1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

NO. 124 

BIOASSAY OF 

PIPERONYL SULFOXIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 120-62-7 

NCI-CG-TR-124 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 

PIPERONYL SULFOXIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Carcinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

Bethesda, Maryland 20014
 

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

Public Health Service
 

National Institutes of Health
 

DHEW Publication No. (NIH) 79-1379
 
ii

 
BIOASSAY OF
 

PIPERONYL SULFOXIDE
 

FOR POSSIBLE CARCINOGENICITY
 

Careinogenesis Testing Program
 

Division of Cancer Cause and Prevention
 

National Cancer Institute
 

National Institutes of Health
 

FOREWORD; This report presents the results of the bioassay of
 

piperonyl sulfoxide conducted for the Carcinogenesis Testing
 

Program, Division of Cancer Cause and Prevention, National Cancer
 

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

Maryland. This is one of a series of experiments designed to
 

determine whether selected chemicals have the capacity to produce
 

cancer in animals. Negative results, in which the test animals do
 

not have a greater incidence of cancer than control animals, do
 

not necessarily mean that the test chemical is not a carcinogen,
 

inasmuch as the experiments are conducted under a limited set of
 

circumstances. Positive results demonstrate that the test
 

chemical is carcinogenic for animals under the conditions of the
 

test and indicate that exposure to the chemical is a potential
 

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

animal carcinogens requires a wider analysis.
 

CONTRIBUTORS; This bioassay of piperonyl sulfoxide was conducted
 

by Frederick Cancer Research Center (FCRC) (1), Frederick, 

Maryland, operated for NCI (2) by Litton Bionetics, Inc., 

Kensington, Maryland (3). 

The manager of the bioassay at FCRC was Dr. D. Creasia. The
 

program manager was Dr. B. Ulland, and the toxicologist was Dr. E.
 

Gordon. Ms. S. Toms was responsible for management of data, Mr.
 

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

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

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

computer services. Necropsies were performed by Drs.
 

B. Ulland, R. Schueler, R. Bail, and R. Cardy. Histopathologic
 

evaluations for rats were performed by Dr. R. A. Renne (4,5), and
 

the histopathologic evaluations for mice were performed by Dr. C.
 

E. Gilmore (4).
 

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

Mason Research Institute (6). Statistical analyses were performed
 

ill
 
by Dr. J. R. Joiner (7) and Ms. P. L. Yong (7), using methods
 

selected for the bioassay program by Dr. J. J. Gart (8). The
 

chemicals used in this bioassay were analyzed at Frederick Cancer
 

Research Center by Dr. W. Zielinsky (l). The results of these
 

analyses were reviewed by Dr. C. W. Jameson (7) and Ms. P. M.
 

Wagner (7). The structural formula was supplied by NCI.
 

This report was prepared at Tracor Jitco (7) under the direction
 

of NCI. Those responsible for the report at Tracor Jitco were Dr.
 

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

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

R. L. Schueler, pathologist; Dr. G. L. Miller, Ms. M. S. King, Ms.
 

L. A. Waitz, and Mr. W. D. Reichardt, bioscience writers; and Dr.
 

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

Ms. P. J. Graboske.
 

The following scientists at NCI (2) were responsible for
 

evaluating the bioassay experiment, interpreting the results, and
 

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

Jr., Dr. J. Fielding Douglas, Dr. Dawn G. Goodman (9), Dr. Richard
 

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

Thomas W. Orme, Dr. Robert A. Squire (10), Dr. Sherman F. Stinson,
 

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

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

Maryland.
 

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

Prevention, National Cancer Institute, National Institutes of
 

Health, Bethesda, Maryland.
 

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

Maryland.
 

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

Herndon, Virginia.
 

(5)	 Now with Battelle Pacific Northwest Laboratories, Battelle
 

Boulevard, Richland, Washington.
 

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

Rockville, Maryland.
 

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

Maryland.
 

iv
 
(8)	 Mathematical Statistics and Applied Mathematics Section,
 

Biometry Branch, Field Studies and Statistics, Division of
 

Cancer Cause and Prevention, National Cancer Institute,
 

National Institutes of Health, Bethesda, Maryland.
 

(9)	 Now with Clement Associates, Inc., 1010 Wisconsin Avenue,
 

N.W., Suite 660, Washington, D. C.
 

(10)	 Now with the Division of Comparative Medicine, Johns Hopkins
 

University, School of Medicine, Traylor Building, Baltimore,
 

Maryland.
 
vi

 
SUMMARY
 

A bioassay of technical-grade piperonyl sulfoxide for possible
 

carcinogenicity was conducted by administering the test chemical
 

in feed to Fischer 344 rats and B6C3F1 mice.
 

Groups of 50 rats of each sex were administered piperonyl
 

sulfoxide in the diet at one of several doses, either 1,500 or
 

3,000 ppm for the males and either 3,000 or 6,000 ppm for the
 

females, for 105 weeks. Matched controls consisted of 20
 

untreated rats of each sex. All surviving rats were killed at
 

the end of the period of administration of the test chemical.
 

Groups of 50 male mice were administered one of two doses, either
 

350 or 700 ppm, for 104 or 105 weeks. Groups of 50 female mice
 

were initially administered one of two doses, either 700 or 1,400
 

ppm. Due to excessive weight depression in the dosed female
 

mice, the doses for this sex were reduced after week 20 to 200
 

and 600 ppm, respectively, and administration of the test
 

chemical at the lower doses was continued for 84 or 85 weeks.
 

The time-weighted average doses for the females were 295 and 754
 

ppm. Matched controls consisted of 20 untreated mice of each
 

sex. All surviving mice were killed at the end of the period of
 

administration of the test chemical.
 

Mean body weights of dosed groups of rats and mice of each sex
 

were lower than those of corresponding control groups, and the
 

depressions in the amount of mean body weight gained were dose
 

related for most or all of the bioassay; the depression in the
 

amount of mean body weight gained was slight, however, in the
 

dosed male rats. Survival of the rats and mice was unaffected by
 

the piperonyl sulfoxide and was 78% or higher in all groups at
 

week 90 of the bioassay; thus, sufficient numbers of dosed and
 

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

development of late-appearing tumors.
 

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

occurred at incidences that were significantly higher in dosed
 

groups than in control groups.
 

In the male mice, hepatocellular carcinomas occurred at
 

incidences that were dose related (P less than 0.001); in direct
 

comparisons, the incidence of these tumors in the high-dose group
 

was significantly higher (P less than 0.001) than that in the
 

control group (controls 6/18, low-dose 31/50, high-dose 46/50).
 

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

technical-grade piperonyl sulfoxide was not carcinogenic for male
 

or female Fischer 344 rats or for female B6C3F1 mice, but was
 

carcinogenic for male B6C3F1 mice, producing an increased
 

incidence of hepatocellular carcinomas.
 

viii
 
TABLE OF CONTENTS
 

Page
 

I. Introduction 1
 

II. Materials and Methods 3
 

A. Chemical 3
 

B. Dietary Preparation 3
 

C. Animals 4
 

D. Animal Maintenance 5
 

E. Subchronic Studies 8
 

F. Chronic Studies 11
 

G. Clinical and Pathologic Examinations 11
 

H. Data Recording and Statistical Analyses 15
 

III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 

B. Survival (Rats) 21
 

C. Pathology (Rats) 23
 

D. Statistical Analyses of Results (Rats) 33
 

IV. Results -Mice 35
 

A. Body Weights and Clinical Signs (Mice) 35
 

B. Survival (Mice) 35
 

C. Pathology (Mice) 37
 

D. Statistical Analyses of Results (Mice) 40
 

V. Discussion 43
 

VI. Bibliography 47
 

APPENDIXES
 

Appendix A Summary of the Incidence of Neoplasms in
 

Rats Administered Piperonyl Sulfoxide in
 

the Diet 51
 

Table Al Summary of the Incidence of Neoplasms in
 

Male Rats Administered Piperonyl Sulfoxide
 

in the Diet 53
 

ix
 
Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Administered Piperonyl Sulfoxide 

in the Diet 57 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered Piperonyl Sulfoxide in 

the Diet 61 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered Piperonyl Sulfoxide 

in the Diet 63 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered Piperonyl Sulfoxide 

in the Diet 66 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered Piperonyl 

Sulfoxide in the Diet 71 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered 

Piperonyl Sulfoxide in the Diet 73 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered 

Piperonyl Sulfoxide in the Diet 78 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered Piperonyl 

Sulfoxide in the Diet 83 

Table Dl Summary of the Incidence of Nonnaoplastic 

Lesions in Male Mice Administered 

Piperonyl Sulfoxide in the Diet 85 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered 

Piperonyl Sulfoxide in the Diet 88 

Appendix E Analyses of the Incidence of Primary 

Tumors in Rats Administered Piperonyl 

Sulfoxide in the Diet 91 

Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered Piperonyl 

Sulfoxide in the Diet 93 

x
 
Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered Piperonyl 

Sulfoxide in the Diet 98 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered Piperonyl Sulfoxide 

in the Diet 103 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered Piperonyl 

Sulf oxide in the Diet 105 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Administered Piperonyl 

Sulf oxide in the Diet 109 

TABLES 

Table 1 Piperonyl Sulfoxide Subchronic Feeding 

Studies in Rats and Mice 9 

Table 2 Piperonyl Sulfoxide

in Rats

 Chronic Feeding Studies 

 12 

Table 3 Piperonyl Sulfoxide Chronic Feeding Studies 

in Mice 13 

FIGURES 

Figure 1 Growth Curves for Rats Administered 

Piperonyl Sulfoxide in the Diet 22 

Figure 2 Survival Curves for Rats Administered 

Piperonyl Sulfoxide in the Diet 24 

Figure 3 Growth Curves for Mice Administered 

Piperonyl Sulfoxide in the Diet 36 

Figure 4 Survival Curves for Mice Administered 

Piperonyl Sulfoxide in the Diet 38 

xi
 
xii

 
I. INTRODUCTION
 

o
II
 

CH3-(CH2) -CH2-S-CH-CH
 

CH.
 

Piperonyl su If oxide
 

Piperonyl sulfoxide (CAS 120-62-7; NCI C02824), is
 

1,2-(methylenedioxy)-4-(2-(octylsulfinyl)propyl)benzene. It is
 

used to enhance the insecticidal properties of the pyrethrins by
 

inhibiting pyrethrin detoxification enzymes, probably microsomal
 

oxidases, in the insect (Metcalf, 1966). Pyrethrins alone
 

produce a transient paralysis, whereas pyrethrins combined with a
 

synergist such as piperonyl sulfoxide are insecticidal (Metcalf,
 

1966).
 

There are no estimates of the amount of piperonyl sulfoxide
 

produced in the United States, since production figures on this
 
chemical are grouped with all other cyclic insecticides and
 

rodenticides (United States International Trade Commission, 1977).
 

The long-term toxicity of piperonyl sulfoxide was investigated by
 

Innes et al. (1969) as a part of a large-scale test of industrial
 

and agricultural chemicals. These investigators, testing mice
 

only, obtained an increased, but not significant, incidence of
 

tumors in mice administered piperonyl sulfoxide, and they
 

categorized this chemical among those that required additional 

testing. On the basis of these preliminary results, piperonyl 

sulfoxide was selected for study in the Carcinogenesis Testing 

Program. 
II. MATERIALS AND METHODS
 

A. Chemical
 

The test chemical used in the bioassay is a commercially
 

available product and was obtained as two 20-kilogram batches of
 

Lot No. 291-MOO-12 technical-grade piperonyl sulfoxide from S. B.
 

Penick and Company, CPC International, Inc., Lyndhurst, New
 

Jersey. According to the manufacturer's specification and assay,
 

the compound contained at least 88% piperonyl sulfoxide plus 12%
 

related compounds. The test chemical is hereinafter referred to
 

as piperonyl sulfoxide.
 

The piperonyl sulfoxide was stored at 5 C until used.
 

B. Dietary Preparation
 

Test diets containing piperonyl sulfoxide were prepared fresh
 

every 1 to 1-1/2 weeks in 6- to 12-kilogram batches at
 

appropriate doses. A known weight of the chemical was first
 

mixed with an equal weight of powdered Wayne® Sterilizable Lab
 

Meal (Allied Mills, Inc., Chicago, 111.), using a mortar and
 
pestle. The mixing was repeated with second and third additions
 

of feed, and final mixing was performed with the remaining
 

quantity of feed for a minimum of 15 minutes in a Patterson-Kelly
 

twin-shell blender. Uniformity of the mixtures was established
 

by comparative analysis of samples taken from three different
 

locations within the blender. A study of the stability of the
 

test chemical at 6,000 ppm in the diet showed a loss of only
 

0.25% per day.
 

The diets were stored at 7 C in plastic bags during the 1 to
 

1-1/2 weeks it was used.
 

C. Animals
 

Male and female Fischer 344 rats and B6C3F1 mice were obtained
 

from the Frederick Cancer Research Center (Frederick, Md.) as
 

4-week-old weanlings, all within 3 days of the same age. The
 

animals were quarantined within the test facility for 2 weeks and
 

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

a system that averaged the weights per cage for a given species
 

and sex. For use in the chronic study, the male rats were
 

required to weigh 90 to 105 g, averaging at least 100 g; the
 

female rats, 80 to 95 g, averaging at least 90 g; the male mice,
 
18 to 22 g, averaging at least 19.5 g; and the female mice, 17 to
 

21 g, averaging at least 18.5 g. Individual animals were
 

identified by ear punch.
 

D. Animal Maintenance
 

The animals were housed in polycarbonate cages (Lab Products
 

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

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

suspended from aluminum racks (Scientific Cages, Inc., Bryan,
 

Tex.) and were covered by nonwoven polyester-fiber 12-mil-thick
 

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

was Absorb-dri hardwood chips (Northeastern Products, Inc.,
 

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

Lab Meal with 4% fat, provided ad_ libitum in suspended stainless
 

steel hoppers and replenished at least three times per week.
 

Water, acidified to pH 2.5, was supplied ad libitum from glass
 

bottles and sipper tubes (Lab Products, Inc.) suspended through
 

the tops of the cages.
 

The cages were sanitized at 82-88 C in a tunnel-type cagewasher
 

(industrial Washing Machine Corp., Mataway, N. J.) twice per
 

week, using the detergents, Clout® (Pharmacal Research
 
Laboratories, Greenwich, Conn.) or Oxford D'Chlor (Oxford
 

Chemicals, Atlanta, Ga.). The contaminated bedding was disposed
 

of through an enclosed vacuum line that led to a holding tank
 

from which the bedding was fed periodically into an incinerator.
 

The feed hoppers were sanitized twice per month in the same
 

equipment. The glass bottles and sipper tubes were sanitized at
 

82-88 C in a tunnel-type bottle washer (Consolidated Equipment
 

Supply Co., Mercersburg, Pa.) three times per week, using a
 

Calgen Commercial Division detergent (St. Louis, Mo.). The racks
 

for the cages were changed and sanitized at or above 82 C in a
 

rack washer (Consolidated Equipment Supply Co.) once per month,
 

using the Calgen Commercial Division detergent, and the filter
 

paper was changed at the same time.
 

The air in the animal rooms was regulated automatically at a
 

temperature of 22-24 C and a relative humidity of 45-55%.
 

Nonrecirculated air was passed through a filter of 65% efficiency
 

and a bag filter of 95% efficiency at the intake and through a
 

"Z"-type roughing filter of 30% efficiency and a bag system of
 

90-95% efficiency at the exhaust (American Air Filters,
 

Louisville, Ky.; Mine Safety Appliances, Pittsburgh, Pa.). The
 

rate of movement allowed 15 changes of room air per hour. The
 

air pressure was maintained negative to a clean hallway and
 
positive to a return hallway. Fluorescent lighting was provided
 

on a 12-hour-per-day cycle.
 

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

on feeding studies of the following chemicals:
 

(CAS 103-33-3) azobenzene
 

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

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

on feeding studies of the following chemicals:
 

(CAS 298-00-0) methyl parathion
 

(CAS 128-66-5) C.I. vat yellow 4 (amanthrene)
 

(CAS 103-33-3) azobenzene
 

(CAS 20941-65-5) ethyl tellurac
 

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

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

(CAS 85-44-9) phthalic anhydride
 

All procedures involving animals receiving any one of the test
 

chemicals were separated as much as possible from procedures
 

involving animals receiving another test chemical in the same
 

room.
 

E. Subchronic Studies
 

Subchronic feeding studies were conducted to estimate the maximum
 

tolerated doses (MTD's) of piperonyl sulfoxide, on the basis of
 

which two concentrations (hereinafter referred to as "low" and
 
"high" doses) were selected for administration in the chronic
 

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

sex were administered feed containing piperonyl sulfoxide at one
 

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

species and sex were administered a basal diet only. The doses
 

used for the rats in the subchronic studies were 4,600, 6,800,
 

10,000, 14,700, and 21,500 ppm; the doses used for the mice in
 

the subchronic studies were 1,500, 2,200, 3,200, 4,600, 6,800,
 

10,000, 14,700, and 21,500 ppm. At a later date, a second series
 

of subchronic studies were conducted on female mice, using doses
 

of 50, 100, 200, 300, 600, 1,200, 1,600, and 2,400 ppm. The
 

period of administration of the test chemical was 7 weeks,
 

followed by 1 week of additional observation. Each animal was
 

weighed twice per week.
 

Table 1 shows the number of animals dying at each dose and the
 

number of weeks on study when deaths occurred; the table also
 

shows the mean body weights of dosed animals at week 7, expressed
 

as percentages of mean body weights of controls at week 7. At the
 

end of the subchronic studies, all animals were killed by C0_
 

inhalation and necropsied. The lowest dose at which
 

histopathologic findings were observed was 21,500 ppm in the male
 

and female rats. At this dose, there were moderate to marked
 
Table 1. Piperonyl Sulfoxide Subchronic Feeding 

Studies in Rats and Mice 

Male Female 

Mean Weight Mean Weight 

Mortality at Week 7 Mortality at Week 7 

Number Week on as % of Number Week on as % of 

(ppm) Dead Study Control Dead Study Control 

RATS 

4,600 93 104 

6,800 83 90 

10,000 76 94 

14,700 1 3 68 80 

21,500 2 5 46 60 

MICE 

1,500 74 73 

2,200 81 90 

3,200 81 83 

4,600 76 89 

6,800 2 8 73 2 8 8 4 

10,000 5 6 4 8 

14,700 5 2 5 2 

21,500 5 2 5 2 

50 101 

100 99 

200 97 

300 104 

600 101 

1,200 91 

1,600 89 

2,400 82 
increases in splenic hematopoiesis. No clinical or
 

histopathologic findings were reported for the mice.
 

Doses required to induce 10% depression in body weight were taken
 

as the major criteria for MTD's. These were estimated as
 

follows: first, least squares regressions of mean body weights
 

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

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

weights of dosed groups at day 49 relative to weights of
 

corresponding control groups were plotted against the logarithms
 

of the doses, and least squares regressions fitted to the data
 

were used to estimate the doses required to induce 10% depression
 

in weight.
 

Using these calculations, the low and high doses for chronic
 

studies using male rats were set at 1,500 and 3,000 ppm; using
 

female rats, 3,000 and 6,000 ppm; using male mice, 350 and 700
 

ppm; and using female mice, 700 and 1,400 ppm. The findings at
 

gross or histopathologic examination did not affect the selection
 

of the doses.
 

10
 
F. Chronic Studies
 

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

feeding studies are shown in tables 2 and 3. Due to excessive
 

weight depression in the dosed female mice, doses for the low-


and high-dose groups were reduced to 200 and 600 ppm,
 

respectively, after week 20.
 

G. Clinical and Pathologic Examinations
 

All animals were observed twice daily for signs of toxicity.
 

Clinical examination and palpation for masses were performed each
 

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

Sick, tumor-bearing, and moribund animals were observed daily.
 

Moribund animals and animals that survived to the end of the
 

bioassay were killed by asphyxiation using CO- and necropsied.
 

Necropsies were also performed on all animals found dead, unless
 

precluded by autolysis or severe cannibalization.
 

The pathologic evaluation consisted of gross and microscopic
 

examination of major tissues, major organs, and all gross
 

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

11
 
Table 2. Piperonyl Sulfoxide Chronic Feeding Studies in Rats
 

Sex and Initial Sulfoxide Time on
 

Test No. of in Diet(b) Study
 

Group Animals(a) (ppm) (weeks)
 

Male
 

Matched-Control 20 0 105
 

Low-Dose 50 1,500 105
 

High-Dose 50 3,000 105
 

Female
 

Matched-Control 20 0 105
 

Low-Dose 50 3,000 105
 

High -Dose 50 6,000 105
 

(a)	 Al animal l s were 6 weeks of age when placed on study.
 

(b)	 Test and control diets were provided ad libitum 7 days per
 

week.
 

12
 
Table 3. Piperonyl Sulfoxide Chronic Feeding Studies in Mice
 

Sex and Initial Sulfoxide Time on Time-Weighted
 

Test No. of in Diet(b) Study Average Dose(c)
 

Group Animals (a) (ppm) (weeks) (ppm)
 

Male
 

Matched-Control 20 0 105

 

Low-Dose 50 350 104-105

 

High-Dose 50 700 104 

Female
 

Matched-Control 20 0 105
 

Low-Dose 50 700 20
 

200 85 295
 

High-Dose 50 1,400 20
 

600 84 754
 

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

(b) Test and control diets were provided ad libitum 7 days per week.
 

(c) Time-weighted average dose = S(dose in ppm x no. of weeks at that dose)
 

E(no.of weeks receiving each dose)
 

13
 
embedded in paraffin, sectioned, and stained with hematoxylin and
 

eosin. The following tissues were examined microscopically:
 

skin, lungs and bronchi, trachea, bone marrow, spleen, lymph
 

nodes (submandibular), thymus, heart, salivary glands (parotid,
 

sublingual, and submaxillary), liver, pancreas, esophagus,
 

stomach (glandular and nonglandular), small and large intestines,
 

kidney, urinary bladder, pituitary, adrenal, thyroid,
 

parathyroid, pancreatic islets, mammary gland, testis, prostate,
 

tunica vaginalis, mammary gland, uterus, ovary, brain (cerebrum
 

and cerebellum), and all tissue masses. Peripheral blood smears
 

also were made for all animals, whenever possible.
 

A few tissues from some animals were not examined, particularly
 

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

been in advanced states of cannibalization or autolysis. Thus,
 

the number of animals from which particular organs or tissues
 

were examined microscopically varies, and does not necessarily
 

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

group.
 

14
 
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 appropriate statistical
 

techniques described in this section. Those analyses of the
 

experimental results that bear on the possibility of
 

carcinogenicity are discussed in the statistical narrative
 

sections.
 

Probabilities of survival were estimated by the product-limit
 

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

report in the form of graphs. Animals were statistically
 

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

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

causes were not statistically censored. Statistical analyses for
 

15
 
a possible dose-related effect on survival used the method of Cox
 

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

extensions of Cox methods for testing for a dose-related trend.
 

One-tailed P values have been reported for all tests except the
 

departure from linearity test, which is only reported when its
 

two-tailed P value is less than 0.05.
 

The incidence of neoplastic or nonneoplastic lesions has been
 

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

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

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

the denominators included only those animals for which that site
 

was examined histologically. However, when macroscopic
 

examination was required to detect lesions prior to histologic
 

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

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

denominators consist of the numbers of animals necropsied.
 

The purpose of the statistical analyses of tumor incidence is to
 

determine whether animals receiving the test chemical developed a
 

significantly higher proportion of tumors than did the control
 

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

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

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

16
 
dose level. When results for a number of dosed groups (k) are
 

compared simultaneously with those for a control group, a
 

correction to ensure an overall significance level of 0.05 may be
 

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

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

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

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

where the Fisher exact P values are shown.
 

The Cochran-Armitage test for linear trend in proportions, with
 

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

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

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

one-tailed 0.05 level of significance. Unless otherwise noted,
 

the direction of the significant trend is a positive dose
 

relationship. This method also provides a two-tailed test of
 

departure from linear trend.
 

A time-adjusted analysis was applied when numerous early deaths
 

resulted from causes that were not associated with the formation
 

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

first tumor was observed were excluded by basing the statistical
 

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

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

17
 
such an early tumor was found, comparisons were based exclusively
 

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

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

obtained, the standard procedures for analyses of the incidence
 

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

followed.
 

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

incidence of tumors. Curves of the proportions surviving without
 

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

The week during which an animal died naturally or was sacrificed
 

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

methods of comparing these curves were used for two groups;
 

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

groups. The statistical tests for the incidence of tumors which
 

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

noted, in the direction of a positive dose relationship.
 

Significant departures from linearity (P less than 0.05,
 

two-tailed test) were also noted.
 

The approximate 95 percent confidence interval for the relative
 

risk of each dosed group compared with its control was calculated
 

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

relative risk is defined as p,./p where p is the true
 L t- C.
 

18
 
binomial probability of the incidence of a specific type of tumor
 

in a dosed group of animals and p is the true probability of
 

the spontaneous incidence of the same type of tumor in a control
 

group. The hypothesis of equality between the true proportion of
 

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

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

of unity represent the condition of a larger proportion in the
 

dosed group than in the control.
 

The lower and upper limits of the confidence interval of the
 

relative risk have been included in the tables of statistical
 

analyses. The interpretation of the limits is that in
 

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

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

control group would be within the interval calculated from the
 

experiment. When the lower limit of the confidence interval is
 

greater than one, it can be inferred that a statistically
 

significant result (P less than 0.025 one-tailed test when the
 

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

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

than unity, but the upper limit is a greater than unity, the
 

lower limit indicates the absence of a significant result while
 

the upper limit indicates that there is a theoretical possibility
 

19
 
of the induction of tumors by the test chemical, which could not
 

be detected under the conditions of this test.
 

20
 
III. RESULTS - RATS
 

A. Body Weights and Clinical Signs (Rats)
 

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

of corresponding matched controls, and the depressions in the
 

amount of mean body weight gained were dose related during most
 

of the bioassay (figure 1). The amounts of mean body weight
 

gained by the dosed male rats were equal to or lower than those
 

of the controls from week 15 to the end of the bioassay.
 

Fluctuation in the growth curve may be due to mortality; as the
 

size of a group diminishes, the mean body weight may be subject
 

to variation. Wasting and alopecia occurred at low incidences in
 

some of the dosed groups and may have been related to
 

administration of the test chemical. Other clinical signs,
 

including corneal opacity and tissue masses, occurred at
 

comparable incidences in dosed and control groups.
 

B. Survival (Rats)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female rats administered piperonyl
 

21
 
450­ D

a 

A A 

§ 

5 

@8A 

A 

(9

ui QA 

,A 

>­ Z50­

Q 

O 

CO 

ZOOMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

O O 

D 

8 A O ° > 250- D Q a g o D A A n O

CO a g ft a a

a aa a ^> 200­

FEMALE RATS 

D MATCHED CONTROL 

"11 O LOW DOSE 

A HIGH DOSE 

SO 

TIME ON STUDY (WEEKS) 

Figure 1. Growth Curves for Rats Administered Piperonyl Sulfoxide in the Diet 

22 
sulfoxide in the diet at the doses of this bioassay, together
 

with those of the matched controls, are shown in figure 2. The
 

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

not significant in either sex.
 

In male rats, 39/50 (78%) of the high-dose group, 36/50 (72%) of
 

the low-dose group, and 13/20 (65%) of the control group were
 

alive at the end of the study. In females, 36/50 (72%) of the
 

high-dose group, 40/50 (80%) of the low-dose group, and 18/20
 

(90%) of the control group were alive at the end of the study.
 

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

development of late-appearing tumors.
 

C. Pathology (Rats)
 

Histopathologic findings on neoplasms in rats are summarized in
 

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

are summarized in Appendix C, tables Cl and C2.
 

Several inflammatory, degenerative, and proliferative lesions
 

commonly observed in aged Fischer 344 rats occurred with
 

approximately equal frequency in dosed and control rats. These
 

23
 
- o­ ­ A^ferH-^, 

i

A* '• ' A- T 

OL\, , 

I 6n 

> A­

) 

3 

D 

(O 

O

H 

050 _ 

m 

0 

oc 

0.20­

0.10­

A

O

[3

MALE RA TS 

 HIGH DOSt 

 LOW DOSE 

 MATCHED CCNTROI 

0­

0 15 30 45 60 75 90 IDS 

TIME ON STUDY (WEEKS) 

kk 

(j-~ TZ ~ TL 

&'• %. 

A-: 

\i 

URVIVAL

p = S - .

c 

CO 

O 

H 

.J 

3

CD 

0 

a. 

 0.40­

0.20­

0.10­

FEMALE R ATS 

A HIGH DOSE 

O LOW DOSE 

r3 MATCHED CC NTROL 

0­

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves for Rats Administered Piperonyl Sulfoxide in the Diet 

24 
lesions included multifocal alveolar macrophage aggregates in
 

lung parenchyma; chronic nephritis with scarring, tubular
 

dilatation, and tubular regeneration; ectasia of lymph sinuses in
 

the mesenteric lymph nodes; and testicular atrophy.
 

Other nonneoplastic proliferative lesions included hyperplasia of
 

the follicular cells and C cells of the thyroid, adrenal medulla
 

and cortex, parathyroid, islet cells of the pancreas, and
 

hepatocytes.
 

With regard to liver lesions, the term "focal hyperplasia" was
 

used in this study to indicate the presence of one or more foci
 

of hepatocytes with increased cytoplasmic basophilia and a slight
 

increase in the amount of nuclear chromatin. Many of these
 

hepatocytes also had a slight increase in nuclear : cytoplasmic
 

ratio when compared with adjacent normal hepatocytes, and,
 

infrequently, mitotic figures or hepatocytes with double nuclei
 

were observed. These foci of hyperbasophilic hepatocytes were
 

thought to represent areas of hyperplasia and were diagnosed as
 

such in this study. Compression of adjacent hepatic parenchyma
 

was minimal or absent. These lesions are similar morphologically
 

to those described by Squire and Levitt (1975) as "basophilic
 

foci."
 

25
 
Lesions classified as "hepatocytotnegaly" consisted of foci of
 

enlarged hepatocytes, many of which contained large, vesicular
 

nuclei and numerous fine cytoplasmic vacuoles, which gave the
 

cytoplasm a "ground glass" appearance. Distortion of lobular
 

architecture in these foci was minimal, and trabeculae were
 

continuous with adjacent normal hepatocytes. These lesions
 

correspond morphologically to those described by Squire and
 

Levitt (1975) as "eosinophilic foci," "ground glass foci," or
 

"clear cell foci."
 

The lesion in one high-dose and that in one low-dose male rat,
 

both classified as hepatocellular carcinomas, were present in
 

livers which were also diffusely infiltrated by neoplastic
 

lymphoreticular cells. In both of these liver sections, most
 

hepatocytes were larger than normal, with increased cytoplasmic
 

basophilia and large, vesicular nuclei. However, in both cases
 

there was a large, compressing nodule in which the hepatocytes
 

were even larger and more basophilic than the surrounding
 

parenchyma, with loss of normal trabecular architecture. Both of
 

these lesions were interpreted as malignancies of hepatocytes,
 

arising in livers that were undergoing a diffuse proliferative
 

process. The relationship of these lesions to the diffusely
 

infiltrating malignant lymphomas involving the same livers is not
 

known.
 

26
 
Endocrine tissues were the most frequent sites of neoplasms in
 

both dosed and control rats in this study. Interstitial-cell
 

tumors of the testis were observed in nearly all male rats in all
 

groups; a high spontaneous incidence of this tumor is
 

characteristic of aged Fischer 344 rats. Adenomas of the
 

pituitary were also found at a high incidence in all groups,
 

especially females. Other endocrine neoplasms observed included
 

follicular-cell and C-cell tumors of the thyroid, carcinomas of
 

the parathyroid, islet-cell tumors of the pancreas, and medullary
 

and cortical tumors of the adrenal.
 

In some proliferative endocrine lesions, differentiation between
 

benign and malignant neoplasms was difficult. C-cell lesions of
 

the thyroid were classified as adenomas when the proliferating C
 

cells were present in nodular masses that widely separated the
 

thyroid follicles and distorted the follicular architecture. In
 

some of the larger adenomas, the C cells were present in
 

interlacing bundles of elongated, spindling cells, rather than
 

the polyhedral to spherical shape characteristic of normal C
 

cells. When invasion of thyroid capsule, adjacent tissues, or
 

vessels was present, or when metastasis was detected, the lesion
 

was classified as C-cell carcinoma.
 

Follicular-cell neoplasms occurred less frequently than C-cell
 

27
 
neoplasms. The follicular-cell adenomas appeared microscopically
 

as well-circumscribed masses composed of enlarged follicles lined
 

by hyperbasophilic follicular cells which were increased in
 

number per unit area by papillary infolding of simple cuboidal or
 

columnar epithelium into the follicular lumen and by stratification of follicular cells surrounding the lumen. Distinct
 

compression of adjacent normal thyroid parenchyma, with some
 

evidence of fibrous encapsulation, was present. Follicular-cell
 

lesions were classified as carcinoma based upon the presence of
 

anaplasia and histologic arrangement in disorderly nests and/or
 

sheets. Areas with papillary patterns were also present.
 

Fibrous stroma often intermingled with, but did not encapsulate,
 

follicular-cell carcinomas.
 

Two adrenal tumors were composed of neural tissue and
 

microscopically resembled tumors described in the literature as
 

"ganglioneuromas" (Todd et al., 1970). One of the neoplasms also
 

had a component of small, basophilic cells reminiscent of
 

neuroblastoma. This neoplasm was classified as a
 

ganglioneuroblastoma.
 

The diagnosis of pheochromocytoma was made when the adrenal
 

medullary lesion was present as a discrete mass that compressed
 

adjacent normal adrenal parenchyma. These neoplasms were
 

28
 
composed of sheets, nests, and/or cords of polyhedral to
 

spherical cells with abundant, slightly basophilic cytoplasm and
 

large nuclei with abundant chromatin. Islet-cell adenomas
 

appeared as discrete, encapsulated nodules of islet cells that
 

compressed the adjacent normal pancreas.
 

The malignant lymphomas are summarized below. The majority of
 

the malignant lymphomas were composed of undifferentiated
 

lymphoreticular cells. The spleen was the organ most frequently
 

involved and most severely affected by this neoplasm; the red
 

pulp and marginal zones of the white pulp were filled with
 

undifferentiated lymphoreticular cells and splenomegaly was often
 

detected grossly. The liver and lung also were frequently
 

involved with large numbers of neoplastic lymphoreticular cells
 

present in the hepatic sinusoids and alveolar septa. Involvement
 

of peripheral blood, lymph nodes, and other tissues was observed
 

in some cases. There were also animals in which increased
 

numbers of undifferentiated lymphoreticular cells were present in
 

the spleen, liver, and/or blood smear, but the degree of change
 

was not sufficient for diagnosis of malignant lymphoma. These
 

lesions were classified as lymphoid hyperplasia. The incidence
 

of malignant lymphoma and lymphoid hyperplasia is summarized
 

below.
 

29
 
Males Females 

Low High Low High 

Control Dose Dose Control Dose Dose 

Number of animals
 

necropsied 20 50 48 20 50
 50
 

Malignant Lymphoma 2 12 4 2 512
 

Lymphoid Hyperplasia 3 0 4 2 1 4
 

The increased incidence of malignant lymphoma in the dosed rats
 

when compared with controls is difficult to evaluate. This is a
 

frequently occurring neoplasm in aged Fischer 344 rats (Davey and
 

Moloney, 1970; Moloney et al., 1970; Sass et al., 1975), and the
 

incidence observed in test animals in this study does not exceed
 

that observed in control groups of Fischer 344 rats from several
 

other bioassay studies evaluated in this laboratory. Further,
 

the average age at which the neoplasm was observed is not
 

strikingly different in dosed and control rats in this study
 

(mean age of rats with malignant lymphoma at necropsy was as
 

follows: control males, 104 weeks; low-dose males, 106 weeks;
 

high-dose males, 92 weeks; control females, 93 weeks; low-dose
 

females, 107 weeks; and high-dose females, 100 weeks). For these
 

reasons, the increased incidence of lymphomas in the dosed rats
 

in this study was not considered to be sufficient evidence for
 

classifying the compound as a carcinogen.
 

Proliferative pulmonary lesions were observed rather infrequently
 

30
 
in both dosed and control groups. Differentiation between
 

adenomas and carcinomas was based on degree of anaplasia, mitotic
 

index, size of the neoplasm, and presence of apparent invasion of
 

adjacent pulmonary parenchyma in carcinomas, as opposed to mere
 

compression of adjacent parenchyma, and thus, a more discrete
 

lesion in adenomas.
 

The most frequently occurring neoplasm of the reproductive tract,
 

other than the previously mentioned interstitial-cell tumor of
 

the testis, was the endometrial stromal polyp of the uterus
 

(controls 0/20, low-dose 5/50, high-dose 3/47). This lesion was
 

present as a discrete mass protruding into the lumen of the
 

uterus, lined by endometrium, and sometimes associated with
 

suppurative endometritis and/or cystic endometrial hyperplasia.
 

The stroma was usually proliferating in a rather loosely woven
 

pattern, with numerous small vessels interspersed among stromal
 

cells. Although this lesion was observed only in dosed animals
 

in this study, it is a frequently occurring spontaneous tumor in
 

aged female Fischer 344 rats (Davey and Moloney, 1970) and was
 

not considered to have been induced by the test chemical.
 

In addition to the stromal polyps, two malignant neoplasms were
 

observed in the uterus, one an endometrial stromal sarcoma and
 

the other an adenocarcinoma.
 

31
 
Four carcinomas of the preputial gland were observed in low-dose
 

male rats, and one carcinoma of the clitoral gland was observed
 

in a control female.
 

The most common neoplasms of the mammary gland were fibroadenomas; these occurred in both males and females, were often
 

multiple, and were seen in both dosed and control groups.
 

In the transitional epithelium of the urinary tract, one neoplasm
 

was observed, a well-differentiated transitional-cell carcinoma
 

of the urinary bladder in a high-dose male rat. This tumor
 

consisted of a papillary mass lined by thickened transitional
 

epithelium in which some cells had hyperchromatic nuclei. The
 

supporting stroma also contained numerous large cells with large
 

vesicular nuclei, which were interpreted as neoplastic cells.
 

There was no normal bladder tissue attached to the neoplasm; thus
 

it was impossible to determine if invasion of adjacent normal
 

bladder submucosa was present. Hyperplasia of transitional
 

epithelium of the renal pelvis, bladder, and urethra were
 

observed infrequently in both dosed and control rats.
 

Various other types of malignant and benign neoplasms were
 

observed at low incidences in sections of skin and subcutis, bone
 

marrow, and in other organs and tissues throughout the body. No
 

32
 
apparent difference in incidence of these neoplasms between dosed
 N
 

and control groups was present.
 

Based on the histopathologic examination, there were instances in
 

this study where neoplastic or hyperplastic lesions occurred only
 

in dosed animals, or with increased frequency in dosed animals
 

when compared with control groups. However, the nature,
 

incidence, and severity of the lesions observed provide no clear
 

evidence of carcinogenic effect of piperonyl sulfoxide in Fischer
 

344 rats under the conditions of this bioassay.
 

D. Statistical Analyses of Results (Rats)
 

Tables El and E2 in Appendix E contain the statistical analyses
 

of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

The results of the Cochran-Armitage test for positive
 

dose-related trend in incidences of tumors and those of the
 

Fisher exact test comparing the incidences of tumors in the dosed
 

groups with those in the control groups in the positive direction
 

are not significant in either sex.
 

33
 
Significant results in the negative direction are observed in the ,
 

incidences of adenoma of the pituitary and of astrocytoma of the
 

brain in male rats and in the incidence of fibroadenoma of the
 

mammary gland in female rats; the incidences of these tumors in
 

the control groups exceed those in the dosed groups.
 

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

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

this indicates the absence of significant positive results. It
 

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

the incidence of fibroadenoma of the mammary gland in female
 

rats) has an upper limit greater than one, indicating the
 

theoretical possibility of the induction of tumors by piperonyl
 

sulfoxide, which could not be detected under the conditions of
 

this test.
 
IV. RESULTS - MICE
 

A. Body Weights and Clinical Signs (Mice)
 

Mean body weights of both dosed male and dosed female mice were
 

lower than those of corresponding matched controls, and the
 

depressions in the amount of mean body weight gained were dose
 

related throughout the bioassay (figure 3). In comparison with
 

their respective controls, the depressions in the amount of mean
 

body weight gained were greater for the dosed females than for
 

the dosed males. Fluctuation in the growth curve may be due to
 

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

weight may be subject to variation. Alopecia occurred at low
 

incidences in most of the dosed groups, and may have been related
 

to administration of the test chemical. Corneal opacity occurred
 

only in one low-dose male mouse. Tissue masses occurred at
 

comparable incidences in dosed and control groups.
 

B. Survival (Mice)
 

The Kaplan and Meier curves estimating the probabilities of
 

survival for male and female mice administered piperonyl
 

35
 
s s ; " 8 

A A 

40- D g A 

I­

(9 

i 

Ul 

>. 30- 0

38 Q 

8 

I J 

E 20} 

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 50 60 70 80 90 100 110 

TIME ON STUDY (WEEKS) 

a o 

o o o o 

ODD Dotes lowered 

for female mice 

at week 20. 

5 40 

S 

> 30 

Q

O

IO 

5 20­

FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

10 20 30 40 BO 70 80 90 100 110 

TIME ON STUDY (WEEKS) 

Figure 3. Growth Curves for Mice Administered Piperonyl Sulfoxide in the Diet 

36 
sulfoxide in the diet at the doses of this bioassay, together
 

with those of the matched controls, are shown in figure 4. The
 

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

not significant in either sex.
 

In male mice, 44/50 (88%) of the high-dose group, 43/50 (86%) of
 

the low-dose group, and 13/20 (65%) of the control group were
 

alive at week 104. In females, 41/50 (82%) of the high-dose
 

group, 40/50 (80%) of the low-dose group, and 16/20 (80%) of the
 

control group were alive at week 104.
 

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

development of late-appearing tumors.
 

C. Pathology (Mice)
 

Histopathologic findings on neoplasms in mice are summarized in
 

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

are summarized in Appendix D, tables Dl and D2.
 

The most common neoplasm in male mice was hepatocellular
 

carcinoma; the incidence was increased in relation to the doses
 

of piperonyl sulfoxide:
 

37
 
45 60 75 

TIME ON STUDY (WEEKS) 

0­ - — TjJn j. 0.90­

%^ 1 A; 

ito— :-c ] 

> 

CO 

o 

I­

< 0.40­

m 

o 

tc 

FEMALE VI ICE 

0.20­ A HIGH DOSE

 

O LOW DOSE 

0.10­ c3 MATCHED CC NTBni 

n- — , 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves for Mice Administered Piperonyl Sulfoxide in the Diet 

38 
Control Males Low-Dose Males High-Dose Males
 

6/18 (33%) 31/50 (62%) 46/50 (92%)
 

Metastases to the lungs occurred in one control male, four
 

low-dose males, and one high-dose male. Hepatocellular
 

carcinomas were also found in 1/19 (5%) control females, 3/50
 

(6%) low-dose females, and 6/50 (12%) high-dose females.
 

The hepatocellular carcinomas had a wide morphologic spectrum,
 

varying from moderately well to very poorly differentiated. Some
 

occurred as relatively small expanding and invasive nodules;
 

others were large masses involving most of the liver.
 

The next most common tumor was malignant lymphoma. The incidence
 

was approximately equal in males and females and slightly higher
 

in controls than in dosed mice. Many of these tumors were widely
 

disseminated to involve several organs and form tissue masses.
 

They accounted for most of the premature deaths.
 

Tumors of the lung (adenocarcinomas and alveolar-cell adenomas)
 

were found in 22% of the control mice, 12% of the low-dose mice,
 

and 9% of the high-dose mice.
 

Most other proliferative or neoplastic lesions were of single
 

39
 
occurrence or very low incidence. Carcinomas of the pancreatic
 

islets were found in a single high-dose male and single high-dose
 

female. There were metastases of the tumor to the lungs and
 

liver in the male. An osteosarcoma of the femur with metastases
 

to the lung was present in a low-dose female.
 

In addition to the proliferative lesions, there was a scattering
 

of inflammatory and degenerative changes in some mice in each
 

group. These included focal mineralization of the brain,
 

myocarditis, focal hepatic necrosis, cystic pancreatic ducts,
 

ovarian cysts, and cystic endometrium. There was no apparent
 

difference in the incidence of these changes among the three
 

groups.
 

In conclusion, based on the histopathologic examination, the
 

incidence of hepatocellular carcinomas in male B6C3F1 mice is
 

associated with the administration of the test chemical under the
 

conditions of this bioassay.
 

D. Statistical Analyses of Results (Mice)
 

Tables Fl and F2 in Appendix F contain the statistical analyses
 

40
 
of the incidences of those primary tumors that occurred in at
 

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

in one or more than one group.
 

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

positive dose-related trend in the incidence of hepatocellular
 

carcinoma is significant (P less than 0.001) and the result of
 

the Fisher exact test comparing the incidence of this tumor in
 

the high-dose group with that in the control group is also
 

significant (P less than 0.001). The Fisher exact comparison of
 

the incidence in the low-dose group with that in the control
 

group indicates a P value of 0.034, which is above the 0.025
 

level required for significance when the Bonferroni inequality
 

criterion is used for multiple comparison. The statistical 

conclusion is that the incidence of hepatocellular carcinoma in 

male mice is associated with the administration of piperonyl 

sulfoxide.
 

In females, the results of the Cochran-Armitage test and of the
 

Fisher exact test are not significant. A significant trend in
 

the negative direction is observed in the incidence of lymphoma
 

in male mice. The incidence of this tumor in the control group
 

exceeds the incidences in the dosed groups.
 

41
 
42

 
DISCUSSION
 

Dose-related depressions in the amount of mean body weight gained
 

occurred in both rats and mice administered technical-grade
 

piperonyl sulfoxide under the conditions of the bioassay; the
 

depression in the amount of mean body weight gained was slight,
 

however, in the male rats. Low incidences of wasting and
 

alopecia among the dosed rats and of alopecia among the dosed
 

mice may have been related to administration of the test
 

chemical. Survival of the rats and mice was unaffected by the
 

piperonyl sulfoxide and was 78% or higher in all groups at week
 

90 of the bioassay; thus, sufficient numbers of dosed and control
 

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

late-appearing tumors.
 

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

occurred at incidences that were significantly higher in dosed
 

groups than in control groups.
 

In the male mice, hepatocellular carcinomas occurred at
 

incidences that were dose related (P less than 0.001); in direct
 

comparisons, the incidence of the tumors in the high-dose group
 

was significantly higher (P less than 0.001) than that in the
 

43
 
control group (controls 6/18 or 33%, low-dose 31/50 or 62%,
 

high-dose 46/50 or 92%).
 

The acute oral LD,-n of piperonyl sulfoxide for rats (strain not
 

specified) has been reported as 2 g/kg (NIOSH, 1976) and for mice
 

(strain not specified) as 12 g/kg (Suspected Carcinogens, 1976).
 

In tests for tumorigenicity (NTIS, 1968; Innes et al., 1969), it
 

was reported that when the chemical was administered at 46.4
 

mg/kg by stomach tube for 3 weeks, then in the diet at 111 ppm
 

for 18 months to hybrid mice (C57BL/6 x C3H/Anf and C57BL/6 x
 

AKR), an elevated incidence of reticulum-cell sarcomas (P = 0.01)
 

was observed, but additional evaluation was proposed. Certain
 

structural congeners of piperonyl sulfoxide (safrole, isosafrole,
 

and dihydrosafrole) have been reported to be carcinogenic in rats
 

(Osborne-Mende1) and mice (C57BL/6 x C3H/Anf and C57BL/6 x AKR),
 

inducing tumors of the liver, esophagus, or lung, depending on
 

the species and sex (Long et al., 1963; Hagan et al., 1965; Innes
 

et al., 1969).
 

Piperonyl sulfoxide is used commercially with pyrethrins. This
 

bioassay, however, tests the carcinogenicity of commercial
 

technical-grade piperonyl sulfoxide alone, and no conclusions can
 

be drawn from the data in this report as to the possible
 

44
 
carcinogenic effects of the combination of the test compound with
 

pyrethrins.
 

It is concluded that under the conditions of this bioassay,
 

technical-grade piperonyl sulfoxide was not carcinogenic for male
 

or female Fischer 344 rats or for female B6C3F1 mice, but was
 

carcinogenic for male B6C3F1 mice, producing an increased
 

incidence of hepatocellular carcinomas.
 

45
 
46

 
VI. BIBLIOGRAPHY
 

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

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

Berenblum, I., ed., Carcinogenicity Testing; A Report of the
 

Panel £f Carcinogenicity of the Cancer Research Commission of
 

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

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

Soc. B34;187-220, 1972.
 

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

London, 1970, pp. 48-52.
 

Davey, F. R. and Moloney, W. C., Postmortem observations on
 

Fischer rats with leukemia and other disorders. Lab. Invest.
 

23:327-334, 1970.
 

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

significance tests, confidence limits and adjustments for
 

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

Hagan, E. C., Jenner, P. M., Jones, W. I., Fitzhugh, 0. G.,
 

Long, E. L., Brouwer, J. G., and Webb, W. K., Toxic properties
 

of compounds related to safrole. Toxicol. Appl. Pharmacol.
 

2:18-24, 1965.
 

Innes, J. R. M., Ulland, B. M., Valeric, M. G., Petrucelli, L.,
 

Fishbein, L., Hart, E. R., Pallotta, A. J., Bates, R. R., Falk,
 

H. L., Gart, J. J., Klein, M., Mitchell, I., and Peters, J.,
 

Bioassay of pesticides and industrial chemicals for
 

tumorigenicity in mice: a preliminary note. J_._ Natl Cancer
 

Inst. 4:2(6): 1101-1106, 1969.
 

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

incomplete observations. J. Amer. Statist. Assoc. 53:457-481,
 

1958. ~
 

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

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

Biomed. Res. 7^:230-248, 1974.
 

Long, E. L., Nelson, A. A., Fitzhugh, 0. G., and Hansen, W. H.,
 

Liver tumors produced in rats by feeding safrole. Arch Path
 

7^:595-604, 1963.
 

47
 
Metcalf, R. L., Insecticides. In: Kirk-Othmer Encyclopedia £f_
 

Chemical Technology, Vol. 11, Interscience Publishers, New
 

York, 1966, pp. 685-686"!
 

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

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

Moloney, W. C., Boschetti, A. E., and King, V. P., Spontaneous
 

leukemia in Fischer rats. Cancer Res. 30:41-43, 1970.
 

National Institute for Occupational Safety and Health, Registry
 

£f Toxic Effects £f Chemical Substances, Rockville, Maryland,
 

1976, p. 170.
 

National Technical Information Service, Evaluation of
 

Carcinogenic, Teratogenic, and Mutagenic Acitvities £f Selected
 

Pesticides and Industrial Chemicals, Vol. I. Carcinogenic
 

Study, U."S. Department of Commerce, PB-223 159~ August, 1968,
 

p. 64.
 

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

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

induced by different numbers of administrations of benzo(a)
 

pyrene and ferric oxide. Cancer Res. 3_2:1073-1081, 1972.
 

Sass, B., Rabstein, L. S., Madison, R., Nims, R. M., Peters, R.
 

L., and Kelloff, G. J., Incidence of spontaneous neoplasms in
 

F344 rats throughout the natural life-span. J. Natl Cancer
 

Inst. 54:1449-1456, 1975.
 

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

classification of specific hepatocelluar lesions in rats.
 

Cancer Res. 35_: 3214-3223, 1975.
 

Suspected Carcinogens, 2nd Edition. A Subfile of the NIOSH
 

Registry of Toxic Effects of Chemical Substances, National
 

Institute for Occupational Safety and Health, Cincinnati, Ohio,
 

1976, p. 36.
 

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

Biometrika 62:679-682, 1975.
 

Todd, G. C., Pierce, E. C., and Clevinger, W. G.,
 

Ganglioneuroma of the adrenal medulla in rats. Pathologia
 

Veterinaria 7:139-144, 1970.
 
United States International Trade Commission, Pesticides and
 

related products. Synthetic Organic Chemicals - United States
 

Production and Sales, 1976, USITC Publication833, United
 

States International Trade Commission, Washington, B.C., 1977.
 

49
 
50

 
APPENDIX A
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

RATS ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

51
 
52

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

ANIMAL S MISSING 2 

ANTBAL S NECEOPSIED 20 50 08 

ANIMAL S EXAMINE D HISTOPATHOLOGICiLLY 20 50 48 

INTEGUMENTAR Y SYSTEM 

*SKIN (20) (50) (48) 

TRICHOFPITHELION A 1 <2X) 1 (2X) 

KEBATOACANTHOM A 1 (5X) 

*SUBCUT TISSUE (20) (50) (48)
 

FIBFOMA 2 (4X)<- 1 (2*)
 

FIBROSARCOMA 2 (4*)
 

OSTEOSARCOMA, METASTATIC 1 (2X)
 

NEURILEMOMA 1 (2X)
 

RESPIRATOR Y SYSTE M 

#LUN G (20) (50) (48) 

SARCOMA, NOS, METASTATIC 1 (2*) 

OSTSOSAPCOMA, HETASTATIC 

HEMATOPOIETIC SYSTEM 

*MULTIPLF ORGAN S (20) (50) 

MAtlG.LYMPHOHA, UNDIFFER-TYPE 2 (10%) 11 (22X) 

MALIG.LYMPHONA , LYBPHOCYTIC TYPE 1 (2X) 

*BONE MARRO W (20) (50) 

SARCOMA , NOS 

CIRCULATORY SYSTEM
 

*HEAPT (20) (50) (18)
 

# NUMB5F OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS N3CROPSIED
 

<- MULTIPLE OCCURRENCE OF MORPHOLOGY IN THE SAME ORGAN TISSUES IS COUNTED ONCE ONLY
 

53 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE 

DIGESTIVE SYSTE M 

OLIVER (20 ) (50) 

HEPATOCELLULA R CARCINOM A 1 (2%) 

SARCOMA , NOS , METASTATIC 

URINAR Y SYSTE M 

IKIDNF Y (20) (50)

 

SARCOMA , NOS , METASTATIC 

tURINAR Y BLADDE R (18) (47 )

 

TRANSITIONAL-CELL CARCINOM A 

ENEOCRIN E SYSTE M 

#PITUITAR Y (18) (45 ) 

ADENOMA , NO S 6 (33% ) 4 (9SS) 

#ADRENA L (20) (50) 

PHEOCHPOMOCYTOM A 1 (5% ) 3 (6%) 

SARCOMA , NOS , METASTATIC 

#THYROI D (20) (49) 

FOLLTCULAR-CEL L ADENOM A 

FOLLICULAR-CEL L CARCINOM A 1 (5%) 

C-CELL ADENOM A 2 (10% ) 3 (6$) 

((PARATHYROI D (18) (43 ) 

CARCINOMA , NOS 1 (2% ) 

tPANCREATT C ISLETS (19 ) (50) 

ISLET-CELL ADENOM A 2 (11% ) 2 (4% ) 

REPRODUCTIV E SYSTE M 

*MAMMAP Y GLAN D (20) (50) 

FIBROM A 1 (2%) 

FIBPOADENOM A 1 (5%) 1 (2%) 

*PREPUTIA L GLAN D (20 ) (50 ) 

CARCTNOMAj,NO S 4 ±d%L 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 * NOHBEF OF ANIMALS N5CPOPSIED
 

HIGH D 

(<»7) 

1 

1 

(<*8) 

1 

(46) 

1 

(44 ) 

4 

(48) 

3 

1 

(48) 

1 

3 

C*6) 

(47 ) 

3 

048) 

1 

(«8) 

OSE 

(2%) 

(2*) 

(2«) 

(21) 

(9%) 

(6%) 

(2*) 

(2% ) 

(6% ) 

(6%) 

(2%) 

54
 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

OTFSTIS (20) (50) (48) 

INTERSTITIAL-CEL L TUHOR 16 (80%) U6 (92S) 43 (99%) 

NERVOU S SYSTEM 

SEP.AIN (20) (50) (<*8) 

GLIOtU, NOS 1 (2X) 

ASTROCYTOM A 2 (10%) 

SPECIA L SENS E ORGAN S 

*ZYMBAL' S GLAN D (20) (50) (48) 

SQUAMOU S CELL CARCINOM A 1 (5X) 

BOSCULOSKELETAL SYSTEM 

*VERTEBRA (20) (50) (48)
 

OSTEOSAPCOMA 1 (2*)
 

*SKFLETAL MUSCLE (20) (50) (48)
 

OSTEOSARCOMA, METASTATIC 1 (2X)
 

BOCY CAVITIES
 

*PERITONEU « (20) (50) (48) 

MESOTH^LIOMA , NOS 1 (2«) ^ ~f —-,____ . — — —— —— — — __ v— *_ ~••*. _ «__ _ •_ ^_• — _.««.___ *•,___• — — — — TMTM^— — — — ——— —^ — — —» — ••—— — — — — ••*• 

A LL OTHER SYSTEMS 

NONE 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

NATURA L DEATHS 4 8 5 

MORIBUN D SACRIFICE 3 6 4 

SCHEDULED SACRIFICE 

ACCIDENTALL Y KILLED 

TERMINA L SACRIFICE 13 36 39 

ANIMA L MISSING 2 

# NUMBE R OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S HECSOPSIBD 

55 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMO R SUHHAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS* 19 U8 U6 

TOTAL PRIMARY TUMORS 36 85 70 

TOTAL ANIMALS WITH BENIGN TUMORS 18 <»7 UH 

TOTAL BENIGN TUMORS 29 64 61 

TOTAL ANIMALS WITH MALIGNANT TUMORS 7 19 6 

TOTAL MALIGNAN T TUMORS 7 21 8 

TOTAL ANIMAL S WITH SECONDARY TUHORSt 2 

TOTAL SECONDARY TUMORS 7 

TOTAL ANIMAL S HITH TUMORS UNCERTAINBENIGN OR MALIGNANT 1 

TOTAL UNCERTAIN TUMORS 1 

TOTAL ANIMAL S WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMOBS: ALL TUMORS EXCEPT SECONDARY TUMORS 

* SECONDARY TUMORS: HETASTATIC TUMORS OR TUMORS INVASIVE INTO AN iDJlCSHT ORGAN 

56 
TABLE A2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

ANIMAL S INITIALL Y IN STUDY

ANIMAL S NT1CROPSIED

ANIMAL S EXAMINE D HISTOPATHOLOGICALL Y

INTEGUMENTAR Y SYSTEM 

*SKIN

TRICHOEPITHELIOB A

*SUBCUT TISSUE

FIBROM A

FIBROSARCOB A

RESPIRATORY SYSTEM 

#LUN G

ALVEOLAR/BRONCHIOLA R ADENOM A

ALVEOLAR/BRONCHIOLA B CARCINOM A

C-CELL CARCINOMA, METASTATIC

HEMATOPOIETI C SYSTEM 

*«ULTIPLE ORGAN S

MALIGNAN T LYMPHOMA , NOS

MALIG.LYMPHOMA , UNDIFFER-TYPE

fSPLEEN

MALIGNAN T LYMPHOMA , NOS

#CERVICA L LYMP H NODE

C-CELL CARCINOMA , HETASTATIC

CIPCULATOR Y SYSTEM 

NOVE 

DIGESTIV E SYSTEM 

NONF 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 20 50 50 

 20 50 50 

 20 50 50 

 (20) (50) (50) 

1 (2%) 

 (20) (50) (50) 

1 (2%) 

1 (2%) 

 (20) (50) (50) 

2 (10% ) 

1 (2X) 

1 (2%) 

 (20) (50) (50) 

1 (5%) 2 (««) 

5 (10*) 10 (20%) 

 (20) (U9) (50) 

1 (5%) 

 (19) (50) (50) 

1 (2%) 

t NUMBE R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S N^CROPSIED 

57 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

UFINAR Y SYSTEM 

NON E 

ENDOCRINE SYSTEM 

#PITUITAR Y (20) (50) (48) 

ADENOMA , NOS 7 (35%) 7 (14%) 12 (25%) 

#ADRFNA L (20) (50) (50) 

COPTICAL ADENOH A 1 (2%) 

GANGLIONEURON A 1 (5%) 

GANGLIONEUROBLASTON A 1 (2*) 

•THYROID (19) (50) (50) 

FOLLTCULAR-CEL L ADENOM A 1 (2X) 

FOLLICOLAR-CEL L CARCINOM A 1 (2%) 

C-CELL ADENOM A 

C-CELL CARCINOM A 

#PANCREATI C ISLETS 

ISLET-CELL ADENOM A 

REPRODUCTIVE SYSTEM 

*MAWMAR Y GLAN D 

FIBROADENOM A 

*CLITOEAL GLAN D 

CARCINOMA , NOS 

*VAGINA 

ENDOMETRIA L STROMAL

• UTERU S 

4 (8%) 

1 (2%) 1 (2*) 

(20) (i»7) (47) 

1 (5%) 1 (2%) 

(20) (50) (50) 

U (20%) 1 (2%) 

(20) (50) (50) 

1 (5% ) 

(20) (50) (50) 

 SARCOMA , IN V 1 (2X) 

(20 ) (50) (U7 ) 

^NDOMKTRIAL STROMAL POLYP 5 (10%) 3 (6S) 

SNDOnETRIAL STROMAL SARCOMA 1 (2%) 

SCERVIX UTERI (20) (50) (47)

 

STROMA L POLY P 1 (2%)

 

#UTERUS/ENDOMETRIU M (20) (50) (47)

 

ADENOCAfCINOMA x _NO S 1_J2%1 

* NUMBE R O F ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBE R OF ANIMAL S NECROPSIE D 

58 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NERVOU S SYSTE M 

SPECIAL SENSE ORGANS 

NCN 5 

MUSCOLOSKFLETA L SYSTEM 

*WtJSCL E OF NEC K

C-CELL CARCINOMA , INVASIVE

 (20) (50) (50) 

1 (2%) 

BOD Y CAVITIE S 

NONE 

A L L OTHE R

NONE 

 SYSTEM S 

ANIMA L DISPOSITION SUMMAR Y 

ANI1AL S INITIALL Y IN STUD Y

NATURA L DEATHS

MORIBUN D SACRIFICE

SCHEDULED SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINA L SACRIFICE

ANIMA L MISSIN G 

JL INCLUDES_AUTOLYZED_ANIMALS 

 20

 2 

 18

 50

6 

«

 HO

 50 

8 

6 

 36 

# NUMBE R

* NUMBE R

 OF ANIMAL S

 OF ANIMAL S

 KITH TISSUE

 NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

59 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMO R SUMNAE Y 

TOTAL ANIMAL S WITH PRIMAR Y TOHORS» 12 2U 27 

TOTAL PRIMAR Y TUMORS 18 29 3H 

TOTAL ANIMAL S WITH BENIGN TUHOHS 11 18 16 

TOTAL BENIGN TUMORS 15 20 18 

TOTAL ANIMAL S WITH MALIGNAN T TUMORS 2 8 15 

TOTAL MALIGNAN T TUHORS 3 9 16 

TOTAL ANIMAL S WITH SECONDARY TUMORS* 2 

TOTAL SECONDAR Y TUMORS <t 

TOTAL ANIMAL S WITH TUMORS UNCERTAINBENIGN OR MALIGNAN T

 

TOTAL UNCERTAIN TUMORS 

TOTAL ANIMAL S WITH TUHOHS ONCERTAINPRIMAR Y OR MET&STATIC

 

TOTAL UNCERTAIN TUMORS 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

* SECONDARY TUMORS: METASTATIC TOMORS OR TUHORS IH?ASIYE INTO AN ADJACENT ORGAN 

60 
APPENDIX B
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 

MICE ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

61
 
62

 
TABLE B1. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

" " 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

50 

ANIMAL S MISSIN G 1 

ANIMAL S NECPOPSIED 18 50 50 

ANIMALS EXAMINE D HIS! ^PATHOLOGICALLY 18 50 50 

ANIMAL S INITIALL Y IN STUDY 20 50 

INTEGUMENTAR Y SYSTEM 

RESPIRATOR Y SYSTE M 

#LUN G (18) (50) (50) 

ISLET-CELL CARCINOMA , METASTATIC 1 (2*) 

HEPATOCELLULAR CARCINOMA, METAST 1 (6%) 4 (8X) 1 (2») 

ALVEOLAR/BRONCHIOLA R ADENOM A 3 (17% ) 6 (12X) 3 (6*) 

ALVEOLAR/BRONCHIOLA R CARCINOM A 3 (17% ) 14 (8%) 4 (8*) 

HEMATOPOIETIC SYSTEM 

*MULTIPL E ORGAN S (18) (50) (50) 

MALIGNAN T LYMPHOMA , NOS 3 (17% ) 5 (10%) 2 (<»*) 

MALIG.LYMPHOMA , HISTIOCYTIC TYPE 1 (2%) 1 (2X) 

*BLOOD (18) (50) (50) 

LYMPHOCYTIC LEUKEMIA 1 (6%) 

#MFSENTERIC L. NODE (18) (l*9) (50) 

ISLFT-CELL CARCINOMA , METASTATIC 1 (2*) 

HEPATOCELLULA R CARCINOMA , METAST 1 (2%) 

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

tSMAL L INTESTIN E (18) (1*9) (50) 

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

#THYMUS (17) (U9) (US)
 

HEPATOCELLULAR CARCINOMA, METAST 1 (2%)
 

CIRCULATORY SYSTEM
 

__NONE
 

# NUMBER OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

63
 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIV E SYSTEM 

#LIVE F

ISLFT-CSLL CARCINOHA , METASTATIC

HEPATOCELLULA R CARCINOM A

HF.MANGIOSARCOP A

 (18)

 6 (33%)

 (50)

 31

 1

 (62%)

 (2%)

 (50) 

1 (2%) 

 «6 (92%) 

3 (6%) 

UFINAF Y SYSTE M 

NON E 

ENDOCRIN E SYSTEM 

•PITUITAR Y

ADENOMA , NOS

tTHYP.OID

FOLLICULAR-CEL L ADBNOH'A

•PANCREATI C ISLETS

ISLET-CFLL CARCINOMA

 (16)

 1

 (17)

 (17)

 (6«) 

 (50)

 (t6)

 1 (2%) 

 («7)

 (49) 

 (50) 

 (50) 

1 (2%) 

REPRODUCTIV E

NON E 

 SYSTE M 

NEFVOU S SYSTEM 

NON F 

SPECIA L SENS E ORGAN S 

*EYE/LACRIMA L GLAN D

ADENOMA , NOS

 (18)

 1 (6% )

 (50) (50) 

3 (6%) 

MUSCULOSKELETA L SYSTEM 

NONE . 

• NUMBE R

* NUMBE R

 OF ANIMAL S 9ITH TISSUE

 O F ANIMAL S NECROPSIED 

 EXAMINE D MICROSCOPICALL Y 

64 
TABLE 81. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

BODY CAVITIE S 

*ABDONINA I CAVIT Y (18) (50) (50) 

NT-UPOFIBROSAHCOM A 1 (2*) 

A L L OTHER SYSTEM S 

SI^E UNKNOW N 

LIPOK A 3

 

ANIMA L DISPOSITION SUMMAR Y 

ANTMAL S INITIALL Y IN STUD Y 2 0 50 50

 

NATUPA L DEATH S 6 7 6

 

MORIBUN D SACRIFICE 1 

SCHEDULE D SACRIFICE 

ACCIDENTALL Y KILLE D 

TERMINAL SACRIFICE 13 13 H3
 ANIMA L MISSING 1
 

3) INCLUDES AUTOLYZED ANIMALS
 

TUMOR SUMMAR Y
 

TOTAL ANIMALS WITH PRIBARY TUMORS* 1<» 39 47
 

TOTAL PRIMARY TUMORS 20 55 63
 

TOTAL ANIMALS WITH BENIGN TUMORS 5 9 6
 

TOTAL BENIGN TUMORS 5 10 6
 

TOTAL ANIMALS WITH MALIGNANT TUMORS 12 38 «7
 

TOTAL MALIGNANT TUMORS 15 i»5 57
 

TOTA L ANIMAL S WITH SECONDARY TUMORS* 1 "» 2 

TOTAL SECONDAR Y TUMORS 1 5 5 

TOTAL ANIMAL S WITH TUHOHS UNCERTAINBENIG N OF MALIGNAN T

 

TOTAL UNCERTAI N TUMORS

 

TOTAL ANIMAL S WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC

 

TOTAL UNCERTAI N TOMORS 

* PRIMAR Y TUMORS : ALL TUHOfiS EXCEPT SECONDARY TUMORS 

* SECONDAR Y TUMORS : METASTATIC TUMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

65 
TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

50 

ANIMAL S MISSING 1 

ANIMAL S NECHOPSIE D 19 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 19 50 50 

ANIMAL S INITIALL Y IN STUDY 20 50 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATORY SYSTEM
 

fLUNG (18) (48) («9)
 

ALVEOLAR/BRONCHIOLAB ADEHOHA 1 (6%) 1 (2X)
 

ALVEOLAR/BRONCHIOLAR CARCINOMA 1 (6%) 2 <«%) 1 (2X)
 

HEMATOPOIETIC SYSTEM
 

*MULTIPLE ORGANS (19) (50) (50)
 

MALIGNANT LYMPHOMA, NOS 4 (21 X) 10 (20%) 5 (10%
 

MALIG. LYMPHOHA, HISTIOCYTIC TYPE 2 {<»%)
 

*BLOOD (19) (50) (50)
 

LYMPHOCYTIC LEUKEHIA 1 (5%)
 

*BONE MARROW (18) (50) («J8)
 

OSTEOSARCOHA 1 (2%)
 

#SPLEEN (18) (50) (48)
 

MALIGNANT LYMPHOBA, HOS 1 (2%) 1 (2%)
 

fLIVER (18) (50) (l»9)
 

MALIGNANT LYMPHOHA, HOS 1 (6*) 1 (2%)
 

•SHALL INTESTINE (18) (49) (47)
 

MALIGNANT LYMPHOHA, HOS 1 (2%)
 

#THYMOS (17) (43) (47)
 

MALIGNAHT LYMPHOHA, HOS 1 (2%)
 

CIRCULATORY SYSTEH
 

NONE .
 

t NUMBER OF ANIMALS WITH TISSOE EXAHIHBD MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

66 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

DIGESTIVE SYSTEM 

#LIVER (18) (50) (49)
 

HEPATOCELLULAR CARCINOMA 1 (6%) 3 (6X) 6 (12X)
 

OSTEOSARCOMA, METASTATIC 1 (2%)
 _ _ _ — —.___ _ —_ —«._ «._ ——_____.—____ _ —._._._._ — ___- . _______ _ —— _ — •—* — — _ — _ — —^-— — —•— «.—*»*•— — 

URINAR Y SYSTEH 

tKIDNE Y (18) (50) (49) 

TUBULAF-CEL L AEENOH A 1 (2X) 

OSTEOSARCOHA, METASTATIC 1 (2X) 

ENCOCRINE SYSTE M 

#PITUITAR Y (17) (HI) (15) 

ADENOMA , HOS 3 (6X) 

tTHYHOI D (18) (W5 ) (U7) 

ADENOCARCINOMA , NOS 1 (2X) 

FOLLICULAR-CELL ADENOM A 1 (2%) H (9S) 

• PANCREATIC ISLETS (18) (H6) (47) 

ISLET-CELL CARCINOMA , INVASIV E 1 (2%) 

REPRODUCTIVE SYSTEM 

*MAMMAR Y GLAND (19) (50) (50) 

MYOEPITHELIOBA 1 (5*) 1 (2X) 

FIBROADENOM A 1 (2%) 

#UTERUS/ENDOHETRIUHi (17) (47) (46) 

CARCINOMA,NO S , 1 (2«) 

#OVAP Y (17) (46) (47) 

FOLLICULAR-CELL ADENOMA 1 (6*) 

JBVOUS SYSTEM 

JONE . , 

NUMBR E OF ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

NUMBE R OF ANIMAL S NECROPSIED 

67 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL SENSE ORGAN S 

*EYE/LACRIMA L GLAN D

ADENOMA , NOS

 (19) (50)

 1 (2%) 

 (50) 

NUSCULOSKELETA L SYSTEM 

NON E 

BOG Y CAVITIES 

NON E 

A LL OTHER SYSTEMS 

SITE UNKNOW N 

LIPOMA 1 2 3 

ANIMA L DISPOSITION SUMMAR Y 

ANIMAL S INITIALLY IN STUDY

NATURA L DEATH9

MORIBUN D SACRIFICE

SCHEDULE D SACRIFICE 

ACCIDENTALL Y KILLED 

TERMINA L SACRIFICE

ANIMA L MISSING

 20

 3 

 16

 1 

 50

9 

1 

 HO

 50 

9 

 tt1 

f NUMBFR

* NUMBE R

 OF ANIMAL S WITH TISSUE

 OF ANIMAL S NECROPSIED 

 EXAMINED MICROSCOPICALL Y 

68 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUM0 3 SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUMORS* 10 23 25 

TOTA L PRIMAH Y TUMORS 12 26 29 

TOTAL ANIMAL S WIT H BENIGN TUMORS U 8 9 

TOTAL BENIG N TUMORS It 8 10 

TOTAL ANIMAL S WITH MALIGNAN T TUMORS 7 16 18 

TOTAL MALIGNAN T TUMORS 8 18 19 

TOTA L ANIMAL S WITH SECONDARY TUMORS* 1 1 

TOTAL SECONDAR Y TUMORS 2 1 

TOTAL ANIMAL S WIT H TUMORS UNCERTAINPENIG N O P MALIGNAN T

 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMAL S WITH TUMORS UNCERTAINPRIMAR Y OR METASTATIC

 

TOTAL UNCERTAI N TUMORS 

* PRIMAR Y TUMORS: ALL TUHORS EXCEPT SECONDARY TUHORS 

* SECONDAR Y TUMORS : METASTATIC TUMORS OR TDHORS INVASIVE INTO AH ADJACENT ORGAN 

69 
70

 
APPENDIX C
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN RATS ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

71
 
72

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

ANI1ALS MISSIN G 2 

ANIMAL S NECROPSIE D 20 50 48 

ANIMAL S EXAMINE D HISTO PATHOLOGIC ALL Y 20 50 48 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE (20) (50) (48)
 

CYST, NOS 1 (2%)
 

INFLAMMATION, GRANULOMATOUS 1 (2%)
 

GRANULOMA, NOS 1 (2%)
 

RESPIRATORY SYSTEM
 

tLONG (20) (50) (48)
 

CONGESTION, ACUTE 1 (2%)
 

EDEHA, NOS 1 (2%)
 

BRONCHOPNEUMONIA, NOS 1 (2%)
 

INFLAMMATION, GRANULOMATOOS 1 (2%)
 

ALVEOLAR MACROPHAGES 1 (5%) 4 (8X)
 

tLUNG/ALVEOLI (20) (50) (48)
 

INFLAMMATION, SUPPURATIVE 1 (2X)
 

HYPERPLASIA, EPITHELIAL 2 (10%) <• (8X) 3 (6%)
 

HEMATOPOIETIC SYSTEM
 

•SPLEEN (20) (50) (48)
 

FCTOPIA 1 (5%) 2
 

ABSCESS, NOS 1 (2X)
 

FIBROSIS, FOCAL 2 (M)
 

INFARCT, NOS 1 (5%)
 

HYPERPLASIA, LYMPHOID 2 (10%)
 

HEMATOPOIESIS
 * 1 (2%)
 

tlYHPH NODE (20) (49) (48) 

CONGESTION, NOS 1 (2%) 

f NDHBER OF ANIHALS KITH TISSUE EXAHIHED BICROSCOPICALLY
 

* NUHBER OF ANIHALS HECROPSIED
 

73
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

#MANDIBULA R L. NODS 

DILATATION , NO S 

*MESENTEPIC L. NODE 

DILATATION , NO S 

CISCHLATOP Y SYSTE M 

*H?AP T 

FIBROSIS , MULTIFOCA L 

iH^ART/ATPIU M 

THROMBOSIS , NOS 

MYOCARDIU M 

INFLAMMATION , SUPPURATIV E 

FIBROSI S 

NECROSIS , FOCAL 

DIGESTIV E SYSTE M 

*LIVSR 

INFLAMMATION , NECROTIZIN G 

INFLAMMATION , GEANULOBATO O S 

NECROSIS , NOS 

NECROSIS , FOCA L 

NECROSIS , DIFFUSE 

METAMORPHOSI S FATTY 

HEPATOCYTOMEGAL Y 

HYPFRPLASIA , FOCAL 

*BILE DUCT 

HYPEPPLASIA , NO S

 

(•PANCREA S 

PFRIARTERITI S 

SPANCREATI C ACINU S 

ATROPHY , NO S 

• STOMAC H 

fJLCJERj,. NOS 

4 HU1BE P OF ANIMAL S WITH TISSUE

* NIIMBF R O F ANIMAL S NECROPSIE D 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(20) (49) (<>8) 

3 (6%) 

(20) (49) (U8) 

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

(20) (50) (48) 

1 (5%) 

(20) (50) (U8) 

1 (5%) 1 (2%) 

(20) (50) (48) 

1 (2«) 

2 (10%) 2 («) 3 (6X) 

1 (2*) 

(20) (50) (47) 

1 (2X) 

1 (2%) 

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

1 (2%)

1 (5%) 5 (10%) 3 (6%) 

2 (10% ) 3 (6X) 4 (9%) 

4 (20%) K (8X) 4 (9%) 

(20) (50) (48) 4 (20% )

 8 (16X) 6 <13X) 

(19) (50) (47) 

1 (2») 

(19) (50) (47) 

2 (11*) 15 (30%) 7 (15%) 

(20 ) (50) (48) 

1 J2S.L 

 EXAMINE D MICROSCOPICALL Y 

74 
TABLE C1. MALE RATS: IMONNEOPLASTIC LESIONS (CONTINUED)

 

—«_«__»——_ 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

INFLAMMATION , ACUTE 1 (2*)

 

INFLAMMATION , CHRONIC 1 (2*)

 

EROSIO N 1 (2*)

 

tGASTRIC MOCOSA (20) (50) (48)

 

ACANTHOSI S 1 (2X) 

#COLON (19) (47) (45) 

PAPASITISM 2 (4X) 

URINAR Y SYSTE M 

tKIDNE Y (20) (50) (48) 

INFLAMMATION , SUPPURATIV E 1 (2X) 

ABSCESS , NOS 1 (2X) 

INFLAMMATION , CHRONIC 12 (60%) 22 (44X) 31 (65X) 

#URINAF Y BLADDE R (18 ) (47 ) (46) 

HYPERPLASIA , EPITHELIA L 1 (6«) 1 (2%)

 

*URETHR A (20) (50) (48)

 

HYPERPLASIA , EPITHELIAL 1 (5X) 

ENDOCRIN E SYSTE M 

#ADRENA L CORTE X (20 ) (50) (48) 

HYPERPLASIA , FOCA L 1 (2X) 

#AD?ENA L MFDULL A (20 ) (50) (48) 

HYFERPLASIA , FOCAL 2 (10%) 4 (8X) 5 (10X) 

tTHYROI D (20) (49) (48) 

CYSTIC FOLLICLES 1 (2X) 

HYPERPLASIA , C-CELL 2 (10$) 13 (27*) 12 (25X) 

HYPERPLASIA , FOLLICULA R-CELL 3 (6X) 

((PARATHYROI D (18) (43) (46) 

HYPERPLASIA , FOCAL 1 (2X) 1 (2X) 

#PANCPEATI C ISLETS (19 ) (50) (47) 

HYPEEPLASIA , NO S 1 (5% ) 2 (4X) 2 (4X) 

REPRODUCTIV E SYSTEM 

* MAMMAR Y GLAN D (20) (50) (48)

 

CYST, NOS 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBFP OF ANIMAL S NECROPSIED
 

75 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

INFLAMMATION, ACUTE 1 (2*) 

INFLAMMATION, CHROMIC FOCAL 1 (2)1) 

*PREPUTIAL GLAND (20) (50) (48)

 

ABSCESS, NOS 1 (2%) 3

 

METAPLASIA, SQUAMOUS 2 (4S)

 

tPROSTATE (19) (50)

 

INFLAMMATION, SOPPUEATIVE 1 (2%)

 

INFLAMMATION, CHRONIC FOCAL 1 (2%)

 

tTESTIS (20) (50)

 

CALCIFICATION, FOCAL 1 (2*)
 

ATROPHY, NOS 1 (5X)

 

*EPIDIDYMI S (20) (50) (48)
 

GRANOLOMA , SPERMATIC 

*SCROTUM (20) (50) (48)
 

NECROSIS, FOCAL 2 (4X) 2 {<**)
 

NERVOUS SYSTEM
 

#BRAIN/MENINGES (20) (50) (48)
 

HEMORRHAGE 1 (2»)
 

#BRAIN (20) (50) (48)
 

HEMORRHAGE 1 (5X) 2 (4«) 2 (4«)
 

_ ______ NECROSIS ______ __________ , FOCA ________ L 1 __ ______ __^___________ ^ __*___•_««•__.B.*»^««^W«^V^—•
 (2X)
 

SPECIAL SENSE ORGANS
 

*EYE/CRYSTALLINE LESS (20) (50) (18)
 

__ ___________ CALCIFICATION _ __ __._^.___ , FOCAL_ ________^ 1 _ ^^^^__ _____ _ __________________ _ __*^^_^^^««^«__ (2%)
.
 

nnSCOLOSKSLETAL STSTEH 

NONE 

BODY CAVITIES 

*ABDOMINAL CAVITY (20) (50) (48) 

t NUMBER OF ANIMALS WITH TISSUE EIAHINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

76
 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

*PL?UR A

FTBFOSI S

 (20) (50) (US) 

1 (2%) 

A L L QTHF 8 SYSTEM S 

*HULTIP1F ORGAN S

HYPEPPLASIA , LYMPHOI 0

ADIPOS E TISSUE 

NECROSIS, FOCAL

 (20)

 1 (5%)

 (50)

 1 

 (U8) 

4 (8*) 

SPECIA L MORPHOLOG Y SUMMAR Y 

ANIMA L MISSING/N O NECROPSY

t NUMBE R OF ANIMAL S WITH TISSDE

* NUMBE R OF ANIMAL S NECBOPSIED 

 EXAMINE D MICROSCOPICALLY 

2 

77 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED PIPERONYL SULFOXIDE

ANIMALS INITIALLY IN STUDY


ANIMALS NECROPSIED


ANIMALS EXAMINED HISTOPATHOLOGIC ALLY


INTEGUMENTARY SYSTEM
 

NONE
 

RESPIRATORY SYSTEM
 

•LUNG/BRONCHOS


INFLAMMATION, SUPPURATIVE
 

tLUNC-


EDEMA, NOS
 

INFLAMMATION, FOCAL GBANOLOMATOU
 

PERIVASCULITIS
 

ALVEOLAR MACROPHAGES


#LUNG/ALVEOLI


ALVEOLAR MACROPHAGES
 

HYPERPLASIA, EPITHELIAL
 

HEMATOPOIETIC SYSTEM
 

#MESENTERIC L. NODE


DILATATION, NOS
 

HEMORRHAGE
 

NECROSIS, NOS
 

CIRCULATORY SYSTEM
 

#HEAFT
 

FIBROSIS, FOCAL 

#HEART/ATRIU M 

MATCHED
 

CONTROL
 

 20
 

 20
 

 20
 

 (20)
 

 (20)
 

1 (5%)
 

 (20)
 

 (19)
 

(19 ) 

(19 ) 

 IN THE DIET 

LOW DOSE HIGH DOSE
 

50 50
 

50 50
 

50 50
 

(50)	 (50)
 

1 (2«)
 

(50)	 (50)
 

2 (4%)
 

1 (2*)
 

1 (2%)
 

i* (8X) 3 (6%)
 

(50) (50)
 

1	 <2X)
 

1 (2S)
 

(50)	 (50)
 

3 (695)
 

1 (2X)
 

1 (2*)
 

(50) (50) 

(50) (50) 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

78
 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

#MYOCARDIUM
 

INFLAMMATION, NOS
 

INFLAMMATION, SOPPURATIVE
 

FIBROSIS
 

#ENDOCARDIOM
 

INFLAMMATION, SOPPORATIVE
 

DIGESTIVE SYSTEM
 

#LIVER
 

INFLAMMATION, CHRONIC DIFFUSE
 

GRANULOMA, NOS
 

NECROSIS, NOS
 

NECROSIS, FOCAL
 

METAMORPHOSIS FATTY
 

HEPATOCYTOMEGALY
 

HYPERPLASIA, FOCAL
 

*BILE DUCT
 

HYPERPLASIA, NOS
 

tPANCREAS
 

INFLAMMATION, CHRONIC FOCAL
 

tPANCREATIC ACINOS
 

ATROPHY, NOS
 

tSTOMACH
 

ULCEE, NOS
 

#GASTRIC MOCOSA
 

HEMORRHAGE
 

HYPERPLASIA, FOCAL
 

KGAST8IC SUBMUCOSA
 

SDEMA, NOS
 

#COLON
 

INFLAMMATION, NECROTIZING
 

ORINAR Y SYSTE M 

#KIDNE Y

# NUMBE R OF ANIMAL S KITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

'

MATCHED 

CONTROL LOW DOSE HIGHD IOSE 

(19) (50) (50) 

1 (2%) 

1 (2%) 

1 (5%) 

(19) (50) (50) 

1 (2*) 

(20) (48) (50) 

1 (2%) 

1 (5%) 3 (6%) 

2 (4%) 1 (2%) 

2 (4%) 

2 (4%) 3 (6%) 

2 (4%) 12 (24%) 

12 (60%) 11 (23%) 5 (10%) 

(20) (50) (50) 

8 (16%) 4 (8%) 

(20) (47) (47) 

1 (2%) 

(20) (47) (47) 

3 (15%) 8 (17%) 6 (13%) 

(20) (50) (49) 

1 (2%) 

(20) (50) (49) 

1 (2%) 

1 (2*) 

(20) (50) (49) 

1 (2%) 

(19) (47) (47) 

1 (2%) 

 (20) (50) (50) 

 EXAMINE D MICROSCOPICALL Y 

79 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

INFLAMMATION , CHRONIC FOCAL 

fPENA L PAPILL A 

NECROSIS , NOS 

#KIDNEY/PELVI S 

DILATATION , NOS 

INFLAMMATION , SUPPDRATIV E 

HYPERPLASIA , EPITHELIAL 

• URINAR Y BLADDE R 

CALCULUS , NOS 

HEMORRHAG E 

INFLAMMATION , HEMORRHAGI C 

INFLAMMATION , CHRONIC 

HYPERPLASIA , EPITHELIAL 

ENCOCRIN E SYSTEM 

tADRENA L CORTEX 

NECROSIS, FOCAL 

HYPERPLASIA , FOCAL 

#ADPENA L MEDULL A 

HYPFRPLASIA , FOCAL 

tTHYROI D 

HYPERPLASIA , C-CELL 

HYPERPLASIA , FOLLICULAR-CELL 

REPRODUCTIV E SYSTEM 

*CLITORA L GLAN D 

CYSTIC DUCTS 

ABSCESS, NOS 

HYPEPPLASIA , NOS 

METAPLASIA , SQUAMOUS 

*UTERUS/ENDOMETRIU H 

HYPERPLASIA , CYSTIC 

METAPLASIA , SQUAMOOS 

#OVARY/PAROVARIA R 

N.EC.R2SIJS.«._.fiOS 

* NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

1 (2X) 

(20) (50) (50) 

1 (2X) 

(20) (50) (50) 

1 (2X) 

2 (<*X) 

1 (5X) 2 C»X) 

(19) (47) (HH) 

1 (2X) 

1 (2X) 

1 (2X) 

1 (2X) 

2 (<*X) 1 (2X) 

(20) (50) (50) 

1 (2X) 

1 (5X) 1 (2X) 2 (UX) 

(20) (50) (50) 

2 (<»X) 

(19) (50) (50) 

8 (U2X ) 17 (3UX) 1U (28X) 

1 (2X) 

(20) (50) (50) 

1 (2X) 

1 (2X) 1 (2X) 

1 (5X) 2

 (<»X) 

(20) (50) (HI) 

2 (<»X) 

1 (2X) 

(20) (50) (U7) ___.— _ 1_12SL_ 

 EXAMINED MICROSCOPICALLY 

80 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NECROSIS, FOCAL 1 (5%) 1 (2S) 

NERVOU S SYSTEM 

• BRAI N (20) (50) (50) 

HEMORRHAG E 1 (2X) 

SPECIAL SENSE ORGAN S 

NONE 

HOSCDLOSKELETAL SYSTEH 

NON E 

BODY CAVITIES 

*PERITOHEOM (20) (50) (50) 

NECROSIS, FOCAL 1 (2*) 

A LL OTHER SYSTEMS 

*MULTIPLE ORGAN S (20) (50) (50) 

HYPERPLASIA , LYMPHOID 2 (10%) 1 (2%) 4 (8%) 

W — ——— — — »»— — — —•—.•— _•»•••_ _*»«»_ •••«.,•.—••—«••——— •»•. • •» — — — «• ••••V^W««»««. •—^^^— ^^— — — »•••— . 

SPECIAL BORPHOLOGY SOHHARY 

NO LESION REPORTED 2 3 3 

t NOHBER OF ANIMALS ilTH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NBCROPSIBD 

81 
82

 
APPENDIX D
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 

IN MICE ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

83
 
84

 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S

ANIMAL S

ANIMAL S

ANIMAL S

 INITIALLY IN STUDY

 KISSING

 NECROPSIED

 EXAMINE D HISTOPATHOLOGICALLY

 20

 1 

 18

 18

 50

 50

 50

 50 

 50 

 50 

IN7FGUHENTA9. Y SYSTEM 

*SKIN (18) (50) (50) 

CYST, NOS 1 (2X) 

ABSCESS, NOS 1 (2X) 

RESPIRATOR Y SYSTEM 

HONE __ __ _ — __ _ — _ «.___ — —-.___,. —f_ — — —— — — — — — _—• — _ ___—— — _— —— __ _ _ .._—______..._.....»_.______.. 

HEMATOPOIETIC SYSTEM 

*SPLEEN (18) (50) («9) 

TNFARCT, NOS 1 (2%) 

HYPERPLASIA , RETICOLOH CELL 2 («X) 1 (2X) 

HYPERPLASIA , LYMPHOI D 1 (2X) 1 (2X) 

HEMATOPOIESI S 5 (28%) 16 (32%) 2 (UX) 

tMESENTERI C L. NODE (18) (49) (50) 

HYPERPLASIA , LYMPHOI D 1 (2X) 

CIRCULATOR Y SYSTEM 

tMYOCAPDTU M (18) (50) (U9) 

INFLAMMATION , NOS 1 (2X) 

INFLAMMATION , FOCAL 2 (UX ) 

DIGESTIV E SYSTEM 

tLIVH (18) (50) (50) 

NJCFOSJSi_ FOCAL 2_JH31 1_ 

# NUMBF R OF ANIMAL S HITH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBE R OF ANIMAL S NECROPSIED 

85 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

HEPATOCYTOMEGALY
 

HYPERPLASIA, NODULAR
 

ANGIECTASIS
 

*PANCREAS
 

CYSTIC DUCTS
 

fSTOHACH
 

INFLAMMATION, NOS
 

URINARY SYSTEM
 

•KIDNEY
 

INFLAMMATION, INTERSTITIAL
 

HYPERPLASIA, LYMPHOID
 

ENDOCRINE SYSTEM
 

•PITUITARY
 

CYST, NOS
 

#THYROID
 

FOLLICULAR CYST, NOS
 

tTHYPOID FOLLICLE
 

CYST, NOS
 

tPANCREATIC ISLETS
 

HYPERPLASIA, SOS
 

REPRODUCTIVE SYSTEM
 

IPROSTATE
 

ABSCESS, NOS
 

*SEKIHAL VESICLE
 

CAST, NOS
 

#TESTIS
 

TNFARCT, NOS
 

MATCHED
 

CONTROL
 

3 (17%)
 

1 (6X)
 

(17)
 

(18)
 

(18)
 

(16)
 

(17)
 

(17)
 

(17)
 

(18)
 

(18)
 

3 (17%)
 

(18)
 

LOW DOSE
 

5 (10X)
 

(i»7)
 

(50)
 

(50)
 

1 (2%)
 

2 («X)
 

(50)
 

1 (2X)
 

(U6)
 

1 (2X)
 

(46)
 

2 («)
 

(HI)
 

3 (6X)
 

(U9)
 

(50)
 

« (8X)
 

(19)
 

1 <2X)
 

HIGH DOSE
 

1 (2X)
 

3 (6X)
 

(50)
 

1 (2X)
 

(U9)
 

1 (2%)
 

(50)
 

(19)
 

(50)
 

(50)
 

1 (2X)
 

(50)
 

(50)
 

1 (2*)
 

(50)
 

U (8%)
 

(50)
 

1 12%L
 

I NUMBER OF ANIMALS WITH TISSUE EXAHINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECBOPSIED
 

86
 
TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NERVOU S SYSTE M 

*BPAI N

MINERALIZATIO N

 (18)

 l» (22%)

 (50)

 5 (10*)

 (50) 

 10 (20*) 

SPECIA L

NON E 

 SENSE ORGAN S 

MUSCULOSKELETA L

NON ? 

 SYSTEM 

BODY CAVITIE S 

*PERICSPDI[J M

INFLAMMATION , NOS

 (18) (50) (50) 

1 (2*) 

A L L OTHER

NON E 

 SYSTEM S 

SPECIAL 10PPHOLOGY S0HHAR Y 

NO LESION REPORTED

ANIMA L MTSSING/NO NECROPSY

AUTOLYSIS/N O NECROPSY

* NUMBE P OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S HECROPSIED 

 EIAHIHED

 1

 1 

1 

 MICROSCOPICALLY 

2 1 

87 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET 

_ II •• inn •« II • •• • in ——> 

MATCHED 

CONTROL LOW DOSE 

ANIMAL S 

ANIMAL S 

ANIMAL S 

ANIMAL S 

INITIALL Y IN STUDY 

MISSING 

NFCROPSIED 

EXAMINE D HISTOPATHOLOGICALLY 

20 

1 

19 

19 

50 

50 

50 

INTEGUMENTAR Y SYSTEM 

NONE 

RESPIRATOR Y SYSTEM 

NONE 

HEMATOPOIETIC SYSTEM 

tSPLEEN (18) (50) 

AMYLOIDOSIS 

HYPERPLASIA, LYMPHOID 1 (6%) 4 (8«) 

HEMATOPOIESIS 11 (61%) 28 (56S) 

#LYMP H NODE (18) (49) 

CYST, NOS 1 (2X) 

*HESENTEBIC L. NODE (18) (49) 

HYPERPLASIA. LYMPHOID 1 (2%) 

tTHYMUS (17) (<»3) 

HYPERPLASIA, LYMPHOID 1 (6%) 

CIRCULATORY SYSTEH 

tMYOCARDIO M (18) (U7) 

INFLAMMATION , NOS 

DIGESTIVE SYSTEM 

ILIVBR (18) (50) _ NECROSIS,, F,QC.ftL_ l.IfiXl 

t NDMBEfi OF ANIMALS BITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSI1D 

HIGH DOSE 

50 

50 

50 

(48) 

1 (2«) 

1 (2X) 

9 (19X) 

(49) 

<<»9) 

1 (2X) 

(47) 

2 («X) 

(49) 

1 (2*) 

(49) 

1-12SL. 

88 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

CYTOPIASMIC VACUOLIZATIOH 1 (6X) 2	 1 (2X) HEPATOCYTOMEGALY

 1 (2X) HYPERPLASIA, NODULAR

 2 (11X) 7 (14X) 8 (16X) 

*BILT: DUCT (19) (50) (50) INFLAMMATION , NOS

 3 (6%) 

* PANCREA S (18) CYSTIC DUCTS

 1 (6*) 2 («X) 

*PAMCSEATIC DUCT (18) (146)
 

CYST, NOS 1 (2X)
 

#STON.».CH (18) (50) (U9)

 

INFLAMMATION, NOS 2 <4X)
 

#COLON (18)
 

GPANULOMA, NOS 1 (2X)
 

URINARY SYSTFf!
 

*KTDHEY (18) (50) (49)
 

GLOMERULOSCL2ROSIS, NOS 1 (21)
 

TNFAPCT, NOS 1 (2*)
 

HYPERPLASIA, LYMPHOID 1 (2X)
 

•URINARY	 BLADDER (18) (45) (45)
 

LYMPHOCYTIC INFLAMMATORY INFILTF 1 (2«)
 

ENDOCRINE SYSTEM
 

tFITUITARY (17) (l»7) (45)
 

CYST, NOS 1 (2») 1 (2X)
 

tTHYROID (18) (15) (17)
 

CYSTIC FOLLICLES 1 (2%)
 

FOLLICULAR CYST, NOS 1 (6X) 1 (2X)
 

(fTHVPOID FOLLICLE (18) (45) (47)
 

CYST, NOS 4 (9X) 2 (4%)
 

REPRODUCTIVE SYSTEM
 

«UTFRUS (17) (47) (46)
 

CISlx_Ji2S 1 i_j2.S.L­

« NtIMB'!:P OF ANIHALS KITH TISSUE EXABINED MICROSCOPICALLY
 

* NUMBER OF ANIMALS NECROPSIED
 

89 
TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

INFLAMMATION , NOS

POLYPOID HYPERPLASI A

*UTFSUS/FNDONETRIUM

CYST, NOS

HYPERPLASIA , CYSTIC

•CVAPY


CYST, NOS


MULTIPLE CYSTS


NERVOUS SYSTEM
 

*BEAIS

MINTEALIZATIO N

INFLS«WATION , NOS

SPECIA L SENSE ORGAN S 

NONE 

MUSCHLOSKFLFTA L SYSTEM 

NONT 

BODY CAVITIE S 

NON F 

A LL OTHEE SYSTEM S 

NONE 

SPECIAL MORPHOLOGY SUMMAR Y 

NO LESION REPORTED

ANIMA L MISSIHG/NO NECROPSY

* NUMBE R OF ANIMAL S WITH TISSUE

* NUMBE R OF ANIMAL S NECROPSIED 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

2 (H%) 1 (2*) 

1 (2«) 1 (2*) 

 (17) (U7 ) (<J6) 

9 (53*) 19 (UOX) 11 (2H%) 

1 (2«) 

 (17) (U6) («7)
 

8 (47%) 8 (17X) 9 (19«)
 

1 (2S)
 

 (18) («9) (U7) 

5 (28%) 12 (2«X) H (9*) 

• 1 (2S) 

1 2 6 

1 

 EXAMINE D MICROSCOPICALL Y 

90 
APPENDIX E
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN RATS ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

91
 
92

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

Administered Piperonyl Sulfoxide in the Diet (a)
 

Topography: Morphology


Hematopoietic System: Lymphoma (b)


P Values (c,d,)


Relative Risk (£)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


VO
 Pituitary: Adenoma, NOS (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

 Control
 

 2/20 (10)
 

 N.S.
 

 96
 

 6/18 (33)
 

P = 0.026(N)
 

96
 

Low
 

Dose
 

12/50 (24)
 

N.S.
 

2.400
 

0.614
 

20.902
 

10
 

4/45 (9)
 

P = 0.026(N)
 

0.267
 

0.066
 

1.005
 

100
 

High
 

Dose
 

4/48 (8)
 

N.S.
 

0.833
 

0.133
 

8.776
 

77
 

4/44 (9)
 

P = 0.028(N)
 

0.273
 

0.067
 

1.027
 

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

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Adrenal: Pheochromocytoma (b) 1/20 (5)	 3/50 (6)
 

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

Relative Risk (f) 1.200
 

Lower Limit 0.106
 

Upper Limit 61.724
 

Weeks to First Observed Tumor 105	 105
 

VO	 Thyroid: C-cell Adenoma (b) 2/20 (10) 3/49 (6)
 

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

Relative Risk (f) 0.612
 

Lower Limit 0.078
 

Upper Limit 6.996
 

Weeks to First Observed Tumor 105 105
 

High
 

Dose
 

3/48 (6)
 

N.S.
 

1.250
 

0.110
 

64.251
 

105
 

3/48 (6)
 

N.S.
 

0.625
 

0.079
 

7.137
 

105
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Matched Low
 

Topography: Morphology Control Dose
 

Pancreatic Islets:
 

Islet-cell Adenoma (b) 2/19 (11) 2/50 (4)
 

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

Relative Risk (f) 0.380
 

Lower Limit 0.030
 

Upper Limit 5.009
 

Weeks to First Observed Tumor 105 105
 

VO
 

(Jt
 Preputial Gland: Carcinoma, NOS (b) 0/20 (0) 4/50 (8)
 

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

Relative Risk (f) Infinite
 

Lower Limit 0.386
 

Upper Limit Infinite
 

Weeks to First Observed Tumor 77
 

High
 

Dose
 

3/47 (6)
 

N.S.
 

0.606
 

0.077
 

6.913
 

0/48 (0)
 

—
 

—
 

—
 

—
 

105 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Testis: Interstitial-cell Tumor (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Brain: Astrocytoma (b)
 

P Values (c,d)
 

Departure from Linear Trend (e)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

16/20 (80)
 

N.S.
 

83
 

2/20 (10)
 

P = 0.026(N)
 

P = 0.044
 

83
 

Low High 

Dose Dose 

46/50 (92) 43/48 (90) 

N.S. N.S. 

1.150 1.120 

0.929 0.900 

1.451 1.456 

77 69 

0/50 (0) 0/48 (0) 

N.S. N.S. 

0.000 0.000 

0.000 0.000 

1.345 1.400 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

(a) Dosed groups received 1,500 or 3,000 ppm.
 

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

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

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

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

test for the comparison of that dosed group with the matched-control group when P less than
 

0.05; otherwise, not significant (N.S.) is indicated.
 

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

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

comparison.
 

vo
 

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

Administered Piperonyl Sulfoxide in the Diet (a)
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma (b)
 

P Values (c.d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Hematopoietic System: Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low 

Control Dose 

2/20 (10) 0/50 (0) 

N.S. N.S. 

0.000 

0.000 

1.345 

105 — 

2/20 (10) 5/50 (10) 

N.S. N.S. 

1.000 

0.184 

10.007 

82 102 

High
 

Dose
 

1/50 (2)
 

N.S.
 

0.200
 

0.004
 

3.681
 

105
 

12/50 (24)
 

N.S.
 

2.400
 

0.614
 

20.902
 

81 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued) 

Matched Low High 

Topography: Morphology Control Dose Dose 

Pituitary: Adenoma, NOS (b) 7/20 (35) 7/50 (14) 12/48 (25) 

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

Relative Risk (f) 0.400 0.714 

Lower Limit 0.144 0.318 

Upper Limit 1.187 1.875 

Weeks to First Observed Tumor 82 86 100 

Thyroid: C-cell Adenoma 0/19 (0) 5/50 (10) 1/50 (2)
 

or Carcinoma (b)
 

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

Relative Risk (f) Infinite Infinite
 

Lower Limit 0.501 0.021
 

Upper Limit Infinite Infinite
 

Weeks to First Observed Tumor 82 102 81
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Topography: Morphology


Mammary Gland: Fibroadenoma (b)


P Values (c,d)


Departure from Linear Trend (e)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor


o
 

o
 

Uterus or Cervix Uteri:
 

Endometrial Stromal Polyp (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Matched
 

 Control
 

 4/20 (20)
 

P = 0.002(N)
 

 P = 0.036
 

 105
 

 0/20 (0)
 

 N.S.
 

Low
 

Dose
 

1/50 (2)
 

P = 0.021(N)
 

0.100
 

0.002
 

0.944
 

105
 

6/50 (12)
 

N.S.
 

Infinite
 

0.667
 

Infinite
 

High
 

Dose
 

0/50 (0)
 

P = 0.005(N)
 

0.000
 

0.000
 

0.427
 

—
 

3/47 (6)
 

N.S.
 

Infinite
 

0.266
 

Infinite
 

Weeks to First Observed Tumor 100 93
 
Table E2. Analyses of the Incidence of Primary Tumors in Female Rats
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

(a) Dosed groups received 3,000 or 6,000 ppm.
 

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

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

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

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

test for the comparison of that dosed group with the matched-control group when P less than
 

0.05; otherwise, not significant (N.S.) is indicated.
 

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

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

comparison.
 

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

APPENDIX F
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 

IN MICE ADMINISTERED PIPERONYL SULFOXIDE IN THE DIET
 

103
 
104

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

Administered Piperonyl Sulfoxide in the Diet (a)
 

Topography: Morphology
 

Lung: Alveolar/Bronchiolar
 

Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Lung: Alveolar/Bronchiolar
 o
 

Ul Adenoma or Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched
 

Control
 

3/18 (17)
 

N.S.
 

105
 

6/18 (33)
 

N.S.
 

103
 

Low
 

Dose
 

4/50 (8)
 

N.S.
 

0.480
 

0.093
 

3.058
 

104
 

10/50 (20)
 

N.S.
 

0.600
 

0.243
 

1.776
 

104
 

High
 

Dose
 

4/50 (8)
 

N.S.
 

0.480
 

0.093
 

3.058
 

98
 

7/50 (14)
 

N.S.
 

0.420
 

0.147
 

1.346
 

98
 
(continued) 

Topography:

Table Fl.

 Morphology

 Analyses of

Administered

 the Incidence of Primary Tumors

 Piperonyl Sulfoxide in the Diet

Matched 

 Control 

Low 

Dose 

 in Male

 (a) 

 Mice 

High 

Dose 

Hematopoietic System: Lymphoma (b) 5/18 (28) 8/50 (16) 3/50 (6) 

P Values (c,d) P = 0.014(N) N.S. P = 0.026(N) 

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.576 

0.200 

2.012 

0.216 

0.039 

1.012 

Weeks to First Observed Tumor 23 81 104 

Liver: Hemangiosarcoma (b) 0/18 (0) 1/50 (2) 3/50 (6) 

o p Values (c,d) N.S. N.S. N.S. 

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.020 

Infinite 

Infinite 

0.227 

Infinite 

Weeks to First Observed Tumor 105 102 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Topography; Morphology
 

Liver: Hepatocellular Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Eye/Lacrimal Gland: Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

Control Dose Dose 

6/18 (33) 31/50 (62) 46/50 (92) 

P less than P = 0.034 P less than 

0.001 0.001 

1.860 2.760 

0.966 1.570 

4.545 4.868 

71 92 83 

1/18 (6) 0/50 (0) 3/50 (6) 

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

0.000 1.080 

0.000 0.096 

6.729 55.565 

105 103 
Table Fl. Analyses of the Incidence of Primary Tumors in Male Mice
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued;
 

Matched Low High
 

Topography: Morphology Control Dose Dose
 

Site Unknown: Lipoma (b) 0/18 (0) 3/50 (6) 0/50 (0)
 

P Values (c,d) N.S. N.S.
 —
 

Relative Risk (f) Infinite
 —
 Lower Limit 0.227
 —
 Upper Limit Infinite
 —
 

Weeks to First Observed Tumor 105
 —
 

(a) Dosed groups received 350 or 700 ppm.
 o
 

00
 

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

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

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

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

test for the comparison of that dosed group with the matched-control group when P less than
 

0.05; otherwise, not significant (N.S.) is indicated.
 

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

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

comparison.
 

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

Administered Piperonyl Sulfoxide in the Diet (a)
 

Matched Low
 

Topography: Morphology Control Dose
 

Lung: Alveolar/Bronchiolar
 

Adenoma or Carcinoma (b) 2/18 (11) 2/48 (4)
 

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

Relative Risk (f) 0.375
 

Lower Limit 0.030
 

Upper Limit 4.932
 

Weeks to First Observed Tumor 105 98
 

M Hematopoietic System: Lymphoma or
 O
 Lymphocytic Leukemia (b) 5/19(26) 12/50 (24)
 

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

Relative Risk (f) 0.912
 

Lower Limit 0.360
 

Upper Limit 2.959
 

Weeks to First Observed Tumor 88 51
 

High
 

Dose
 

2/49 (4)
 

N.S.
 

0.367
 

0.029
 

4.834
 

102
 

10/50 (20)
 

N.S.
 

0.760
 

0.284
 

2.547
 

85
 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Topography: Morphology
 

Liver: Hepatocellular Carcinoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Pituitary: Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

Control Dose Dose 

1/18 (6) 3/50 (6) 6/49 (12) 

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

1.080 2.204 

0.096 0.302 

55.565 99.144 

105 105 104 

0/17 (0) 3/47 (6) 0/45 (0) 

N.S. N.S. — 

Infinite 

0.229 — 

— Infinite — 

105 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

Topography: Morphology


Thyroid: Follicular-cell Adenoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Site Unknown: Lipoma (b)


P Values (c,d)


Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

Weeks to First Observed Tumor
 

Matched Low High 

 Control Dose Dose 

 .0/18 (0) 1/45 (2) 4/47 (9) 

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

Infinite Infinite 

0.022 0.372 

Infinite Infinite 

105 102 

 1/19 (5) 2/50 (4) 3/50 (6) 

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

0.760 1.140 

0.043 0.101 

43.961 58.635 

105 105 60 
Table F2. Analyses of the Incidence of Primary Tumors in Female Mice
 

Administered Piperonyl Sulfoxide in the Diet (a)
 

(continued)
 

(a) Dosed groups received time-weighted average doses of 295 or 754 ppm.
 

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

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

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

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

test for the comparison of that dosed group with the matched-control group when P less than
 

0.05; otherwise, not significant (N.S.) is indicated.
 

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

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

comparison.
 

(f) The 95% confidence interval of the relative risk between each dosed group and the control group.
 
Review of the Bioassay of Piperonyl Sulfoxide* for Carcinogenicity
 

by the Data Evaluation/Risk Assessment Subgroup
 

of the Clearinghouse on Environmental Carcinogens
 

August 31, 1978
 

The Clearinghouse on Environmental Carcinogens was established in
 

May, 1976, in compliance with DHEW Committee Regulations and the Provisions of the Federal Advisory Committee Act. The purpose'of the
 

Clearinghouse is to advise the Director of the National Cancer Institute
 

(NCI) on its bioassay program to identify and to evaluate chemical
 

carcinogens in the environment to which humans may be exposed. The
 

members of the Clearinghouse have been drawn from academia, industry,
 

organized labor, public interest groups, State health officials, and
 

quasi-public health and research organizations. Members have been
 

selected on the basis of their experience in carcinogenesis or related
 

fields and, collectively, provide expertise in chemistry, biochemistry,
 

biostatistics, toxicology, pathology, and epidemiology. Representatives
 

of various Governmental agencies participate as ad hoc members. The
 

Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is charged
 

with the responsibility of providing a peer revn'w of reports prepared
 

on NCI-sponsored bioas^ays of chemicals studied tor carcinogenicity. It
 

is in this context that the below critique is given on the bioassay of
 

Piperonyl Sulfoxide for carcinogenicity.
 

The primary reviewer agreed with the conclusion in the report that
 

Piperonyl Sulfoxide was not carcinogenic in either sex of rats or female
 

mice, but produced an increased incidence of hepatocellular carcinomas
 

in treated male mice under the conditions of test. He briefly described
 

the experimental design and noted the incidence of hepatocellular
 

carcinomas in the control and treated male mice.
 

A motion was approved unanimously that the report on the bioassay
 

of Trimethylthiourea be accepted as written.
 

Members present were:
 

Arnold L. Brown (Chairman), University of Wisccmsin Medical School
 

Joseph Highland, Environmental Defense Fund
 

Michael Shimkin, University of California at San Diego
 

Louise Strong, University of Texas Health Sciences Center
 

Subsequent to this review, changes may have been made in the
 

bioassay report either as a result of the review or other
 

reasons. Thus, certain comments and criticisms reflected in
 

the review may no longer be appropriate.
 

113
 

•&U.S. GOVERNMEN T PRINTIN G OFFICE : I 978-2B I "2 1 7/32 64
 

DHEW Publication No. (NIH) 79-1379