·
 

In male rats, for C-cell adenomas of the thyroid, the Fisher
 

exact test comparing the high dose to the pooled control was significant (P - 0*031)· The Cochran-Armitage test and the Fisher exact test
 

comparing low dose to control did not confirm this finding· Because
 

of the poor survival in the high dose male rats, an additional analysis was conducted based upon males that survived at least 52 weeks;
 

however, no statistically significant results were found. Because of
 

the lack of supporting results and because only two such tumors were
 

found in the high dose group, there was insufficient statistical
 

evidence to conclude that the tumor incidence was associated with the
 

administration of nitrofen·
 

To provide additional insight, 95 percent confidence intervals
 

on the relative risk have been estimated and entered in the tables
 

based upon the observed tumor incidence rates· In many of the intervals shown in Tables 3 and 4, the value one is included; this indicates the absence of statistically significant results· It should
 

also be noted Inat many of the confidence intervals have an upper
 

limit greater than one, indicating the theoretical possibility of a
 

33
 
significantly increased rate of tumor incidence induced in rats by
 

nitrofen that could not be established under the conditions of this
 

test.
 

34
 
IV. CHRONIC TESTING RESULTS: MICE
 

A· Body Weights and Clinical Observations
 

As indicated in Figure 3, weight gain of high dose male mice was
 

depressed relative to low dose male mice. Mean body weights of male
 

controls were close to those of low dose males but occasionally fell
 

as low as those of high dose males. A dose-related depression in body
 

weight gain was evident for female mice.
 

During the first year of the bioassay, patterns of appearance
 

and behavior were generally comparable for treated and control mice
 

except that body sores and alopecia (usually associated with fighting)
 

were observed as early as week 2 in the treated males. Other clinical
 

signs often observed in group-housed laboratory mice were noted at a
 

comparable rate in control and treated groups· These signs included
 

a hunched appearance, penile, vulvar, or anal irritation, squinted or
 

reddened eyes, palpable nodules, and rough or stained fur.
 

Beginning in week 54 and until termination of the study, bloating or pronounced abdominal distension was displayed by an increasing
 

number of treated mice, particularly in the females. Necropsy of
 

these animals revealed liver tumors that were subsequently diagnosed
 

as hepatocellular carcinomas.
 

B. Survival
 

The estimated probabilities of survival for male and female mice
 

in the control and nitrofen-treated groups are shown in Figure 4.
 

35
 
TIME ON TEST (WEEKS) 

FIGURE 3 

GROWTH CURVES FOR NITROFEN CHRONIC STUDY MICE 

36 
TIME ON TEST (WEEKS) 

FIGURE 4 

SURVIVAL COMPARISONS OF NITROFEN CHRONIC STUDY MICE 

37 
For male mice the Tarone test did not indicate a significant
 

positive association between dosage and mortality· The survival of
 

the control group was unexpectedly poor, as only 10 percent survived
 

until the end of the test compared to 34 percent of the high dose and
 

54 percent of the low dose male mice· Sixty percent of the control
 

mice were dead by week 67, leaving only eight animals at risk from
 

late-developing tumors· No common cause for these early deaths could
 

be detected, but 11 of the 12 had amyloidosis at multiple sites and
 

chronic inflammation of the kidney·
 

For female mi.ce, again, the Tarone test did not indicate a significant positive association between dosage and mortality· Survival
 

was adequate in all groups as 62 percent of the high dose, 54 percent
 

of the low dose, and 85 percent of the control female mice survived
 

until the end of the study·
 

C· Pathology
 

Histopathologic findings on neoplasms in mice are tabulated in
 

Appendix Β (Tables Bl and B2); findings on nonneoplastic lesions are
 

tabulated in Appendix D (Tables Dl and D2)·
 

Long-term dietary intake of nitrofen was associated with a high
 

incidence of hepatocellular carcinoma in both sexes and at all dose
 

levels· This liver tumor occurred in 4/20 (20 percent) control males,
 

36/49 (73 percent) low dose males, 46/48 (96 percent) high dose males,
 

36/41 (88 percent) low dose females, and 43/44 (98 percent) high dose
 

females· These were primarily confined to the liver, but a few
 

38
 
metastasized· The hepatocellular carcinomas were characterized
 

microscopically by proliferating eosinophilic or basophilic swollen
 

hepatocytes forming liver plates usually one or more cells in thickness and by compressed adjacent parenchyma· Although most tumors
 

were relatively well-differentiated, occasional anaplastic lesions
 

were composed of intensely basophilic staining hepatocytes forming
 

pseudo-acini and thick blunted liver plates.
 

Hemangiosarcoma of the liver occurred in 1/49 (2 percent) low
 

dose males, 2/48 (4 percent) high dose males, and 4/44 (9 percent)
 

high dose females. Also considered spontaneous were subcutaneous
 

fibrosarcomas which were Observed in 8/44 (18 percent) low dose males
 

but not in high dose males, matched controls or treated females·
 

In the urinary bladder, 2/40 (5 percent) high dose males had
 

transitional-cell carcinomas, while 1/41 (2 percent) high dose
 

females had a transitional-cell papilloma. These are normally very
 

infrequent tumors in the urinary bladder of this strain. However,
 

the low incidence of these lesions does not provide adequate evidence for a carcinogenic effect.
 

The increased incidence of hepatocellular carcinomas in all
 

groups of mice receiving nitrofen in the diet provides histopathologic evidence of carcinogenicity in B6C3F1 mice of both sexes.
 

D. Statistical Analyses of Results
 

The results of the statistical analyses of tumor incidence in
 

mice are summarized in Tables 5 and 6. The analysis for every type
 

39
 
___ 

TOPOGRAPHY:MORPHOLOGY
 

Subcutaneous Tissue: Fibroma
 

 ValuesC
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)4
 

Lower Limit
 

upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Subcutaneous Tissue: Fibrosarcoma
 

F Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)*1
 

Lower Limit
 

upper Limit
 

Weeks to First Observed Tumor
 

Subcutaneous Tissue: Fibroma or Fibrosarcoma
 

F Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)*
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN MALE MICE TREATED WITH NITROFEN*
 

POOLED MATCHED
 

CONTROL CONTROL
 

0/74(0.00)	 0/20(0.00)
 

N.S. N.S.
 

 - 0.020
 

3/74(0.04)	 0/20(0.00)
 

N.S. N.S.
 

p<0.001 p<0.001
 

— .
 

3/74(0.04)	 0/20(0.00)
 

N.S.	 N.S.
 

p<0.001	 p<0.001
 

_
 

- —
 —
 

—
 

LOW
 

DOSE
 

2/44(0.05)
 

N.S.
 

Infinite
 

0.494
 

Infinite
 

Infinite
 

0.140
 

Infinite
 

92
 

8/44(0.18)
 

 - 0.040**
 « 0.014*
 

4.485
 

1.142
 

24.852
 

Infinite
 

1.086
 

Infinite
 

75
 

10/44(0.23)
 

 - 0.016**
 

 - 0.003*
 

5.606
 

1.536
 

29.925
 

Infinite
 

1.412
 

Infinite
 

75
 

HIGH
 

DOSE
 

0/46(0.00)
 

N.S.
 

— _
 

_ —
 

0/46(0.00)
 

N.S.
 

0.000
 

0.000
 

2.673
 

0/46(0.00)
 

N.S.
 

0.000
 

0.000
 

2.673
 

---·
 
TOPOGRAPH?: MORPHOLOGY
 

Hematopoietic System: Lymphoma^
 

 Valuesc
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma
 

 Valuesc
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)0
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Urinary Bladder: Transitional-Cell Carcinoma
 

 Valuesc
 

Relative Risk (Pooled Control)4
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

TABLE 5 

(CONTINUED) 

POOLED MATCHED 

CONTROL CONTROL 

1/74(0.01) 0/20(0.00) 

N.S. N.S. 

_ 

— — 

9/74(0.12) 4/20(0.20) 

p<0.001 P< 0.001 

 - 0.016 

62 

0/69(0.00) 0/20(0.00) 

 ­ 0.051 N.S. 

LOW
 DOSE
 

1/44(0.02)
 

N.S.
 

1.682
 

0.022
 

129.041
 

Infinite
 

0.025
 

Infinite
 

58
 

36/49(0.73)
 

P< 0.001**
 

P< 0.001*
 

6.041
 

3.285
 

11.623
 

3.673
 

1.616
 

11.735
 

58
 

0/47(0.00)
 

N.S.
 

HIGH
 

DOSE
 

0/46(0.00)
 

N.S.
 

0.000
 

0.000
 

29.957
 

46/48(0.96)
 

t< 0.001**
 

P< 0.001*
 

7.880
 

4.927
 10.091
 

4.792
 

2.360
 

8.369
 

57
 

2/40(0.05)
 

N.S.
 

Infinite
 

0.510
 

Infinite
 

Infinite
 

0.153
 

Infinite
 

92
 
TABLE 5
 

(CONCLUDED)
 

POOLED MATCHED LOW HIGH 

TOPOGRAPHYiMORPHOLOGY CONTROL CONTROL DOSE DOSE 

All Sites: Hemangiosarcoma 0/74(0.00) 0/20(0.00) 1/44(0.02) 4/48(0.08) 

 Values0 = 0.010 ­ 0.074 N.S. - 0.022* 

Relative Risk (Pooled Control)d Infinite Infinite 

Lower Limit 0.090 1.417 

Upper Limit Infinite Infinite 

Relative Risk (Matched Control)d Infinite Infinite 

Lower Limit 0.023 0.402 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor 92 92 

^osed groups received time-weighted average doses of 2348 and 4696 ppm in feed.
 

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

Beneath the incidence of each of the controls is the probability level for the Cochran-Armitage test for dose-related trend in proportions when it is
 

below 0.05, otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath the dose group
 

incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group with the pooled control group (*) and
 

the matched control group (**) when either is below 0.05, otherwise N.S. - not significant.
 

(N) Less incidence in the dose group (s) than in a control group results in a negative indication.
 

Relative Risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence interval for that
 

relative risk.
 
TABLE 6
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT
 

SPECIFIC SITES IN FEMALE MICE TREATED WITH NITROFENa
 

TOPOGRAPHY :MORPHOLOGY
 

Hematopoietic System: Lymphoma1

*
 

 Values0
 

Relative Risk (Pooled Control)
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Liver: Hepatocellular Carcinoma15
 

 Values0
 

Departure from Linear Trend
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

b
 All Sites: Hemangiosarcoma
 

 Values0
 

Relative Risk (Pooled Control)d
 

Lower Limit
 

Upper Limit
 

Relative Risk (Matched Control)d
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

POOLED
 

CONTROL
 

11/80(0.14)
 

 - 0.056(N)
 

0/80(0.00)
 

p<0.001
 

p<0.001
 

—
 

-,—
 

—
 

—
 

—
 

—
 

2/80(0.03)
 

 - 0.032
 

MATCHED
 

CONTROL
 

2/20(0.10)
 

N.S.
 

92
 

0/19(0.00)
 

p<0.001
 

P< 0.001
 

1/18(0.06)
 

N.S.
 

—
 

87
 

LOW
 

DOSE
 

3/40(0.08)
 

N.S.
 

0.545
 

0.102
 

1.913
 

0.750
 

0.095
 

8.508
 

91
 

36/41(0.88)
 

P < 0.001**
 

P < 0.001*
 

Infinite
 

24.549
 

Infinite
 

Infinite
 

6.116
 

Infinite
 

62
 

0/41(0.00)
 

N.S.
 

0.000
 

0.000
 

6.570
 

0.000
 

0.000
 

8.171
 

HIGH
 

DOSE
 

2/45(0.05)
 

N.S.
 

0.323
 

0.036
 

1.390
 

0.444
 

0.035
 

5.844
 

81
 

43/44(0.98)
 

P < 0.001**
 

P < 0.001*
 

Infinite
 

30.137
 

Infinite
 

Infinite
 

7.434
 

Infinite
 

64
 

5/44(0.11)
 

N.S.
 

4.545
 

0.777
 

45.880
 

2.045
 

0.258
 

94.395
 

87
 

^osed groups received time-weighted average doses of 2348 and 4696 ppm in feed.
 

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

c

Beneath the incidence of each of the controls is the probability level for the Cochran-Armitage test for dose-related tiend in proportions when
 

it is below 0.05, otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath the
 

dose group incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group with the pooled control
 

group (*) and the matched oontrol group (**) when.either is below 0.05, otherwise N.S. - not significant.
 

(N) Less incidence in the dose group (s) than in a control group results in a negative indication.
 

Relative Risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence interval for that
 

relative risk.
 
of tumor that was observed in more than 5 percent of any of the
 

nitrofen-dosed groups of either sex is included·
 

Two control groups were used for statistical analyses: the
 

control group originally assigned to the nitrofen bioassay in the
 

experimental design (designated in this section as the "matched11
 

control group) and a pooled control group that combined the controls from the studies of nitrofen, chlorobenzilate, endosulfan, and
 

mexacarbate· Each of the control groups had 20 mice· The control
 

mice used for the pool were of the same strain, were housed in the
 

same room, were tested concurrently for at least one year, and were
 

diagnosed by the same pathologists·
 

A high incidence of hepatocellular carcinoma was observed in
 

dosed mice. For both sexes the Cochran-Armitage test indicated a
 

highly significant (P < 0.001) positive association between dosage
 

and tumor incidence when compared with either the matched controls or
 

the pooled controls. The departure from linear trend was significant
 

because of the extremely high incidence in both high and low dose
 

groups. For both sexes, both the high dose and low dose groups had a
 

significantly (P < 0.001) higher incidence of hepatocellular carcinomas than either of the controls. In all cases the lower limit of
 

the confidence interval on the relative risk was greater than the
 

value one. Finally, in the historical controls on B6C3F1 mice (compiled to date for this specific laboratory by the NCI Bioassay Program) hepatocellular carcinomas were found in 13/180 (7 percent) of
 

44
 
the males and 4/180 (2 percent) of the females, substantially lower
 

rates than observed in the dosed mice.
 

Based upon these results the statistical conclusion is that
 

nitrofen had a carcinogenic effect on the livers of B6C3F1 mice at
 

the dose levels of this experiment·
 

Hemangiosarcomas were also noted in both male and female mice.
 

The Cochran-Armitage test showed a significant positive association
 

between increased dose and elevated tumor incidence for both males
 

(P = 0.010) and females (P = 0.032) when compared to the pooled
 

controls. The Fisher exact test showed a significantly (P = 0.022)
 

higher incidence in the high dose than in the pooled control for
 

males, but for females the results were not significant.
 

Based on these results the statistical conclusion is that the
 

incidence of hemangiosarcomas in male B6C3F1 mice was associated with
 

the administration of nitrofen at the dose levels of this experiment.
 

In low dose male mice the Fisher exact tests showed a significantly higher incidence of either fibromas or fibrosarcomas of the
 

subcutaneous tissue than in the pooled control (P = 0.014); the
 

comparison of matched control to low dose was not significant under
 

the Bonferroni criterion. It was questionable, however, whether this
 

tumor was dose-related since none of these tumors were observed in
 

the high dose group.
 

45
 
V. DISCUSSION
 

Under the conditions of this study the administration of nitro-


fen was associated with a highly significant incidence of hepatocellular carcinomas and an elevated incidence of hemangiosarcoma of the
 

liver in mice of both sexes. In female rats, nitrofen was associated
 

with an increased incidence of carcinomas of the pancreas, and various
 

tumors of the reproductive system· Because of an accelerated early
 

mortality, the number of male rats at risk from late-developing tumors
 

was inadequate for meaningful statistical analyses·
 

In mice of both sexes, the incidence of hepatocellular carcinoma
 

showed a highly significant positive association with concentration
 

of nitrofen in the diet· The incidences of hepatocellular carcinomas
 

for each dosed group were highly significant when compared to either
 

control group. A statistically significant positive association
 

between dosage and incidence of hemangiosarcomas of the liver was
 

observed in mice of both sexes· The incidence in the high dose male
 

mice, but not female or low dose male mice, was significantly higher
 

than controls.
 

The incidence of carcinomas of the pancreas had a statistically
 

significant positive association with concentration of nitrofen in
 

diets of female rats· The high dose group had a statistically significant incidence of carcinoma of the pancreas when compared to the
 

pooled controls. The fact that this is an unusual neoplasm in the
 

46
 
Osborne-Mendel rat is further indication that occurrence of this
 

tumor is related to administration of nitrofen·
 

The results of this study indicate that nitrofen is a liver
 

carcinogen, causing hepatocellular carcinomas in B6C3F1 mice of both
 

sexes and hemangiosarcoma of the liver in male mice. In addition,
 

the compound is carcinogenic to female Osborne-Mendel rats, causing
 

an increased incidence of pancreatic carcinomas· Survival of the
 

high dose male rats was not adequate for meaningful statistical analysis of tumor incidence.
 

47
 
VI. BIBLIOGRAPHY
 

Armitage, P., Statistical Methods in Medical Research, Chapter 14.
 

J. Wiley & Sons, New York, 1971.
 

Berenblum, I·, editor, Carcinogenicity Testing. International Union
 

Against Cancer, Technical Report Series, Vol. 2. International
 

Union Against Cancer, Geneva, 1969.
 

Chemical Abstracts Service, The Chemical Abstracts Service (CAS) Ninth
 

Collective Index, Volumes 76-85, 1972-1976. American Chemical
 

Society, Washington, D.C., 1977.
 

Cox, D.R., Analysis of Binary Data, Chapters 4 and 5. Methuen and
 

Co., Ltd., London, 1970.
 

Cox, D.R., "Regression Models and Life-Tables.11

 Journal of the Royal
 

Statistical Society, Series "B" 34:187-220, 1972.
 

Doroshenko, G.V., "Hygenic Characteristics of Working Conditions and
 

Health Status of Persons Handling a Complex of Pesticides in
 

Gardening." Gigiena Truda I Professional-nye Zabolevaniya
 

2_:12~14, 1975.
 

Environmental Protection Agency, Compendium of Registered Pesticides,
 

Vol. I supplement. June 30, 1975.
 

Gart, J.J., "The Comparison of Proportion: A Review of Significance
 

Tests, Confidence Limits, and Adjustments for Stratification."
 

International Statistical Institute Review 39:148-169, 1971.
 

Kaplan, E.L., and P. Meier, "Nonparametric Estimation from Incomplete
 

Observation." Journal of the American Statistical Association
 

5^:457-481, 1958.
 

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

"Carcinogenesis Bioassay Data System." Computers and Biomedical
 

Research 7:230-248, 1974.
 

Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book
 

Co., New York, 1966.
 

Reuber, M.D., and E.L. Glover, "Cirrhosis and Carcinoma of the Liver
 

in Male Rats Given Subcutaneous Carbon Tetrachloride." Journal
 

of the National Cancer Institute 44:419-423, 1970.
 

48
 
Saffiotti, U., R. Montesano, A.R. Sellakumar, F. Cefix, and D.G.
 

Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced
 

by Different Numbers of Administration of Benzo (a) Pyrene and
 

Ferric Oxide·11 Cancer Research 32:1073-1079, 1972.
 

Stanford Research Institute, 1975 Directory of Chemical Producers,
 U.S.A. Menlo Park California, 1975.
 

Tarone, R.E., "Tests for Trend in Life-Table Analysis." Biometrika
 

£2:679-682, 1975.
 

Weed Science Society of America, "Nitrofen." Herbicide Handbook.
 Champaign, Illinois, 1974.
 

49
 

APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN RATS TREATED WITH NITROFEN
 

TABLE Al

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH NITROFEN 

HIGH DOSE
 01-M062
 

50
 

50
 

50
 

(50)
 

(50)
 

1 (2%)
 

(SO)
 

(50)
 

(50)
 

ANIMALS INITIAL!.! I» STUD!
 

ANIMALS NECR0PS1SD
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

IaTEGUMuNIARY SYSTEM
 

•SKIN
 

PAPILLOMA, NOS
 

•SUBCUT TISSUE
 

FIBROMA
 

FIBROSARCOMA
 

FIBROUS HISTIOCYTOMA, MALIGNANT
 

LIPOMA
 

RESPIRATORY SYSTEfl
 

• LUNG
 

ADENOCARCINOMA, NOS, METASTATIC
 

CORTICAL CARCINOMA, METASTATIC
 

FIBROUS HISTIOCYTOMA, METASTATIC
 

MIXED TOMOR, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

•SPLEEN
 

FIBROSARCOMA, METASTATIC
 

FIBROUS HISTIOCYTOMA, METASTATIC
 

HEMANGIOMA
 

HEMANGIOSARCOMA
 

CIRCULATORY bYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

«LIVER
 

FIBROUS HISTIOCYTOMA, METASTATIC
 

CONTROL(VEH) LOW DOSE
 01-M060 01-H061
 

20 50
 

20 50
 

** 20 50
 

(20)	 (50)
 

2 (4%)
 

(20)	 (50)
 

1 (S%) 2 (4%)
 2 (10%) 1 (2%)
 

1 (2%)
 2 (4%)
 

(20)	 (50)
 

1 (2%)
 

1 (2%)
 1 (2%)
 

1	 (5%)
 

(20)	 (50)
 

1 (2%)
 1 (2%)
 

1 (5%)
 2 (10%) 2 (4%)
 

(20)	 (50)
 

1 (2%)
 

# NUMBER OF AAISAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

A-3 
TABLE Al (CONTINUED)
 

HIGH DOSE
 

01-M062
 

(**8)
 

(49)
 

(50)
 

(48)
 

(44)
 

1 (2*)
 

(47)
 

2 (4*)
 

(50)
 

(49)
 

1 (2*)
 

«PANCREAS
 

FIBROSARCOMA, METASTATIC
 

«COLOA
 

ADENOMATOUS POLYP, NOb
 

URINARY SISTER
 

•KIDNEY
 

CORTICAL CARCINOMA, METASTATIC
 

MIXED TUHOR, MALIGNANT
 

«URINARY BLADDER
 

TRANSITIONAL-CELL CARCINOMA
 

FIBROUS HISTIOCYTOMA, METASTATIC
 

ENDOCRINE SYSTEM
 

«PITUITARY
 

CHROMOPHOBE ADENOMA
 

«ADRENAL
 

CORTICAL CARCINOMA
 

MIXtfD TUMOR, METASTATIC
 

•THYROID
 

FOLLICULAR-CELL ADENOMA
 

FOLLICULAR-CELL CARCINOMA
 

C-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

«MAMMARY GLAND
 

ADENOCARCINOMA, NOS
 FIBROADENOMA
 

«TESTIS
 

INTERSTITIAL-CELL TUMOh
 

NERVOUS SYSTEM
 

NONE
 

CONTROL(VEH)
 

01-M060
 

(18)
 

(20)
 

(20)
 

1 (5*)
 

(20)
 

(20)
 

2 (10*)
 

(20)
 

1 (5*)
 

(20)
 

1 (5*)
 

1 (**>
 

(20)
 

1 (5*>
 

(20)
 

LOW DOSE
 

01-M061
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

1 (2*)
 2 (4*)
 

(49)
 

1 (2%)
 

1 (2%)
 

(46)
 

9 (20%)
 

(50)
 

1 (2*)
 

(50)
 

7 (14*)
 

1 (2*)
 

1 (2%)
 

(50)
 

2 (4*)
 

2 (4*)
 

(50)
 

2 (4*)
 

t NUMBER OF ANIMALS WlTrf TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

A-4
 
TABLE Al (CONTINUED)
 

CONTROL (VEH) LOW DOSE HIGH DOSE
 01-H060 01-M061 01-M062
 

SPECIAL SENSE ORGANS
 

NOSE
 

MUSCULOSKELETAL SYSTEIi
 

NONE
 

BODY CAVITIES
 

•MEDIASTINUM	 (20) (50) (50)
 

FIBROSARCOMA, METASTATIC 1 (2*)
 

*ABDOHINAL CAVITY (20) (50) (50)
 

FIBROSARCOMA 1 (2%)
 

^MESENTERY (20) (50) (50)
 

FIBROSARCOMA, METASTATIC 1 (2*)
 

ALL OTHER SYSTEMS
 

nous
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STJI>Y 20 50 50
 

NATURAL DEATH* 10 19 35
 

MORIBUND SACRIFICE 1 1
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE 9 30 15
 

ANIftAL MISSING
 

 INCLUDES AUTOLYZED AHIHALS
 

# NUMBER OF ANIMALS WITii TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NEChOPSIED
 

A-5
 
TABLE Al (CONCLUDED) 

CONTROL(7EH) LOW DOSB HIGH DOSE
 01-H060 01-H061 0t-H062
 

TUMOR SUMMARY
 

TOTAL AS1BALS WITH PRIMARY TUMORS* 10 30 n
 lOfAL PRIMARY TUSORS 12 40 5
 

TOTAL ANIMALS WITH BBRItiS TUMOBS 5 23 4
 TOTAL BENIGN THROBS 5 28 5
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 7 11
 TOTAL MALIGNANT TUHOBS ? 12
 

TOTAL ANIMALS WITH SECONDABY TUMORS» 1 α
 TOTAL SECONDARY TtTHOBS 2 11
 

TOTAL ANIMALS WITH TUflORS UHCURTAINB£NIb9 OR «ALIGNAST
 

TOTAL UNCiiBTAlii TUSOBS
 

TOTAL ANIMALS WITH TUMORS UNCERTAIN-


PBIttARr OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

* ΡΚΙΛΑβ TUMORS: ALL TUflORS EXCEPT SBCONDAItZ TUMORS
 

» SECONDARY PUSORS: METASTATIC TUMORS OB TUHOBS INVASIVE ΙΚTO AN ADJACENT OBGAN
 

A-6
 
TABLE A2

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH NITROFEN 

HIGH DOSE
 01-FG64
 

50
 

50
 

50
 

(50)
 

1 (2%)
 

1 (2%)
 

(50)
 

(50)
 

7 (14%)
 

1 (2%)
 

(50)
 

3 (b%)
 

(50)
 

a (8%)
 

1 (2%)
 

(19)
 

1 (2%)
 

ANIMALS INITIALLY IN STUDY
 

ANIMALS HECR0PS1ED
 

ANIMALb EXAMINED HISTOPATHOLOGICALLY
 

INTEGUMENTARY SYSTBB
 

*SKIN
 

SQUAMOUS CBLL CARCISOSA
 

KERATOACANTHOMA
 

*SUBCUT TISSUE
 

FIBROSA
 

RESPIRATORY SYSTEM
 

#LUNG
 

CARCINOMA, NOS, METASTATIC
 

SQUAMOUS CELL CARCINOMA, BETASTA
 

ADENOCARCINOMA, NOS, METASTATIC
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS
 

MALIG.LYMPHOMA, HISTIOCYTIC TYPE
 

*SPLEEN
 

CARCINOMA, NOS, METASTATIC
 

SQUAMOUS CELL CARCINOMA, METASTA
 

GRANULOSA-CELL CARCINOMA, METAST
 HEMANGIOSARCOSA
 

#UTERUS
 

MALIG.LYMPHOMA, LYMPHOCYTIC TYPE
 

CIRCULATORY SYSTEM
 

NONE
 

CONTROL(VEH)
 01-F060
 

20
 

20
 

** 20
 

(20)
 

(20)
 

2 (10%)
 

(20)
 

(20)
 

(20)
 

1 (5%)
 

(20)
 

A-7 

LOR DOSE
 01-F06J
 

50
 

50
 

50
 

(50)
 

(50)
 

(SO)
 

2 (4%)
 

1 {2%)
 

(50)
 

(50)
 

1 (2*)
 1 (2%)
 

(50)
 

# NUMBER OF ANIMALS WITH TISSU E EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECFOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 
TABLE A2 (CONTINUED) 

CONTROL (VEd) LO» DOSE
 01-FOoO 01-P06^
 

DIGESTIVE SYSTEM
 

«LIVEh (20) (50)
 

CARCINOMA, NOS, METASTATIC 2 (4%)
 

•PANCREAS	 (20) (50)
 

CARCINOMA,NOS 2 (4%)
 

SQUAMOUS CELL CARCINOMA, ME1ASTA 1 (2%)
 

•ESOPHAGUS	 (20) (50)
 

SQUAMOUS CELL CARCINOMA, METASTA 1 (2%)
 

«STOMACH (20) (49)
 

CARCINOMA, aOb, METASTATIC 2 (4%)
 

SQUAMOUS CELL CARCINOMA, MEI'ASTA 1 (2%)
 

•SMALL INTESTINE	 (19) (50)
 

CARCINOMA, NOS, METASTATIC
 

S^UASOUS CELL CARCINOMA, METASTA 1 (2%)
 

•LARGE INTESTINE	 (20) (49)
 

LARCIdQMA, NOS, METASTATIC
 

URINARY SYSTEM
 

•KIDNEY	 (20) (50)
 

CARCINOMA, NOS, METASTATIC 1 (2%)
 

TUBULAR-CELL ADENOCARCINOMA
 

MIXED TUMOK, MALIGNANT ib%)
 

HAMARTOMA
 

«UfilNAhY dLADDEft (18)
 

CARCIuUMA, nOS, METASTATIC 1 (2%)
 

EHDOHETRIAL STROMAL SARCOMA, SSI 1 (2%)
 

ESDOCRI2IE SYSTEM
 

•PITUITARY	 (18) (49)
 

CHROMOPriObL ADENOMA 3 (17%) 15 (31%)
 

«ADRENAL (^0) (50)
 

CAriCIaOMA, aOb, METASTATIC
 

PtlEOCHKOMOCiTOMA
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NKCROPSIED
 

A-8
 

HIGH DOSE
 01-F004
 

(50)
 

3 (o%)
 

(50)
 

7 (14%)
 

(39)
 

(50)
 

7 (14%)
 

(49)
 

5 (10%)
 

(49)
 

3 (6%)
 

(50)
 

2 (4%y
 

1 (2%)
 

1 {2%)
 

1 (2%)
 

(49)
 

2 (4*)
 

(48)
 

18 (3b*)
 

(49)
 

2 (4%)
 

1 (2%)
 
HIGH DOSS
 

01-F064
 

(50)
 

U (8%)
 

1 (2*)
 

1 (2%)
 

(50)
 

(50)
 

4 (8%)
 

12 (24*)
 

(50)
 

 ah
 

(49)
 

2 (4*)
 

1 (2%)
 1 (2*)
 

(49)
 

2 f4*)
 

4 (s£y
 

(50)
 

«THYROID
 FOLLICULAR-CBLL ADENOMA
 

FOLLICULAR-CELL CARCINOMA
 

C-C2LL AuSuOflA
 C-CEJUL CARCINOMA
 

«PANCREATIC ISLETS
 ISLSr-CELL ADENOMA
 

REPRODUCTIVE SYSTEM
 

•MAMMARY GLAND
 

ADENOCARCINOMA, SOS
 

PlbitOiJA
 

FIBROADENOMA
 

«VAGINA
 CARCINOMA, MOS, METASTATIC
 

SgUASOUS CELL CARClUOHA
 

«UTERUS
 

CARCliiiOnA, bOS.ttETA STATIC
 SyuASOUS CELL CASCIHQftA
 

ADENOCARCINOMA, «OS
 ENDOMETRIAL STROMAL POLYP
 

EbUOnsrRIAL STROMAL SARCOMA
 

«OVARY
 

CARCINOMA, ΝOS, METASTATIC
 

SU.UAUOUS CELL CARClNOilA, METASTA
 

CXSTADENUCARCINOMA, NOS
 

GRANOLOSA-CELL TUMOR
 

GRANULOSA-CELL CARCIHOllA
 

NERVOUS SYSTEtl
 

UOhL·
 

SPECIAL SENSE ORGANS
 

*EΥΕ
 

SQUAMOUS CELL PAPILLOMA
 

t NUflBEK OF ANIMALS WITH TISSUE

* ttOAb£R OF AjIrtALS uECROPSIED 

TABLE A2 (CONTINUED) 

CONTROL(VEH)
 01-F060
 

(19)
 

1 (5%)
 

1 (5%)
 

(20)
 

(20)
 

1 (5*)
 

f (35*)
 

(20)
 

(20)
 

2 (10*)
 

(20)
 

y (**)
 

(20)
 

 EXARINED BICROSCOPICALLY 

A-9 

LOW DOSE
 

01-F063
 

(<*9)
 

2 (4%)
 

1 (2*)
 2 (4*)
 

1 (2%)
 

(50)
 

1 (2%)
 

(50)
 

3 (6%)
 

1 (21)
 10 (20*)
 

(50)
 

(50)
 

1 (2%)
 

1 (2%)
 3 (6*)
 

1 (2%)
 

(50)
 

1 (2*)
 

1 (2*)
 

1 (2%)
 

1 (2%)
 

(50)
 

1 (2*)
 
MUSCULOSKELETAL SYSTEM
 

•SKELETAL	 MUSCLE
 

SQUAMOUS CELL CARCINOMA,


bOJY CAVITIES
 

•AbDOMINAL	 CAVITY
 

HE»ANGIOSARCOMA
 

•MESENTERY
 

CARCINOMA, NOS, METASTATIC
 

ALL OTflSR SYSTEMS
 

NONL·
 

ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATHS
 

MORIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TfchMINAL SACRIFICE
 

AJtIMAL MlbSIHG
 

 INCLUDES AUPOLYZBD AiilBALS
 

TABLE A2 (CONTINUED) 

 METASTA
 

CONTROL(VSH)
 01-F060
 

(20)
 

(^0)
 

(20)
 

20
 

4
 

lb
 

LOW DOSE
 01-F063
 

(50)
 

(50)
 

(50)
 

1 (2%)
 

50
 

12
 

1
 

37
 

HIGH DOSE
 01-F064
 

(50)
 

1 (2%)
 

(50)
 

1 (2*)
 

(50)
 

2 (4%)
 

50
 

22
 

28
 

# NUMbEK OF ANIMALS KITH TISSUE EXAMINED MICROSCOPICALLY
 

• SUMtihK OF ANIMALS NECROPSIED
 

Α-IO
 
TABLE A2 (CONCLUDED)
 

TUBOR SUMMARY
 

TOTAL ASIBALS WITH PRIBABY TUBORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIGN TUMORS
 

TOlAL BENIGN TUMORS
 

TOTAL ANIMALS WITH BAL1GNANT TUBORS
 

TOTAL MALIGuAiiT TUMORS
 

CO8TROL(f£H)
 01-F060
 

14
 

19
 

13
 

15
 

4
 

4
 

TOTAL ANIMALS WITH SECONDARE TUHORSi 1
 

TOTAL SECONDARY TUBORS 1
 

TOTAL ANIMALS WITH TUBORS UNCERTAINBLNIGH OR ftALIGNANT
 

TOTAL UNCBRTAIi* TUMORS
 

TOTAL ANIMALS WITH TUBORS UNCERTAINPRIBAR? OR METASTATIC
 

TOTAL UNCERTAIN TUftORS
 

LOW DOSE HIGH DOSE 01-F06J Q1-F064 

31 42 

47 66 

26 25 

35 39 

10 20 

12 23 

5 8 

19 44 

4 

4 

• PRIftAR* TUMORS: ALL TUMORS EXCEPT SECONDARY TOMORS
 

# SECONDARY TUSORS; METASTATIC TUftORS OR TUMORS IHVASIVB INTO AN ADJACENT ORGAH
 

Α-II
 

APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS
 

IN MICE TREATED WITH NITROFEN
 

TABLE Bl

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH NITROFEN 

HIGH DOSE
 

02-B069
 

bO
 

1
 

46
 

46
 

(46)
 

(46)
 

(46)
 

1 (2%)
 

1 (2%)
 

(46)
 

(47)
 

2 (4%)
 

(48)
 

46 J96*l
 

ANIMALS INITIALLY In STUD!
 

ANIMALS SISSIHG
 

AM13ALS aECROPSIED
 ANIMALS EXAMINED HISTOPATHOLOGICALLY
 

IflTutoUMfiHTARY SYSTEfl
 

•SKI»
 

LYMPHANGIOflA
 

•SOBCUT TISSUE
 

FIBROSA
 

FIBROSARCOMA
 

HESANGIOPBRICYTOBA, NOS
 

h£UROFIBROMA
 

INSPIRATORY SYSTEM
 

• LUNG
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALVUuLAR/BRONCHlOLAR ADENOMA
 

HEMATOPOIETIC SYSTLH
 

•MULTIPLE	 ORGAHS
 

ulALIi,.LiftPHOeA, HISTIOCIllC 1YPE
 

fSPLEEN
 

H£ftANGIOSARCOBA
 

CliiCULATORY SJSTEfl
 

NONf,
 

DIGESTIVE SYSTEM
 

«LIVER
 

HEPATOCELLULAR CARCINOMA
 

« »UBbEK O¥ ASIfiALS WITH TISSUE

• i»UMur.fi OF ANIMALS ASCROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

COATROL(?EH)
 

02-B06?
 

20
 

** 2 0
 

** 20
 

(20)
 

1 (5%)
 

(20)
 

(20)
 

1 (5*)
 

(20)
 

(20)
 

(20)
 

4 (20*)
 

LOW DOSE
 

02-H068
 

bO
 

44
 

44
 

(44)
 

(44)
 

2 (5%)
 

8 (18%)
 

1 (2%)
 

1 (2%)
 

(48)
 

t (2%)
 

1 (2%)
 

(49)
 

(49)
 

36 J?3*l^
 

 hXAHlKED MICROSCOPICALLY 

B-3 
TABLE Bl (CONTINUED) 

CONTROL(VEH) 02-B067 LOU DOSE 02-B068 

HEfJAHGIOSARCOBA 1 (2*) 

•blLE bUCl (20) 

BILE DUCT CAHC1HOBA 1 (2%) 

URINARY SYSI'B!!
 

•URINARY	 BLADDER (20) (47)
 

TttANSlTiONAL-CSLL CAfiCINOBA
 

LHDOCfilNi. SYSTEB
 

NONE
 

fitfJfhODUCTIVB SYSTEM
 

#PROSTATE (18) (42)
 

PAPILLOMA, JtfOS 1 (2%)
 

NtRVOUS SYSI£R
 

SOMi!
 

SPECIAL SENSE ORGANS
 

MONL
 

aascULOSKELETAL SYSTEB
 

NONt
 

BODY CAViXI£S
 

NONE
 

ALL OTHER SYSTEBS
 

NOKK
 

OP ANIMALS WITH TISSUE BXAflINED ftICROSCOPICALLY 

NUMBER UP A^ilAALS AiECftOPSlED 

B-4 

HIGH DOSE
 02-B069
 

2 (4%)
 

(46)
 

(40)
 

2 (5%)
 

(28)
 
TABLE Bl (CONCLUDED)
 

CONTROL (VEH)
 

AalMAL DISPOSITIOI SUMMARX
 

ANIMALS INITIALLY IH STUDY
 NATURAL DEATH*
 

MORIBUND SACRIFICE
 

SCHEDDLED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

0 INCLUDES ADTOLYZED AMBALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS*
 

TOTAL PRIMARY TUMORS
 

TOTAL ANIMALS WITH BENIbN TUMORS
 

I'OTAL tiENIGN rUBORS
 

TOTAL ANIMALS WITH MALIGNANT TUMORS
 

TOTAL MALIGNANT TUMORS
 

TOTAL ANIMALS WITH SECONDARY TUMORSt
 

1OTAL SECONDARY TUHORb
 

TOTAL ANIMALS WITH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

1OTAL UNCERTAIN TUMORS
 

TOTAL ABIMALS WITH TUMORS UNCERTAIN-


PRxSARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

02-M067
 

20
 

Iβ
 

2
 

b
 

6
 

2
 

2
 

4
 

LOW DOSE HlbU DOSS
 

02-M068 02-M069
 

&0 50
 22 32
 

1
 

27 17
 

1
 

38 46
 

53 53
 

5 t
 b 1
 

38 46
 47 52
 

1
 

1
 

1
 

1
 

* PRIMARY TUMORS: ALL TUMURS EXCEPT SECONDARY TUMORS
 

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

B-5
 
TABLE B2 

THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH NITROFEN 

CONTROL(VEd) LOW DOSS
 02-P0t>7 02-F070
 

AtflMALS INITIALLY IK STUDY 20 50
 

AN1XALS 3ISS1NG 1
 

ASIMALb SECROPSIED 39
 ** 2 0
 

ANIMALS EXAMINED HISTOPATHOLOG1CALLY ** 20 39
 

INTEGOSEHTARY SYSTEM
 

NUN iS
 

INSPIRATORY SYST2M 

•LUNb 

HEPATOCELLULAR CARCIN

ALVKOLAR/bRONCHlOLAR

OMA, METAST 

 ADENOMA 

(20) (38) 

1 (3*) 

HEMATOPOIETIC SYSTEM
 

•MULTIPLE	 ORGANS (20) (39)
 

MALlG.LYMPHOMA, LYMPHOCYTIC TYPE 1 (5%) 2 (5%)
 

MAL1G.LYMPHOMA, HISTIOCYTIC TYPE 1 (5%)
 

tSPLEED (Iβ) (39)
 

HEWA^GIOSARCOMA 1 (<>*)
 MALIG.LYMPHOMA, HISTIOCYTIC TYPE t (3%)
 

frtfcSJSMTLRIC L. MODE (20) (40)
 

MALlG.LYMPttOAA, HISTIOCYTIC TYPE
 

CIKCULATORY SYSTEM
 

»O*£
 

DIGESTIVE SYSTEM
 

• LlVr,R	 (19)
 

HEPATOCELLULAR CARC1HOHA 36 (86%)
 hLttANGIOSARCOHA
 

* NUMBSH OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* hUSa£tv OF ANIMALS NhCEOPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

B-6
 

SIGH DOSE
 02-P071
 

50
 

2
 

43
 

43
 

(<•*>
 

2 (4*)
 

(*3)
 

1 (2%)
 

(43)
 

(45)
 

1 (2*)
 

(**»>
 43 (98%)
 

4 I9%1
 
TABLE B2 (CONTINUED) 

UK1NARY SfSTfiH
 

9UKINAR* BLADDER
 

TfiAN^ITIONAL-CELL PAPILLOMA
 

ENDOCKINE SYSTEIS
 

#TH5fR01D
 

FOLL1C0LAR-CELL CARCIliOMA
 

KEPKO0UCT1VE SJfSTEfl
 

*QVhki
 

CISTADENOKA, HOS
 

HERVOOb SYSrKH
 

NONE
 

SPECIAL SENSE 0B6AMS
 

NONE
 

HUSCULOSKSLJSTAL SYSXEfl
 

NONE
 

bODl LAV1TISS
 

•AbDOHIMAL CAVITY
 

llLnANGIOSABCO9A
 

ALL OPHEB SYSTEBS
 

NONE
 

« NUHBEK OF ANlhALS WITH TISSUE

* aU&BEK OF AiilAAL^ 4»ECROPSXED 

CONTBOL(VEH)
 02-F067
 

(19)
 

(20)
 

(19)
 

(20)
 

 EXAMINED MICROSCOPICALLY 

B-7 

LOW DOSE
 02-F070
 

(35)
 

(35)
 

1 (3%)
 

1
 

(37)
 

1 (3%)
 

(39)
 

HIGH DOSE
 02-F071
 

(41)
 

1 (2*)
 

(39)
 

0*0)
 

(43)
 

1 (2*>
 
ANIMAL DISPOSITION SUMMARY
 

ANIMALS INITIALLY IN STUDY
 

NATURAL DEATH@
 

MOPIBUND SACRIFICE
 

SCHEDULED SACRIFICE
 

ACCIDENTALLY KILLED
 

TERMINAL SACRIFICE
 

ANIMAL MISSING
 

TABLE B2 (CONCLUDED)
 

COHTROL(VEri)
 02-F067
 

20
 

3
 

17
 

d INCLUDES AUToOLYZED ANIMALS
 

TUMOR SUMMARY
 

TOTAL ANIMALS WITH PRIMARY TUMORS* 3
 

TOTAL PRIMARY TUKORS 3
 

TOIAL AilMALS WITH BESIfeJi TUMORS
 

lo£AL BENIGN TUSORb
 

TOTAL ANIMAL S WITH MALIGNANT TUMORS 3
 

TOTAL MALIGNANT TUKORS 3
 

TOTAL ANIMALS WITH SECONDARY TUMORS#
 

TOTAL SECONDARY TUMORS
 

TOTA L ANIMALS W1TH TUMORS UNCERTAINBENIGN OR MALIGNANT
 

TOIAL UNCERTAIN TUKORS
 

TOTAL ANIMALS WITH TUMORS UNLERTAIN ­

PRIMARY OR METASTATIC
 

TOTAL UNCERTAIN TUMORS
 

• PRIMARY TJMORS: ALL TUMORS EXCEPT SECONDARY TUMORS
 

LOW DOSE HIGH DOSE
 02-F070 02-F071
 

50 50
 

22 17
 

1
 

26 31
 

1 2
 

36 43
 

42 51
 

2 1
 

2 1
 

36 43
 

40 50
 

2
 

2
 

# bi.C0N»>ARY TUMOKS: METASTATIC TUMORS UR TUMORS INVASIVE INTO AN ADJACENT ORGAN
 

B-8
 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN RATS TREATED WITH NITROFEN
 

TABLE CI 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS TREATED WITH NITROFEN 

CONTROL (VEB) LOW DOSE HIGH DOSE
 01-11060 01-M061 01-M062
 

ANIMALS INITIALLY IN STUDY 20 50 50
 ANIMALS NECROPSIED 2 0 50 50
 **
 ASIflALS EXAMINED HISTOPATHOLOG1CALLY ** 20 50 50
 

INTEGUMENTARY SYSTEM
 

•SKIH	 (20) (50) (50)
 

INFLAMMATION, NOS 1 (2%)
 METAPLASIA, OSSEOOS 1 (2%)
 

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

INFLAMMATION, GRANULOMATOUS 1 (2*)
 NECROSIS, FAT 1 (2%)
 

RESPIRATORY SYSTEM
 

•TRACHEA	 (20) (50) (48)
 

INFLAMMATION, NOS 2 (4*)
 

*LUNG (20) (50) (50)
 

THROMBOSIS, NOS 1 (2%)
 EDEMA, NOS 1 (2*)
 HEMORRHAGE 2 (4*) 8 (16%)
 

INFLAMMATION, NOS 1 (2%)
 PNEUMONIA, CHRONIC MURINE 13 (65*) 35 (70*) 17 (34*)
 

FIBROSIS, FOCAL 1 (2*)
 

CALCIUM DEPOSIT 1 (2%)
 

HEMATOPOIETIC SYSTEM
 

#BONE BARROW (20) (50) (48)
 

METAMORPHOSIS FATTY 2 (4*)
 

#SPLhLN (20) (50) (50)
 

THROMBUS, ORGANIZED 1 (2%)
 HEMATOPOIESIS 2 (4*)
 

CIRCULATORY SYSTEM
 

•HEART	 (20) (50) (49)
 THROMBUS- ORGANIZED 2 (*%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

• DUMBER OF AiilMALS NECROPSIED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS 

C-3 
TABLE CI (CONTINUED) 

MEDIAL CALCIFICATION
 

CALCIUa DEPOSIT
 

•MYOCARDIUM
 

INFLAMMATION, NOS
 

FIBROSIS
 

DEGENERATION, NOS
 

•AORTA
 

MEDIAL CALCIFICAT1O*
 

•CORONARY ARTERY
 

IIBDIAL CALCIP1CATION
 

•MESENTERIC ARTER*
 

MEDIAL CALCIFICATION
 

CALCIUM DEPOSIT
 

DIGESTIVE SYSTEM
 

•LIVER
 

THROMBUS, ORGANIZED
 

INFLAMMATION, FOCAL
 

NECROSIS, NOS
 METAMORPHOSIS FATTY
 

FOCAL CELLULAR CHANGE
 ANGlLCTASIS
 

tLIVJsR/CBNTRILOBDLAR
 

NECROSIS, hOS
 

•bILE DUCT
 

DILATATION, NOS
 

HYPERPLASIA, ttOS
 

•PANCREAS
 

INFLAMMATION, NOS
 

PERIARTERITIS
 

ATROPHY, NOS
 

ATROPHY, FOCAL
 

•STOMACH
 

ULCER, POCAL
 

CALCIUM DEPOSIT
 

HYPERKERATOSIS
 

* NUMfcEit OF ANIMALS WITH TISSUE

* NUMBER Of ANIMALS &BCROPSIED 

CONTROL (VEH) LOB DOSS
 01-H060 01-M061
 

1 (2*)
 2 (10t) 1 (2*)
 

(20)	 (50)
 

2 (4*)
 

1	 (5*) 2 (4%>
 1 (2*)
 

(20)	 (50)
 

3 (IS*) 6 (12*)
 

(20)	 (50)
 

(20)	 (50)
 

1	 (5%) 3 (6*)
 1 (2*)
 

(^0) (50)
 

2 (**)
 1 (5%)
 

3 (15*) (18*)
 

7 (14%)
 

(20) (50)
 

(20) (50)
 

1 (2%)
 

7 (35*) 9 (18%)
 

(18)	 (50)
 

1 (6*) 1 (2%)
 

1 (6*) 1 (2%)
 

2 («»%)
 

1 (2%)
 

(20)	 (50)
 

1 (2%)
 

3	 (15*) 4 (8%)
 

1 (2%)
 

 EXAMINED MICROSCOPICALLY 

C-4 

HIGH DOSE
 01-M062
 

1 (2*)
 

(49)
 

1 (2*)
 

1 (2*)
 

(50)
 

(50)
 

1 (2*)
 

(50)
 

(50)
 

1 (2*)
 

1 (2*)
 

1 (2*)
 

1 (2*)
 

(50)
 

21 (42*)
 

(50)
 

3 (6*)
 

(<»8)
 

(50)
 

1 (2*)
 
»GOLUtt
 

NEttJkTODIASlS
 

UH1NAKX SYSTEM
 

•KIDMEX
 

dfDROHBPHROSIS
 PYELONEPHRITIS, *OS
 

ISFLAttMATION, CHROMIC
 

CALC1UR DEPOSIT
 

CALCIPICATIOM, «OS
 

•URISIABY BLADDER
 

CALCULUS, MOS
 HSnORRttAGS
 IBPLAMIATION, HEMOBBHAlilC
 

IRPLARHAT1OW, CHROK1C
 

HYPERPLASIA, EPITHELIAL
 

POLYP, 1SPLABBATORT
 

EMDOCfilUS SYSTSS
 

tPITUITARY
 ANG1KCTAS1S
 

•ADBSMAL
 

HBHORRHAGE
 HfPLAKHATION, CHRO81C
 

AMGIECIAS1S
 

•ADRENAL COBTEX
 

DEGENEHAT1ON, MOS
 

•THYROID
 

FOLLICULAR CYST, HOS
 

HIPKRVLA3IA, C-CBLL
 

•PARATflYhOID
 HYPERPLASIA, SOS
 

REPRODOCTIVE SYSTBS
 

#PROSTAT£
 HEMORRHAGE
 

•

 JiUflbt'R OP ANIMALS WITH TISSUE

* NUKBEfc OP ANIMALS MECEOPSIfiD 

TABLE CI (CONTINUED) 

CONTROL (VBH) LOW DOSS
 01-W060 01-B061
 

(20)	 (50)
 

1 (5%)
 

(20)	 (50)
 

1 (2%)
 5 (10%)
 

13 (65%)	 37 (74%)
 

2 (10%) 4 (8%)
 

(20)	 (49)
 

1 (2%)
 

2 (4*)
 

1 (2%)
 

1 (2%)
 

(20)	 (*6)
 

3 (7%)
 

(20) (50)
 

1 (2%)
 2 (4%)
 

(20)	 (50)
 

3 (6%)
 

(20) (50)
 

10 (20%)
 

1 (2%)
 

(3)	 (3)
 3 (100%) 3 (100%)
 

(18) (44)
 

 EXABIRED ftlCROSCOPICALLY 

C-5 

HIGH DOSB
 01-8062
 

(49)
 

1 (2%)
 

(50)
 

1 (2%)
 2 (4%)
 

28 (56%)
 

1 (2%)
 

1 (2%)
 

(48)
 

1 (2%)
 2 (4%)
 

1 (2%)
 

1 (2%)
 

(44)
 

(49)
 

1 (2%)
 

(49)
 

(47)
 

3 (6*)
 

(34)
 

4 (12%)
 
INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

•SEMINAL	 VESICLE
 HEaO&KHAGE
 

INFLAMMATION, NOS
 

•TESTIS
 

HEMORRHAGE
 

INFLAMMATION, NOS
 PERIARTERITIS
 

DEGENERATION, NOS
 

ATROPHY, NOS
 

•EPIDIDYMIS
 

HEMORRHAGE
 INFLAMMATION, NOS
 

-LaFLAMMATlOJi, CHRONIC
 

FloftOSIS
 

NECROSIS, FAT
 

•SCROTUM
 

HEMORRHAGE
 

NERVOUS SYSTEM
 

•BRAIN
 

HYDROCEPHALUS, NOS
 

HEMORRHAGE
 

INFLAMMATION, NOS
 

AbSCfiSS, ROS
 

CALCIUM DEPOSIT
 

•PINEAL BODY
 

FIBROSIS
 

CALCIFICATION, NOS
 

i>PSCIAL SENSE ORGANS
 

•BYE
 

CATARACT
 

MUSCULOSKELETAL SISTEM
 

NONE
 

t rtUfiBER OF ANIMALS WITH TISSUE

• NUMBER OF ANIMALS NECFOPSIED 

TABLE CI (CONTINUED) 

CO»TROL (?EH)
 

01-M060
 

1 (6%)
 

(20)
 

(20)
 

5 (25*)
 

(20)
 

(20)
 

(20)
 

(20)
 

(20)
 

LOW DOSE
 01-FJ061
 

10 (23%>
 

2 (5*)
 

(50)
 

1 (2%)
 

(50)
 

1 (2%)
 

23 (46%)
 

(50)
 

(50)
 

(50)
 

1 (2*)
 t (2*)
 

(50)
 

1 (2*)
 

1 (2%)
 

(50)
 

 EXAMINED MICROSCOPICALLY 

C-6 

HIGH DOSE
 01-H062
 

3 (9*)
 

4 (12*)
 

(50)
 

5 (10*)
 

<» (8*)
 

(49)
 

5 (10*)
 

1 (2*)
 

5 (10*)
 5 (10*)
 

(50)
 

5 (10*)
 2 (4*)
 

2 (4*)
 

1 (2*)
 

3 (6*)
 

(50)
 

* (8*)
 

(49)
 

1 (2*)
 

« (8%)
 

1 (2*)
 

1 (2*)
 

(50)
 

(50)
 

1 (2*)
 
TABLhCl (CONCLUDED) 

CONTHOL(VEH) LOW DOSE HIGH liOSE
 01-ft060 01-M061 01-K062
 

BODY CAVITIES
 

*PKRITO8£Utt (20) (50) (50)
 

IMFLAMHATIG&, NOS 1 &%)
 

•PSRICARDIUS	 (20) (50) (50)
 

INFLAWflATION, NOS 1 (5%) 2 (4%)
 

*flESE»TgfiY (20) (50) (*0)
 PERIARTERITIS 1 (5%) 3 (6*)
 

ALL OTHBR SYSTEttS
 

PERlNEOft
 

HEBORKHAGE 1
 

SPECIAL aORPHOLOGX SUMMARY
 

NO LLSION REPORTED	 t 1
 

f AUSBEE OF AWI9ALS WITH TISSUE EXAfilNED fliCROSCOPICALLY 

* NUfliifiR OF ANIftALS NECROPSIED 

C-7 
TABLE C2 

SUMMARY OF THF INCIDENCE O f NONNEOPLASTIC LESIONS IN FEMALE RATS TREATED WITH NITROFEN 

CONTROL(VEH) LOW DOSL HIbH DOSE
 01-F060 01-F06J 01-J-064
 

ANIMALS INITIALLY IN STUDY 20 50 50
 

ANIMALS HECROPSIED	 20 50 50
 

ANIMALS EXAMINED HISTOPATHOLOGICALLY ** 20 50 50
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (50) (50)
 

HYPERKERATOSIS 1 (5%)
 ACANTHOSIS 1 (5%)
 

*SUBCOT TISSUE (20) (50) (50)
 

HEMATOMA, NOS 1 <S%)
 

RESPIRATORY SYSTEM
 

#LUNG (20) (50) (50)
 

PNEUMONIA, CHRONIC MURINE 12 (60*) 36 (72%) 34 (68%)
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN (20) (50) (50)
 

THROMBOSIS, NOS 1 (2*)
 HEMATOPOIESIS 5 (10*) 6 (12%)
 

#THYMUS (17) (39) (3tt)
 

CYST, NOS 1 (3%)
 INFLAMMATION, NOS
 

CIRCULATORY SYSTSH
 

#MYOCARDIUM (20) (50) (50)
 

FIBROSIS 1 (2*) 3 (6%)
 

DEGENERATION,NOS 1 (2*) 3 (6%)
 

* ENDOCARDIUM	 (20) (50) (SO)
 

HYPERPLASIA, NOS 1 (2%)
 

*AORTA (20) (50) (50)
 

MEDIAL CALCIFICATION 2 <4%1
 

# NUMBER Of AN1MALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OP ANIMALS NECROPS1ED 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

C-8
 
TABLE C2 (CONTINUED) 

COiXlKOL(VEH) LOW DOSE
 U1-F060 U1-F06J
 

DIGEbl'lVE SYSTEM
 

•SALIVARi GLAND	 (42)
 

CYST, NOS
 

INPLASMATIOSi, CHRONIC
 

#LIVEtv (20) (SO)
 

INFLAMMATION, SOS 1 (^%)
 

INFLAKflATIOS, FOCAL
 

ABSCESS, hOS 1 (5%)
 

PlbROSIS, FOCAL
 

POLIOSIS KEPAT1S
 

SEI'ASORPEOSIS FATTY 1 (2%)
 FOCAL CELLULAR CHANGE 1 (2%)
 HYPERPLASIA, NOS 1 (5%)
 HYPERPLASIA, CYSTIC
 

ANG1£CTASIS 1 (2%)
 

*LIVSR/CENTRILOBULAR (20) (50)
 

NECROSIS, JMOS 2 (4%)
 

•»ILE DUCT	 UO) (SO)
 

DILATATION, NOb
 HYPERPLASIA, SOS 3 (15%) 9 (1b*)
 

HYPERPLASIA, CXSriC
 

iSTOrtACH (20) (49)
 

ULCER, FOCAL 2 (10%) 3 (6%)
 

tCOLOR (ZO) (49)
 

NEflAToDIAblS 1 (5*) 2 (4*)
 

URINAki bYSTEn
 

»KID1EJ (20) (SO)
 

iliUHOiiEPHROSIS 1 (2%)
 

PYELONEPHRITIS, NOb z (10*)
 

INFLArtflATION, CHROKi^, 4 (20%) 27 (54%)
 

CALCIUM DEPOSIT
 

ENDOCKINE SYSTEft
 

•ADRENAL	 (20) (SO)
 

ANblECTASIS 2 (10%) 3 (6%)


# ND.1fci,a OF ANIKALS WITH TISSUE EXAMINED &ICRGSCOP1CALLY 

* NUttO^h 0? AiilKALS NaCI-OPblEb 

C-9 

HIbH DOSE
 01-P064
 

(42)
 

1 (2*)
 

1 (2*)
 

(SO)
 

1 (2%)
 

1 (2%)
 1 (2*)
 

1 (2%)
 2 (4%)
 

1 (2%)
 

1 U*)
 1 (2%)
 

(SO)
 

Z (4*)
 

(SO)
 

1 (2%)
 S (10%)
 

3 (6%)
 

(SO)
 

3 (6%)
 

(49)
 

(SO)
 

2 (4%)
 

24 (48*)
 

1 (2*)
 

(49)
 

 4 (8*)
 
TABLE C2 (CONTINUED) 

COSTKOL(VEri) LOW DOSE HltiH DOSE 

01-F060 01-F063 01-F064 

tAJRfcNAL CORTEX (20) (50) (49) 

DEGENERATION, NOS 2 (10*) 3 (6%) 4 (8%) 

fTHXhOID (19) (49) (50) FOLL1CJLAR C*S1 , SOS 3 (b*) 3 (6%) 

hXi'lS&PLASIA, C-CELL 1 (2%) 2 (4*) 

REPRODUCTIVE SYSTEM 

•VAtilNA (20) (50) (50) INFLAMMATION, NOS 1 (2*) POLYP, INFLAMMATORY 1 (2%) 

#JTisSUS (20) (50) (49) INTUSSUSCEPTION 1 (2*) HYDROSETRA 1 (5%) 8 (16%) 6 (12*) HF?!ORKHAtf£ 1 (2*) INFLAMMATION, HoS 1 (2%) 1 (2*) 

• Ul'EhUS/JSNDO.IETRIOfl (20) (50) (49) HYPERPLASIA, CYSTIC 6 (12*) 1 (2*) 

fOVARY (20) (50) (*»9) 

CiSt, ikOS 1 (S%) 4 (b*) 6 (12*) 

ISyLASMATlON, MUS 1 (2*) 

NiiRVOUS SYSrSB 

#«HiiiN/ntNINGES (-^0) (50) (50)

 

IMFLAnflATIOM, NOS 1 (**>

 

SPECIAL £>£NSi2 ORGANS 

•EYE (20) (50) (50)

 

PiiiNUS 

*E)fE/IRlS (20) (50) (50)

 

lUFLABfiATIOS, NOS 1 (5*)

 

•EYJS/LACRIMAL GLAND (20) (50) (50)

 

INfLArtflATlOS, NOS 1 (2*)

 

MUSCULOSKELETAL SYSTEM 

NONL 

# SUftblSK OF ANIfiALS WITH TISSUE £XAftl»BD BICROSCOFICALLY 

• NOJfbKK OP AHISALS NiiCJiOPSIfiD 

C-IO 
TABLE C2 (CONCLUDED) 

CONTROL (VEA) LOW DOSE HIGH DOSS 

01-F060 01-F06J owoet* 

bOOl CAVITIES 

•PhRlTONEUfl (20) (50) (50) INlfLAttHATlOS, SOS 1 (2*) 

•PERICARDII)*! (20) (50) (50)

 INFLAMMATION, SOS 1 (2%)

 

•HESlsMTERY (^0) (50) (50)

 PERIARTERITIS 1 (2%)

 

ALL OTHER SXSTJSMS 

SPECIAL eO&PHOLOGJ SUHflARl 

SO LSblOH REPORTED 1 1 t 

# NUM&JSR OP ANIMALS WITH TISSUE EXAB1SED MICROSCOPIC ALL! 

• ilUtlbER OF ANIMALS riECROPSlLD 

C-ll 

APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC
 

LESIONS IN MICE TREATED WITH NITROFEN
 

TABLE Dl

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE TREATED WITH NITROFEN 

CONTROL (9Sri) LOW DOSE
 02-H067 02-H068
 

ANIMALS INITIALLY IN STUDY 20 50
 

ANIMALS SISSING
 

ANIMALS NLCROPSIED ** 20 44
 

ANIMALS EXAMINED HISTOPATHOLOG1CALLI 20 44
 

INTEGUMENTARY SYSTEM
 

*SKIN (20) (44)
 

EPIDERMAL INCLUSION CYST 2 (10*) 1 (2*)
 INFLAMMATION, NOS 1 (2%)
 ACANTHOSIS 1 (5%)
 

*SUBCUT TISSUE (20) (44)
 

ABSCESS, NOS 2 (10%) 1 (2*)
 

RESPIRATORY SYSTEM
 

NONE
 

HEMATOPOIETIC SYSTEM
 

*SPLEEN (2 0) (49)
 

AMYLOIDOSIS 15 (75%) 4 (8%)
 

HEMATOPOIESIS 3 (6%)
 

#LYMPH NODE (20) (50)
 

INFLAMMATION, NOS 1 (2%)
 

#MESENTERIC L. NODE (20) (50)
 

LYMPHANGIECTASIS 1 (5%)
 INFLAMMATION, NOS 1 (5%) 1 (2%)
 HYPERPLASIA, NOS 1 (2%)
 

CIRCULATORY SYSTEM
 

*HEART (20) (48)
 

THROMBUS, ORGANIZED 2 (10*)
 

#MYOCARDIUM (20) (48)
 

INFLAMMATION, NOS 2 (10%)
 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED BICROSCOPICALLY
 

* NUMBER OP ANIMALS NECROPSIES
 

**EXCLUDES PARTIALLY AUTOLYZED ANIMALS
 

0-3
 

HIGH DOSE
 02-B069
 

i>0
 

1
 

46
 

46
 

<*">)
 

1 (2%)
 

1 (2*)
 

(«6)
 

(47)
 

2 (4%)
 

(46)
 

(46)
 

(46)
 

(46)
 
TABLE Dl (CONTINUED) 

fKtfbOcARDIUM
 

INFLAMMATION, SOS
 

DlGr.STJ.VE SYSTEM
 

#LIV£R
 

THROMBUS, ORGANIZED
 

AMYLOIDOSIS
 

HYPERPLASIA, SODULAK
 

•PANCREAS
 

THKOMbUS, ORGANIZED
 

INFLAMMATION, NOS
 

PERIARTERITIS
 

CALCIUM DEPOSIT
 

#STOHACH
 HYPERKERATOSIS
 

ACANTHOSIS
 

•cOLON
 

PARASITISM
 

JKINARX bYSTSfl
 

itClDNEY
 

taYDKONEPHROSIS
 

PYELONEPHRITIS, huS
 

INFLAMMATION, NOS
 

INFLAMMATION, CHRONIC
 

AMYLOIDOSIS
 

#UR1NAKY bLADDER
 INFLAftBATlON, NOS
 

LNDGCfilSE SYSTEM
 

NONE
 

KEPKODOCTIVE SYSTEM
 

•PENIS
 

IHPLABMATIOli. NOS
 

# NLJMbER OF ANIMALS *ITh TISSUE

• NUftttE* Of ANIKALS NECROPSIED 

COaTROL(VEH) LOW DOSE
 02-M067 02-H06B
 

(20)	 (*8)
 

2 (10%)
 

(20)	 (49)
 

6 (12%)
 

13 (65*)
 

2 (4%)
 

(i<0 (46)
 

1 (2*)
 1 (2*)
 

1 (2*)
 

1 (2%)
 

(20) (48)
 

1 (2%)
 

1 (2*)
 

(20)	 (49)
 

3 (6%)
 

(20) (50)
 

1 (2%)
 

16 (80*) 3 (6%)
 

1b (75*) 1 (2*)
 

(20)	 (47)
 

1 (2%)
 

(20) (44)
 

* (5*)
 

 EXAMINED MICROSCOPICALLY 

D-4 

HIGH DOSS
 02-M069
 

(46)
 

(48)
 

2 (4*)
 

(43)
 

1 (2%)
 

(46)
 

(44)
 

1 (2%)
 

(46)
 

3 (7*)
 

1 (2%)
 

(40)
 

(46)
 
•PREPUTIAL GLAND
 

INFLAMMATION, SOS
 

•TLSI1S
 

CALC1UH DEPOSIT
 

ATROPHY, NOS
 

•EPIDIDYMIS
 

GRANULOMA, SPERMATlL
 

ATROPHY, NOS
 

NERVOUS SYSTEM
 

NONi.
 

SP2CIAL SENSE ORGANS
 

NOSE
 

MUSCULOSKELETAL SYSTEM
 

HOME
 

BODY CAVITIES
 

•MESENTERY
 

PERIARTERITIS
 

ALL OTHER SYSTEMS
 

HOME
 

SPECIAL MORPHOLOGY SUMMARY
 

MO LESIOH REPORTED
 

ANIMAL MISSING/IO NECROPSY
 

AUTO/MECROPSY/HISTO P£RF
 AUTOLYSIS/NO NECROPSY
 

f MUHBKfi OF ANIMALS WITH TISSUE EXAMINED


• M0flb£fi OF ANIMALS NECROPSIED
 

TABLF Dl (CONCLUDFD)
 

CONTROL(VEH)
 02-tt0o7
 

(20)
 

(18)
 

(20)
 

(20)
 

1
 

1
 

LOB DOSL
 02-M068
 

(44)
 

1 {2%)
 

(49)
 

1 (2%)
 

1 (2*)
 

(44)
 

1 (2*)
 

1 (2%)
 

(44)
 

1 (2*)
 

4
 

6
 

 MICROSCOPICALLY
 

Hlbti DOSE
 O2-MO69
 

(46)
 

(45)
 

1 (2*)
 

(46)
 

(46)
 

1
 

3
 

D-5
 
TABLE D2

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE TREATED WITH NITROFEN 

CONTROL(VEH) LOti DOSE HIGH DOSE
 0^-F067 02-P070 02-P071
 

AAlttALS INITIALLY I» STUDY 20 50 50
 

ANIMALS KISSING 1 2
 

ANI.iALS KLLROPSlfiD ** 20 39 43
 

ANlftALs fcXAflINSl) HISIOPATHOLOGICALLT 20 39 43
 

INX^GUSKNTARY SYS1EH
 

KOBi.
 

RSSPIRA1ORY SYSTEH
 

#Ll)HG (20) (38) (45)
 

PNEUftONIA, CHRONIC KdhINiS 1 (2%)
 

rili«AroP01£TIC SYSi*23
 

#SPLi:£8 (18) (39) (43)
 

ti£»ATOPOIhiSlS 1 (3*)
 

#LUttbAH LYMPH NODE (20) (40) (45)
 

HYPERPLASIA, NOS 1 (5*)
 

ifltSESTKRlC L. KODf (20) (10) (45)
 

LIHPUA«tGI£CTASIS 1 (3%) 3 (7%)
 

INFLAflflAriOM, NOS 2 (5%)
 

AN61BCTASIS 1 (5*)
 

fRi.NAL LY^PM NODE (20) (40) (45)
 

HYPERPLASIA, MOS 1 (5%)
 

C1PCULATOHY SYSTEM
 

NOM£
 

DIGEbTIVfi SYSTEM ­

tLIVEfi (19) (41) (44)
 

CYST. HG£ 1 (211
 

« MUSbEfi OP AJilflALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NOflBER OP AHIflALS HEChOFSlED 

**EXCLUDES PARTIALLY AUT0LYZED ANIKALS 

D-6 
TABLE D2 (CONTINUED) 

THROMbUS, ORGANIZED
 

INFLAMMATION, NOS
 

CALCIUfl DEPOSIT
 

iPANCREAS
 

DIL&TATION/DUCTS
 

CXST , NOS
 

I bF LA a B AT I Oil, NOS
 

ATROPHY, NOS
 

fPANCREATIC DUCT
 

l/ILATATIOi*, NOS
 

HYPERPLASIA, NOS
 

#brottACH
 

HYPERKERATOSIS
 

ACANTHOSIS
 

URINARY SYSTEM
 

•KIDNEX
 

HYDRONEPHROSIS
 

INFLAMMATION, CHROhlC
 

PIGMENTATION, NOS
 

EtfDOCRlflE SYSTEM
 

NONE
 

REPRODUCTIVE SYSTEM
 

fUl'EKUS
 

HYi>ROMETRA
 

THEOrtBOSIS, NOS
 

lNFLAMMATION, NOS
 

• JTKROS/ESDOflETRIIJrt
 

INFLAMMATION, NOS
 

HYPERPLASIA, CYSTIC
 

•OVARY/OVIDUCT
 

INFLAMMATION, NOS
 

•OVARY
 

CYST- NOS
 

t NUMBER OF ANIMALS WITH TISSUE

• NJMBER OF ANlflALS NECROFSIED 

CONTROL(VEH)
 

02-F06?
 

(20)
 

1 (5*)
 

1 (5%)
 

(20)
 

U0)
 

(20)
 

(19)
 

2 (1U)
 

(19)
 

7 (37*)
 

4 (21%)
 

(19)
 

1 (5%)
 

(19)
 

8 (42%)
 

 EXAMINED MICROSCOPICALLY 

D-7 

LOW DOSE
 02-F070
 

3 (7%)
 

2 (5%)
 

(37)
 

1 (3%)
 

2 (5%)
 

(37)
 

1 (3%)
 

(39)
 

(40)
 

1 (3*)
 

1 (3*)
 

(37)
 

4 (11%)
 

b (14%)
 

(37)
 

3 (8%)
 

(37)
 

2 (5%)
 

(37)
 

10 (27%)
 

HIGH DOSE
 02-F071
 

2 (5%)
 

4 (9*)
 

(38)
 

2 (5%)
 

1 (3%)
 

(38)
 

1 (3%)
 

(42)
 

3 (7%)
 3 (7%)
 

(44)
 

2 (5%)
 

1 (2%)
 

(40)
 

7 (18%)
 

1 (3%)
 

(**0)
 

1 (3%)
 

(*0)
 

(40)
 

4 (10%)
 
TABLE D2 (CONCLUDED) 

CONTfiOL(?EH) 02-P06? LOW DOSE 02-F070 riluH DOSE 02-F071 

INFLAMMATION, NOS 7 (47%) 13 (3b%) 

CAiXlUrt DEPOSIT 1 (3%> ANbIBLTASIS 1 (3*) 

NEitVOJS SYSTEM
 

NONE
 

SPECIAL SENSE ORGANS
 

NONE
 

MUSCULOSKELETAL SXSlfca
 

NONE
 

BODY CAVITIES
 

•P£RilONEUtt	 (20) (39) (*3)
 

iN*LAMMAlION, NOS 1 (5%) 1 (3%)
 

ALL 01HE& SYSTEMS
 

NONE
 

SPECIAL AORPHOLOGY SUftMAR*
 

NO LEi>ION REPORTED 1 1
 

ANIMAL rtISSlNG/NO NEChOPSX 1 2
 AUro/NECfiOPSX/HISlO PtKlf 1
 

AOTOLISIS/SO 8SCROPSY 10 5
 

* NURBfcK OF ANIrtALS WITti TISSUE EXAMINED MICROSCOPICALLY 

* NUaB^R OP ANIMALS NJiChOPSlED 

* U S GOVERNMENT PRINTING OFFICE 1978-260-899 3202 

D-8 

DHEW Publication No. (NIH) 78-826