1979 

National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

NO. 120 

BIOASSAY OF 

PIPERONYL BUTOXIDE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 51-03-6 

NCI-CG-TR-120 

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

Public Health Service 

National Institutes of Health 

BIOASSAY OF
 


PIPERONYL BUTOXIDE
 


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) 79-1375
 

ii

 
BIOASSAY OF
 


PIPERONYL BUTOXIDE
 


FOR POSSIBLE CARCINOGENICITY
 


Carcinogenssis 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 butoxide 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 car­


cinogen, 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 butoxide 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 the
 


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


computer services. Histopathologic evaluations for rats were
 


performed by Dr. R. A. Renne (4,5), and the h is topatho logic
 


evaluations for mice were performed by Dr. C. E. Gilmore (4). 

The diagnoses included in this report represent the 

interpretations of Drs. Renne and Gilmore. 

ill
 

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


Mason Research Institute (6). Statistical analyses were 

performed 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 (1). The results of
 


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


M. Wagner (7).



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)	 Careinogenesis Testing Program, Division of Cancer Cause and
 


Prevention, National Cancer Institute, National Institutes of
 


Health, Bethesda, Maryland.
 


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


Maryland.
 


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


Herndon, Virginia.
 


(5)	 No wit w h Battelle Pacific Northwest Laboratories, Battelle
 


Boulevard, Richland, Washington.
 


iv
 

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


Rockville, Maryland.
 


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


Maryland.
 


(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 butoxide for possible 

carcinogenic ity 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 

butoxide in the diet at one of two doses, either 5,000 or 10,000 

ppm, for 107 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 mice of each sex were initially administered 

piperonyl butoxide at one of two doses, either 2,500 or 5,000 

ppm. After week 30, the doses for the mice were reduced to 500 

and 2,000 ppm, respectively, and administration of the test 

chemical at the lowered doses was continued for 82 weeks. The 

time-weighted average doses for the mice were either 1,036 or 

2,804 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 body weights were dose related. Survival of the 

rats and mice was unaffected by the piperonyl butoxide and was 

80% or greater 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 female rats, lymphomas occurred at incidences that were 

dose related (P = 0.007); in a direct comparison, the incidence 

of the tumor in the high-dose group was higher (P = 0.020) than 

that in the control group (controls 1/20, low-dose 7/50, 

high-dose 15/50). However, the incidence of lymphomas, 

leukemias, and reticuloses in historical-control female Fischer 

344 rats at the same laboratory was 19/191 (10%). These 

historical-control groups include one with an incidence of 

animals with lymphoma or leukemia of 7/20 (35%) and another with 

an incidence of 6/20 (30%). Thus, the incidence of lymphomas in 

the control female rats of the present bioassay may have been 

abnormally low, and the occurrence of the higher incidence in the 

dosed groups cannot be clearly related to administration of 

piperonyl butoxide. 

vii 
In the male mice, adenomas of the lacrimal gland occurred at 

incidences that were dose related (P = 0.023), but in direct 

comparisons the incidences in the individual dosed groups were 

not significantly higher than that in the control group (controls 

0/20, low-dose 0/49, high-dose 4/50); thus, the occurrence of 

this tumor in the male mice was not clearly related to 

administration of the test chemical. 

It is concluded that under the conditions of this bioassay, 

piperonyl butoxide was not carcinogenic for Fischer 344 rats or 

B6C3F1 mice. 

viii 
TABLE OF CONTENTS
 


I. Introduction 1



II. Materials and Methods 3



A. Chemical 3



B. Dietary Preparation 4



C. An imals 4



D. Animal Maintenance 5



E. Subchronic Studies 7



F. Chron ic Stud ies 10
 

G. Clinical and Pathologic Examinations 13
 


H. Data Recording and Statistical Analyses 14
 


III. Results - Rats 21
 

A. Body Weights and Clinical Signs (Rats) 21
 


B. Survival (Rats) 21
 

C. Pathology (Rats) 24
 

D. Statistical Analyses of Results (Rats) 31
 


IV. Results -Mice 33
 

A. Body Weights and Clinical Signs (Mice) 33
 


B. Survival (Mice) 33
 

C. Pathology (Mice) 36
 

D. Statistical Analyses of Results (Mice) 38
 


V. Discussion 41
 

VI. Bibliography 45
 

APPENDIXES
 


Appendix A Summary of the Incidence of Neoplasms in
 


Rats Administered Piperonyl Butoxide in the
 


Diet 49
 

Table Al Summary of the Incidence of Neoplasms in
 


Male Rats Administered Piperonyl Butoxide in
 


the Diet 51
 

ix
 

Table A2 Summary of the Incidence of Neoplasms in 

Female Rats Administered Piperonyl Butoxide 

in the Diet 55 

Appendix B Summary of the Incidence of Neoplasms in 

Mice Administered Piperonyl Butoxide in 

the Diet 59 

Table Bl Summary of the Incidence of Neoplasms in 

Male Mice Administered Piperonyl Butoxide 

in the Diet 61 

Table B2 Summary of the Incidence of Neoplasms in 

Female Mice Administered Piperonyl Butoxide 

in the Diet 65 

Appendix C Summary of the Incidence of Nonneoplastic 

Lesions in Rats Administered Piperonyl 

Butoxide in the Diet , 69 

Table Cl Summary of the Incidence of Nonneoplastic 

Lesions in Male Rats Administered 

Piperonyl Butoxide in the Diet 71 

Table C2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Administered 

Piperonyl Butoxide in the Diet 75 

Appendix D Summary of the Incidence of Nonneoplastic 

Lesions in Mice Administered Piperonyl 

Butoxide in the Diet 79 

Table Dl Summary of the Incidence of Nonneoplastic 

Lesions in Male Mice Administered 

Piperonyl Butoxide in the Diet 81 

Table D2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Mice Administered 

Piperonyl Butoxide in the Diet 85 

Appendix E Analyses of the Incidence of Primary 

Tumors in Rats Administered Piperonyl 

Butoxide in the Diet 89 
Table El Analyses of the Incidence of Primary Tumors 

in Male Rats Administered Piperonyl 

Butoxide in the Diet 91 

Table E2 Analyses of the Incidence of Primary Tumors 

in Female Rats Administered Piperonyl 

Butoxide in the Diet 97 

Appendix F Analyses of the Incidence of Primary Tumors 

in Mice Administered Piperonyl Butoxide 

in the Diet 101 

Table Fl Analyses of the Incidence of Primary Tumors 

in Male Mice Administered Piperonyl Butoxide 

in the Diet 103 

Table F2 Analyses of the Incidence of Primary Tumors 

in Female Mice Administered Piperonyl 

Butoxide in the Diet 108 

TABLES 

Table 1 Piperonyl Butoxide Subchronic Feeding 

Studies in Rats and Mice 9 

Table 2 Piperonyl Butoxide Chronic Feeding 

Studies in Rats 11 

Table 3 Piperonyl Butoxide Chronic Feeding 

Studies in Mice 12 

FIGURES 

Figure 1 Growth Curves for Rats Administered 

Piperonyl Butoxide in the Diet 22 

Figure 2 Survival Curves for Rats Administered 

Piperonyl Butoxide in the Diet 23 

Figure 3 Growth Curves for Mice Administered 

Piperonyl Butoxide in the Diet 34 

Figure 4 Survival Curves for Mice Administered 

Piperonyl Butoxide in the Diet 35 

xi
 
xii

 
I. INTRODUCTION

 

CH3CH2CH2CH2OCH2CH2OCH2CH2OCH2 

Piperonyl butoxide 

Piperonyl butoxide (CAS 51-03-6; NCI C02813), 5-((2-(2­

butoxyethoxy)ethoxy)-methyl-6-propyl-l,3-benzodioxole, is used to 

enhance the insecticidal properties of the pyrethrins, by 

blocking the pyrethrin detoxification enzymes in the insect 

(Metcalf, 1966). Pyrethrins alone produce a very rapid knockdown 

of insects, followed by substantial recovery, whereas addition of 

a synergist such as piperonyl butoxide decreases the insecticidal 

dose of pyrethrin (Metcalf, 1966). Piperonyl butoxide is also 

formulated with synthetic pyrethrin analogues, such as allethrin 

and tetramethrin (Stanford Research Institute, 1976). 

Piperonyl butoxide has commercial importance as a synergist for 
insecticides. Approximately 1 million pounds of this chemical 

were used in the United States in 1974, of which 80% was used in 

commercial, domestic (house and garden), and industrial 

establishments, and 20% on livestock and poultry (Ayers and 

Johnson, 1976). 

The acute oral LD^ of piperonyl butoxide has been reported as 

11.5 ml/kg for rats of unspecified strain (Draize et al., 1948) 

and as between 7.5 and 10.0 gtn/kg for Wistar rats (Sarles et al., 

1949); that for mice (strains not specified) has been reported as 

8.3 ml/kg (Draize et al., 1948) and as 3,800 rag/kg (Kenaga and 

Allison, 1969). 

The long-term toxicity of piperonyl butoxide was investigated by 

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

and agricultural chemicals. These investigators did not obtain a 

significant incidence of tumors in either the (C57BL/6 x 

C3H/Anf)Fl or the (C57BL/6 x AKR)F1 hybrid mice, the only 

species tested, and they categorized this chemical among those 

that probably required further testing. On the basis of these 

preliminary results, piperonyl butoxide was selected for study in 

the Carcinogenesis Testing Program. 
II. MATERIALS AND METHODS
 


A. Chemical
 


The test chemical used in the bioassay was obtained as a



91-kilogram batch of Lot No. 5 technical-grade piperonyl butoxide
 


from Niagara Chemical Company, FMC Corporation, Middleport, New
 


York. Gas chromatographic (gc) analysis performed at Frederick
 


Cancer Research Center, Frederick, Maryland, showed three major
 


components and some minor ones totaling less than 1% each. As
 


identified by the manufacturer, the three major components and
 


their respective percentages of the total gc peak area were
 


piperonyl butoxide, 88.4%; butyl carbitol, 2.1%; and an isomer of
 


piperonyl butoxide, 2.4%. The infrared absorption spectra were
 


identical with that of an authentic standard. The
 


technical-grade piperonyl butoxide as described above will
 


hereinafter be referred to in this report as piperonyl butoxide.
 


The piperonyl butoxide was stored at 7 C until used.
 

B. Dietary Preparation
 


Test diets containing piperonyl butoxide were prepared fresh each
 


week in 6- to 12-kilogram batches at appropriate doses. A known
 


weight of the chemical was first mixed with an equal weight of
 


autoclaved 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.
 


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 housed within the test facility for 2 weeks and then
 


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


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., Gar fie Id, 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 autoclaved 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.
 


Contaminated bedding was disposed of through an enclosed vacuum
 

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

periodically into an incinerator. The cages were sanitized at 

82-88 C in a tunnel-type cagewasher (industrial Washing 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 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 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 

t ime. 

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 999-81-5) chlorocholine chloride 

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

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

on feeding studies of the following chemicals: 

(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
 


E. Subchronic Studies
 


Subchronic feeding studies were conducted to estimate the maximum
 


tolerated doses (MTDs) of piperonyl butoxide, 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 butoxide 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 14,700, 21,500,
 


31,500, 46,000, and 68,000 ppm; the doses used for the mice in
 


the subchronic studies were 4,600, 6,800, 10,000, 14,700, 21,500,
 


31,500, 46,000, and 68,000 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 the 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
 


CO- inhalation and necropsied. The lowest doses at which
 


clinical and histopathologic findings were observed were 21,500
 


ppm in the male and female rats and 14,700 ppm in the male and
 


female mice. In the rats, interstitial pneumonia, perivascular
 


lymphoplasmacytosis, and peribronchiolar lymphocytosis were
 


observed, but were not considered to be related to administration
 


of the test chemical. In the mice, extramedullary hematopoiesis
 


was observed in one male and five females, polypoid nuclei were
 


observed in livers of two males and one female, and slight
 


vacuolization of hepatocytes was observed in four females.
 


8


Table 1



Piperonyl Butoxide Subchronic Feeding Studies in Rats and Mice
 


(ppra)
 


RATS
 


14,700
 


21,500
 


31,500
 


46,000
 


68,000
 


MICE
 


4,600
 


6,800
 


10,000
 


14,700
 


21,500
 


31,000
 


46,000
 


68, 000
 


Male
 


Mortality
 


Numb er Week on
 


Dead Study
 


4 8



5 1



1 8



4 8



5 2



5 2



5 2



Mean Weight
 


at Week 7



as % of
 


Control
 


94
 


73
 


60
 


60
 


91
 


87
 


76
 


85
 


76
 


Female
 


Mortality
 


Number Week on
 


Dead Study
 


3 8



5 1



1 8



4 8



4 8



5 2



5 2



Mean Weight
 


at Week 7



as % of
 


Control
 


95
 


76
 


68
 


38
 


94
 


93
 


76
 


72
 


87
 

Ten percent depression in body weight was taken as the major
 


criterion for estimation of MTDs. The doses required to produce
 


this response were determined by the following procedure: first,
 


least squares regressions of mean body weights versus days on
 


study were used to estimate mean body weights of each of the
 


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


dosed groups at day 49 relative to weights of corresponding
 


control groups were plotted against 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.
 


When these data were evaluated in conjunction with the clinical
 


and histopathologic findings, the low and high doses for chronic
 


studies using rats were set at 5,000 and 10,000 ppm, and those
 


for chronic studies using mice were set at 2,500 and 5,000 ppm.
 


F. Chronic Studies
 


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


feeding studies are shown in tables 2 and 3. Doses administered
 


to the mice were reduced after week 30 as indicated.
 


10
 

Table 2. Piperonyl Butoxide Chronic Feeding Studies in Rats
 


Sex and 


Test 


Group 


Male
 


Matched-Contro1 
 20 0 107
 


107
 


107
 


Low-Dose 
 50 5,000 

High -Dose 
 50 10,000 

Female
 


Matched-Contro1 
 20 0 107
 


107
 


107
 


Low-Dose 
 50 5,000 

High -Dose 
 50 10,000 

Piperonyl
 


Initial Butoxide Time on
 


No. of in Diet (b) Study
 


Animals (a) (ppm) (weeks )



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


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


11
 

Table 3.


Sex and 


Test 


Group 


Male 


Matched-Control 
 20	 
 0 112
 


Low-Dose 
 50 
 2,500 30
 


High-Dose 
 50	 
 5,000 30
 


Female 

Matched-Control 20 

0 112 

Low-Dose 50 2,500 30 

High-Dose 50 5,000 30 

 Piperonyl Butoxide Chronic Feeding Studies in Mice
 


Initial 


No. of 


Animals (a)


Piperonyl 


Butoxide Time on 


in Diet (b) Study 


 (ppm) (weeks ) 


500 82 


2,000 82 


500 82 

2,000 82 

Time-Weighted 


Average Dose (c) 


(ppm) 


1,036
 


2,804
 


1,036 

2,804 

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

(b) Test and control diets were provided ad^ libitum. 

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

£(no. of weeks receiving each dose) 

12 
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. 

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

bioassay were killed by asphyxiation using C02 and necropsied. 

Necropsies were also performed on all animals found dead, unless 

precluded by autolysis or severe cannibalization. 

The pathologic evaluation consisted of gross and microscopic
 


examination of major tissues, major organs, and all gross
 


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


embedded in paraffin, sectioned, and stained with hematoxylin and
 


eosin. The following tissues were examined microscopically:
 


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


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


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


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


and large intestine, kidney, urinary bladder, pituitary, adrenal,
 


thyroid, parathyroid, testis, prostate, mammary gland, uterus,
 


ovary, brain (cerebrum and cerebellum), and all tissue masses.
 


Peripheral blood smears also were made for all animals, whenever
 


possible.
 


13
 

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 auto lysis. Thus,
 


the number of animals from which particular organs or tissues
 


were examined microscopically varies, and does not necessarily
 


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


group.
 


H. Data Recording and Statistical Analyses
 


Pertinent data on this experiment have been recorded in an auto­


matic 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 section.
 


14
 

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 

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

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

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

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

except the departure from linearity test, which is only reported 

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

The incidence of neoplastic or nonneoplastic lesions has been 

given the ratio as of the number of aniirsls 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. 

15 
The purpose of the statistical analyses of tumor incidence is to 

determine whether animals receiving the test chemical developed a 

significantly higher proportion of tumors than did the control 

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

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

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

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

compared simultaneously with those for a control group, a 

correction to ensure an overall significance level of 0.05 may be 

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

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

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

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

where the Fisher exact P values are shown. 

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

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

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

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

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

from linear trend. 

16 
A time-adjusted analysis was applied when numerous early deaths 

resulted from causes that were not associated with the formation 

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

first tumor was observed were excluded by basing the statistical 

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

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

such an early tumor was found, comparisons were based exclusively 

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

the first tumor was 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. 

17 
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
 


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
 


18
 

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 greater than unity, the lower 

limit indicates the absence of a significant result while the 

upper limit indicates that there is a theoretical possibility of 

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

detected under the conditions of this test. 

19 
20

 
III. RESULTS - RATS
 


A« Body Weights and Clinical Signs (Rats)
 


A dose-related depression in the mean body weights of both dosed 

male and female rats, when compared with corresponding matched 

controls, was observed throughout the bioassay (figure 1). 

However, the depressions in the mean body weights of the low- and 

high-dose groups were slight during the first 50 weeks. Wasting 

among the rats and redness of the skin and mucous membranes 

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 butoxide 

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. 

21 
a a 

o a 

A O o 

A 

D a a n a 

Q Q a @ 

a

MALE RATS 

DMATCHED CONTROL 

OLOW DOSE 

AHIGH DOSE 

I I I i 

40 50 60 70 

TIME ON STUDY (WEEKS) 

I 

O 

O 300­

o o o 8

m A 

2 D a a 

s n a S 

FEMALE RATS 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 

TIME ON STUDY (WEEKS) 

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

22
°""*.j 0 90 * *'*-*-ljh,

tk°63^ 

s < ^ > 

ff n 60 " 

CO 

u. 

° ncn 

3 

CD nAn 

CO 

0 

CC 

MALE RA1rs 

0.20-1 AHIUH UOSt 

OLOW DOSE 

CD MATCHED CCWTum 0.10­

Q 

0 15 30 45 60 75 90 105 12 

TIME ON STUDY (WEEKS) 

•-••­• •-••-• •-••-•a

'^1 — ^. 

A­— ^.^ "£>"&&.­

1 

URVIVAl

p £ 8 c 

<a 

O 

> a5°­

Ij 

1 0.40­

m 

O 

EC 

FEMALE R ATS 

0.20­ A HIGH DOSE

 

O LOW DOSE 

0.10- C] MATCHED CCNTRni 

nTIME ON STUDY (WEEKS) 

Figure 2. Survival Curves For Rats Administered Piperonyl Butoxide in the Diet 

23 
In male rats, 37/50 (74%) of the high-dose group, 36/50 (72%) of 

the low-dose group, and 15/20 (75%) of the controls were alive at 

termination of the study. In females, 40/50 (80%) of the 

high-dose group, 41/50 (82%) of the low-dose group, and all 20 of 

the controls were alive at termination 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.
 


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
 


24
 

such in this study. They did not compress adjacent hepatic
 


parenchyma. These lesions are similar morphologically to those
 


described by Squire and Levitt (1975) as "basophilic foci."
 


Lesions classified as "hepatocytomegaly" 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."
 


Lesions classified as "neoplastic nodules" had many similarities
 


to the previously described proliferative hepatocytic lesions.
 


However, the lesions classified as neoplastic nodules were larger
 


and contained more distinct abnormality of lobular architecture;
 


liver cords at the periphery of the neoplastic nodules were
 


oriented perpendicular to cords of adjacent normal hepatic
 


parenchyma, and distinct compression of adjacent normal liver was
 


evident.
 


Endocrine tissues were the most frequent sites of neoplasms in
 


25


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, islet-cell 

tumors of the pancreas, and pheochromocytomas and cortical 

carcinomas 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 a C-cell carcinoma. 

Follicular-cell neoplasms occurred less frequently than C-cell 

neoplasms. The follicular-cell adenomas appeared microscopically 

26 
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. Follicularcell lesions were classified as carcinomas 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. 

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 

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

spherical cells with abundant, slightly basophilic cytoplasm arid 

large nuclei with abundant chromatin. Islet-cell adenomas 

appeared as discrete, encapsulated nodules of islet cells that 

compressed the adjacent normal pancreas; diagnosis of islet-cell 

carcinoma in a high-dose male rat was based on invasion of the 

capsule surrounding the neoplasm. 

27 
Two neoplasms were observed in the intestinal tract. A malignant
 


spindle-cell tumor was present in the wall of the cecum of a



high-dose male rat. This was a large neoplasm that extended into
 


the mucosa; the diagnosis of leiomyosarcoma was based on the
 


location in the wall and the cellular morphology. A



hemangiosarcoma was present in the wall of the large intestine of
 


a low-dose female rat.
 


Malignant lymphomas occurred rather frequently. Most of these
 


neoplasms were composed of relatively undifferentiated
 


lymphoreticular cells and involved numerous organs and tissues
 


throughout the body. The organ most frequently affected was the
 


spleen; also frequently affected were the liver, lymph nodes,
 


thymus, and lungs. There was only one malignant lymphoma in the
 


control group of female rats in this study; this is a relatively
 


common tumor in Fischer 344 rats. Evidence of leukemia (masses
 


of neoplastic lymphoreticular cells in vessel lumens) was seen in
 


some cases.
 


Proliferative pulmonary lesions were observed rather infrequently
 


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
 


28
 

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. 

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. 

Mesothelioma of the tunica vaginalis was observed in sections of 

testis from four male rats, all of which were from dosed groups. 

One generalized peritoneal mesothelioma was present in a low-dose 

male rat. 

Squamous-cell carcinomas were observed in the preputial gland of 

a high-dose male rat and in the cliteral gland of a high-dose 

female. 

The most common neoplasms of the mammary gland were 

29 
fibroadenomas; these occurred only in females, were often
 


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


Various other types of malignant and benign neoplasms were
 


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


in other organs and tissues throughout the body. No apparent
 


difference in incidence of these neoplasms between dosed and
 


control groups was present.
 


Numerous inflammatory, degenerative, and proliferative lesions
 


commonly observed in aged Fischer 344 rats occurred with
 


approximately equal frequency in dosed and control rats. These
 


lesions included chronic tracheitis; 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; testicular
 


atrophy; and C-cell hyperplasia of the thyroid.
 


Other nonneoplastic proliferative lesions included hyperplasia of
 


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


parathyroid, endometrium, mammary epithelium, and hepatocytes.
 


Based on the histopathologic examination, there were instances in
 


this study in which neoplastic or hyperplastic lesions occurred
 


30
 

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 butoxide 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. 

In female rats, the result of the Cochran-Armitage test for 

positive dose-related trend in the incidence of lymphomas is 

significant (P = 0.007). The results of the Fisher exact test 

show that the incidence in the high-dose group is significantly 

higher (P = 0.020) than that in the control group. The 

historical record of the incidence of lymphomas and leukemias in 

female rats at this laboratory to date is 19/191(10%). These 

historical-control groups include one with an incidence of 

animals with lymphoma or leukemia of 7/20 (35%) and another with 

an incidence of 6/20 (30%). The statistical conclusion suggests 

31 
that the incidence of lymphomas in female rats may be associated
 


with the administration of piperonyl butoxide; however, this
 


conclusion may be due to the lower than usual incidence in the
 


control group compared with the historical data.
 


Significant results in the negative direction are observed in the
 


incidences of neoplastic nodules of the liver and of adenomas or
 


carcinomas of the pituitary in the male rats; the incidences of
 


these tumors in the control group exceed those in the dosed
 


groups.
 


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


in the tables, the value of one is included; this indicates the
 


absence of significant positive results. It should also be noted
 


that each of the intervals has an upper limit greater than one,
 


indicating the theoretical possibility of the induction of
 


tumors, which could not be detected under the conditions of this
 


test.
 


32
 

IV. RESULTS - MICE
 


A. Body Weights and Clinical Signs (Mice)
 


A dose-related depression in the mean body weights of both dosed
 


male and female mice, when compared with corresponding matched
 


controls, was observed throughout the bioassay (figure 3).
 


Alopecia occurred at low incidences in the dosed groups of
 


females and may have been related to administration of the test
 


chemical. Corneal opacity and 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 butoxide 

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, 41/50 (82%) of the high-dose group, 42/50 (84%) of 

33 
Doses lowered for 

male mice at week 30. 

Qtrnu 

OQOOO 

DD 

°g* O A

 Q D 

° e A ° 

D 

%\ 

o o 

I 

O s uj 

>­

Q 

O 

D 

DD O O 

O AO , 

O 

A 

A A 

co 

<

Ul 

s y 

MALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

-1— —r­ 40 50 60 70 

TIME ON STUDY (WEEKS) 

D" 

D D 

] 

Doses lowared O O DD 

for female mice 

«week30. H mnnnn 

PjTnTtttHf 0 D A A AA ° O ° 

O 

D D 

A A A 

UJ

* ­ D 0 O 

o o A 

Q ,D O A O 

m 

z ago AOA 

DD0 O A 

A A 

UJ 

S 20. 

FEMALE MICE 

D MATCHED CONTROL 

O LOW DOSE 

A HIGH DOSE 

40 50 60 70 100 110 

TIME ON STUDY (WEEKS) 

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

34 
3 

0.20­

0.10­

0.90­

_J 0.70­

> 

in 

°0.50­

K 

< 

m 

O 

oc 

a. o.30­

0.20­

0.10­

A 

O 

C3

 HIGH DOSb 

 LOW DOSE 

 MATCHED CC NTRni 

45 60 75 

TIME ON STUDY (WEEKS) 

! ; 

A 

( ) 

A 

— 

I 

 -O - •» • 

\ 

a ^ 

0-. 

Oi

O 

( 

"

A

A-.. 

~1 

>£l 

1 

A--A 

FEMALE MICE 

A HIGH DOSE 

O LOW DOSE 

CD MATCHED CC NTnm 

TIME ON STUDY (WEEKS) 

Figure 4. Survival Curves For Mice Administered Piperonyl Butoxide in the Diet 

35 

PROBA WIVAL 

pU>

 f 3 C 

J 

0 < ! S 

U) 

0

 0.50­

h -] MALE MICE 

j l

. .-..-..-..-......-] •-•--• •-• •-• -.... D

""iS 1 .O-.-l J, ** o- *^- -i ­ A"tV 

^ 
the low-dose group, and 17/20 (85%) of the control group survived
 


to termination of the study. In females, 34/50 (68%) of the
 


high-dose group, 35/50 (70%) of the low-dose group, and 15/20
 


(75%) of the control group lived to termination of the study.
 


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 hepatocelluar
 


carcinoma. There were 10/20 in control males, 17/50 in low-dose
 


males, and 20/50 in high-dose males. Metastases were found in
 


two mice from each of these groups. There was one hepatocelluar
 


carcinoma in a control female, two in low-dose females, and five
 


in high-dose females. Metastases to the lungs were found in one
 


high-dose female.
 


The next most common tumors were of lymphoreticular origin
 


36
 

(malignant lymphoma and reticulum-cell sarcoma). Their 

incidences were as follows: males — controls 4/20, low-dose 

11/49, high-dose 6/50; females — controls 8/20, low-dose 14/47, 

high-dose 9/50. Many of these tumors were widely disseminated, 

involving several organs and forming tissue masses. 

One low-dose male had a widely disseminated round-cell tumor that 

was diagnosed as a mast-cell tumor. 

Hemangiosarcomas were found in one control male, one control 

female, three low-dose males, two low-dose females, and in one 

male and one female in the high-dose groups. Both of the tumors 

in the low-dose females and the one in a high-dose male were in 

multiple organs. 

There were occasional alveolar-cell adenomas and adenocarcinomas 

of the lung. They were distributed with approximately equal 

frequency between males and females and between dosed and control 

groups. 

Most other proliferative or neoplastic lesions were of single 

occurrence or very low incidence with approximately equal 

distribution between the dosed and control groups. There were 

37 
four adenomas of the lacrimal gland in high-dose males; none were
 


found in any of the female mice.
 


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, nephritis, focal hepatic necrosis, pancreatic
 


atrophy, cystic endometrium, and ovarian cysts.
 


Based on the histopathologic examination, there were no
 


differences in the incidences of neoplastic or nonneoplastic
 


lesions between dosed and control mice; it is concluded that the
 


dietary administration of piperonyl butoxide was not carcinogenic
 


in B6C3F1 mice under the conditions of this bioassay.
 


D. Statistical Analyses of Results (Mice)
 


Tables Fl and F2 in Appendix F contain the statistical analyses
 


of the incidences of those primary tumors that occurred in at
 


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


in one or more than one group.
 


38
 

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

positive dose-related trend in the incidence of adenomas of the 

lacrimal gland is significant (P = 0.022), but the results of the 

Fisher exact test are not significant. 

A significant dose-related trend in the negative direction is 

observed for the incidence of lymphomas in female mice; the 

incidence of this tumor in the control group exceeds the 

incidences in the dosed groups. 

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

in the tables, the value of one is included; this indicates the 

absence of significant positive results. It should also be noted 

that each of the intervals has an upper limit greater than one, 

indicating the theoretical possibility of the induction of tumors 

by piperonyl butoxide, which could not be detected under the 

conditions of this test. 

39 
40

 
V. DISCUSSION
 


Dose-related depressions in mean body weight occurred during most 

or all of the bioassay in both rats and mice administered 

piperonyl butoxide. Redness of the skin and mucous membranes and 

wasting among the rats and alopecia among the mice occurred at 

low incidences in some of the dosed groups and may have been 

related to administration of the test chemical. Survival of the 

rats and mice was unaffected by the piperonyl butoxide and was 

80% or greater 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 female rats, lymphomas occurred at incidences that were 

dose-related (P = 0.007); in a direct comparison, the incidence 

of the tumor in the high-dose group was higher (P = 0.020) than 

that in the control group (controls 1/20, low-dose 7/50, 

high-dose 15/50). However, the incidence of lymphomas, 

leukemias, and reticuloses in historical-control female Fischer 

344 rats at the same laboratory was 19/191 (10%). These 

historical-control groups include one with an incidence of 

animals with lymphoma or leukemia of 7/20 (35%) and another with 

incidence of 6/20 (30%). Thus, the incidence of lymphomas in the 

41 
control female rats of the present bioassay may have been 

abnormally low, and the occurrence of the higher incidence in 

the dosed groups cannot be clearly related to administration 

of the piperonyl butoxide. 

In the male mice, adenomas of the eye or lacrimal gland 

occurred at incidences that were dose related (P 0. 02 3) ' 

but in direct comparisons the incidences in the individual 

dosed groups were not significantly higher than that in 

the control group (controls 0/20, low-dose 0/49, high-dose 

4/50) thus, the occurrence of this tumor in the male 

mice was not c 1 early re 1 a ted to administration of the 

test chemical. 

When piperonyl butoxide was administered in the diet for 2 

years to Wistar albino rats at doses as high as 25, 000 ppm 

(Sarles and Vandegrift, 1952), no evidence was found for 

carcinogenicity. When the chemical was administered at 464 

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

1,112 ppm for 18 months, to hybrid mice (C57BL/6 x C3H/Anf 

and C57BL/6 x AKR), an elevated incidence of reticulum-cell 

sarcoma was observed p 0.05) but was not significant 

at the selected criterion of P 0.01 (NTIS, 1968; Innes 

et al., 1969) . No evidence of carcinogenicity was 

observed when neonatal Swiss mice were given subcutaneous 

injections of 0.1 ml of a 5% solution of the chemical in 

42 
redistilled tricaprylin at days 1 and 7 and 0.2 ml at days 14 and
 


21; however, when the chemical was injected subcutaneously into
 


the neonatal mice in combination with Freon 112, hepatomas
 


developed at a statistically significant incidence (Epstein et
 


al., 1967). Certain structural congeners of piperonyl butoxide
 


(safrole, isosafrole, and dihydrosafrole) have been reported
 


carcinogenic for rats (Osborne-Mandel) and mice, (C57BL/6 x



C_H/Anf and C57BL/6 x AKR) inducing tumors of the liver,
 


esophagus, or lung, depending on species and sex (Long et al.,
 


1963; Hagan et al., 1965; Innes et al., 1969).
 


Piperonyl butoxide is used commercially with pyrethins. This
 


bioassay, however, tests the carcinoginicity of technical-grade
 


piperonyl butoxide alone, and no conclusions can be drawn from
 


the data in this report as to the possible carcinogenic effects
 


of the combination of the two chemicals.
 


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


was no convincing evidence that piperonyl butoxide was
 


carcinogenic for Fischer 344 rats or B6C3F1 mice.
 


43
 

44

 
VI. BIBLIOGRAPHY
 


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


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


Ayers, J. H. and Johnson, 0. H., Insecticides In: Chemical
 


Economics Handbook, Stanford Research Institute, Menlo Park,
 


Calif., 1976, sec. 573.30071.
 


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


Panel on Carcinogenicity ojf the Cancer Research Commission o_f the
 


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


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


Soc. B 34;187-220, 1972.
 


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


1970, pp. 48-52.
 


Draize, J. H., Alvarez, E., Whitesell, M. F., Woodard, G., Hagan,
 


E. C., and Nelson, A. A., Toxicological investigations of
 


compounds proposed for use as insect repellents. J. Pharmacol.
 


Exp. Therap. 93:26-39, 1948.
 


Epstein, S. S., Joshi, S., Andrea, J., Clapp, P., Falk, H., and
 


Mantel, N., Synergistic toxicity and Carcinogenicity of 'Freons1



and piperonyl butoxide. Nature 214:526-528, 1967.
 


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. ^: 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. 42:1101-1114, 1969. 

45
 

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

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

1958. 

Kenaga, E. E., and Allison, w. E., Connnercial and experimental 

organic insecticides. Bull. Entomol. Soc. Amer. 15 (2):85-148, 

1969. 

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

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

Biomed:. Res. 2_: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. 

22.:595-604, 1963. 

Metcalf, R. L., Insecticides. In: Kirk-Othmer Encyclopedia of 

Chemical Technology, Vol. !l' 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. 

National Technical Information Service, Evaluation of 

Carcinogenic, Teratogenic, and Mutagenic Activities of Selected 

Pesticide and Industrial Chemicals, Vol. Carcinogenic Study, 

U. S. Department of Commerce. PB-223 159. August, 1968, p. 64. 

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

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

induced by different numbers of administrations of benzo(a) 

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

Sarles, M. P. and Vandegrift, W. B., Chronic oral toxicity and 

related studies on animals with the insecticide and pyrethrum 

synergist, piperonyl butoxide. Amer. J, Trop. Med. ~ 

!:862-883, 1952. 

Sar 1es, M. P • , Dove , W. E • , and Moore, D. H., Acute toxicity and 

irritation tests on animals with the new insecticide piperonyl 

butoxide. Am. :!...:_ Trop. Med. 29:151-166, 1949. 

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

classification of specific hepatocellular lesions in rats. 

Cancer Res. 35:3214-3223, 1975. 

46 
Stanford Research Institute, Stage I Chemical Dossier. 

Research Institute, Menlo Park, Calif., April 1976. 

 Stanford 

Tarone, R. E., Tests for 

Biometrika 62:679-682, 1975. 

 trend in life table analysis. 

47
 

48

 
APPENDIX A



SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 


RATS ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


49
 

50

 
TABLE A1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMALS INITIALLY IN STUDY 20 50 50 

ANIMAL S N<!CROPSIED 20 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 2 0 50 50 

INTEGUMENTARY SYSTEM
 


*SKIN (20) (50) (50)
 


KSRATOACANTHOMA 1 (5X) 1 (2X)
 


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


PIBPOMA 2 (UX) 3 (6%)
 


FIBROSARCOMA 1 (2*) 1 (2X)
 


LIPOMA 1 (2X)
 


LIPOSARCOMA 1 (2X)
 


NEURILEMOMA, MALIGNANT 1 (2X)
 


RESPIRATORY SYSTEM
 


tLUNG (20) (48) (50) 

ALVJOLAR/BRONCHIOLAR ADENOM A 2 (4X) 

ALVEOLAE/B80NCHIOLAR CARCINOM A 1 (5X) 1 (2X) 

HEMATOPOIETIC SYSTEM 

*MULTIPLE ORGANS (20) (50) (50) 

MALIGNAN T LYMPHOMA , NOS 2 (10X) 3 (6X) 3 (6X) 

«ALIG.LYMPHOHA, UNDIPFER-TYPE 7 (35X) 12 (2«X) 9 (18X) 

MALIG.LYMPHOHA , LYHPHOCYTIC TYPE 1 (2X) 

CIRCULATOP Y SYSTEM 

*HEAH T , (20) (50) (50) 

ALVEOLAR/BEONCHIOLAR CA, INVASIV 1 (5X) 

DIGESTIVE SYSTEW 

*LIVE3 (20) (50) (49) 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 


* NUMBER OF ANIMALS NECROPSIED
 


51
 

TABLE A1. MALE RATS: NEOPLASMS

#CECOH 

LEIOHYOSARCOMA 

ORINAR Y SYSTEM 

NONE 

ENDOCRIN E SYSTEM 

#PITUITAR Y 

CARCINOMA , NOS 

ADENOMA , NOS 

CHHOMOPHOBE ADENOM A 

tADRFNA L 

CORTICAL ADENOM A 

PHEOCHROMOCYTOHA 

tTHYROI D 

FOLLICOLAR-CELL ADENOM A 

FOLLICOLAR-CELL CARCINOMA 

C-CELL ADENOMA 

C-CELL CARCINOMA 

#PANCREATIC ISLETS 

ISLET-CELL ADENOMA 

ISLET-CELL CARCINOMA 

REPRODUCTIVE SYSTEM 

•PREPUTIAL GLAN D 

SQUAHOUS CELL CARCINOM A 

#TESTIS 

INTERSTITIAL-CELL TUBOR 

HERVOUS SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

fiONE 

f NUMBER OF ANIMAL S HITH TISSUE

* NUMBER OF ANIMAL S NECROPSIED 

 (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

(20) (U9) (U8) 

1 (2X) 

(19) (l»9) (t8) 

1 (2X) 

H (21X ) 

1 (5X) 7 (14X) 6 (13X) 

(20) (50) (50) 

1 (5X) 

2 (10%) « (8X) 1 (8*) 

(20) («9) (50) 

2 (4X) 

1 (5%) 

1 (2X) 

5 (10*) 

1 (2%) 

1 (2X) « (8X) 

(18) 

1 (6*) 

(HH) 

U (9*) 

(U8) 

2 (HI) 

1 (2X) 

(20) (50) (50) 

1 (2*) 

(20) (49) (50) 

19 (95%) U8 (98X) 46 (92X) 

 EXAMINED MICROSCOPICALLY 

52 
TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NUSCUIOSKISLETA L SYSTEM 

NOSE 

v v ——•—• ———.———•-».••———————————•—•«•— «•»<•__••_«•• •»_••»•»»•• *« — •—••••i——•••••«•••»••—••«•••.



BODY CAVITIES
 


*PLEURA (20) (50) (50)



ALVEOLAR/B30NCHIOLAR CA, INVASIV 1 (5«)
 


*TUNICA VAGINALIS (20) (50) (50)



UESOTHELIOMA, NOS 2 (H%) 2 (H%)

 ••• W«F••. ._««__« KI_>.VV«»«*V«IB« BM^IK •• __ ^« •«^••• « KB«*»_« •*_••••——••«.«.•••»— •^•••••^—^^••^•— I



ALL OTHER SYSTEMS
 


'MULTIPLE ORGANS (20) (50) (50)



NESOTH?LIOMA, NOS 1 (2»)
 


ANIMAL DISPOSITION SUMMARY
 


ANIMALS INITIALLY IN STUDY 20 50 50
 


NATURAL DEATHS 4 11 8



MORIBUND SACRIFICE 1 3 5



SCHEDULED SACRIFICE
 


ACCIDENTALLY KILLED
 


TERMINAL SACRIFICE 15 36 37
 


ANIMAL MISSING
 


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


53
 

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HI6H DOSE 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIHAE Y TUMORS* 20 50 H9 

TOTA L PRIMAR Y TUMORS 1*2 9U 92 

TOTAL ANIMAL S WITH BENIG N TUMORS 19 «9 47 

TOTAL BENIG N TUMORS 30 72 69 

TOTAL ANIMAL S HITH MALIGNAN T TUMORS 10 17 19 

TOTAL MALIGNAN T TUMORS 10 19 21 

TOTAL ANIMAL S WITH SECONDARY TDMORSf 1

TOTAL SECONDARY TUMORS 2

TOTAL ANIMAL S WITH TUHORS UNCERTAINBENIGN O R MALIGNANT 2 3 2 

TOTAL UNCERTAIN TUMORS 2 3 2 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN- 

PRIMAR Y OR METASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUHORS 

I SECONDARY TUHORS: HETASTATIC TUMORS OR TUHORS INVASIVE INTO HI ADJACENT ORGAN 

54 
TABLE A2. 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH D 

ANIMAL S INITIALL Y IN STUDY 20 50 50 

ANIMAL S NECPOPSIED 20 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 20 50 50 

INTEGUMENTAR Y SYSTEM 

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

ADENOCARCINOMA , NOS 1 (2X) 

FIBROM A 1 

NEURILEMOM A 1 

RESPIRATOR Y SYSTE M 

tLUN G (20) (50) (50) 

SQUAMOU S CELL CARCINOMA 1 

ADENOCARCINOMA , NOS, MBTASTATIC 1 (2X> 

HEKATOPOIETI C SYSTEM 

#EP.AI N (20) (50) (50) 

MALIGNAN T RETICULOSIS 1 

*MULTIPL F ORGAN S (20) (50) (50) 

MALIGNAN T LYMPHOHA , NO S 1 (2«) 6 

MALIG.L'YNPHOMA , UNDIFFER-TYPE 1 (5X) 5 (10X) 9 

1ALIGNAN T LYMPHOBA . HIXE D TYPE 1 (2X) 

#SPLEE N (18) (50) (50) 

HEMANGIOSARCOM A 1 

CIRCULATOR Y SYSTE M 

#HEAB T (20) (50) (50) 

APENOCARCINOMA , NOS, METASTATIC 1 (2X) 

DIGESTIV E SYSTE M 

#CECU1 (19) (50) (US) 

HKMA.MIO.SARC, SJIS.fi l_J2Si, 

# NUMSt P OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NU«BE F OF ANIMAL S NECROPSIED 

OSE 

(2X) 

(2X) 

(2X) 

(2X) 

(12X) 

(18%) 

(2X) 

55 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

URINARY SYSTEH
 


NONE
 


ENDOCRINE SYSTEM
 


tPITUITAF Y 

CARCINOMA , NOS 

CHROHOPHOBE ADENOMA 

#ADRENA L 

CORTICAL CARCINOM A 

PHEOCHROHOCYTOMA 

#THYROID 

FOLLICDLAR-CELL CARCINOM A 

C-CELL ADENOSA 

C-CELL CARCINOM A 

#PANCREATI C ISLETS

 

ISLET-CELL ADENOM A 

REPRODUCTIVE SYSTEM
 


*MAMMARY GLAND
 


CARCINOMA, NOS
 


FIBROMA
 


FIBROADENOMA
 


*CLITORAL GLAND
 


SQOABOUS CELL CARCINOMA
 


f UTERUS
 


ENDOMETRIAL STROHAL POLYP
 


NEPVOUS SYSTEM
 


#BPAIN
 


EPENDYMOM A 

SPECIAL SENSE ORGAN S 

__J!21LE 

# NUMBFR OF ANIMAL S WITH TISSUE 

* NUMBE R OF ANIMAL S NECROPSIED 

MATCHED
 


CONTROL LOW DOSE
 


(19) 

(48) 

1 (5%) 1 (2*) 

8 (42%) 11 (23%) 

(20) (50) 

1 (5%) 

1 (2%) 

(20) (50) 

4 (20*) 

4 (8%) 

2 (4%) 

(20) 

(49) 

1 (5%) 

(20)	 	 (50)
 


1 (2%)
 


1 (2%)
 


3 (15%)	 	 8 (16X)
 


(20)	 	 (50)
 


(20)	 	 (49)
 


4 (20%) 7 (14%)
 


(20) 

(50) 

1 (2%) 

 EXAMINE D MICROSCOPICALLY 

HIGH DOSE
 


(49) 

14 (29%) 

(50)

2 (4%) 

(i»9) 

1 (2X) 

2 (ttX) 

1 (2*) 

(46) 

(50)
 


1 (2%)
 


2 (4%)
 


(50)
 


1 (2X)
 


(47)
 


3 (6%)
 


(50) 

56 
TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) -• — — — — — — — — — — — — — — — — — —— — — — ——————_—_—.——•——————______———__———— — — — — __—_.•.— _—«.»•.....».., 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MUSCULOSKEIETA L SYSTEM 

*SKKLETA L MUSCLE (20) (50) (50) 

SARCOMA , NOS 1 (2*) 

*MUSCLE OF NECK (20) (50) (50)
 


RHABDOMYOSARCOMA 1 (2X)
 


BODY CAVITIES
 


*MEniASTTNUM (20) (50) (50)
 


SQUANOUS CELL CARCINOMA, INVASIV 1 (2S)
 


ALL OTHER SYSTEMS
 


NONE
 


AVI!UL DISPOSITION SUMMARY '



ANIMALS INITIALLY IN STUDY 20 50 50
 


NATURAL DEATH9 5 8



MORIBUND SACRIFICE H 2



SCHEDULED SACRIFICE
 


ACCIDENTALLY KILLED
 


TERMINAL SACRIFICE 20 <»1 <*0
 


ANIMA L MISSING
 


* NUMBER OF ANIMALS UITH TISSUE EXAMINED MICROSCOPICALLY
 


* NUMBER OF ANIMALS NECROPSIED
 


57
 

TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y TUHORS* 15 33 33 

TOTAL PRIMAR Y TUHORS 23 U7 U8 

TOTAL ANIMAL S WITH BENIGN TUMORS 1« 21 21 

TOT?L BENIGN TUHORS 20 32 2U 

TOTAL ANIMAL S WITH MALIGNAN T TUHORS 3 14 21 

TOTAL MALIGNAN T TUHORS 3 15 2« 

TOTAL ANIMALS WITH SECONDARY TUMORSI 1 1 

TOTAL SECONDAR Y TUHORS 2 1 

TOTAL ANIMALS WITH TOHORS UNCERTAIN- 

BENIGN OR MALIGNAN T 

TOTAL UNCERTAI N TUHORS 

TOTAL ANIMAL S WITH TUMOBS UNCERTAIN- 

PRIMAR Y OR METASTATIC 

TOTAL UNCERTAI N TUHORS 

* PRIMAR Y TUHORS: ALL TUHOHS EXCEPT SECONDARY TUMORS 

« SECONDAR Y TUHORS : METASTATIC TDHORS OR TUHORS INVASIV E INTO AN ADJACENT ORGAN 

58 
APPENDIX B



SUMMARY OF THE INCIDENCE OF NEOPLASMS IN
 


MICE ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


59
 

60

 
TABLE B1.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET

 

ANIMAL S 

ANIMAL S 

ANIMAL S 

ANIMAL S 

INITIALL Y IN STUDY 

MISSIN G 

NECROPSIED 

EXAMINE D HISTOPATHOLOGICALLY 

INTEGUMENTARY SYSTEM
 


*SU3CUT TISSUE
 


LIPOMA
 


HEHANGIOSARCOMA
 


NEUROFI PRO SARCOMA
 


RESPIRATORY SYSTEM
 


ttLUNG
 


HEPATOCELLULAR CARCINOMA, METAST
 


ALVEOLAR/BRONCHIOLAR 1DBNOMA
 


ALVEOLAR/BRONCHIOLAR CARCINOMA
 


HENANGIOSARCOMA, METAST4TIC
 


HEMATOPOIETIC SYSTEM 

*MOLTIPLE ORGAN S 

MALIGNAN T LYMPHOHA , NOS 

MALIG.LYMPHOMA , LYBPHOCYTIC TYPE 

1ALIG.LYMPHOSA , HISTIOCYTIC TYPE 

MAST-CEL L SARCOM A 

*SUSCUT TISSUE 

MALIG.LYMPHOMA , HISTIOCYTIC TY FE 

*SPLEEN 

HFMANGIOSAHCONA
 


MALIG.LYMPHOMA , LYNPHOCYTIC TYPE 

*P!ESFNTEHIC L. NODE 

HEPATOCELLULAR CARCINOMA, METAST 

MATCHED 

CONTROL 

20 

20 

20 

(20) 


2 (10%) 


1 (5%) 


(20) 


2 (10%) 


2 (10%) 


3 (15%) 


(20) 

1 (5%) 

(20) 

1 (5%) 

(20) 

(20) 

1 (5%) 

LOW DOSE 

50 

1 

49 

49 

(U9) 


3 (6%) 


1 (2*) 


(50) 


2 (4%) 


3 (6%) 


3 (6%) 


(49) 

3 (6%) 

1 (2%) 

1 (2%) 

(49) 

1 (2%) 

(49) 

1 (2%) 

1 (2%) 

(49) 

HIGH DOSE 

50 

50 

50 

(50) 


2 (<U) 


0»8)


2 (4%) 


6 (13%) 


2 (4%) 


1 (2X) 


(50) 

1 (2%) 

3 (6%) 

1 (2%) 

(50) 

(19) 

(49) 

# NUMBF R OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALLY 

* NHMB^ H OF ANIMAL S NECROPSIED 

61 
TABLE 81. MALE MICE: NEOPLASMS (CONTINUED) 

===~==~==~~~=====~~====~======~=~=======================================:: 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

fSMALL INTESTINE ( 20) (50) (50) 


MALIG.lYMPHOMA, LYMPHOCYTIC TYPE 3 (151) q (81) 


M'LIG.LYMPHOMA, HISTIOCYTIC TYPE 1 ( 21) 


CIRCULATORY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

HIV'ER (20) (50) (50) 

HEPATOCELLULAR CARCINOMA 10 (501) 17 (Jql) 20 (ltOI) 

HEM ANGIOSARCOMA 1 (51) 1 (21) 1 (21) 

IES0PHAG'JS (20) (50) (50) 


SQUAMOUS CILL CARCINOMA 1 (51) 


UF.INHY SYSTEM 

liKIDNEY (50) (50) 


HEP,TOCP.LLULAR CARCINOMA, METAST 


ENCOCRINE SYSTEM 

tTHYROID 

ADENOCARCINOMA, 

fOLLICULAR-CELl 

REPRODUCTIVE SYSTEM 

NONF 

NERVOUS S YSTE!! 

NONE 

(20) (50) 

NOS 2 (ltl) 

ADENOMA 1 (51) 

SPECIAL SENSE ORGANS 

*lY!/LACRIMAL GLAND (20) (q9) (50) 

--A.l2112.11L.-li.Q~------------------------------------..a!~...lUL 

t NU!!BEF OF ANIMALS 'ITH TISSUE Ell!INBD !ICROSCOPIClLLY 

* NUMBER OF ANIMALS NECROPSIED 

6~ 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MOSCULOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

NONE 

ALL OTHER SYSTEMS 

NONE 

ANIMAL DISPOSITION SUMMARY 

ANIMALS INITIALLY IN STUDY 

NATURAL DEATHS 

MORIBUND SACRIFICE 

SCHEDULED SACRIFICE 

ACCIDENTALLY KILLED 

TERMINAL SACRIFICE 

ANIMAL MISSING

 20

 3

 17

 50

 7

 42

 1 

 50 

9 

 U1 

# NUMBER 

* NUMBE R

 OF ANIMAL S BITH TISSUE

 OF ANIMAL S MECROPSIED 

 EXAMINE D MICROSCOPICALLY 

63 
TABLE B1. MALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

Cl 1NTROL LOW DOSE HIGH DOSE 

TUMO R SUMMAR Y 

TOTAL ANIMAL S WITH PRIHAR Y 

TOTAL PRIHARY TOHORS 

 TOHORS* 16 

26 

32 

H <4 

31 

43 

TOTAL ANIMAL S WITH BENIGN 

TOTAL BENIGN TOHORS 

 TOMORS U 

5 

9 

9 

10 

12 

TOTAL ANIMAL S WITH HALIGNANT 

TOTAL HALIGNANT TOHORS 

 TOHORS 15 

21 

27 

35 

27 

31 

TOTAL ANIMAL S WITH SECONDARY 

TOTAL SECONDARY TOHORS 

 TOHORS* 2 

<t 

2 

2 

3 

3 

TOTAL ANIHALS WITH TOMORS ONCERTAINEEHIGN OR HALIGNANT 

TOTAL ONCERTAIN TOHORS 

TOTAL ANIHALS WITH TOHORS ONCERTAINPRIHARY OR METASTATIC 

TOTAL ONCERTAIN TOHORS
 


* PRIMARY TOHORS: ALL TOHORS EXCEPT SECONDARY TOHORS
 


# SECONDARY TUHORS: HETASTATIC TOHORS OR TOHORS INVASIVE INTO AH ADJACENT ORGAN
 


64
 

TABLE B2.

 

SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALLY IN STUDY 

ANIMALS MISSING 

ANIMALS NECROPSIED 

ANIMALS EXAMINE D HIS1!^PATHOLOGICALLY 

20 

20 

20 

50 

3 

47 

47 

50 

50 

50 

INTEGUMENTARY SYSTEM
 

*SUBCUT TISSUE
 

FIBROSARCOMA
 

LIPOMA
 

(20) 

1 (5%) 

1 (5X) 

(47) 

1 (2X) 

(50) 

1 (2*) 

RESPIRATORY SYSTEM 

#LUNG
 

HEPATOCELLULAR CARCINOMA, METAST
 

ALVEOLAR/BRONCHIOLAR ADENOMA
 

ALVEOLAR/BRONCHIOLAR CARCINOMA
 

FIBROSARCOMA, HETAST&TIC
 

OSTEOSAFCOMA, METASTATIC
 

(20)
 

2 (10%)
 

(47)
 

H (9%)
 

2 (4X)
 

(18)
 

1 (2X)
 

2 (4X)
 

2 (<4X)
 

1 (2X)
 

HFMATOPOIETIC SYSTEM 

*MULTIPLE ORGAN S 

MALIG.LYMPHOMA , 

MALIG.LYMPHOHA , 

 LYMPHOCYTIC

 HISTIOCYTIC

 TYPE 

 TYPE 

(20)
 

3 (15X)
 

(47)
 

2 (UX)
 

6 (13X)
 

(50)
 

5 (10X)
 

2 (4X)
 

*SUBCOT TISSUE 

MALIG.LYMPHOMA, 

MALIG.LYMPHOMA , 

 LYMPHOCYTIC TYPE 

 HISTIOCYTIC TYPE 

(20)
 

1 (5X)
 

2 (10X)
 

(U7)
 

2 (4X)
 

fHESENTESIC L. NODK 

HALIG.LYHPHOHA . 

MALIG.LYMPHOMA , 

 LYHPHOCYTIC TYPE 

 HISTIOCYTIC TYPE 

(20)
 (46)
 

1 (2X)
 

1 (2X)
 

(44)
 

#SMALL INTESTINE 

MALIG.LYMPHOMA , 

MALIG.LYMPHOMA , 

 LYMPHOCYTIC TYPE 

 HISTIOCYTIC TYPE 

(19)
 

1 (5X)
 

(46)
 

1 (2X)
 

(48)
 

1 (2X)
 

#THYMU S 

ZIB.B2S&R.C.QJ1A 

(18)
 (44)
 (42)
 

* NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY
 

* NUMBEE OF ANIMALS NECROPSIED
 

65
 

TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

MALIG.LYMPHOMA ,

MALIG.LYMPHOMA ,

 LYMPHOCYTIC TYPE

 HISTIOCYTIC TYPE

 1 (6*) 1 (2«) 

1 (2X) 

CIRCULATOR Y SYSTEM 

#H£AHT

FIBROSARCOMA

 (19) (47) (48)
 

1 (2%)


DIGESTIVE SYSTEM
 

#LIVER

HEPATOCELLULAR CAHCINOBA

HEMANGIOSARCOMA

 (20)

 1 (5X)

 1 (5%)

 (47)

 2 (H%)

 (<»8)
 

5 (10X)
 

1 (2«)
 

*PANCPEAS

ADENOCAFCINOMA. NOS

 (17) (46) (»6)
 

1 (2*)
 

URINARY SYSTEM
 

NONE
 

ENDOCRINE SYSTEM
 

•PITUITARY	 

ADENOMA, NOS 

#ADRENA L 

CORTICAL ADENOMA 

CORTICAL CARCINOMA 

tTHYROID 

FOLLICULAR-CEL L ADENOM A 

#PANCRPATI C ISLETS 

ISLET-C-ELL ADENOM A 

(19 ) 

(20) 

(20) 

1

(17) 

 (5«) 

1

(U5 ) 

1

 (2%) 

(2%) 

(U5) 

(HI) 

1 (2X) 

(47) 

2

(46) 

1 (2*) 

REPRODUCTIV E SYSTE M 

*MAMMAR Y GLAN D (20) (47) (50) 

# NUMBE R

* NUMBE R

 OF ANIMAL S

 O F ANIMAL S

 WIT H TISSUE

 NECROPSIED 

 EXAMINE D MICROSCOPICALLY 

66 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

«».___ — _____.. _ __„___ _ __ _ — ..__ __ ... _ _ _ «_..__„. _____ _ _— ————— — — —— — ——. — —_ — _—.—.——.••..-•-.— •— ——. — — • 

#OVAFY (19) (17) (39)
 


THKCOMA 1 (2%)



NERVOUS SYSTEM
 


NONF
 


SPECIAL SENSE OFGANS
 


NONE
 


dUSCULOSKELETAL SYSTEM
 


*BONE (20) (47) (50) 

OSTEOSARCOM A 1 (2%) 

____________________^.___ — ________ _ _________.______ — —— •_———._ _ — —— __ ______—_ _ — — — — — —, 

BOCY CAVITIES 

NON ^ 

ALL OTHER SYSTEMS
 


*MULTIPLE ORGANS (20) (47) (50)
 


SARCOMA, NOS 1 (2%)



HEMANGIOSAECOMA 2



LOWER LEG
 


OSTFOSAPCOMA 1



ANIMAL DISPOSITION SUMMARY
 


ANIMALS INITIALLY IN STDDY 20 50 50

 

NATURAL DEATHS 15

 

MORIBUND SACRIFICE 1

SCHEDULED SACRIFICE
 


ACCIDENTALLY KILLED
 


TERMINAL SACRIFICE 15 35

 

ANIMAL MISSING 3

a_INCLUDES_AUTOLYZED_ANIHALS 

f NUMBER OF ANIMAL S WITH TISSUE EXAMINE D MICROSCOPICALL Y 

* NUMBER OF ANIMALS NECROPSIED 

67 
TABLE B2. FEMALE MICE: NEOPLASMS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

TUMOR SUMMAR Y 

TOTAL ANIMAL S WITH PRIMAR Y

TOTAL PRIMAR Y TUMORS

 TUMORS*

 .

 15

 16

 22

 30

 20 

 27 

 ANIMAL S WITH 4 

TOTAL BENIGN TUMORS 4 8 5 

TOTAL ANIMAL S WITH MALIGNAN T TUMORS 12 20 17 

TOTAL MALIGNAN T TUMORS 12 22 22 

TOTAL ANIMAL S WITH SECONDARY TUMORS* 3 

TOTAL SECONDARY TUMORS l» 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN- 

BENIGN OR MALIGNAN T 

TOTAL UNCERTAI N TUMORS 

TOTAL ANIMAL S WITH TUMORS UNCERTAIN- 

PRIMAR Y OR HETASTATIC 

TOTAL UNCERTAIN TUMORS 

* PRIMAR Y TUMORS: ALL TUMORS EXCEPT SECONDARY TUMORS 

# SECONDARY TUHORS: HETASTATIC TUHORS OR TOHORS INVASIVE INTO AN ADJACENT ORGAN 

TOTAL BENIGN TUMORS 5 7 

68 
APPENDIX C



SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 


IN RATS ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


69
 

70

 
TABLE C1.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALIY IN STUDY 20 50 50 

ANIMAL S NECROPSIED 20 50 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALLY 20 50 50 

INTEGUMENTAR Y SYSTEM 

*SKIN (20) (50) (50) 

DERMA L INCLUSION CYST 1 (5%) 

INFLAMMATION , SUPPORATIVE 1 (5%) 

GRANULOMA , NOS 1 (2*) 

RESPIRATOR Y SYSTEM 

• LUNG , (20) (U8) (50) 

ALVEOLA R MACROPHAGES 1 (2*) 1 (2%) 

HYPERPLASIA , ALVEOLAR EPITHELIUM 1 (5*) 5 (10X) 5 (10*) 

HEMATOPOIETIC SYSTEM 

#SPLEEN (19) (49) (U9) 

TCTOPIA 1 (5%) 

INFARCT, FOCAL 1 (2*) 

#PANCREATI C L.NOD E (20) (50) (50) •DILATATION, NOS 1 (5*) 

IMESENTERI C L. NODE (20) (50) (50) 

DILATATION, NOS 12 (2H%) 32 (64*) 

CIRCULATORY SYSTEM 

#H3ART/ATRIO M (20) (50) (50) 

THROMBOSIS, NOS 1 (2*) 1 (2*) 

#MYOCASDIU M (20) (50) (50)' 

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

* NUMBER OF ANIMALS NECROPSIED 

71 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

*— TM-u-TM-""-TM-TM-"n''TM-~" "•— ~



MATCHED
 


CONTROL LOW DOSE
 HIGH DOSE
 


FIBROSIS, FOCAL
 	 1 (2X)
 


DIGESTIVE SYSTEM
 


#LIVER
 (20) (50) (49)
 


NECROSIS, FOCAL
 1(2X) 


METAMORPHOSIS FATTY
 2(4X) 1 (2X)
 


HEPATOCYTOMEGALY
 5(25X) 10(20X) 10 (20X)i
 


HYPERPLASIA, FOCAL
 1 (5X) 1 (2X) 


*BILE DUCT
 (20) (53) (50)
 


HYPERPLASIA, NOS
 14 (70 X) 2(4X) 


" HYPERPLASIA', FOCAL
 T (2X) 


•PANCREAS
 	 (18) C"») (48) 


FIBROSIS, FOCAL
 1(6X) 


PERIARTERITIS
 3 (17X) 1 (2X) 1 (2X) 


tPANCREATIC ACINUS
 (18) (44) (48) 


ATROPHY, NOS
 3 (17X) 3 (7X) 4 (8X) 


fSTOMACH
 (19) (50) (50) 


ULCER, .NOS
 1 (2X) 


HLCER, FOCAL
 1 (2X) 2 (4X) 


NECROSIS, FOCAL
 1 (2X) 


•LARGE INTESTINE
 (20) (49)
 (48) 


NEMATODIASIS
 1 (2X)
 


HYPERPLASIA, LYMPHOID
 1 (5X)
 


URINARY SYSTEM
 


tKIDNEY
 (20) (50)
 (50)
 


CYST, NOS
 1 (2X)
 


INFLAMMATION, CHRONIC
 18 (90X) 41 (82X)
 45 (90X)
 


PERIABTERITIS
 1 (2X)
 


HYPERPLASIA, TOBULAR CELL
 1 (2X)
 


#KIDNEY/COPTEX
 (20)	 (50)
 (50)
 


NECROSIS, FOCAL
 1 (2X)
 


tKIDNEY/PELVIS
 	 (20) (50)
 (50)
 


t NUMBER OF ANIMAL S WITH TISSUEEXAMINED MICROSCOPICALLY * NUMBER OF ANIMAL S NECROPSIED 

72 
TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 


CONTROL LOW DOSE
 


•URINARY	 BLADDER (18) (46)
 


HYPSRPLASIA, EPITHELIAL
 


ENDOCRINE SYSTEM 

•PTTUITAPY 

HEMORRHAG E 

(19) 

1 (5%) 

(49) 

3 (6%) 

•ADRENAL 

METAPLASIA, OSSEOUS 

(20) (50) 

1 (2%) 

•ADRENAL CORTEX 

HYPERPLASIA, FOCAL 

(20) (50) 

tADRENA L MFOULLA 

HYPERPLASIA, FOCAL 

(20) (50) 

3 (6%) 

#THYROID 

HYPERPLASIA, C-CELL 

(20) 

8 (40%) 

(49) 

10 (20%) 

•PARATHYROID 

HYPERPLASIA, NOS 

(18) (46) 

•PANCREATIC ISLETS 

HYPERPLASIA, NOS 

(18) 

1 (6%) 

(44) 

REPRODUCTIVE SYSTEM
 


*PREPUTIAL GLAND (20) (50)
 


CYST, NOS
 


INFLAMMATION, GRANOLONATOOS 1 (2%)
 


HYPERPLASIA, NOS 2 (<*«)
 


•PROSTATE	 	 (18) (HI)



INFLAMMATION, SUPPURATIVE 2 (11%) 8 (17%)
 


HYPERPLASIA, EPITHELIAL
 


•TESTIS	 	 (20) (49)
 


PFRIARTERITIS
 


*EPIDIDYMI S 

(20) (50) 

* NUMBER OF ANIHALS WITH TISSUE EXABISED HICROSCOPICALLY
 


* NUMBER OF ANIMALS HECROPSIED
 


HIGH DOSE
 


(50)
 


2 (U%)
 


(48)
 


2 (4%)
 


(50)
 


(50)
 


1 <2X)
 


(50)
 


5 (10%)
 


(50)
 


12 (24%)
 


C»2)
 


1 (2«)
 


(48)
 


(50)
 


1 (2%)
 


(49)
 


17	 (35%)
 


1 (2%)
 


(50)
 


1 (2%)
 


(50) 

73
 

TABLE C1. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

NERVOU S SYSTEM 

*BRAI N (20) («9) (49) 

HEMORRHAG E 2 (10%) 3 (6X) 

CORPOR A AHYLACE A 1 (2X) _—. —— — — _ _ — ____ _ ..__..«___«.. _____«.__.._ _..__....,•_«___ _____ _ ...— ___•*—«._••«._••••••_*«•»•••_••< 

SPECIAL SENSE ORGANS
 


*EYE (20) (50) (50)
 


CATARACT 3 (15S)
 


MUSC3LOSKELETAL SYSTEB
 


NONE
 


BODY CAVITIES
 


*MESENTERY (20) (50) (50)



PERIARTERITIS 1 (2X)
 


ALL OTHER SYSTEHS
 


ADIPOSE TISSUE
 


INFLAMMATION, FOCAL 3 2

 — — __ — _-» __ _ __ •_ __.__.__•_•_.._•«_ _v«»_^...^a..««^^__^___v_ «<• — — ••^____«^«.»»^^««—••••• — — 

SPECIAL MORPHOLOG Y SOMBABY 

NONE 

* NUMBE R OF ANIMAL S KITH TISSUE EXAMINE D MICROSCOPICALLY * NUMBE R OF ANIH&LS NECROPSIED 

74 
TABLE C2.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

ANIMALS INITIALLY IN STUDY

ANIMALS NECROPSIED

ANIHALS EXAMINED HISTOPATHOLOGICALLY

 20 

 20 

 20 

50 

50 

50 

50 

50 

50 

INTEGUMENTAR Y SYSTEM 

NON* 

RPSPIRATORY SYSTEM 

*LUNG 

ALVEOLAE MACROPHAGES 

HYPERPLASIA, ALVEOLAR EPITHELIUM 

(20) 

1 (5%) 

(50) 

6 (12%) 

3 (6%) 

(50) 

3 (6%) 

6 (12%) 

HEMATOPOIETIC SYSTEM
 

fBONE HA B ROB 

HYPERPLASIA, HEHATOPOIETIC 

(20) (U9) (U9)
 

1 (2%)
 

tSPLEFN 

NECROSIS, FOCAL 

HEMATOPOIESIS 

(18) (50) 

1 (2%)
 

3 (6%) 

(50)
 

1 (2%)
 

tMESFNTERIC L. NODE 

DILATATION, NOS 

INFLAMMATION, PYOGRANULOMATOUS 

NECROSIS, NOS 

(20) (50) 

5 (10%) 

1 (2%)
 

1 (2%)
 

(50)
 

18 (36%)
 

CIRCULATORY SYSTEM
 

NONE
 

DIGESTIVE SYSTEM
 

*LIVER (20) (50) (19)
 

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

75
 

TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 

CONTROL LOW DOSE HIGH DOSE
 

METAMORPHOSIS FATTY 

HEPATOCYTOMEGALY 

HYPERPLASIA, FOCAL 

HEHATOPOIESIS 

2 (10X) 

13 (65X) 

2 (4X)
 

5 (10X) 

28 (56X) 

1 (2X)
 

10 (20X)
 

12 (2«X)
 

*BILE DUCT 

HYPERPLASIA, NOS 

(20) (50) 

1 (2X) 

(50)
 

1 (2X)
 

#PANCREATIC ACINUS 

ATROPHY, NOS 

(20) 

1 (5X) 

(49) 

5 (10X) 

(46)
 

3 (7X)
 

•LARG?	 INTESTINE 

NEMATODIASIS 

(19) (50) 

1 (2X) 

(48)
 

1 (2X)
 

URINARY SYSTEM
 

*KIDNEY 

PYELONEPHRITIS SOPPURATIVE 

INFLAMMATION, CHRONIC 

PERIARTERITIS 

(20) 

8 (UOX) 

1 (5X)
 

(50) 

1 <2X)
 

25 (50X) 

(50)
 

26 (52X)
 

#KIDNEY/PELVIS 

HYPERPLASIA, EPITHELIAL 

(20) (50) (50)
 

1 (2X)
 

#URINAHY BLADDER 

HYPERPLASIA, EPITHELIAL 

(19) (U8) 

1 (2X)
 

(46)
 

ENDOCRINE SYSTEM
 

#PITUITARY 

CYST, NOS 

(19) (48) 

1 (2X)
 

(49)
 

•ADRENAL COPTEX 

NECROSIS, NOS 

NECROSIS, FOCAL 

CYTOWBGALY 

(20) (50) 

1 (2X)
 

1 <2X)
 

1 (2X)
 

(50)
 

tADRENAL MEDULLA 

FOSINOPHILIC INFILTRATE 

HYPERPLASIA, FOCAL 

(20) (53) 

1 (2X)
 

1 (2X)
 

(50)
 

tTHYROID	 (20) (50)
 

—!!£12SL_


* NUMBER OF ANIMALS HITH TISSOE EXAMINED MICROSCOPICALLY
 

» NUMBER OF ANIMALS NECROPSIED
 

76
 

TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

HYPEEPLASIA , C-CELL 

HYPERPLASIA , FOLLICOLAR-CELI 

tPARATHYHOI D 

HYPFRPLASIA , NOS 

REPRODUCTIV E SYSTEM 

*MAMMAR Y LOBULE 

HYPERPLASIA , NOS

 

HYPERPLASIA , FOCAL 

tUTERUS/ENDOBETRIDM 

HYPERPLASIA, NOS 

HYPERPLASIA, CYSTIC 

NERVOU S SYSTEM 

NONE 

SPECIAL SENSE ORGANS 

*EYE 

CATARACT 

BUSCULOSKELETAL SYSTEH 

NONE 

BODY CAVITIES 

NONE * 

ALL OTHEB SYSTEHS 

^MULTIPLE ORGANS 

BACTERIAL SEPTICEHIA 

ADIPOSE TISSUE 

t NUMBER OF ANIMALS WITH TISSUE * NUHBEB OF AHIHALS NECROPSIED 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

 11 (55X) 22 (UUX ) 22 («5«) 

2 (4X) 

 (18) (45) (43) 

1 (2«) 

 (20) (50) (50) 

1 (5%) 1 (2«) 

1 (2%) 

 (20) (49) (ft?) 

1 (2*) 

1 (5«) 1 (2«) 1 (2*) 

 (20) (50) (50) 

1 (2K) 

 (20) (50) (50) 

1 (2X) 

 EIAMMBD MICROSCOPICALLY 

77 
TABLE C2. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL MORPHOLOG Y SUMMAR Y 

NO LESION EEPOBTED 

* NU1BEB OF ANIMALS HITH TISSUE EXAMINED MICROSCOPICALLY
 


* NUMBEP OF ANIMALS NECROPSIED
 


78
 

APPENDIX D



SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS
 


IN MICE ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


79
 

30

 
TABLE 01.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

„____, n

 "" rm-m-TM—TM 

MATCHED 

CONTROL LOW DOSE HIGH D OSE 

ANIBALS INITIALLY IN STUDY 20 50 50 

ANIBALS HISSING 1 

1NIBALS NECROPSIED 20 U9 50 

ANIBALS EXAMINE D HISTOPATHOIOGICALLY 20 49 50 

INTEGDHENTAB Y SYSTEB 

*SKIN (20) <U9 ) (50) 

ALOPECIA 2 (4X) 2 C»X) 

RESPIRATORY SYSTEM 

NONE 

HEBATOPOIETIC SYSTEM
 


*SPLEEN
 (20)
 (49)
 C»9)
 


HYPERPLASIA, LYBPHOID
 2 (10X)
 1 (2X)
 3 (6X)
 


HEBATOPOIESIS
 6 (30 X)
 5 (10X)
 1 (2X)
 


IBANDIBULAR L. NODE
 (20)
 (49)
 (49)
 


HYPERPLASIA, LYHPHOID
 1 (2X)
 


IBESENTERIC L. NODE
 (20)
 (49)
 (49)
 


HYPERPLASIA, RETICULOH CELL
 1 (2X)
 1 (2X)
 


HYPERPLASIA, LYBPHOID
 1 (2X)
 


HEBATOPOIESIS
 4 (20 X)
 1 (2X)
 


ITHYMUS
 (20)
 (47)
 (48)
 


ATROPHY, NOS
 2 (4X)
 


HYPERPLASIA, LYMPHOID
 1 (5X)
 1 (2X)
 3 (6X)
 


CIRCULATORY SYSTEM
 


tMYOCARDIUM
 (20)
 (50)
 (50)
 


ISZ1A1HA1I01U BQS. _
 _1 1211
 2_iiSL_



t NOMBER OF ANIBAL S ilTH TISSUEEXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECROPSIED 

81 
TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED
 


CONTROL LOW DOSE HIGH DOSE
 


DIGESTIVE SYSTEM
 


#LIVER (20) (50) (50)
 


NECROSIS, NOS 1 (2X)
 


NECROSIS, FOCAL 1 <5X) 2 («X)
 


HYPERPLASIA, NODULAR 1 (5X) 3 (6X) 5 (10X)
 


ANGIECTASIS 1 (5X)
 


HEMATOPOIESIS 1 (5X)
 


•GALLBLADDER	 	 (20) (49) (50)
 


CALCULUS, NOS 1 (5X)
 


•BILE DUCT (20) (49) (50)
 


INFLAMMATION, NOS 2 (10X) 3 (6X)
 


•PANCREAS	 	 (20) (47) (49)
 


CYSTIC DUCTS 1 (2X)
 


ATROPHY, NOS 1 (2X)
 


tSTOMACH (20) (49) (50)
 


INFLAMMATION, NOS 2 (10X)
 


#PEYERS PATCH (20) (50) (50)
 


HYPERPLASIA, NOS 1 (2X)
 


URINARY SYSTEM
 


#KIDNEY (20) (50) (50)
 


HYDRONEPHROSIS 1 (2X)
 


INFLAMMATION, INTERSTITIAL 1 (5X) 2 («)
 


INFLAMMATION, CHRONIC 1 (2X)
 


PERIVASCULITIS 1 (2X)
 


INFARCT, NOS 1 (2X)
 


ENDOCRINE SYSTEM
 


#THYROID (20) (49) (50)
 


CYSTIC FOLLICLES 1 (5X)
 


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


#PABATHYROID (15) (33) (3«)
 


CYSTi £OS ' 1_13.IL­


* NUMBER OF ANIMALS SITH TISSUE EXAMINED MICROSCOPICALLY
 


* NOMB?F OF ANIMALS NECROPSIED
 


82
 

TABLE D1. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

•PANCREATIC ISLETS 

HYPEHPLASIA, NOS 

(20) 

3 (15X) 

(47) 

3 (6«) 

(49) 

2 (4)1) 

REPRODUCTIVE SYSTE M 

*HAMMAR Y GLAN D 

HYPEFPLASIA , CYSTIC 

*PREPUTIAL GLAN D 

CYST, NOS 

*SEHINAL VESICLE 

CAST, NOS 

ITESTIS 

ATROPHY, NOS 

(20) 

1 

(20) 

(20) 

6

 (20)

 (5X) 

 (33S) 

(49) 

2 (4X) 

(49) 

11 (22S) 

 (48) 

(50) 

(50) 

2 (4)1) 

(50) 

10 (20X) 

 (50) 

1 (2«) 

NERVOUS SYSTEM 

IBRAIN 

HINERALIZATION

 (20) (50)

 4 (8X)

 (49) 

8 (16X) 

SPECIAL SENSE ORGANS 

NONE 

MUSCOLOSKELETAL SYSTEM 

NONE 

BODY CAVITIES 

*PLBURA 

INFLAMMATION, NOS

 ' (20)

 1 (5S) 

 (49) (50) 

ALL OTHER SYSTEMS 

* NUMBER 

* NUMBER

 OP ANIMALS »ITH TISSUE

 OF ANIMALS NECROPSIED 

 EXAMINED MICROSCOPICALLY 

83
 

TABLE 01. MALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

SPECIAL HOBPHOLOGY SOSHUBY 

NO LESIOM REPORTED 1 10 

I NUNBEB OP ANIMAL S BITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMALS NECROPSIED 

84 
TABLE 02.

 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE 

ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET 

» — ——_•_.».•__•_._*._ ._ _*._..• _-TM -M.I11 _ J_ _« 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

ANIMAL S INITIALL Y IN STUDY 20

 50 50 

ANIMU S MISSING 3 

ANIMALS N'CROPSIED 20 «7 50 

ANIMAL S EXAMINE D HISTOPATHOLOGICALL Y 20 U7 50 

INTFGOMFNTAB Y SYSTEM 

»SKI N (20) (U7) (50) 

1LOPECIA 3 (6X) 3 (6%) 

RESPIRATOR Y SYSTEM 

*IUN G (20) (47) (<»8) 

ATFL^CTASIS 1 (5«) 2 (H%) 

CONGESTION. NOS 3 (6%) 

HENATOPOIHTIC SYSTEM 

iSPLEEN (19) («7) (46) 

HYPERPLASIA , LYMPHOID tt (9X) a (9X) 

HEMATOPOIESIS 8 (U2% ) 8 (17«) 3 (7«) 

•LYMPH NODE 

CYST, NOS 

•KESENTEPIC L.

CYST. HOS 

HYPE»PLASIA ,

*THYMUS 

HYPE'PLASIA,

(20) (»6) (<•«) 

1 (2X) 

 NODE (20) («6.) (HH) 

 LYBPHOID 

1

1

 (5X)

 (5X)

 1 (2X) 

3 (7X) 

(18) (HH) (»2) 

 LYMPHOID 2 (11X) 1 (2X) 

CTPC'JLATORY SYSTEM 

NOME 

DIGESTIVE SYSTEM
 


(20)
 


.2-JHI1.
 


* NOME?1? 0? ANIMAL S WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBER OF ANIMAL S NECBOPSIEO 

85 
TAILE D2. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED)

 

HYP'RPIASIA, NODULAR
 


HESATOPOIESIS
 


•HEPATIC CAPSULE
 


INFLAMMATION. NOS
 


•GALLBLADDER
 


HYPERPIASIA, HOS
 


•BILE	 DUCT
 


INFLAMMATION, NOS
 


•PANCREAS
 


CYSTIC DUCTS
 


ATPOPHY, HOS
 


•STOMACH
 


INFLAMMATION, NOS
 


•SHALL INTESTINE
 


POLYP'



• PITERS PATCH 

HYPEKPLASIA, NOS 

URINAR Y SYSTEM 

• KIDNEY
 

PERIVASCULITIS
 

HYPERPLASIA, LYSPHOID
 

ENDOCRINE SYSTEM
 


• PITUITARY
 

AHGIECTASIS
 

• THYROID 

CYSTIC FOLLICLES 

•PANCREATIC ISLETS 

HYPERPLASIA, NOS 

REPRODUCTIVE SYSTEH 

• UTERUS 

UZUJUUtliaH*_112S 

MATCHED 

CONTROL 

3 (15*) 

1 (5*) 

(20) 

1 (5X) 

(20) 

1 (5%) 

(20) 

2 (10%) 

(17) 

1 (6%) 

(20)
 


3 (15*)
 


(19)
 


1 (5%)
 


(19)

 (20)


 1 (5%)
 


(19)


 1 (5%)
 


(20)

 (17)

 (20) 

LOW DOSE HIGH DOSE 

7 (15%) 4 (8%) 

(U7) (48) 

(50) 

(50) 

5 (11%) 1 (2%) 

(46) 

1 (2%) 

(47) 

(U8) 

2 1 (2%) 

 (U6 ) (U8) 

1 (2%) 

 (47)	 (»8)
 


1 (2%)
 


 (45) (H5)
 


 (l»») (l»7) 

1 (2%) 5 (11%) 

 (46) (46) 

1 (2%) 

 (47) (44) 

i_12SL_ 

• RURBER OP ANIMAL S ilTH TISSUE EXAMINE D MICROSCOPICALLY 

* NUMBER OP ANIMAL S NECROPSIED 

86
 

TABLE 02. FEMALE MICE: NONNEOPLASTIC LESIONS (CONTINUED) 

MATCHED 

CONTROL LOW DOSE HIGH DOSE 

POLYPOID HYPERFLASIA 2 (10*) 

*UTE»US/ENDOMETRIUM (20) (U7 ) (UU ) 

CIST, NOS 9 (U5% ) 21 (45%) 22 (50%) 

HYPERPLASIA, HOS 1 (2%) 

tCVAR Y (19) (U7 ) (39) 

CYST, NOS 5 (26%) 11 (23%) 5 (13%) • — « ———————— V _ _• . _ •.»««_V_ V• —____•_••»__**._ —* ~. _ _~. __ _ _ _ _ __ _ _ _«__ ~————— ————__ ——V —

HERVOOS SYSTEM 

tBRAI N (20) (1*7) (US) 

HINrCALIZATIOH 2 (10%) 9 (19%) 6 (13%) 

INPLAHNATION, FOCAL 1 (2%) 

SPECIAL SENSE ORGANS 

*EYE/LACRIBAL GLAN D (20) (U7 ) (50) 

CYST, NOS 1 (2%) 

MOSCrjLOSKELSTAL SYSTEH 

NONE 

BOEY CAVITIES 

NONE 

ALL OTHEF SYSTEMS 

NONE 

SPECIAL MORPHOLOGY SOHHARY 

NO LESION REPORTED 1 6 8 

ANIMA L MISSING/NO NECROPSY 1 

ACTO/HECROPST/HISTO PERF 1 

* NU1BEH OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMBFR OF ANIMALS NECROPSIED 

87 

APPENDIX E



ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 


IN RATS ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


89
 

90

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

Administered Piperonyl Butoxide in the Diet (a)
 

Topography: Morphology 

Matched 

 Control 

Low 

Dose 

High 

Dose 

Integumentary System: Fibroma 

of the Subcutaneous Tissue (b) 0/20 (0) 2/50 (4) 3/50 (6) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

Infinite 

0.123 

Infinite 

Infinite 

0.250 

Infinite 

Weeks to First Observed Tumor 104 107 

Hematopoietic System: Lymphoma (b) 9/20 (45) 15/50 (30) 13/50 (26)
 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.667 

0.346 

1.478 

0.578
 

0.289
 

1.316
 

Weeks to First Observed Tumor 91 81 77
 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 


Administered Piperonyl Butoxide in the Diet (a)
 


(continued) 

Topography ; Morphology
 


Liver: Neoplastic Nodule (b)
 


P Values (c,d)
 


Departure fron Linear Trend (e)
 


Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


VO Weeks to First Observed Tumor NJ 

Pituitary: Adenoma or
 


Carcinoma, NOS (b)
 


P Values (c,d)
 


Departure from Linear Trend (e)
 


Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor 

Matched
 


Control
 


2/20 (10)
 


P = 0.025(N)
 


P - 0.043
 


98
 


4/19 (21)
 


P - 0.009(N)
 


P = 0.004
 


102 

Low
 


Dose
 


0/50 (0)
 


N.S.
 


0.000
 


0.000
 


1.345
 


—



0/49 (0)
 


P = 0.005(N)
 


0.000
 


0.000
 


0.413
 


High
 


Dose
 


0/49 (0)
 


N.S.
 


0.000
 


0.000
 


1.372
 


—



1/48 (2)
 


P = 0.020(N)
 


0.099
 


0.002
 


0.932
 


103 
Table El. Analyses of the Incidence of Primary Tumors in Male Rats
 


Administered Piperonyl Butoxide in the Diet (a)
 


(continued) 


Matched Low High 


Topography ; Morphology Control Dose Dose 


Pituitary: Chromophobe Adenoma (b) 1/19 (5) 7/49 (14) 6/48 (13) 


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


Relative Risk (f) 2.714 2.375 


Lower Limit 0.393 0.324 


Upper Limit 119.544 106.788 


VO Weeks to First Observed Tumor 107 104 107 


Adrenal: Pheochromocytoma (b) 2/20 (10) 4/50 (8) 4/50 (8) 


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


Relative Risk (f) 0.800 0.800 


Lower Limit 0.128 0.128 


Upper Limit 8.436 8.436 


Weeks to First Observed Tumor 107 57 104 

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


Administered Piperonyl Butoxide in the Diet (a)
 


(continued)
 

Topography: Morphology
 

Matched 

 Control 

Low 

Dose 

High 

Dose 

Pituitary: Chromophobe Adenoma,
 

Adenoma, NOS or Carcinoma (b)
 5/19 (26) 7/49 (14) 7/48 (15) 

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

Relative Risk (f)
 

Lower Limit
 

Upper Limit
 

0.543 

0.176 

1.959 

0.554 

0.180 

1.997 

Weeks to First Observed Tumor
 102 104 103 

Thyroid: Follicular-cell
 


Adenoma or Carcinoma (b)
 0/20 (0) 1/49 (2) 3/50 (6) 


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


Relative Risk (f)
 Infinite Infinite 


Lower Limit
 0.023 0.250 


Upper Limit
 Infinite Infinite 


Weeks to First Observed Tumor 107 81 
Table El Analyses of the Incidence of Primary Tumors in Male Rats
 


Administered Piperonyl Butoxide in the Diet (a)
 


Topography ; Morphology
 


Thyroid: C-cell Adenoma
 


or Carcinoma (b)
 


P Values (c,d)
 


Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor
 


VO Pancreatic Islets: Islet-cell
 
 Ui Adenoma or Carcinoma (b)
 


P Values (c,d)
 


Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor
 


Matched
 


Control
 


1/20 (5)
 


N.S.
 


107
 


1/18 (6)
 


N.S.
 


107
 


Low
 


Dose
 


6/49 (12)
 


N.S.
 


2.449
 


0.332
 


110.166
 


104
 


4/44 (9)
 


N.S.
 


1.636
 


0.181
 


78.690
 


107
 


High
 


Dose
 


4/50 (8)
 


N.S.
 


1.600
 


0.175
 


77.169
 


107
 


3/48 (6)
 


N.S.
 


1.125
 


0.100
 


57.811
 


107
 

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

Administered Piperonyl Butoxide in the Diet (a)
 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Testis: Interstitial-cell Tumor (b) 19/20 (95) 48/49 (98) 46/50 (92) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.031 

0.961 

1.109 

0.968 

0.902 

1.174 

Weeks to First Observed Tumor 96 78 77 

(a) Dosed groups received 5,000 or 10,000 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.
 

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


Administered Piperonyl Butoxide in the Diet (a)
 


Topography: Morphology
 

Hematopoietic System: Lymphoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor
 

Pituitary: Chromophobe Adenoma (b)
 

P Values (c,d)
 

Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor
 


Matched
 


 Control
 


 1/20 (5)
 


P = 0.007
 


 107
 


 8/19 (42)
 


 N.S.
 


107
 


Low
 


Dose
 


7/50 (14)
 


N.S.
 


2.800
 


0.403
 


123.407
 


94
 


11/48 (23)
 


N.S.
 


0.544
 


0.251
 


1.348
 


107
 


High
 


Dose
 


15/50 (30)
 


P = 0.020
 


6.000
 


1.048
 


245.704
 


73
 


14/49 (29)
 


N.S.
 


0.679
 


0.336
 


1.606
 


105
 

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


Administered Piperonyl Butoxide in the Diet (a)
 


(continued)
 


Matched Low High
 


Topography: Morphology Control Dose Dose
 


Thyroid: C-cell Adenoma
 


or Carcinoma (b) 4/20 (20) 6/50 (12) 3/49 (6)
 


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


Relative Risk (f) 0.600 0.306
 


Lower Limit 0.164 0.050
 


Upper Limit 2.659 1.675
 


Weeks to First Observed Tumor 107 107 105
 


VO
 


00
 


Mammary Gland: Fibroadenoma (b) 3/20 (15) 8/50 (16) 2/50 (4)
 


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


Relative Risk (f) 1.067 0.267
 


Lower Limi t 0.295 0.024
 


Upper Limit 5.813 2.190
 


Weeks to First Observed Tumor 107 107 105
 

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

Administered Piperonyl Butoxide in the Diet (a) 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Uterus: Endometrial Stromal Polyp (b) 4/20 (20) 7/49 (14) 3/47 (6) 


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


Relative Risk (f) 0.714 0.319 


Lower Limit 0.211 0.052 


Upper Limit 3.052 1.743 


Weeks to First Observed Tumor 107 107 107 


vo
 


VO
 


(a) Dosed groups received 5,000 or 10,000 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 (M.S.) is
 


indicated. Beneath the incidence of tumors in a dosed group is the probability level for
 


the Fisher exact test for the cj'iparison 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.
 

100

 
APPENDIX F



ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 


IN MICE ADMINISTERED PIPERONYL BUTOXIDE IN THE DIET
 


101
 

102

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


Administered Piperonyl Butoxide in the Diet (a)
 


Matched Low
 


Topography: Morphology Control Dose
 


Integumentary System: Lipoma of
 


the Subcutaneous Tissue (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 112 112
 


o



LO
 


Lung: Alveolar /Bronchiolar
 


Carcinoma (b) 3/20 (15) 3/50 (6)
 


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


Relative Risk (f) 0.400
 


Lower Limit 0.060
 


Upper Limit 2.802
 


Weeks to First Observed Tumor 103 105
 


High
 


Dose
 


2/50 (4)
 


N.S.
 


0.400
 


0.032
 


5.277
 


112
 


2/48 (4)
 


N.S.
 


0.278
 


0.025
 


2.278
 


112
 

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


Administered Piperonyl Butoxide in the Diet (a)
 


(continued)
 


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
 


Control
 


5/20 (25)
 


N.S.
 


103
 


4/20 (20)
 


N.S.
 


112
 


Low
 


Dose
 


6/50 (12)
 


N.S.
 


0.480
 


0.143
 


1.807
 


105
 


10/49 (20)
 


N.S.
 


1.020
 


0.346
 


4.068
 


91
 


High
 


Dose
 


8/48 (17)
 


N.S.
 


0.667
 


0.227
 


2.338
 


112
 


6/50 (12)
 


N.S.
 


0.600
 


0.164
 


2.659
 


90
 

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

Administered Piperonyl Butoxide in the Diet (a) 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

All Sites: Hemangiosarcoma (b) 1/20 (5) 3/49 (6) 1/50 (2) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

1.224 

0.108 

62.958 

0.400 

0.005 

30.802 

Weeks to First Observed Tumor 112 105 112 

o 

Ul Liver: Hepatocellular Carcinoma (b) 10/20 (50) 17/50 (34) 20/50 (40) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.680 

0.379 

1.403 

0.800 

0.462 

1.604 

Weeks to First Observed Tumor 88 83 99 
__ 

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


Administered Piperonyl Butoxide 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
 

Lacrimal Gland: Adenoma, NOS (b)
 

P Values (c,d)
 

Relative Risk (f)
 


Lower Limit
 


Upper Limit
 


Weeks to First Observed Tumor
 


Matched
 


 Control
 


 1/20 (5)
 


 N.S.
 


 112
 


 0/20 (0)
 


P = 0.022
 


Low
 


Dose
 


3/49 (6)
 


N.S.
 


1.224
 


0.108
 


62.958
 


112
 


0/49 (0)
 


N.S.
 


—



—



High
 


Dose
 


0/50 (0)
 


N.S.
 


0.000
 


0.000
 


7.475
 


—



4/50 (8)
 


N.S.
 


Infinite
 


0.386
 


Infinite
 


112
 

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


Administered Piperonyl Butoxide in the Diet (a)
 


(continued)
 


(a) Dosed groups received time-weighted average doses of 1,036 or 2,804 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.
 
 o



(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 Butoxide in the Diet (a)
 


Matched Low
 


Topography: Morphology Control Dose
 


Lung: Alveolar/Bronchiolar
 


Adenoma or Carcinoma (b) 2/20 (10) 6/47 (13)
 


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


Relative Risk (f) 1.277
 


Lower Limit 0.258
 


Upper Limit 12.285
 


Weeks to First Observed Tun.or 111 98
 


o



oo
 


Hematopoietic System: Lymphoma (b) 8/20 (40) 14/47 (30)
 


P Values (c,d) P - 0.030(N) N.S.
 


Relative Risk (f) 0. 745
 


Lower Limit 0.364
 


Upper Limit 1.765
 


Weeks to First Observed Tumor 82 45
 


High
 


Dose
 


2/48 (4)
 


N.S.
 


0.417
 


0.033
 


5.490
 


112
 


9/50 (18)
 


N.S.
 


0.450
 


0.190
 


1.174
 


73
 

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

Administered Piperonyl Butoxide in the Diet (a)
 

(continued) 

Topography: Morphology 

Matched 

Control 

Low 

Dose 

High 

Dose 

Liver: Hepatocellular Carcinoma (b) 1/20 (5) 2/47 (4) 5/48 (10) 

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

Relative Risk (f) 

Lower Limit 

Upper Limit 

0.851 

0.048 

49.164 

2.083 

0.259 

96.358 

Weeks to First Observed Tumor 112 111 107 

(a) Dosed groups received time-weighted average doses of 1,036 or 2,804 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 is 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 is 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 is less than 0.05
 


for any comparison.
 


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


group.
 

110

 
Review of the Bioassay of Piperonyl Butoxide* 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 DREW Committee Regulations and the
 


Provisions of the Federal Advisory Committee Act. The purpose of the
 


Clearinghouse is to advise the Director of the National Cancer Institute
 


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


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


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


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


quasi-public health and research organizations. Members have been
 


selected on the basis of their experience in carcinogenesis or related
 


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


biostatistics, toxicology, pathology, and epidemiology. Representatives
 


of various Governmental agencies participate as ad hoc members. The
 


Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is
 


charged with the responsibility of providing a peer review of reports
 


prepared on NCI-sponsored bioassays of chemicals studied for carcino­


genicity. It is in this context that the below critique is given on
 


the bioassay of Piperonyl Butoxide for carcinogenicity.
 


The primary reviewer said that the staff concluded that Piperonyl
 


Butoxide was not carcinogenic in rats or mice, under the conditions of
 


test. He briefly described the experimental design and noted the
 


increased incidence of lymphomas observed among treated female rats. He
 


said that the neoplasms were not regarded as significant when compared
 


to historical controls. The primary reviewer concluded that the study
 


was adequate and his recommendation was approved unanimously that the
 


report be accepted as written.
 


Members present were:
 


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


Joseph Highland, Environmental Defense Fund
 


Michael Shimkin, University of California at San Diego
 


Louise Strong, University of Texas Health Sciences Center
 


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


bioassay report either as a result of the review or other reasons.
 


Thus, certain comments and criticisms reflected in the review ma>
 


no longer be appropriate.
 


Ill
 


JJU.S . GOVERNMEN T PRINTIN G OFFICE . 1978-281-217/326 9 

DHEW Publication No. (NIH) 79-1375