A Carcinogenicity Bioassay of Isobutyl 2-Cyanoacrylate (IBC) in Fischer-344 Rats Final Report

Larry D. Brown, DVM, LTC, VC Paul W. Mellick, DVM, COL, VC Catherine D. Smith, DVM, MAJ, VC and

Don W. Korte, Jr., PhD, LTC, MSC

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\ Carcinogenicity Bioassay of Isobutyl 2-Cyanoacrylate (IBC) in Fisclier-344 Rats - Final Report (Toxicology Series 187)- Brown el at. . ... . ... , i

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(U) A Carcinogenicity Study of Rats Final Report

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Isobutyi 2-Cyanoacrylate (IBC) in Fischer-344

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Toxicology Series No. 187

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Chronic Toxicity/ Isobutyl 2-Cyanoacrylate, IBC, Bucrylate®,-,’Mammalian Toxicology, Tissue Adhesive; Carcinogenicity Bioassav/ Rat^ ; -

19. ABSTRACT < Continue on reverse if necessary and identify by block number)

This report covers the results from the second year of a two-year ^ 1 inogen i c i t y bioassay of the tissue adhesive, isobutyl 2— cyanoacrylate (IBC). Four hundred seven 6-week-old Fischer-344 rats were randomized into three groups (control, 10 p.1 IBC, and 100 Jil IBC), each group containing both male and female animals. ^The IBC was administered by surgical implantation of the liquid monomer directly onto the ventral capsule of the liver. The monomer was allowed to polymerize before two-layer closure of the abdominal incision. Control animals received 100 ^1 of isotonic saline, also by surgical implantation. All animals were examined daily, and weighed and palpated monthly. „A dose-related increase in the incidence of clinically observed intra-abdominal masses was evident among IBC-treated animals of both sexes. In addition to the masses and the more permanent sequelae of the surgical procedure (xyphoid protuberance, corneal opacity) , the animals

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19 (cont.) ^presented with a variety of transitory clinical signs sporadically throughout the first and second year. These signs were observed in all dose groups, and could not be attributed to compound administration. The weight gain of the two IBC treatment groups was comparable to that recorded for the control group.

Three hundred five animals remained on study at the start of the second year. During the second year, 118 animals died or were sacrificed prematurely. Twenty-one were unscheduled deaths and 97 were unscheduled sacrifices. At the end of the second year, the 187 remaining animals were sacrificed. All animals (118 unscheduled and 187 scheduled) were evaluated by necropsy during the second year. One hundred eighty-four of the animals that received the IBC had fibrotic adhesions between the liver and the omentum, peritoneal membrane, diaphragm, stomach, skin, and/or intestine. These lesions were characterized histologically as foreign body granulomatous reactions. Thirty-one rats treated with IBC developed sarcomas during the second year. The first sarcoma was detected on day 399 after intra-abdominal administration of the test material. Twenty-one of 31 animals with sarcoma died or were sacrificed in a moribund condition, while 10 animals with sarcoma survived until terminal sacrifice. Twenty-seven of 31 animals with sarcoma had grossly detectable masses at necropsy. Four sarcomas were detected only by microscopic examination. The liver was the most frequently affected organ^^ (27 of 31 animals had lesions in the liver) . Four IBC-treated rats (2 low- dose males and 2 high-dose females) had hepatocellular carcinomas of the liver, while controls had none. F-344 leukemia or large granular lymphocyte leukemia (LGLL) occurred in 62 animals distributed among all groups, including controls. Solid-state carcinogenesis mechanisms are the most probable cause for the sarcomas, but humans, unlike rodents, fail to elicit a solid-state carcinogenesis effect. The incidence of hepatocellular carcinomas was twice that reported for rats of the same strain and age, which provides reason for concern. However, the incidence of this tumor was not statistically significant and there was no dose-response pattern by sex. All other gross or histopathological findings were considered incidental to IBC treatment, or were sequelae of the surgical procedure.

Results of this study indicate that the ?? sence of IBC in the abdominal cavity had no effect on survival, weight gain, jr the clinical condition of F- 344 rats during the second year following its implantation. IBC did produce sarcomas in the abdomen of 16% of the animals. The sarcomas were attributed to a solid-state effect which is not present in man. A nonstatistically significant increase in hepatocellular carcinomas was observed in 4 IBC- treated rats but there was no clear evidence that this could be attributed to the IBC implants. ,, o j ^ p

"7'

.-p

ABSTRACT

This report covers the results from the second year of a two-year carcinogenicity bioassay of the tissue adhesive, isobutyl 2-cyanoacrylate (IBC). Four hundred seven 6-week- old Fischer-344 rats were randomized into three groups (control, 10 |il IBC, and 100 ^.1 IBC) , each group containing both male and female animals. The IBC was administered by surgical implantation of the liquid monomer directly onto the ventral capsule of the liver. The monomer was allowed to polymerize before two-layer closure of the abdominal incision. Control animals received 100 |il of isotonic saline, also by surgical implantation. All animals were examined daily, and weighed and palpated monthly. A dose- related increase in the incidence of clinically observed intra-abdominal masses was evident among IBC-treated animals of both sexes. In addition to the masses and the more permanent sequelae of the surgical procedure (xyphoid protuberance, corneal opacity) , the animals presented with a variety of transitory clinical signs sporadically throughout the first and second year. These signs were observed in all dose groups, and could not be attributed to compound administration. The weight gain of the two IBC treatment groups was comparable to that recorded for the control group.

Three hundred five animals remained on study at the start of the second year. During the second year, 118 animals died or were sacrificed prematurely. Twenty-one were unscheduled deaths and 97 were unscheduled sacrifices. At the end of the second year, the 187 remaining animals were sacrificed. All animals (118 unscheduled and 187 scheduled) were evaluated by necropsy during the second year. One hundred eighty-four of the animals that received the IBC had fibrotic adhesions between the liver and the omentum, peritoneal membrane, diaphragm, stomach, skin, and/or intestine. These lesions were characterized histologically as foreign body granulomatous reactions. Thirty-one rats treated with IBC developed sarcomas during the second year. The first sarcoma was detected on day 399 after intra¬ abdominal administration of the test material. Twenty-one of 31 animals with sarcoma died or were sacrificed in a moribund condition, while 10 animals with sarcoma survived until terminal sacrifice. Twenty-seven of 31 animals with sarcoma had grossly detectable masses at necropsy. Four sarcomas were detected only by microscopic examination. The liver was the most frequently affected organ (27 of 31 animals had lesions in the liver) . Four IBC-treated rats (2 low-dose males and 2 high-dose females) had hepatocellular carcinomas of the liver, while controls had none. F-344 leukemia or large granular lymphocyte leukemia (LGLL) occurred in 62 animals distributed among all groups, including controls.

i

Solid-state carcinogenesis mechanisms are the most probable cause for the sarcomas, but humans, unlike rodents, fail to elicit a solid-state carcinogenesis effect. The incidence of hepatocellular carcinomas was twice that reported for rats of the same strain and age, which provides reason for concern. However, the incidence of this tumor was not statistically significant and there was no dose-response pattern by sex.

All other gross or histopathological findings were considered incidental to IBC treatment, or were sequelae of the surgical procedure .

Results of this study indicate that the presence of IBC in the abdominal cavity had no effect on survival, weight gain, or the clinical condition of F-344 rats during the second year following its implantation. IBC did produce sarcomas in the abdomen of 16% of the animals. The sarcomas were attributed to a solid-state effect which is not present in man. A nonstatist ically significant increase in hepatocellular carcinomas was observed in 4 IBC-treated rats but there was no clear evidence that this could be attributed to the IBC implants.

Key Words: Chronic Toxicity, Isobutyl 2-Cyanoacrylate, IBC, Bucrylate®, Mammalian Toxicology, Tissue Adhesive, Carcinogenicity Bioassay, Rat

ii

PREFACE

TYPE REPORT: Chronic Carcinogenicity Bioassay GLP Final Report

TESTING FACILITY:

US Army Medical Research and Development Command Letterman Army Institute of Research Presidio of San Francisco, CA 94129-6800

SPONSOR: US Army Medical Research and Development Command US Army Institute of Dental Research Washington, D.C. 20307-5400

Project Officer: Eric S. Koppelman, COL, DC

PROJECT/WORK UNIT/APC: Tissue Adhesive Project,

35162775A825, WU 015, APC TL11

GLP STUDY NUMBER: 83009

STUDY DIRECTOR: LTC Don W. Korte, Jr., PhD, MSC

Diplomate, American Board of Toxicology

PRINCIPAL INVESTIGATOR: LTC Larry D. Brown, DVM, MPVM, VC

Diplomate, American College of Veterinary Preventive Medicine;

Dipl., American Board of Toxicology

PATHOLOGISTS: COL Paul W. Mellick, DVM, PhD, VC

Diplomate, American College of Veterinary Pathology (ACVP)

MAJ Catherine D. Smith, DVM, VC Diplomate, ACVP

TOXSYS™/DATA MANAGER: Yvonne C. LeTellier, BS

REPORT AND DATA MANAGEMENT:

A copy of the final report, study protocol, retired SOPs, raw data, analytical, stability, and purity data of the test compound, tissues, microslides, and an aliquot of the test compound will be retained in the LAIR Archives.

TEST SUBSTANCE: Isobutyl 2-Cyanoacrylate (IBC), Bucrylate®

INCLUSIVE STUDY DATES (YEAR 2) : 30 January 1985 -

14 February 1986

OBJECTIVE: The objective of this study was to evaluate the

carcinogenic/tumorigenic potential of isobutyl 2- cyanoacrylate in male and female Fischer-344 rats subjected to lifetime (2-year) exposure to the implanted test material.

iii

ACKNOWLEDGMENTS

Richard A. Spieler, RLAT (AALAS) , SGT Thomas W. Johnson, and Nancy J. Smith assisted in the study. SSG James D.

Justus and staff provided animal and facility management. Dianna B. Johnson provided office management and secretarial assistance for this study and preparation of the report. LTC William G. Rodkey, DVM, Diplomate, American College of Veterinary Surgeons, COL William B. Carpenter, DDS, and LTC Eric S. Koppelman, DDS, performed the requisite surgery in support of this project. Histology technicians were Lucille Cote and Marge Henderson. CPT Dan Brogdon, DVM, Ocular Hazards Division, was the veterinary ophthalmology consultant to this study. CPT Gary M. Zaucha, DVM, collated the final report. CPT Robert E. Harris, DVM, provided assistance with collating the final report.

PAUL W. MELLI^K, DVM / DATE COL, VC

Senior Pathologist

C. DAHLEM SMITH, DVM / DATE MAJ, VC

Assistant Study Pathologist

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH PRESIDIO OF SAN FRANCISCO. CALIFORNIA 94129-6800

ATTENTION OF;

SGRD-ULZ-QA 19 January 1990

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 83009

1. This is to certify that in relation to LAIR GLP Study 83009 the following inspections were made:

23 August 1983 17 January 1984 23 January 1984 17 February 1984 26 March 1984 17 May 1984 05 July 1984 02 August 1984 29 January 1986

Protocol Review

Weighing

Weighing/Dosing

Observation/Weighing

Observation

Observation

Observation/Weighing

Weighing

Observation/Necropsy

2. The institute report entitled "A Carcinogenicity Bioassay of Isobutyl 2 -Cyanoacrylate (IBC) in Fischer-344 Rats Final Report," Toxicology Series 187, was audited on 2 January 1990.

to

CAROLYN M. LEWIS Diplomate, American Board of Toxicology

Quality Assurance Auditor

vi

TABLE OF CONTENTS

Volume 1

Page

Abstract . i

Preface . iii

Acknowledgments . iv

Signatures of Principal Scier .ists . v

Report of Quality Assurance Unit . vi

Table of Contents . vii

INTRODUCTION . 1

Objective of Study . 1

MATERIALS . 1

Test Substance . 1

Animal Data . 2

Husbandry . 2

METHODS . 3

Group Assignment/Acclimation . 3

Dosage Levels . 3

Preparation of Compound . 3

Chemical Analysis of IBC . 3

Test Procedures . 4

Clinical Observations . 4

Pathological Examinations . 5

Statistical Analyses . 6

Duration of Study . 6

Changes/Deviations from Protocol . 6

Storage of Raw Data and Final Report . 6

RESULTS . 7

Mortality . . . 7

Clinical Observations . 16

Gross Pathology . 23

Microscopic Pathology . 24

DISCUSSION . 31

vii

TABLE OF CONTENTS (cont.)

Page

CONCLUSION . 34

REFERENCES . 35

APPENDICES . 38

Appendix A. Chemical Data . 39

Appendix B. Animal Data . 4 6

Appendix C. Surgical Report . 48

Appendix D. Historical Listing of Study Events . 54

Appendix E. Glossary of Terms for

Clinical Observations . 57

Appendix F. Individual Animal Histories . 64

Appendix G. Gross Necropsy Observations,

Incidence Summary . 369

Appendix H. Microscopic Observations,

Incidence Summary . 37 9

Appendix I . Glossary of Terms for

Pathology Findings . 402

Appendix J. Pathology Individual Animal Data . 466

OFFICIAL DISTRIBUTION LIST . 1150

viii

A Carcinogenicity Bioassay of Isobutyl 2-Cyanoacrylate (IBC) in Fischer-344 Bats -- Final Report--Brown et al.

INTRODUCTION

Isobutyl 2-cyanoacrylate (IBC) is being evaluated by the U.S. Army Medical Department as a tissue adhesive for use in sutureless wound closure, oral and maxillofacial surgery, and in other surgical procedures. The use of this modality could provide a time-saving and sometimes life-saving dimension to the management of combat wounds. The U.S. Army Institute of Dental Research (USAIDR) has been assigned the mission of evaluating the therapeutic potential of IBC. As part of their mandate, USAIDR has tasked the Toxicology Branch, Letterman Army Institute of Research (LAIR) , to evaluate IBC in a chronic carcinogenicity bioassay.

Results from the first year of the study indicate that IBC has no effect on survival, weight gain, or the clinical condition of rats during the first year following its implantation. The only gross or histopathological finding observed in the first year that was attributed to the IBC treatment was the presence of adhesions and a granulomatous reaction of the liver and those adjacent organs that came in contact with the IBC. No tumors were observed during the first year that could be attributed to IBC treatment (1) .

This report will review salient features of the first year and address findings that occurred during the second year of this study.

Objective of Study

The objective of this study was to evaluate the carcinogenic/tumorigenic potential of isobutyl 2- cyanoacrylate in male and female Fischer-344 rats subjected to lifetime (2-year) exposure of the implanted test material.

MATERIALS

Test Substance

Chemical Name: Isobutyl 2-cyanoacrylate (IBC), Bucrylate®

Chemical Abstract Service Registry No.: 1069-55-2 Molecular Formula: C8HnN02

Brown et al. 2

Molecular Structure:

/COOH CH—C^

Additional information on the test substance is presented in Appendix A.

Animal Data

Two hundred nine male and 210 female 4-week-old Fischer- 344 (CDF) rats were received for this study on 11 Jan 84 from Charles River Breeding Laboratories, Inc., Wilmington, MA. They were identified individually with ear tags numbered 84D00001 to 84D00209 (inclusive) for the males and 84D00226 to 84D00435 (inclusive) for the females. Four males and 4 females were selected randomly for quality control necropsy evaluation at receipt. The animal weights on the day following receipt (12 Jan 84) ranged from 30-57 g. During quarantine, three underweight females and one maloccluded male were submitted to necropsy on 20 Jan 84. Additional animal data are presented in Appendix B.

Husbandry

Animals used in this study were housed at LAIR in the Toxicology Suite, a restricted access facility. Rats were caged individually in stainless steel wire-mesh cages in racks equipped with automatically flushing dumptanks. No bedding was used in any of the cages. The diet, fed ad libitum, consisted of Purina Certified Rodent Chow® Diet 5002 (Ralston Purina Company, St. Louis, MO). Tap water was provided by automatic water valves on a central line. Water was analyzed quarterly for impurities, bacteria, physical and chemical properties, organic residues, pesticides, and heavy metals. The animal room temperature and relative humidity were continuously recorded. The room was maintained at temperatures ranging from 20.0°C to 25.5°C, with a relative humidity range of 35% to 60%, except for short periods (spikes) during which cleaning of the room altered the relative humidity, and on six occasions when increases up to 70-86% occurred for 4-14 hours due to steam outages or servicing of the ventilation system fans. The photoperiod of 12 hours of fluorescent light per day was electronically controlled- Air changes, cage size, and husbandry conformed to National Research Council Institute of Laboratory Animal Resources standards (2) . The LAIR animal facility is

CH,

I 3

HC CHj CH3

Brown et al.--3

accredited by the American Association for Accredited Laboratory Animal Care.

METHODS

This study was performed in accordance with the protocol, applicable amendments, and cited operating procedures including: LAIR Standard Operating Procedure 0P- STX-81, "Chronic Bucrylate Bioassay Procedures within Toxicology GLP Suite and Administrative Areas" (3) ; OP-STX- 73, "Chronic Carcinogen Bioassay" (4); and FDA Nonclinical Laboratory Studies, Good Laboratory Practice Regulations (5) .

Group Assignment/Acclimation

Study rats were randomized on 19 January 1984 into two experimental dose groups of 68 males and 68 females each, and a saline control group of 68 males and 67 females.

Allocation was accomplished by using the Beckman TOXSYS™ Animal Allocation Program (Beckman Instruments, Inc., Somerset, NJ) . The animals were acclimated for 12 days before the day of dosing. During this period they were observed daily for signs of illness.

Dosage Levels

The following test doses were administered: high-dose group, 100 |Xl IBC/animal; low-dose group, 10 p.1 IBC/animal; and vehicle control group, 100 |Xl saline/animal.

Exspar at ion, of Compound

Bucrylate® IBC tissue adhesive (lot 929-252, Ethicon, Inc., Somerville, NJ) was received from the sponsor on 10 January 1984 as 750 0.5 ml scored ampules in individual overwrap sterile bags. The IBC required no preparation prior to implantation. It was stored at LAIR at room temperature under darkness. Dosing of control animals was performed with commercial sterile isotonic (0.9%) saline (Lot No. 56-329-FD- 05, expiration date 1 Sep 86, Abbott Labs, Chicago, IL) .

Chemical Analysis of IBC

Ethicon, Inc., provided data on infrared, chromatographic, and chemical analysis of the IBC (Appendix A). Lot 929-252 contained 99.9% (w/w) total monomer. Before dosing, the LAIR Analytical Chemistry Group verified the identity of the IBC by IR spectroscopy, confirmed the purity

!

Brown et al . 4

by gas chromatography, and demonstrated the stability of the IBC during the dosing period (Appendix A) .

Test Procedures

The fixed volume of neat IBC or saline each animal received was based upon its assigned dosage group, and was administered directly onto the ventral (inferior surface) capsule of the liver. High-dose animals received 100 ill of IBC, low-dose animals received 10 |ll of IBC, and control animals received 100 ill of saline. Rats were 6 weeks of age at dosing. Since the surgical survival rate for implantation of this compound was unknown, all animals, regardless of size, were subjected to the surgical procedure and dosed to ensure that sufficient numbers would be available for inclusion in the chronic phase of the study. Body weights at dosing ranged from 29 g to 123 g, with the mean male and female weights at 94.0 g and 73.8 g, respectively.

The test compound was implanted under sterile conditions in the LAIR Operating Room Suite on 23 and 24 Jan 84. The surgical laparotomy implantation procedure was performed under xylazine/ketamine anesthesia. Following anesthesia, surgical preparation, and midline incision, the animal's liver was exposed by "tenting” the abdominal wall with either ophthalmic retractors or tissue forceps. The IBC was applied to the ventral capsule of the liver, usually the caudate lobe, by using either a sterile tip 100 Jll or 10 }il fixed volume Eppendorf micropipette. The IBC was then allowed to polymerize for 2-3 minutes before closure. All pipettes used in the study were shown to be accurate within ±2%. A two- layer closure of bioresorbable 4-0 Vicryl® (Ethicon, Inc.) and skin staples or skin clips was used. After the animals recovered to sternal recumbency, they were returned to the Toxicology Suite. A complete surgical report is provided in Appendix C.

Clinical Observations

On the day of dosing and during the following 2-week period, animals were checked intermittently throughout the day. During the first postoperative week, 25 animals were removed from the study due to underweight condition and/or complications. Three hundred eighty-two animals recovered satisfactorily and remained on study as of 1 Feb 84. After the animals were stabilized, observations for mortality and signs of toxicity or illness were reduced in frequency to twice daily. An observation was performed according to the following procedure: (a) each morning all animals were observed closely for signs of toxicity or illness without

Brown et al.

disturbing them in the cage; (b) once a week the animals (20% cf them per work day) were removed from their cages and observed; (c) animals were observed after being returned to their cages. A second "walk-through, live/dead check" observation was performed every afternoon with only significant observations recorded. Monthly, a more detailed clinical examination was performed, body weights were obtained, and the abdomens of all animals were gently palpated. These data were recorded electronically on a Beckman TOXSYS™ Data Collection Terminal. Daily observat ions/clinical signs were recorded on written records and significant changes entered into the TOXSYS workstation. TOXSYS software on the LAIR Data General Computers, Models MV8000 and C330, was used to analyze clinical signs and body weight data.

Pathological Examinations

Animals that were moribund at the time of clinical examination were euthanized by pentobarbital overdose, exsanguinated by axillary incision, and necropsied. During the second year, 21 animals died unexpectedly (unscheduled) , and 97 unscheduled sacrifices were required. Additionally, 187 animals were submitted for sacrifice at the end of the second year, 27 Jan - 14 Feb 1986. Gross necropsy examinations were performed, and tissues were collected from each animal in accordance with LAIR OP-STX-32, "General Pathology Procedures", OP-PSG-7, "Necropsy Procedure Gross Examination of Small Laboratory Animals", and OP-PSG-12, "Histopathology Trimming of Rodent Tissues."

The pathologic evaluation consisted of gross and microscopic examination of major organs/tissues and all gross lesions from sacrificed animals and animals found dead. The following organs/tissues were examined microscopically: eyes and lens, skin, subcutaneous tissue, mammary gland, brain (4 levels: anterior cerebral, midcerebral, midbrain, cerebellum) , middle ears, auditory canal, sebaceous gland, trachea, lungs, nasal region, sternum, heart, aorta, salivary glands (parotid, submaxillary, sublingual) , Harderian lacrimal and intraorbital lacrimal glands, exorbital lacrimal glands, liver (2-4 sections of various lobes), pancreas, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, urinary bladder, accessory sex glands (male only prostate, seminal vesicle, coagulating gland, epididymis), testes/ovaries, uterus (horns and body), skeletal muscle (2 sections, longitudinal and cross) , sciatic nerve, tongue, pituitary, thyroid/parathyroid, adrenals, thymus, spleen (2 cross sections), mesenteric lymph nodes, femur (bone marrow), and vertebrae with spinal cord (3

Brown et al. 6

sections: cervical, thoracic, and lumbar). These tissues were preserved in 10% buffered formalin, trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin prior to microscopic examination. Necropsy data were recorded and entered into a Xybion™ (Xybion Medical Systems, Cedar Knoll, NJ) computerized data acquisition program designed for a DEC VAX™ 750 computer. Microscopic findings were entered into the computer directly as microslides were read. Since the Xybion animal-numbering system was incompatible with the TOXSYS numbering system used during the "in-life" phases of the study, the animals had to be renumbered by the Pathology Section as they were necropsied in order to enter them into the Xybion Pathology Data System. A cross reference of animal identification numbers is presented in Appendix J.

Statistical Analyses

Statistical analyses were performed on the study results. TOXSYS System programs were used to determine the group mean animal body weights (EDS002) and the frequencies of clinical signs (EDS057-61) . The Xybion Pathology Data System was used to generate the pathology raw data listings, summary pathology reports, lesion frequency data, and terminal body weights (group mean, standard deviation, Bartlett’s Test, ANOVA, and Dunnett's Test). The incidence of microscopic lesions for each test group were compared to the control group using the Kolmogorov-Smirnov two-tailed test. The 5% (p ^ 0.05) level of significance was used for all tests .

Duration of Study

The "in-life" period for the second year of the study ran from 30 Jan 85 until 14 Feb 86, when the terminal sacrifice was completed. Appendix D is a complete historical listing of study events.

Changes/Devlationa from, Protocol

Performance of this study was in accordance with the protocol and applicable amendments.

Storage of Raw Data and Final Report

A copy of the final report, study protocol and amendments, raw data, relevant SOPs, analytical data for the test compound, and an aliquot of the test compound will be retained in the LAIR Archives.

Brown et al.--7

RESULTS

Mortality

One hundred eighteen animals died or were sacrificed prematurely during the second year of the study (controls, 13 males, 22 females; low dose, 16 males, 19 females; high dose, 27 males, 21 females) . Of these, 21 unscheduled deaths were recorded and 97 animals were euthanized because they were moribund, had infections, had lost weight, or were in poor condition (Tables la - Id) .

TABLE la

Listing of Unschadulsd Dsaths/Euthanixsd* Animals

1 Fab - 30 Apr 85

Toxicology

Dose

Animal No. 84D00-

Date

Clinical Outcome

Sex

Group

376

13

Feb

85

Died in cage

F

High IBC

157

26

Feb

85

Died in cage

M

High IBC

296

15

Mar

85

Sacrificed, large** intra-abdominal mass

F

High IBC

170

23

Apr

85

Sacrificed, alopecia and skin scabbing/scaling (infection control)

M

Control

428

23

Apr

85

Sacrificed, perivaginal fistula & discharge

F

Control

* Animals were sacrificed to conserve tissues in face of imminent death, for infection control purposes, or for humane reasons .

**Large - £ 20 mm in diameter

Brown et al. 8

TABLE lb

Listing of Unscheduled Desths/Euthanized* Animals

1 May - 31 Jul 85

Toxicology Animal No. 84DQQ- _

Date

Clinical Outcome

Sex

Dose

Group

422

7

May

85

Sacrificed, severe dehydration

F

Low IBC

156

9

May

85

Died in cage

M

High IBC

423

10

May

85

Sacrificed, large** intra-abdominal mass

F

High IBC

181

10

May

85

Sacrificed, large intra-abdominal mass

M

High IBC

109

19

May

85

Sacrificed, large intra-abdominal mass

M

High IBC

384

31

May

85

Sacrificed, skin lesions neck (infection control)

F

Control

129

5

Jun

85

Large intra-abdominal

mass

M

High IBC

275

14

Jun

85

Large intra-abdominal

mass

F

High IBC

326

1

Jul

85

Died in cage

F

Low IBC

431

8

Jul

85

Large intra-abdominal

mass

F

High IBC

430

9

Jul

85

Died in cage

F

High IBC

415

17

Jul

85

Pallor with palpable splenic mass

F

Low IBC

193

21

Jul

85

Died in cage

M

High IBC

091

30

Jul

85

Died in cage

M

High IBC

* Animals were sacrificed to conserve tissues in face of imminent death, for infection control purposes, or for humane reasons .

**Large - £ 20 mm in diameter

Brown et al. 9

TABLE lc

Listing of Unscheduled Deaths/Euthanized* Animals

1 Aug - 31 Oct 85

Toxicology Animal No. 84D00-

Date

Clinical Outcome

Sex

Dose

Group

099

1

Aug

85

Large** intra-abdominal mass

M

High IBC

149

1

Aug

85

Emaciated and dehydrated

M

Low IBC

301

1

Aug

85

Malocclusion

F

Control

191

1

Aug

85

Infected subcutaneous mass

M

High IBC

286

2

Aug

85

Pallor with palpable abdominal mass

F

High IBC

388

2

Aug

85

Pallor with palpable abdominal mass, died in cage

F

Control

147

16

Aug

85

Large subcutaneous perihumeral mass

M

Low IBC

407

16

Aug

85

Large subcutaneous mass on thoracic wall

F

Low IBC

279

20

Aug

85

Marked weight loss and perianal staining

F

Low IBC

081

28

Aug

85

Large subcutaneous mass

M

High IBC

426

28

Aug

85

Pallor with weight loss

F

Low IBC

074

30

Aug

85

Large intra-abdominal mass with excessive weight loss

M

High IBC

* Animals were sacrificed to conserve tissues in face of imminent death, for infection control purposes, or for humane reasons .

**Large » t 20 mm in diameter

Brown efc al. 10

TABLE lc (COnt.)

Listing of Unschsdulsd Deaths/Euthanized* Animals

1 Aug - 31 Oct 85

Toxicology

Dose

Animal No. 84D00-

Date

Clinical Outcome

Sex

Group

102

30

Aug

85

Large** intra-abdominal mass and pallor

M

Low IBC

108

30

Aug

85

Skin mass over maxilla

M

High IBC

151

30

Aug

85

Pallor with large intra-abdominal mass

M

High IBC

327

30

Aug

85

Large intra-abdominal mass

F

High IBC

338

30

Aug

85

Large intra-abdominal mass

F

High IBC

180

9

Sep

85

Large intra-abdominal mass with weight loss

M

Low IBC

288

9

Sep

85

Emaciated

F

Control

363

13

Sep

85

Oral discharge

F

Low IBC

389

13

Sep

85

Cardiac murmur with weight loss

F

Control

032

19

Sep

85

Large subcutaneous mass

M

Control

404

23

Sep

85

Died in cage

F

Control

260

25

Sep

85

Enteritis with weight loss

F

Low IBC

059

26

Sep

85

Large subcutaneous mass

M

Low IBC

095

26

Sep

85

Large intra-abdominal mass

M

High IBC

* Animals were sacrificed to conserve tissues in face of imminent death, for infection control purposes, or for humane reasons .

**Large = £ 20 mm in diameter

Brown et

TABLE lc (cont.)

Listing of Unscheduled Deaths/Euthanised* Animals

1 Aug - 31 Oct 85

Toxicology Animal No. 84DQQ- _

Date

Clinical Outcome

Sex

Dose

Group

104

26

Sep

85

Large** intra-abdominal mass

M

High IBC

124

26

Sep

85

Large splenic mass

M

Control

266

3

Oct

85

Large subcutaneous mass on neck

F

Control

069

3

Oct

85

Large intra-abdominal mass with weight loss

M

High IBC

368

8

Oct

85

Severe pallor from hemorrhagic enteritis

F

Control

199

16

Oct

85

Deteriorated condition and large palpable intra-abdominal mass

M

High IBC

278

17

Oct

85

Large subcutaneous mass over humerus

Y

Control

166

17

Oct

85

Large subcutaneous mass

M

Control

084

24

Oct

85

Pallor, weak, depressed

M

Low IBC

080

24

Oct

85

Died in cage

M

High IBC

203

24

Oct

85

Pallor, large intra-abdominal mass

M

Low IBC

424

25

Oct

85