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institute Report No. 298A

Developmental Toxicity Potential ofNitroguanidine in Rabbits

0.. 0

N Valerie G. Coppes, BSCharlotte L. Gomez

Dean K. Magnuson, BS, SP4

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

MAMMALIAN TOXI[COLOGY BRANCH D IDIVISION OF TOXICOLOGY DTL C

NOV 0 8 MS

September 1988 Toxicology Series: 184

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

DIATRIUTION r ATI -

Approved for pablic ra.Imea'I£

UNCLASSIFIEDp SECURITY CLASSIFICATION OF HI PAGE

REPORT DOCUMENTATION PAGE FA o.r70- A~r89

la REPORT SECURITY CLASSIFICATION lb RESTRICTIVE MARKINGS

UNCLASSIFIED ____________________

2a. SECURITY CLASSIFICATION AUTHORITY 3 DiSTRiBUTiONiAVAiLABILITY OF REPORT

Approved for public release;2b. DECLASSIFICATION/DOWNGRADING SCF4DULE Distribution is unlimi ted

4 PERFORMING ORGANIZATION REPORT NUMBER(S) 5 MONITORING ORGANIZATION REPORT NUMBER(S)

LAIR Institute Report No. 298

6a. NAME OF PERFORMING OIRGANIZATION 6b. OFFICE SYMBOL 7&. NAME OF MO iNITORING ORGANIZATIONMammalian Toxicology Br- (if Applicable) US Army Biomedical Research an"'Division of Txclg SGRD-ULE-T Development Laboratory

6C. ADDRESS (City, Start, anld ZIP Code) 7b. ADDRESS (City, State. and ZIP Code)Letterman Army Institute of Research Fort Detrick, MD 21701-5010Presidio, of San Francisco, CA

94129-6800Ba. NAME 09 FUNDING iSPONSORING 8b OFFICE SYMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER

ORGANIZATION (If aJPplCable)U SAMRDC I

5c. ADDRESS (City, State, and ZIP Code) 10 SOURCE OF FUNDING NUMBERSPROGRAM PROJECT TASK WORK UNIT

Fort Detrick, MD 21701-5010 ELEMENT NO. NO. NO. CCESSION NO.

62720A 835 AB tA30391311. TITLE (Include Security Classification)

Developmental Toxicity Potential of Nitroguanidine in Rabbits

12. PERSONAL AUTHOR(S)

Valerie G._Coppes, Charlotte L. Gomez, Dean K. Magnuson, Don W. Korte, Jr.

I I

institute -1FROMv2 5/ 9 /8 6TOl9/2/8 7 1 1988 Septem~ber 15616. SUPPLEMENTARY NOTAniON

Toxicology Series 184

17. COSATI CODES 18. SUBJECT TERMS (Continue on rtvtr if necessary and identify by blo.:k number)FIELD GROUP SUB-GROUP Developmental Toxicity, Teratology, Nitroguanidine,

Rabbi t

19. AASTRACT (Continue on reverse of necessary and identify by block number)

T he potential of nitroguanidine to produce developmental toxicity wasevaluated in pregnant New Zealand White rabbits. Nitroguanidine, suspended4n 1% carboxyrnethylcellulose, was administered at doses of 0, 100, 316, and1000 mg/kg/day by oral gavage on Days 6 through 18 of gestation. Fetuseswere delivered by cesarearl section on 1 ay 29, weighed and examlinedexterally. The soft tissues were examined while the bogty was beingeviscerated for subsequent processing in alizarin red stain for skeletalexamination, Ten dams in the 1000-mg/kg/day group died or were terminated i.na moribund condition following a generalized failure to thrive. The damsadministered 1000 mg/kg/day nitroguariidine exhibited weighnt loss. anddecreased food consumotion. Signs of developmental toxicity associated withnnitroguanidine administration were an increased incidence of resorptions i.r

20 DISTRIBUTION/ AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATIONM UNCLASSIHED/UNLi.TED 0 SAME AS RPT C3 TIC USERS UN;CLAo.,5FIED

22a. NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (lncide i1e oe 2c OFFICE SYMBOLEdwin S. Beatrice, COL MC 415 561-36007770 SGRD-ULZ

DForm 1473, JUN 86 Pre vious editfons art obsoletf. S~ECURITY CLASSIFICArION OF Ti-uS PAGE

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19. ABSTRACT (Continued)all dose groups. Fetuses in the 1000-mg/kg/day group were lighter in weightand had an increased incidence of retarded ossification of the sternebrae,olecranon, patellae, and phalanges. There were no dose-relatedmalformations. On the basis of these findings, we concluded thatnitroguanidine had no teratogenic potential but does have the potential tocause developmental toxicity.

---- ' - 'W ~ , - - -. - -

ABSTRACT

The potential of nitroguanidine to producedevelopmental toxicity was evaluated in pregnant NewZealand White rabbits. Nitroguanidine, suspended in 1%carboxymethylcellulose, was administered at doses of 0,100, 316, and 1000 mg/kg/day by oral gavage on Days 6through 18 of ge3tation. Fetuses were delivered bycesarean section on Day 29, weighed and examinedexternally. The soft tissues were examined while thebody was being eviscerated for subsequent processing inalizarin red stain for skeletal examination. Ten damsin the 1000-mg/kg/day group died or were terminated in a

moribund condition following a generalized falui-e tothrive. The dams administered 1000-mg/kg/daynitroguanidine exhibited weight loss and decreased foodconsumption. Signs of %-velopmental toxicity associatedwith nitroguanidine administration were an increasedincidence of resorptions in all dose groups. Fetuses inthe 1000-mg/kg/day group were lighter in weight and hadan increased incidence of retarded ossification of thesternebrae, olecranon, patellae, and phalanges. Therewere no dose-related maltormations. On the basis ofthese findings, we concluded that nitroguanidine had noteratogenic potential but does have the potential tocause developmental toxicity.

Key Words: Developmental Toxicity, Teratology,Nitroguanidine, Rabbit

iP Acoession For

NTI GRA&t01 DTIC TAI, _

Jv. _ _

iJ: "I ). K.%

PREFACE

TYPE REPORT: Developmental Toxicity Study

TESTTNG FACILITY: US Army Medical Research and Development ComnandLetterman Army Institute of ResearchPresidio of San Francisco, CA 94129-6800

SPONSOR: US Army Medical Research and Development CommandUS Army Biomedical Research and Development LaboratoryFort Detrick, MD 21701-5010Project Officer: Gunda Reddy, PhD

Work Unit 180; APC: TLBO

GLP STUDY NUMBER: 86003

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

PRINCIPAL INVESTIGATOR: Valerie G. Coppes, BS

CO-PRINCIPAL INVESTIGATORS: Charlotte L. GomezDean K. Magnuson, BS, SP4

REPORT AND DATA MANAGEMENT: A copy of the final report, studyprotocol, SOPs, and raw data will beretained in the LAIR Archives.Alizarin specimens will be retained inthe LAIR Pathology Archives.

TEST SUBSTANCE: Nitroguanidine

INCLUSIVE STUDY DATES: 25 September 1986 - 19 February 1987

OBJECTIVE: The purpose of this study was to determine thedevelopmental toxicity potential of nitroguanidine inpregnant New Zealand White rabbits when administeredorally during the period of organogenesis.

~iii

ACKNOWLEDGMENTS

Conrad R. Wheeler, PhD; Virginia L. Gildengorin,PhD; John T. Hixon; MAJ C. Dahlem Smith, DVM; MAJCharles B. Clifford, DVM; MP.J Larry D. Brown, DVM; MAJJohn C. Turni.r, VMD; CPT Harry L. Walker, DVM; Nancy J.Smith; SSG James D. Justus; SGT Paul B. Simboli; SP4Theresa L. Polk; SP4 Scott L. Schwebe; SP4 James J.

Fisher; Obie Goodrich, Jr.; and Richard Katona providedresearch assistance.

iv

I

SIGNATURES OF PRINCIPAL SCIENTISTSINVOLVED IN THE STUDY

We, the undersigned, declare that GLP Study 86003 wasperformed under our supervision, accordiig to the proceduresdescribed herein, and that this report is an accurate record ofthe results obtained.

DON W. KORTE PhD / DATE DEAN K. MAGNUSON, BS / DATEMAJ, MSC SP4 Study Director Co-Principal Investigator

VALERIE G. COPPSD CONRAD R. WHEELER, PhD DATDAC DACPrincipal Investigator Analytical Chemist

CPARLOTTE L. GOVZ d5ATEDACCo-Frincipal Investigator

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129.6800

qEOLY TO

T TENTION OF

SGRD-ULZ-QA 23 September 1988

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance Statement

1. This is to cettify that the protccol for GLP Study 86003

was reviewed on 16 July 1986.

2. The institute report etititled "Developmental Toxicity

Potential of Nitroguaxidine in Rabbits," Toxicology Series184, was audited on 12 August 1988.

CAROL,. M. LEWISChief, Quality Assurance

61

vi

TABLE OF CONTENTS

Page

Abstract ................................................ i

Preface .............................................. iii

Acknowledgments .......................................... iv

Signatures of Principal Scientists ........................ v

Report of Quality Assurance Unit ......................... vi

Table of Cont.ents ....................................... vii

BODY OF REPORT

INTRODUCTION ......................................... 1

Objective of the Study ........................... 1

MATERIALS

Test Substance .................................. 1Vehicle . ........................................ 2Animal Data ..................................... 2Husbandry ........................................ 2

METHODS .............................................. 3

Acclimation ..................................... 3Group Assignment ................................ 3Dose Levels ..................................... 3Compound Preparation and Analysis ............... 3Breeding ........................................ 4Cesarean Section Procedure ...................... 4Observations and Records ......................... 4Schedule of Study Events ......................... 5Statistical Analysis ............................ 5Changes/Deviations .............................. 5Raw Data and Final Report Storage ............... 5

RESULTS

Maternal Data ..................................5Cesarean/Fetal Data ............................. 8Tables ......................................... 1 0

vii

Table of Contents (Continued)

D ISCUSSION .................. ......................... 24

CONCLUSION ............................................ 26

REFERENCES ............................................ 27

APPENDICES ............................................ 29

Appendix A Chemical Data ........................... 31Appendix B Animal Data ............................. 35Appendix C Chemical Analysis ....................... 36Appendix D Schedule of Study Events .............. 30Appendix E Individual Maternal Body Weights ...... 39Appendix F Individual Maternal Food Consumption .. 43Appendix G Individual Maternal Clinical Signs .... 47Appendix H Maternal Gross Necropsy Findings at

Cesarean Section ...................... 60Appendix 1 Individual Ceqtational Data ............ 64Appendix J Fetal Sex, Weight, and Length .......... 68Appendix K Fetal External Examination ............. 72Appendix L Fetal Visceral Examination ............. 95Apperdix M Fetal Skeletal Examination ............. 118Appendix N Incidence of Fetal Examination

Findings ............................. 148Appendix 0 Incidence of Fetal Malformations

and Variations ....................... 152

OFFICIAL DISTRIBUTION LIST ............................ 156

viii

Developmental Toxicity Potential of Nitroguanidine inRabbits -- Coppes et a

INTRODUCTION

Nitroguanidine, a primary component of US Army triple-base propellants, is now produced in a Government-ownedcontractor-operated ammunition plant. The US Army BiomedicalResearch and Development Laboratory (USABRDL), as part of itsmission to evaluate the environmental and health hazards ofmilitary-unique pollutants generated by US Army munitions-manufacturing facilities, conducted a review of thenitroguanidine data base and identified significant gaps inthe toxicity data (1). -he Division of Toxicology, LAIR, wastasked by USABRDL to develop a genetic and mammalian toxicityprofile for nitroguanidine, related intermediates/by-productsof its manufacture, and its environmental degradationproducts. The rabbit developmental toxicity study describedin this report represented one of three studies (a ratdevelopmental toxicity study and rat multigenerationreproductive study are the others) in the reproductivetoxicity assessment being conducted as part of the healtheffects profile of nitroguanidine.

Objiective of the Study

The purpose of this study was to determine thedevelopmental toxicity potential of nitroguanidine inpregnant New Zealand White rabbits when administered orallyduring the period of organoge .esis.

4

MATERIALS

Test Substance

Chemical Name: Nitroguanidine

Chemical Abstracts Service Registry No.: 556-88-?

Toxicology Group Test Compound No.: Phase i TP36APhase II TP036B

Molecular Structure

H2N C = N -N02H2 N-

----------

Coppes et al -- 2

Source: Hercules Aerospace DivisionSunflower Ammunition PlantDeSoto, Kansas

Lot No.: Phase I SOW84KO10-A-001Phase II SOW85FOll-028

Molecular Weight: 104.1

Physical State: White powder

Other test substance information is presented inAppendix A.

The vehicle for nitroguanidine was a 1% solution ofcarboxymethylcellulose sodium salt, high viscosity (SigmaChemical Co., St. Louis, MO). Niuroguanidine is not solublein water at the concentrations tested.Carboxymethylcellulose holds nitroguanidine in a homogeneoussuspension.

Animal Data

New Zealand White rabbits were obtained from ElkhornRabbitry, Watsonville, CA. The study was conducted in twophases due to the number of animals recquired. Seventy-eightnulliparous females, approximately 4 nonths of age uponarrival at LAIR, and eighteen proven breeder males were used.Animals were identified individually by ear tattoo numbers.Additional animal data are presented in Appendix B.

A positive control study with hydroxyurea establishedthe New Zealand White rabbit as a sensitive test system fordevelopmental toxicity studies at LAIR (2). Historic data onmalformations and variants in New Zealand White rabbits arewell documented (3-5).

ti:sandryRabbits were housed individually in stainless steel wire

mesh cages wLth automatic water dispensers. Bedding was notprovided. Animals were fed Purina Certified Rabbit Chow 5322(Ralston Purina Company, St. Louis, MO). Males and nonbredfemales were fed approximately 150 g per day; bred femaleswere given 300 g per day, and their feed consumption wasmonitored. Water (reverse osmosis Technic Central Systems,Series 3 0U) was available ad libitum througnout the study.No contaminants or naturally occurring substances wereexpected to influence tha study. The animal room temperatureranged oetween 140C and 21"C, and the relative humidityranged between 42 and 78 percent (range of hygrothermograph

S-

Coppes et al -- 3

readings recorded in daily room log). The photoperiod was 12hours of light per day.

METHODS

Methods used are described in detail in OP-STX-40"Developmental Toxicity Study" (5) and were in accordancewith Environmental Protection Agency Good Laboratory PracticeStandards (6) and Health Effects Testing Guidelines (7).

Acclimation

Animals were acclimatized for approximately three weeksprior to start of breeding.

Group Assignment

Females were assigned to test groups by the weight-biased, stratified randomization method (OP-STX-78"Stratified Randomization"), which is based on the bodyweight at the start of breeding (8), on the Data GeneralEagle MV8000 computer.

Dose levels tested were 0, 100, 316, and 1000 mg/kg/day.Bred females were dosed daily from Day 6 through Day 18 ofgestation by oral intubation using a syringe equipped with a13-cm length of endotracheal catheter tubing. Dosing wasconducted without sedation or anesthetization of the animals.The dose for each female was based on the Day 6 body weight,and that dose was used throughout the treatment period.Phase I females were dosed from 20 Oct 86 through 15 Nov 86.Phase II females were dosed from 18 Jan 87 through 8 Feb 87.

Compound Preparation and Analysis

Initially, a smooth paste containing nitroguanidine anda small amount of vehicle was prepared in a mortar with apestle. Vehicle was then added gradually until the finalvolume was obtained. The concentrations prepared were 20mg/ml for the 100-mg/kg/day dose, 63.2 mg/ml for the 316-mg/kg/day dose, and 200 mg/ml for the 1000-mgikg/day dose.The dosing suspensions and vehicle control were given at a

6 volume of 5 ml/kg body weight. The vehicle and dosingsuspensions were prepared prior to the start of dosing foreach study phase and refrigerated. Fefore the animals weredosed each day, the containers of dosing preparation wereplaced in a beaker of hot tap water for 15 to 30 minutes tobring the suspensions to room temperature. Chemical analysesfor accuracy and homogeneity of zhe doging suspensions arereported in Appendix C.

Coppes Ct al -- 4

Each female was bred randomly to two males. Mating wasconfirmed by observation of the pair mating. Immediatelyafter the first mating, the female was removed from themale's cage and placed with another male. After the secondmating the bred female was returned to her cage. Day 0 foreach female was the day of mating.

Cesarean Sec-tion Procedure

Dams were weighed and euthanized with sodiumpentobarbi i overdose administered i.v. on Day 29 ofgestation. All females were examined, and nonpregnant oneswere removed from the study. Gravid uteri were examined forimplantations (resorptions and live and dead fetuses). Eachimplantation was assigned an identification letter. Thefetuses, uterus, and ovaries were removed, the corpora luteawere counted, and the dam was examined for gross visceralsigns of toxicity and reweighed. Each fetus was weighed,measured crowi.-to-rump, and examined externally.

Fetuses were placed in 70% ethanol and then carefullyeviscerated. The viscera were examined for anomalies, andthe sex of the fetus was determined. The fetuses were theaprocessed by the alizarin red S staining technique of Crary(9). After processing, the specimens were stored in glycerolwith a few crystals of thymol to inhibit bacterial and moldgrowth.

Observations and Records

nred females were weighed on Days 0, 6, 12, 18, 23, and29. Their feed was weighed, and food consumption wascalculated dily. Females were observed daily from Day 0through Day 29 for clinical signs of toxicity, abortion, orpremature delivery. Date, time, and volume of dosingsuspension administered were recorded during the daily dosingon Days 6 through 18. At cesarea, section, uterine data,gravid body weight, number of corpoza lutea, and results fromgross examination of the dam were recorded. Dams werereweighed after the removal of the gravid uterus to determinethe "Corrected Day 29" weight.

Fetal weignt, crown-to-rump measurement, and externalexamination findings from live fetuses were recorded.Visceral examination findings and sex were recorded. Theskeletons stained by alizarin were examined under lowmagnification on a light box for malformations, alignment,and degree of ossification. The ossified sternebrae, ribs,caudal vertebrae, metacarpals, metatarsals, and phalangeswere counted.

Coppes -' al -- 5

Schedule of Study Events

The study was divided into two phases to allow adequatetime for animal care, fetal processing, and fetalexamination. The historical listing of study events is givenin Appendix D.

Sqtat;istical Analysis

The data were analyzed with BMDP software on a DataGeneral Eagle MV8000 computer (10). Methods used aredescribed by Winer (11). Data from both phases "2re combinedfor analysis. The litter or litter mean was ' . as theexperimental unit. All tests were run at the 0.05 )evel ofsignificance. The maternal body weights, weight changes,food consumption, and fetal weights and lengths were comparedby one-way analysis of variance. Then, if a significant Fvalue occurred, the Newman-Keuls test was applied to thedata. The implantation efficiency, percent resorptions, andpercent live and dead fetuses were compared by thenonparametric Kruskal-Wallis test. If the Kruskal-Wallistest was significant, an appropriate multiple comparison testwas used to determine which groups were different (12). Thenumbers of litters per group with resorptions, litters withdead fetuses, litters containing fetuses with skeletal or anyvariations, and the number of fetuses with skeletal or anyvariations were compared by chi-square analysis, and, ifthese were significant, the Marascuilo's methc' of multiplecomparison was used to determine which groups were different(13). The numbers of fetuses or litters with malformat: nsor external or visceral variations were not comparedstatistically because there were too few.

Changes/Deviations

The study was accomplished according to the protocol andaddenda.

Raw Data and Final Report Storage

A copy of the final report, study protocol, addenda, rawdata, SOPs, and an aliquot of test compound will be retainedin the LAIR Archives. Alizarin specimens will be retained inthe LAIR Pathology Archives.

REST LTS

Maternal Data

The number of sperm-positive females in each group,number of animals that died during the study, and number of

Coppes et al -- 6

animals that were pregnant are presented in Table 1.Nitroguanidine did not affect the pregnancy rate. The numberof litters with resorptions was significantly higher in the100-mg/kg/day and the 1000-mg/kg/day groups in comparison tothe control group. The 316-mg/kg/day group also had anincreased number of litters with resorptions, but the numberof litters was not significant in comparison to the controlgroup.

Six animals from the 1000-mg/kg/day group died during

the study.

86F216, found dead on Day 14, lost 988 g bodyweight from Day 6 to Day 14 and had thick,foamy, granular orange-rust colored urine,convulsions, hypertonia, loss ofconsciousness, shallow, rapid respiration,dehydration, and mucus in the ncse during thetreatment period. Necropsy findings includedcongestion of the lungs and liver and severelymphoid depletion of the thymus and spleen.

86F229, found dead on Day 11, lost 812 g bodyweight from Day 6 to Day 11, had orange-rustcolored urine, was prostrate followingconvulsions, moved stiffly, had red materialin urine during the treatment period, and hada ruptured stomach at necropsy.

86F233, found dead on Day 19, lost 359 g bodyweight from Day 6 to Day 18, and clinicalsigns observed included thick, foamy, orange-rust colored urine, convulsions, tremors, andprostration. Necropsy findings includedbiliary hyperplasia of the lung and slightmultifocal hemorrhage in the brain and lung.

86F315 lost 674 g from Day 6 to Day 11, hadthick, foamy, orange-rust colored urine,hunched posture, hypertonia, was inactive andcool to the touch, and was found dead on Day11. Necropsy findings weremeningoencephalitis of the cerebrum andcerebellum and granulomas of the liver.

Clinical sigis for animal 86F319, found deadon Day 15, were weight loss of 815 g from Day6 to Day 15, thick, foamy urine, red-stainednose and mouth, and hypertonia. Necropsyfindings were renal mineralization, two massesof congealed dosing material in the pyloricarea of the stomach, and an esophageal ruptureat the thoracic inlet.

Coppes et al -- 7

Animal 86F306, found dead on Day 11, had lost857 g from Day 6 to Day 10, moved stiffly, andhad thick, foamy, orange-rust colored urine,hunched posture, and cyanosis. Congestion ofthe lungs was noted at necropsy. Pregnancystatus was not reported for this animal.

Four moribund animals from the 1000-mg/kgiday group wereeuthanized during the study.

Animal 86F209, euthanized on Day 12, had a weightloss of 967 g from Day 6 to Day 12, granular,foamy red urine, red nasal discharge, red-stained hindquarters, hunched posture, andtremors, and it moved stiffly. A bloodyhairball was found under the cage on Day 12.Morphologic diagnosis for this pregnant animalwas acute, multifocal placentitis.

86F293, euthanized on Day 16, had a weightloss of 538 g from Day 6 to Day 16, thick,foamy, orange-rust colored urine from Days 6through 33 but clear yellow urine on Day 14,hunched posture, hypertonia, convulsions,cyanosis, and ataxia. Necropsy of thispregnant animal revealed mild vasculitis ofthe cerebral choroid plexus.

86F286, euthanized on Day 9, lost 599 g bodyweight from Day 6 to Day 9, moved stiffly, andhad thick, foamy urine, red material in urine,red-stained hindquarters, hunched posture, andtremors. A bloody hairball was found underthe cage. At necropsy this bred animal wasnot pregnant and revealed nephrosis.

86F318, also euthanized on Day 16, lost 571 gbody weight from Day 6 to Day 16 and hadthick, foamy, orange-rust colored urine andconvulsions. Necropsy findings on this animalwere hyperplasia of the bile duct and vacuolarchange and hepatocellular loss in the liver.Pregnancy status was not reported for thisanimal.

Clinical siqns, body weights, and food consumption ofanimals not pregnant at necropsy or cesarean section or ofthe two animals whose pregnancy status was not included inthe necropsy results are not included in this report.

Individual maternal body weights and average daily foodconsumption for the pretreatment, treatment, andposttreatment study periods are presented in Appendix E andAppendix F, respectively. Results of maternal body weigrts,

Coppes et al-- 8

weight changes, and food consumption by group are in Table 2.when given at 1000 mg/kg/day, nltroquanidine producedsignificant weight loss and decreased food consumption duringthe treatment period, Days 6 to 18, in comparison to thecontrols. Lower doses of nitroguanidine did notsignificantly affect maternal weight gain or foodconsumption, although there was a trend toward reduced bodyweight gain with increasing dose from Days 6 to 18.

Individual maternal clinical signs are listed inAppendix G. Summaries of clinical signs by dose group duringthe pretreatment (Day 0 through Day 5), treatment (Day 6through Day 18),and posttreatment (Day 19 through Day 29)periods are found in Tables 3a, 3b, and 3c, respectively.Dose-related clinical signs, which occurred with a highfrequency in the 1000-mg/kg/day group during the treatmentperiod, included orange-rust colored urine which was oftenthick and foamy and decreased amount of feces found under thecage. Clinical signs occurring only in the 1000-mg/kg/daygroup included hunched posture, hypertonia, increased startlereflex, and death or moribund condition.

Individual maternal gross necropsy findings at cesareansection are listed in Appendix H, and a summary by group isfound in Table 4. Findings were observed in all dose groups.Cysts on fallopian tubes or dark red fallopian tubes wereobserved most frequently.

Cesarean/Fetal Data

The individual gestational data are listed in Appendix 1I, the mean gestational data by group in Table 5.Nitroguaniaine had no effect on the number of corpora lutea,implantations, and live and dead fetuses. Percent resorptionwas significantly increased in all nitroguanidine dose groupsin comparison to the control.

The number of live males and females per litter and theaverage fetal weight and length per litter are given inAppendix J; the group means are in Table 6. Nitroguanidine .0did not affect the male-to-female ratio. Male and femalefetuses from the 10OC-mg/kg/day dose group were significantlylighter in weight than the controls. There was no sizedifference in the 100- and 316-mg/kg/day dose group fetusesin comparison to the controls.

Individual external examination findings are presentedin Appendix K. A summary by dose group is in Table 7. Theonly variation observed was bloated abdomen in one fetus inthe 1000-mg/kg/day group. Malformations observed werehindpaw ectrodactyly in one 100-mg/kg/day fetus and cleftpalate in one 1000-mg/kg/day fetus.

Coppes et al -- 9

Individual visceral examination findings are in AppendixL, and a summary by dose group is presented in Table 8.Visceral variations occurred in four fetuses. Dilated renalpelvis occurred in one fetus in the 100-mg/kg/day group andin one fetus in the 316-mg/kg/day group. Elongated ovariesoccurred in one 316-mg/kg/day fetus. Enlarged left ventricleof the heart occurred in one 1000-mg/kg/day fetus. The 1000-mg/kg/day fetus with the cleft palate at the externalexamination also had the only visceral malformation in thestudy. The left ureter transversed the midline and ran Jadjacent to the right ureter.

Individual skeletal variations and malformations aredescribed in Appendix M, and a group summary appears in Table9. Skeletal variations occurred in all dose groups. Therewas no difference in the number of litters containing fetuseswith variations, but the number of fetuses with variations inthe 1000-mg/kg/day group was significantly higher incomparison to the control group. Variants most frequentlyseen were reduced ossification of the sternebrae, olecranon,patellae, and phalanges. The only two skeletal malformationswere observed in the fetus with ectrodactyly in the 100-mg/kg/day group and in the fetus with cleft palate in the1000-mg/kg/day group previously described as externalmalformations. Skeletal examination of the fetus withectrodactyly revealed one metatarsal and four phalangesabsent from the right hindpaw, one metatarsal and sevenphalanges absent from the left, and the three phalanges ofthe left forepaw pollex absent.

The individual incidence of external, visceral, andskeletal variations and malformations is found in Appendix N,and the individual incidence of any variation andmalformation is found in Appendix 0. A summary by dose groupof the effect of nitroguanidine on the incidence of fetalmalformations and variations is presented in Table 10. Therewas no significant difference in the rate of malformationsamong the dose groups. The number of fetuses with skeletalvariations was significantly increased in the 1000-mg/kg/daydose group in comparison to the control.

Coppes et al -- 10

TABLE 1

Effect of Nitroguanidine onSurvival and Pregnancy

Nitroguanidine (mg/kg/day)

Examination Finding 0 100 316 1000

Bred females 17 18 16 22

Females that died* 0 0 0 10Nongravid 0 0 0 1Gravid 0 0 0 7Pregnancy not confirmed 0 0 0 2

Females examined on Day 29 17 18 16 12Nonaravid 4 3 1 1Gravid 13 15 15 11

With live fetuses 13 15 15 10With dead fetuses 0 3 1 2With resorptions 3 13t 7 5 tResorptions only 0 0 0 1

Females that were gravid 13 15 15 18

*Includes females that were euthanized in a moribund

condition.tSignificantly different from control by Marascuilo's methodof multiple comparison of proportions, p < 0.05.

~1

Coppes et al.-~I

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Coppes et at -- 12

TABLE 3a

Maternal Clinical Signs* - Pretreatment (Days 0-5)


0 100 316 1000

Number of animals observed 13 15 15 18Number with signs 8 7 5 9

Orange-rust urine 1 2Diarrhea 8 5 4 6Small amount of feces 1Small black feces 1Yellow-stained nose 1 1Yellow stain on top of head 1

*Preqnant females.

Coppes et at -- 13

TABLE 3b

Maternal Clinical Signs* - Treatment (Days 6-18)


0 100 316 1000


Orange-rust colored urine 2 5 7 16Thick/foamy urine 1 4 16Granular/foamy urine 3Clear yellow urine 1Red urine 2Red material in urine 2Small amount of urine 1No urine under cage 1Diarrhea 8 6 8 7Small amount of feces 1 1 9Small feces 1Small black feces 1No feces under cage 1Mucus on feces 1Bloody hair ball under cageRed/yellow granular materialunder cage 1

Red material under cage 1Yellow-stained nose 5 6 5 5Stained perianal region 2 1Brown material on leg/paws 1 1 2Brown material on abdomen/tail 1Yellow stain on top of head 1Red-stained nose or mouth 1 2Orange-stained legs 1 1Red-stained paws, hindquarters 1 2Injured bloody toenails 3

*Fregnant females.

°o |

Coppes et al -- 14

TABLE 3b (Concluded)

Maternal Clinical Signs* - Treatment (Days 6-18)


0 100 316 1000

Clear eye discharge 1Mucus or nasal discharge 2Red nasal discharge 1Blood in mouth at dosing 2 1 3Hair loss from underside 1 3 1Short hair under chin 1Upper front teeth broken 1Strong rabbit/urine odor 1Abscess appeared, drained, healed 1Deprived of water 2Ataxia 1Inactive 2Increased startle reflex 3Convulsions 3Twitching 1Hypertonia 5Hunched posture 4Moved stiffly 2Cyanosis 1Tremors 1Loss of consciousness 1Prostrate iRapid/shallow respiration 1Dehydrated 1Cried out after dosina 1Cool to touch 1Death or euthanized in

moribund condition 6

*Pregnant females.

Coppes et al -- 15

TABLE 3c

Maternal Clinical Signs* - Posttreatment (Days 19-29)


0 100 316 1000


Orange-rust colored urine 2 9Thick/foamy urine 2 6Red urine 1Diarrhea 1 1Small amount of feces 1 3 3 4Small feces 1No feces under cage 1No urine or feces under cage 2Mucus on feces 1Hair in feces 1Yellow-stained nose 2 3 1Yellow-stained forepaws 1Stained perianal region 1Brown material on abdomen/tail 1Mucus or nasal discharge 1 2Hair loss from underside 1Abscess healed 1Deprived of water 2 2Inactive 1Pulling hair for nesting 1Red material under cage 2Clump of mucus under cage 1Convulsions 1Hypertonia 1Prostrate 1Tremors IIncreased salivation 1Death or euthanized inmoribund condition 1

*Pregnant females.

Coppes et al -- 16

TABLE 4

Maternal Gross Necropsy Findings at Cesarean Section


0 100 316 1000

Number examined 13 15 15 11Number with findings 10 11 12 9

Cysts on fallopian tubes 7 6 9 5Dark red fallopian tubes 3 3 4 1Red inflamed fallopian tubes 1Dark spots on ovary 2 2Cysts in adipose tissue 1Fragile uterus 1Small pale liver 1Blotchy or mottled liver 1 1Mass on liverNecrotic lung tissue 1Cyst on lobe of lung 1Mass on lung 1Red mass on pancreas 2 2Dark spots on pancreas 1 1Dilated renal pelvis 1 1 1Spots on kidney 2Cavitation of kidney medulla 1 1Blotchy kidney 1 1Pale kidney 1

S

Coppes et a!.--1

(Nif co w CO

-4)

-H- + i++++ +4+4+4H (1

on 0'ao 0'CC) -

-- 4

O1 +4 + H+1 + +14-

(41

a% LoC mr

E .

W1 4) 0-L)o r

4) c un)rLl ~(

--. 4 -4- (4 0 U

4) 4 C0 %4 04+ N+4 C1i4

-~ >1

U) 4T0 -4 (n C 0 C

4.4o 0 0 C44++ 0

4.Je' LnLnN 0X - 0

4-4 0 -

0 -4 --

u~ 4 401 -J04)4.

W) L) x

(4414 $4 >a (4 -144C 04 -- 1 -)

u0 c14- '-) 0 0 0

S-4--4 0 A.)4) 1 4 ,

Q. ;) 00 C -+

Q 0E4 4) - 1 , C )a

-4 4.) -) 0 W 44 4 4jO) .4

Coppes et a!. - 18

CN - -4 fn r- 1.0 C) C-4

C

- ~ ~ r r-r r(N O

o -I CD -4 f- Ut-C LA Ln

V +1++4 +1 H4 +i+i -H

00

41)4Cp +i -Hr++i +i- -H +i

--4 :3

0 01144++++0" W CVCTC N00C

x 41 r -; lw -4 -

E- V z%W . >4

(NN 0 nmC

4-4 a4

1-444)

U ) 444

U) 41 41)4-4U) U) L1U- .)*

000 -)- -4

4.4 E t4.4.4

4-4 -4 44 U Q u,01Q)1dJ.~~ 0C0C

U M L- to or

4 -4--1 v --

Coppes et al -- 19

TABLE 7

Effect of Nitroguanidine onExternal Malformations and Variations*



Total number 121/13 135/15 131/15 87/10

Malformations

Cleft palate 1/1Ectrodactyly 1/1

Variations

Bloated abdomen 1/1

*Data presented as fetuses/litters.

Coppes et al -- 20

I

TABLE 8

Effect of Nitroguanidine onVisceral Malformations and Variations*



Total number 121/13 135/15 131/15 87/10

Malformations

Cleft palate IitUreter transversed midline 1/it

Variations

Enlarged heart ventricle 1/1Dilated renal pelvis 1/1 1/1Elongated ovaries 1/1

*Data presented as fetuses/litters.tBoth malformaticns occurred in the same fetus.

I

I

fI

Coppes et at -- 21

TABLE 9

Effect of Nitroguanidine onSkeletal Malformations and Variations*



Total number 121/13 135/15 131/15 87/10

Malformations

Ectrodactyly 1/1Cleft palate 1/1

Variations

Sutural bone 1/1 1/1Dot of ossification adjacent

to 7th cervical vertebra 1/1 7/421 vertebra (thoracic,

lumbar, and sacral) 1/1Vertebral arch, centrum

incomplete ossification 1/1Ribs short, not parallel 1/1Rib riot ossified 1/1Mid section of rib not

ossified 1/1Sternebrae

4 ossified 2/2 1/1 1/15 ossified 15/8 21/8 29/9 16/6Split 6/3 4/4 2/2 2/2Partially ossified 10/5 24/8 20/10 15/8Diagonally ossified 2/2 1/1 2/1 2/2Scrambled i/ JFused 1/1Dumbbell shaped 2/2 1/1 1/1 1/1Misshaped 2/2 I/! 1/1Dot of ossification above

first sternebrae 3/3 1/1

*Data presented as fetuses/litters.

A single fetus may have more than one abnormality and,therefore, will be included more than once in this table.

Coppes et at -- 22

TABLE 9 (Concluded)

P Effect of Nitroguanidine onSkeletal Malformations and Variations*



Variations (Continued)

Olecranon not ossified 1/1 15/4 9/3 18/4Patellae not ossified 6/3 14/7 10/5 14/5Pubis not ossified 4/1Nmber of phalanges ossifiedForepaw

14 7/3 17/8 11/5 11/613 1/1 1/1 4/3 4/1

12 1/1 1/111 111/1

Hindpaw11 2/2 1/1 4/2

*Data presented as fetuses/litters.A single fetus may have more than one abnormality and,therefore, will be included more than once in this table.

it

S

Coppes et at -- 23

TABLE 10

Effect of Nitroguanidine on the Incidence

of Fetal Malformations and Variations*


0 100 316 1000

Total number 121/13 135/15 131/15 87/10

Any (External/Visceral/Skeletal)Malformations 1/1 1/1Variations 46/12 65/13 62/14 52t/10

External examinationMalformations 1/1 1/1Variations 1/1

Visceral examinationMalformations 1/1Variations 1/1 2/2 1/1

Skeletal examinationMalformations 1/1 1/1Variations 46/12 64/13 61/14 52t/10

*Data presented as ietuses/litters.tSignificantly different from control by Marascuilo's method ofmultiple comparison of proportions, p < 0.05.

Coppes et al -- 24

DISCUSSION

The health effects of nitroguanidine are beingdetermined because of the Army's decision to incorporatenitroguanidine in its triple-base propellants. Previously,this laboratory showed that nitroguanidine was slightly toxicin rats and mice following acute oral administration, wasnonirritating to the skin and eyes of rabbits, and wasnonreactive in a dermal sensitization study in guinea pigs(14). A subchronic toxicity study in rats with doses as highas a "limit dose" of 1000 mg/kg/day mixed in the diet for 14days produced no definitive toxicological effects (15). Thislack of toxicity was supported by metabolic fate studies thatindicated that nitroguanidine was absorbed 100% followingoral administration and was excreted unchanged in the urine,60-80% within the first 8 hours (16). In a developmentaltoxicity study in rats, nitroguanidine given by oral gavageon gestational days 6 through 15 at 1000 mg/kg/day produceddecreased food consumption, maternal weight loss, and smallerfetuses with an increased incidence of retarded ossificationof the sternebrae, caudal vertebrae, and pubis. Thedevelopmental toxicity no-observed-effect level fornitroquanidine in rats was 316 mg/kg/day (17).

The predominant sign of maternal toxicity observed inthis study was death in ten animals (six animals died andfour moribund animals were terminated) in the 1000-mg/kg/daygroup. It is doubtful that these deaths were attributable toa direct pharmacological effect. Necropsy findings on theten animals were varied with no finding common to all. Thegeneral failure to thrive of these animals suggested that thehigh concentrations of nitroguanidine necessary to administerthe 1000-mg/kg/day dose by oral intubation interfered withthe digestive processes of the animals in this group. Thisis supported by the decreased food consumption and weightloss during the treatment period. Additionally, one animalhad a ruptured stomach, another animal with two masses ofdosing macerial in the stomach had a ruptured esophagus. Thetubing used to administer the test compound was smooth andflexible, not likely to tear healthy tissue. Nitroguanidinealso reduced food consumption and decreased body weight inrats when given by oral gava,e at 1000 mg/kg/day (17).Necropsy findings on several rabbits with convulsionssuggested that Encephalitozoon cuniculi was a possibleetiology. Stress was indicated as a contributing factor inthe death of the animal with severe lymphoid depletion.Possible hemoglobinuria could have occurred as a result ofthe test compound in the animal with nephrosis. The onlydose-related adverse maternal effects which occurred in the100- or 316-mg/kg/day groups were a low frequency incidenceof orange-rust colored urine and thick and foamy urine.

The four primary manifestations of developmentaltoxicity are death of the conceptus, malformation, retarded

Coppes et al -- 25

development, and functional deficit. This study was designedto screen for the first three. In a developmental toxicitytest the fetal examination findings may range in severityfrom slightly retarded development or minor variations tomajor malformations. Retarded development may be transitory,for example, caused by decreased maternal food consumption,and the retarded offspring may catch up quickly after birthor after weaning. Minor variations from normal may not havean adverse effect on the function and quality of life of theoffspring. Major structural malformations, such as malformedor missing organs or limbs, can either be life threatening orseverely limit the functioning and longevity of theoffspring. A test substance is considered developmentallytoxic if, when administered at a dose level that is notovertly maternally toxic, it produces malformations at asignificantly higher incidence than in the controls.Although variations are not as serious as malformations, asignificantly increased incidence of variations, incomparison to the controls, is a sign of some fetal ormaternal toxicity (18). Spontaneous malformations are thosethat occur randomly, usually at low frequency, and are ofunknown cause, and whose incidence is not dose-related.

Nitroguanidine increased the number of litters withresorptions in the 100-mg/kg/day and 1000-mg/kg/day groupsand the percent resorption per litter in all dose groups incomparison to the control group. One dam in the 1000-mg/kg/day group had 100% of implants resorbed. An increasedincidence of resorptions is a manifestation of developmentaltoxicity.

In this study each fetus was examined externally atcesarean section and then for visceral and skeletalabnormalities. The findings on each fetus were described andcategorized as either variations or malformations, dependingon the severity or whether the changes were permanent. Thefinding of 13 ribs (unilateral, bilateral, or rudimentary)was not included in this report because it occurred at a highfrequency and was not dose-related. Those findingscategorized as variations included such transitory findingsas retarded ossification (includes those fetuses with fewerthan six sternebrae, tewer than 15 phalanges per forepaw, andfewer than 12 phalanges per hindpaw ossified) and minordeviations from normal that may or may not be permanent suchas slightly misshapen sternebrae or ribs, extra dots ofossification, dilated renal pelvis, elongated ovaries,enlarged heart ventricle, and bloated abdomen. Findings ofmore serious consequence that were categorized asmalformations were cleft palate, displaced ureter, andectrodactyly.

The retarded development in the 1000-mg/kg/day groupresulted in fetuses that were significantly lighter in weightand had an increased incidence of skeletal variations in

Coppes et al -- 26

comparison to the controls. This retarded development couldbe attributed to maternal toxicity rather than to a directeffect of nitroguanidine on the fetus. The 1000-mg/kg/daygroup dams lost weight and consumed less food than thecontrols during the treatment period.

The malformations observed in this study are consideredspontaneous because they are not dose-related and occurred ata low frequency. One dam in the 1000-mg/kg/day group hadnasal discharge from Days 7 through 29, and marked necrosisof the lung was observed at cesarean section. The fetus withmultiple malformations (cleft palate and displaced ureter),the fetus with bloated abdomen at external examination, andthe fetus with enlarged heart ventricle at visceralexamination were in her litter. The malformations andvariations which occurred in this litter could be attributedto the compromised condition of the dam. Two fetuses (one inthe 100-mg/kg/day group and one in the 1000-mg/kg/day group)out of a total of 474 fetuses in the study were malformed.The incidence of spontaneous malformations in this study issimilar to that published by Palmer (3) and historicalcontrols from this laboratory (2) and is not.attributed tonitroguanidine.

CONCLUSION

There was no evidence of nitroguanidine producingteratogenicity (malformations) in rabbits under conditions ofthis study. Nitroguanidine, administered at a dose levelthat does not produce overt maternal toxicity, has thepotential to cause developmental toxicity (increasedresorptions).

Coppes et al -- 27

REFERENCES

1. Kenyon KF. A data base assessment of environmental fate

aspects of nitroguanidine. Frederick, MD: US Army Medical

Bioengineering Research and Development Laboratory, 1982.

DTIC No. ADA125591.

2. Coppes VG, Speckman CL, Korte DW. Developmental

toxicity potential of hydroxyurea, a positive control, in

rabbits. Toxicology Series 145. Presidio of San Francisco,

CA: Letterman Army Institute of Research, 1988. Institute

Report No. 269.

3. Palmer AK. Spontaneous malformations of the New Zealand

White rabbit: The background to safety evaluation tests.

Lab Anim 1968;2:195-206.

4. Palmer AK. Sporadic malformations in laboratory animals

and their influence on drug testing. Adv Exp Med Biol

1972;27:45-60.

5. Developmental Toxicity Study. Standard Operating

Procedure OP-STX-40. Presidio of San Francisco, CA:

Letterman Army Institute of Research, 15 Aug 1985.

6. Environmental Protection Agency. Toxic Substances

Control, Good Laboratory Practice Standards (40 CFR 792).

Final Rule, 29 Nov 1983, (48 FR 53922-44).

7. Environmental Protection Agency. Health Effects Testing

Guidelines (40 CFR 798). Final Rule, 27 Sep 1985, (50 FR

39433-4).

8. Stratified Randomization. Standard Operating Procedure

OP-STX-78. Presidio of San Francisco, CA: Letterman Army

Institute of Research, 2 Dec 1983.

9. Crary DD. Modified benzyl alcohol clearing of alizarin-

stained specimens without loss of flexibility. Stain Technol

1962;37:124-5.

10. Dixon WJ, ed. BMDP statistical software. Berkeley:

University of California Press, 1981.

11. Winer BJ. Statistical principles in experimental

design. New York: McGraw-Hill, 1971.

12. Hollander M, Wolfe DA. Nonparametric statistical

methods. New York: Wiley, 1973.

13. Marascuilo LA. Large-sample multiple comparisons.

Psychol Bull 1966;65:280-90.

Coppes et al --- 28

14. Hiatt GFS, Morgan EW, Brown LD, Lewis CM, Johnson YC,Mullen L, Bauserman JW, Okerberg CV, Lollini LO, Korte DW.Acute toxicity of guanidine nitrate and nitroguanidine. In:1985 Joint Army-Navy-NASA-Air Force Safety and EnvironmentalProtection Subcommittee Meeting. Chemical PropulsionInformation Agency Publication 436. Laurel, MD, 1985:321-30.

15. Morgan EW, Ho B, Brown LD, Lewis CM, Tillctson JA,Lollini LO, Korte DW. Subchronic toxicity and metabclism ofnitroguanidine in the rat. In: 1985 Joint Army-Navy-NASA-Air Force Safety and Environmental Protection SubcommitteeMeeting. Chemical Propulsion Information Agency Publication436. Laurel, MD, 1985:331-40.

16. Ho B, Tillotson JA, Kincannon LC, Simboli PB, Korte DW.The fate of nitroguanidine in the rat. Fundam Appl Toxicol1988; 10:453-8.

17. Coppes VG, Orner GA, Korte DW. Developmental. toxicitypotential of nitroguanidine in rats. Toxicology Series 174.Presidio of San Francisco, CA: Letterman Army Institute ofResearch, 1988. Institute Report No. 257.

18. Schwetz BA. Monitoring problems in teratology. In:Gralla EJ, ed. Scientific considerations in monitoring andevaluating toxicological research. Washington, DC:Hemisphere Publishing Co., 1981:183-4.

p

ICoppe5 et al -- 29

Appendix A Chemical Data .................................. 31Appendix B Animal Data .................................... 35Appendix C Chemical Analysis ............................. 36Appendix D Schedule of Study Events ...................... 38Appendix E Individual Maternal Body Weights ............ 39Appendix F Individual Maternal Food Consumption ........ 43Appendix G Individual Maternal Clinical Signs ........... 47Appendix H Maternal Gross Necropsy Findings at

Cesarean Section ............................ 60Appendix I Individual Gestational Data ................... 64Appendix J Fetal Sex, Weight, and Length ............... 68Appendix K Fetal External Examination .................... 72Appendix L Fetal Visceral Examination .................... 95Appendix M Fetal Skeletal Examination ................... 118Appendix N Incidence of Fetal Examination Findings..... 148Appendix 0 Incidence of Fetal Malformations and

Variations .................................. 152

Coppes et al -- 31

* Appendix A: CHEMICAL DATA

Chemical name: Nitroguanidine (NGu)

Other listed names: Guanidine, Nitro; alpha-Nitroguanidine;I beta-Nitroguanidine

LAIR Code: Phase I: TP036APhase II: TP036B

Structural formula:

H2N'-C = N-N02H 2N~~NO

Molecular formula: CH4N402

Molecular weight: 104.1

pH range of dosing suspensions: 6.7 - 7.4(l)

Physical state: White Powder

Melting point: 2320 C (2 )

Source: Hercules Aerospace DivisionSunflower Ammunition PlantDeSoto, Kansas

Lot No. Phase I: SOW84KO10-A-001Phase II: SOW85FOl1-028

Coppes et al -- 32

Appendix A (Cont.): CHEMICAL DATA

Analytical data/purity: The major peaks in the infraredspectrum of the compound wereobserved at 3450, 3396, 3342, 3278,3201, 1666, 1634, 1525, 1404, 1314,1151, 1045, 782 cm-. (3) Thespectrum obtained for the testcompound in our lab was identical tothe spectrum for TP36B ( 4 )and tothe Sadtler standard spectrum fornitroguanidine(5 ). HPLC showed onlyone peak (retention time 4.9 min forTP36A (6); 4.8 min for TP36B) (7)The conditions employed were asfollows: column, Brownlee RP-18 (4.6x 250 mm); solvent, 10% methanol-90%water; flow rate, 0.7 ml/min; oventemperature, 50*C; monitoringwavelength, 265 nm.

Stability: Stable in 1% carboxymethylcellulose for at leastfour months (see Appendix C)

References :

1. Wheeler CR. Nitrocell.ulose-Nitroguanidine ProjectsLaboratory Notebook #85-12-022, p. 26. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.

2. Fedoroff BT, Sheffield OE. Encyclopedia of explosivesand related itemds. Vol V. Dover, New Jersey: PicatinnyArsenal 1975: G154.

3. Wheeler CR. Nitrocellulose-Nitroguanidine Projects.Laboratory Notebook #85-12-022, p. 22-23. Letterman ArmyInsitute of Research, Presidio of San Francisco, CA.

4. Ibid., pp. 58-9.

5. Sadtler Research Laboratory, Inc. Sadtler standardspectra. Philadelphia: The Sadtler Research Laboratory,Inc., 1962: Infra-red spectre .;m #21421.

6. Wheeler CR. Nitrocellulose-Nitroguanidine ProjectsLaboratory Notebook #8S-12-022, pp. 24-25. Letterman ArmyInstitute of Research, Presiiio of San Francisco, CA.

7. Ibid., pp.33-5.

Coppes et al -- 33

Apipedix A (Cont.) CHEMICAL DATA

DESUION SHEll FOR EXPLOSIVES, CHEMICALS, ETC -tooON.S.5. 102.109 AR 335 - I

TM1 FRMm, DA TE

UsAml ree Sunflower Army Ammunition Plant October 9. 1984

"al = 5ca t Ca m , DeSoto. K nasas 66018 A , ALA

At~~ ~~ 9:100I-A it roguanid ite

Hercules Aerospace Division. Hercules Inc. DAAA-09-77-C-4016 CLIN 0295

SOU34KO1OAOOI r 1 ... 25 pounds7U =An PICTUIIObP9; SPI11A TQaO a1Od 0 ~' OSunfloer Army Amunition Plant .41-_N_ 45 d. 17 July 1964

5~t IMMN Z0 N. 19)$CRIP11 WOF MaITJOIAL

Analysis

Property Mi. Max.

Purity. 2 99.0 99.20Asb Content. Z 0.30 0.12pM Value 4.5 7.0 6.0Acidity (" H2SO4), 0.06 0.0

Toral Voletiles, 2 O.25 0.16

Sulfates (As N2S04 ), Z - 0.20 0.17Iipurities. R20 Insoluble. 2 0.20 0.02

FarticlA Size. microns 3.4 6.0 3.5

.color White.istesCy Crystalline, Free Flowing

a Coobined average@ of sampling taken in accordance with MIL-N-00494B. Pars. 4.4.3.2.

ITO PackagingS Level C Fiber drums per specification DOT 2IC602) This lot wae manufactured 5 October 1984 and is submitted as First Article In coapliance

with Paragraph 4.3 of NIL-N-00494.

3) Cuaridia. Nitrate supplied by SKi-A"erican oesch was used In manufacture of this lot.

i I • tlel iON0N ltIlr.I ,tAMPLINGCONA O i '} OT ' I ^0 . &~u ftffi'L COMPL-t$ -. 1. Al.. $PtC;F,¢I ION1

Hercules Aerospace Division sOYdII1M(*N1I AND it CIITIPiIO TIuI AND CORICT.I .ooo.. ., q7, ./ .

rculae Aerospace Division '.,Z

FtIt Alove uusCRIeGI LoftAR HN5 1 As CCEPTD \4*0 ?me COMMANDER

ANACOM 21-i. esse FOAN I AVG 11 - '; ) JI.

I I

Coppes et al -- 34

Appendix A (Cont.): CHEMICAL DATA

Inp

DESCRIPTION SHEET FOR EXPLOSIVES, CHEMICALS, ETC " "5 of,.t M 33 is 1206 2

C ee ISunflower Amy A zunition Plant .1 26. 1985OI &m All mSLtIm DeSoco, Kansas 66018fl e ChIemilL CU IlA1| lSO -mgQa IIUmg)gdn46StIut.16. ELL. UM

Hercules Aerospace Company . DAAA09- 7 6 -C- 016

I •g 5"Wood As. 6 S O8'T &O9WIGV £rgFDTg-SOW85FOIL-028 -"i 1 10,950 pounds

I LAE e &ulCTUl6D MF1Cj PICA VON ap 0 koa~'.'. No.Sunlower Army Ammunition Plant MIL-N-004945 dtd. 17 ialq 1984

.~ ~ ~ UIM O g .our. F MAIIAL

TEST REQUIREMENT-SIWT AVERAGEMAX'--9- 0 "o.30o-% ?, 0 0o-0. L 0~l.,% 0o2o% 6, o,

MIN-'" to 00% - - ,.5 - - 34

Ii

L O T f l . A T 0 U . P U R I T Y A S I ) # H C I 0 i l i ' ( -F 5 23I i l

OWS011-028 415-534 i

-028 537-548-028 6-20-85 8-4 557-566 * * 5 * * 0-028 568-572-028 574-576TOTAL DRUMS 1 219

*Tescing for requirecent at reduced frequency per HIL-STL-1235A.

Sampling and testing in accordance with MZL-N-00494B and MIL-STD-1235A. Mtn test result

reported is an average of shift samples on the date the lot was packed.

1) Palklng: Level C - Fiber drum per specification DOT21C60.2) Interfix number 011 identities lots manufactured vith Sunflower prouced gusnidinenitrate,3) The average bulk density' for Lot SOW85FOll-028 is 0.262 gm/cc so dearuimned by Method

201.3 at MIL-STU-650.

Saf. ... couteo of .__ A.p6le Wat55h. & COUPL.,S 'If 4

Hercules Aerospace CompanyTITINGQ CouCu:yea 6f ~gi~ Q~.1vAauar in

Hercules Aerospace Company A ; R j,. ,-Y .

[-1 464.4 OIICII1tO I l.OTlS A1 W156T ACCIPtlO

\Chief, QA Division

| -.

Coppes et al -- 35

Appendix B: ANIMAL DATA

Species: Rabbit

Strain: New Zealand White (albino)

Source: Elkhorn RabbitryWatsonville, CA

Sex: 78 Females and 18 Males

Age:

Males: Proven breeders, approximately 5 1/2 months oldat arrival at LAIR.

Females: Nulliparous, approximately 4 months at arrivalat LAIR, approximately 5 months at start ofbreeding.

Animal identification numbers:

Males: 86F241 - 86F258

Females: Phase I: 86F201 - 86F240Phase II: 86F283 - 86F322

Phase I Phase II

Weight range (kg) at start of breeding:

Males 3.8- 4.7 4.2 - 4.7Females 3.1 - 4.3 3.1 - 4.3

LI

Coppes et al -- 36

Appendix C: CEMICAL ANALYSIS

HOMOGENEITY*

A suspension of nitroguanidine (200 mg/mi, 300 ml) wasprepared in 1% carboxymethylcellulose. This suspension wassubsequently used to prepare two more dilute suspensions ofapproximately 60 mg/ml (20 ml) and 20 mg/ml (20 ml) in 20-mlvials. The suspensions were stirred well, and aliquots of 1 mlwere removed from the top, middle, and bottom layers of eachsuspension. The aliquots were transferred to either 500- or 1000-ml volumetric flasks and diluted to volume with watex. After onemore dilution (see table below) the optical absorbance at 264 nmwas determined.

The concentration of the original suspension was thencalculated using the dilution and absorbance data. A comparisonof the individual values to the mean value of the appropriategroup showed no deviation larger than 3%.

Target Ar,2a ist 2nd Absorbance Concen-Concentration Saffple Dilution Dilution at tration

mg/ml ml ml 264 nm mg/ml

top 1.305 23.420 middle 500 5 1.304 23.4

bottom 1.302 23.4

top 1.021 73.460 middle 1000 10 1.043 75.0

bottom 1.076 77.3

top 1.150 206.6200 middle 1000 25 1.163 209.0

bottom 1.135 203.9

*Wheeler CR. Nitrocellulose-Nitroguanidine Projects. LaboratoryNotebook #85-12-022, p. 27-29. Letterman Army Institute ofResearch. Presidio of San Francisco, CA.

Coppes et al -- 37

-- 0 9)coo 0% C)0CO-

40) c :3.~- 0 %C :) DC ( n

410)~ 70 C -C - -

(nC 0 c) w0.f

0.. -,q -4 >1 -4 CIO'OCIOOM N(N CO N

V)r'- 41O~ 0

0 ko C)

V) u* 41 a)~ ) (

o 41- 3c- aE - C14 CO( w C: -1

-4 4.0 r= I

C) 4-1 E-- a 4(v

0 EW3: C - -4 w:3 S: CN 0 Ad 0C m

0-0 w -4 I0 4)0 o vH -14 0O~liw--I a ) 0 r- 44 0

M - .q0 M- 4. cuC:)N0 0 a I w ' w4 044 ( ( (i a)- 0 .1. cl rfClOC a I CC .- ) 4

dg -, D ) L)G) 4- Q E(N0 4C Dr CD r. (a 4 '.)

44 =EaC 4-4 SW .. .. " a 0

:3 CVm 1 w0-4--4 0 -4 4J -

,C 0 40 Q) 0 0 41 >,b~~L >J0 0 Cue-(0 ~ 4

--H 4 ~ 0 W -J 0) 0- 4 -4 $4H0 4 - C -4 r_04

Q., (.4 N -4 (0C041 0 - ~0 0 0 C

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Coppes et al -- 38

Appendix D: SCHEDULE OF STUDY EVENTS

DATE EVENT

23 Jul 86 Date protocol approved.

25 Sep 86 Male and female rabbits for Phase Iarrived at LAIR.

14 - 28 Oct 86 Phase I breeding.

20 Oct - 15 Nov 86 Phase I females dosed.

12 - 26 Nov 86 Cesarean sections, Phase I females.

18 Dec 86 Female rabbits for Phase II arrivedat LAIR.

12 - 21 Jan 87 Phase II breeding.

18 Jan - 8 Feb 87 Phase II females dosed.

10 - 19 Feb 87 Cesarean sections, Phase II females.

Coppes et al -- 39

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Coppes et al -- 43

Appendix F

IND IVIDUAL MATERNALFOOD CONSUMPTION*

Control Animals

Days of Gestation

MaternalID 1-6 6-18 18-29

86F212 254t 237 24286F214 229 133 20486F218 248 164 14186F231 233 201 15286F235 204 239 16386F236 215 227 22586F237 243t 145 7886F297 209 172 15286F303 255 231 20686F304 213 170 13386F309 269 222 19386F316 242 177 10386F321 247 222 156

*Average daily consumption in g.

tReflects 4 days of data.

Coppes et al -- 44

Appendix F (Cont.)

INDIVIDUAL MATERNALFOOD CONSUMPTION*

100 mg/kg/dayNitroguanidine Animals

Days of Gestation


86F202 244 206 14286F203 267 202 25386F210 237 195 16086F225 234 169 13486F226 201 174 19086F230 234 210 18086F232 298t 246 14386F239 279f 230 19786F292 285 237 19686F295 214 191 17286F300 247 200 15286F308 176 117 6686F310 190 162 16786F311 265 237 19886F320 232 194 146

*Average daily consumption in g.TReflects 4 days of data.

Coppes et al -- 45

Appendix V (Cont.)



Days of Gestation


86F215 242 196 194

86F21 7 238 184 210

86,F220 236 211 163

86F223 220 188 134

86F22 4 237 219 189

86F22 7 231 208 166

86F234 261 262 219

86F23 8 226 171 180

86F284 271 238 180

86F285 206 178 177

86F298 214 173 157

86F301 273 190 187

86F313 190 132 13186F31 4 286 212 21B86F322 216 171 169

*Average daily consumPtion in g. 9

9

Coppes et al -- 46

Appendix F (Cont.)



Days of Gestation

MaternalI 1-6 6-18 18-29

86F209 245 71= §86F216 276 94= §86F221 < 221 18486F228 299 226 18986F229 257 105** §86F233 247 163 §86F240 242 197 18286F283 254 168 20686F288 285 118 21686F290 220 63 12186F293 214t 73tt §86F294 250 138 22886F296 267 169 14586F302 207 110 14686F312 298 131 22486F315 185 7** §86F317 160 65 14386F319 206 16§§ §

*Average daily consumption in g.tReflects 4 days of data.§Animal died.<<Missing data.-Reflects 7 day.s of data.**Reflects 5 days of data.ttReflects 10 days of data.§§Reflects 9 days of data.

Coppes et al -- 47

Appendix G: INDIVIDUAL MATERNAL CLINICAL SIGNS

Control Animals

Maternil StudyID Day(s) Date(s) Signs

86F212 4 23 Oct 86 Diarrhea6 25 Oct 86 Orange-rust colored urine7 26 Oct 86 Yellow-stained nose

10 29 Oct 86 Diarrhea10-20 29 Oct-8 Nov 86 Yellow-stained nose

15 3 Nov 86 Diarrhea

86F214 6 20 Oct 86 Diarrhea10 24 Oct 86 Orange-rust colored urine

13-14 27-28 Oct 86 Blood in mouth at dosing16-17 30-31 Oct 86 Small feces

86F218 7 26 Oct 86 Yellow-stained nose8 27 Oct 86 Diarrhea

86F231 0-29 20 Oct-29 Nov 87 Normal

86F235 5 26 Oct 86 Diarrhea

86F236 2 23 Oct 86 Diarrhea4-5 25-26 Oct 86 Diarrhea6 27 Oct 86 Blood in mouth at dosing8 29 Oct 86 Diarrhea

10-15 31 Oct-5 Nov 86 Diarrhea12-14 2-4 Nov 86 Brown material on legs

16 6 Nov 86 Yellow-stained nose17-19 7-9 Nov 86 Diarrhea

18 8 Nov 86 Yellow-stained nose23-24 13-14 Nov 86 Diarrhea

28 18 Nov 86 DiarrheaS

86F237 9 31 Oct 86 Hair loss on inside thighs12 3 Nov 86 Hair loss on inside thighs15 6 Nov 86 H~ir loss on inside thighs

16-26 7-17 Nov 86 h ._r loss on inside thighsand arm pits

27-29 18-20 Nov 86 Small amount of feces29 20 Nov 86 Hair loss on underside

S

Coppes et al -- 48

Appendix G (Cont.): INDIVIDUAL MATERNAL CLINICAL SIGNS

Control Animals

Maternal StudyID Day(s) Date(s) Signs

86F297 2-4 17-19 Jan 87 Diarrhea

7-8 22-23 Jan 87 Diarrhea

86F303 3 17 Jan 87 Diarrhea

86F304 3 17 Jan 87 Diarrhea9-10 23-24 Jan 87 Diarrhea

86F309 2-3 17-18 Jan 87 Diarrhea8 23 Jan 87 Diarrhea

86F316 3 23 Jan 87 Diarrhea8-11 28-31 Jan 87 Yellow-stained nose

13 2 Feb 87 Yellow-stained nose16 5 Feb 87 Small amount of feces

86F321 13 2 Feb 87 Diarrhea13-14 2-3 Feb 87 Yellow-stained nose

24 13 Feb 87 Yellow-stained nose28 17 Feb 87 Yellow-stained nose

Coppes et al -- 49

Appendix G (Cont.): INDIVItUAL MAT~kNAL CLINICAL SIGNS

100 mg/kg/day Nitruguanidine Animals


86F202 9-il 24-26 Oct 86 Orange-rust colored urine11 26 Oct 86 Yellow-stained nose25 9 Nov 86 Small amount of feces

86F203 7 23 Oct 86 Blood in mouth at dosing10 26 Oct 86 Orange-rust colored urine

10-11 26-27 Oct 86 Yellow-stained nose13-19 29 Oct-4 Nov 86 Stained perianal

14 30 Oct 86 Diarrhea

86F210 4 20 Oct 86 Diarrhea7 23 Oct 86 Diarrhea

Yellow-stained nose9 25 Oct 86 Orange-rust colored urine

10 26 Oct 86 Yellow-stained nose13-20 30 Oct-5 Nov 86 Yellow-stained nose

86F225 0-29 21 Oct-19 Nov 86 Normal


6-7 22-23 Oct 86 Stained perianal8 24 Oct 86 Orange-rust colored urine

10 26 Oct 86 Orange-rust colored urine

86F230 5 25 Oct 86 Orange-rust colored urine8-15 28 Oct-4 Nov 86 Hard round 1 cm diameter

mass under chin16 5 Nov 86 Mass broke open exposing

thi-k cream-colored pus;abs-ess drained

17 6 Nov 86 Abscess drained18-19 7-8 Nov 86 Abscess healing

86F232 3-4 25-26 Oct 86 Diarrhea10-11 1-2 Nov 86 Diarrhea

12 3 Nov 86 Yellow-stained nose28 19 Nov 86 Small amount of feces

Coppes et al -- 50


100 mg/kg/day Nitroguanidine Animals


86F239 10-11 1-2 Nov 86 Thick, foamy urine10-12 1-3 Nov 86 Diarrhea

15 6 Nov 86 Orange-rust colored urine

86F292 5 17 Jan 87 Diarrhea

86F295 0-29 13 Jan-11 Feb 87 Normal

86F300 3 17 Jan 87 Diarrhea7 21 Jan 87 Yellow-stained nose

7-8 21-22 Jan 87 Diarrhea9 23 Jan 87 Clear discharge from eye

12 26 Jan 87 Clear discharge from eye14-19 28 Jan-2 Feb 87 Yellow-stained nose

86F308 14 29 Jan 87 Small amount of fecesDeprived of water

24-25 8-9 Feb 87 Small feces24-26 8-10 Feb 87 Small amount of feces25-28 9-12 Feb 87 Inactive27-28 11-12 Feb 87 Nasal discharge

86F310 3 18 Jan 87 Yellow-stained nose13 28 Jan 87 Deprived of water

86F311 6 25 Jan 87 Yellow-stained nose8-25 27 Jan-13 Feb 87 Yellow-stained nose

86F320 0-29 29 Jan-18 Feb 87 Normal

i

Coppes et al -- 51




86F215 9 23 Oct 86 Blood in mouth at dosing

11-12 25-26 Oct 86 Orange-rust colored urine

86F217 12 26 Oct 86 Yellow-stained nose

86F220 9 24 Oct 86 Orange-rust colored urine10 25 Oct 86 Diarrhea11 26 Oct 86 Yellow-stained nose

11-12 26-27 Oct 86 Orange-rust colored urine


10-11 26-27 Oct 86 Orange-rust colored urine24 9 Nov 86 Pulling hair for nesting

86F224 6 29 Oct 86 Diarrhea6-12 29 Oct-4 Nov 86 Hair loss inside theighs

8 31 Oct 86 Thick/foamy urine10 2 Nov 86 Thick/foamy urine19 11 Nov 86 Thick/foamy urine27 19 Nov 86 Small amount of feces

)eprived of water

86F227 4 23 Oct 86 Yellow-stained top of head6-17 25 Oct-5 Nov 86 Yellow-stained top of head

17 5 Nov 86 Diarrhea18 6 Nov 86 Yellow-stained nose

86F234 6-14 1-9 Nov 86 Short hair under chin7-14 2-9 Nov 86 Hair loss from thigh and

between front legs

86F238 8-11 31 Oct-3 Nov 86 Diarrhea19 11 Nov 86 Thick/foamy urine27 19 Nov 86 Small amount of feces

Deprived of water

Coppes et al -- 52




86F284 7 19 Jan 87 Thick/foamy urine9-11 21-23 Jan 87 Orange-rust colored urine

10 22 Jan 87 Blood in mouth at dosingRed-stained nose and paws

11 23 Jan 87 Diarrhea13-16 25-28 Jan 87 Orange-rust colored urine

21 2 Feb 87 Orange-rust colored urine

86F285 9 23 Jan 87 Orange-rust colored urineThick/foamy urine

12 26 Jan 87 Orange-rust colored urine

86F298 4 17 Jan 87 Diarrhea7 20 Jan 87 Upper front teeth broken

10-11 23-24 Jan 87 Orange-rust colored urine13 26 Jan 87 Thick/foamy urine

13-16 26-29 Jan 87 Orange-rust colored urine16 29 Jan 87 Diarrhea

18-19 31 Jan-l Feb 87 Orange-rust colored urine18-20 31 Jan-2 Feb 87 Hair in feces

86F301 2-3 17-18 Jan 87 Diarrhea

86F313 6-9 25-28 Jan 87 Brown material on forepaw12 31 Jan 87 Brown material on forepaw13 1 Feb 87 Diarrhea14 2 Feb 87 Brown material on forepaw15 3 Feb 87 Brown material on hindlegs18 6 Feb 87 Brown material on hindlegs25 13 Feb 87 Small amount of feces

86F314 4 23 Jan 87 Diarrhea8 27 Jan 87 Hair loss from inside leg

1i 30 Jan 87 Hair loss from inside leg13-14 1-2 Feb 87 Hair loss from inside leg

15 3 Feb 87 Orange stained fore leg

86F322 11 31 Jan 87 Orange-rust colored urn e14 3 Feb 87 Yellow-stained nose17 6 Feb 87 Blood in mouth at dosing

!S

Coppes et al -- 53




86F209 1 16 Oct 86 Diarrhea7 22 Oct 86 Granular/foamy urine

9-10 24-25 Oct 86 Granular/foamy urine10 25 Oct 86 Red-stained hindquarters

10-11 25-26 Oct 86 Red urine11 26 Oct 86 Yellow-srai .-d nose

Red-sta:ned. nose12 27 Oct 86 Red nas.-,,l discharge

Hunche.' w)stureT-emo sMo,.-.. T LfflyIncrea3-, startle reflexBloody hairball found

under cageEuthanized in moribund

condition

*1

Coppes et al -- 54



Maternal StudyID Day(s) Date (s) Signs

86F216 4 23 Oct 86 Small amount of fecesSmall black feces

7 26 Oct 86 Small amount of fecesSmall black fecesYellow-stained noseGranular urine

8 27 Oct 86 Nasal discharge10 29 Oct 86 Convulsions

Injured bloody toenails11 30 Oct 86 Hair loss from groin

Thick/foamy urineStrong rabbit/urine odor

12 31 Oct 86 Orange-rust colored urineTwitchingRed-stained yellowish

granular material undercage

ConvulsionsLoss of consciousnessMucus in noseRapid/shallow respiration

12-13 31 Oct-i Nov 86 Red-stained pawsRed-stained perianalHypertonia

13 1 Nov 86 DehydratedInactiveYellow-stained noseStrong rabbit/urine odorCried out after dosing

procedure14 2 Nov 86 Death

86F221 7 29 Oct 86 Yellow-stained nose8-19 30 Oct-10 Nov 86 Orange-rust colored urine

Thick/foamy urine10-i 1-2 Nov 86 Diarrhea

1i 2 Nov 86 Brown material on hindpaws21-23 12-14 Nov 86 Orange-rust colored urine

Coppes et al -- 55p Appendix G (Cont.): INDIVIDUAL MATERNAL CLINICAL SIGNS



86F228 0-3 20-23 Oct 86 Diarrhea5 25 Oct 86 Orange-rust colored urine6 26 Oct 86 Yellow-stained nose7 27 Oct 86 Diarrhea

7-14 27 Oct-3 Nov 86 Granular/foamy urine8-20 28 Oct-9 Nov 86 Orange-rust colored urine

12 1 Nov 86 Diarrhea15-19 4-8 Nov 86 Thick/foamy urine

16 5 Nov 86 Diarrhea17 6 Nov 86 Orange-stained forepaw18 7 Nov 86 Diarrhea

86F229 5 25 Oct 86 Orange-rust colored urine6-7 26-27 Oct 86 Yellow-stained nose7 27 Oct 86 Red urine

Red material in urine9 29 Oct 86 Convulsions

Prostrate10 30 Oct 86 Moved stiffly

Orange-rust colored urineInjured bloody toenails

11 31 Oct 86 Death

86F233 5 26 Oct 86 Diarrhea9 30 Oct 86 Thick/foamy urine

9-16 30 Oct-6 Nov 86 Orange-rust colored urine11-13 1-3 Nov 86 Diarrhea11-16 1-6 Nov 86 Thick/foamy urine

14 4 Nov 86 Increase startle reflex18 8 Nov 86 Small amount of feces19 9 Nov 86 Increased salivation

Mucus on noseConvulsionsProstrateTremorsNo urine or feces under

cageRed material under cageDeath

Coppes et al -- 56




86F240 7-10 30 Oct-2 Nov 86 Thick/foamy urine8-10 31 Oct-2 Nov 86 Orange-rust colored urine

10-13 2-5 Nov 86 Diarrhea12 4 Nov 86 Orange-rust colored urine

Thick/foamy urine14-19 6-11 Nov 86 Thick/foamy urine14-20 6-12 Nov 86 Orange-rust colored urine

15 7 Nov 86 Diarrhea17-18 9-10 Nov 86 Diarrhea

27 19 Nov 86 DiarrheaDeprived of waterSmall amount of feces

86F283 3-4 17-18 Jan 87 Diarrhea7-19 21 Jan-2 Feb 87 Orange-rust colored urine

Thick/foamy urine

86F288 7-18 20-31 Jan 87 Thick/foamy urine9-14 22-27 Jan 87 Orange-rust colored urine

16 29 Jan 87 Small amount of feces18 31 Jan 87 Orange-rust colored urine19 1 Feb 87 Red material under cage

19-20 1-2 Feb 87 Small amount of feces20-24 2-6 Feb 87 Orange-rust colored urine

86F290 2-3 14-15 Jan 87 Diarrhea7-8 19-20 Jan 87 Mucus on feces8 20 Jan 87 Increased startle reflex

8-17 20-29 Jan 87 Thick/foamy urine9-14 21-26 Jan 87 Orange-rust colored urine14-18 26-30 Jan 87 Diarrhea15-18 27-30 Jan 87 Brown material on abdomen

and tail19 31 Jan 87 No feces/urine under cage20 1 Feb 87 Mucus on feces

Clump of mucus under cage21 2 Feb 87 No feces/urine under cage23 4 Feb 87 Red urine

23-25 4-6 Feb 87 Brown material on abdomenand tail

Coppes et al -- 57




86F293 6-7 18-19 Jan 87 Thick/foamy urine8-13 20-25 Jan 87 Orange-rust colored urine9-11 21-23 Jan 87 Thick/foamy urine

13 25 Jan 87 Thick/foamy urine14 26 Jan 87 Clear yellow urine

14-16 26-28 Jan 87 Small amount of feces15 27 Jan 87 Orange-rust colored urine

Thick/foamy urineHunched postureConvulsionsInjured bloody toenailsCyanosis

15-16 27-28 Jan 87 AtaxiaHypertonia

16 28 Jan 87 Euthanized in moribundcondition

86F294 2 15 Jan 87 Diarrhea4 17 Jan 87 Diarrhea

8-9 21-22 Jan 87 Thick/foamy urine11-19 24 Jan-i Feb 87 Thick/foamy urine11-22 24 Jan-4 Feb 87 Orange-rust colored urine

14 27 Jan 87 Diarrhea

86F296 7-19 26 Jan-7 Feb 87 Orange-rust colored urineThick/foamy urine

8 27 Jan 87 Hunched posture9 28 Jan 87 Hypertonia

22 10 Feb 87 Orange-rust colored urine

86F302 7-16 20-29 Jan 87 Thick/foamy urine8 21 Jan 87 Diarrhea

8-14 21-27 Jan 87 Orange-rust colored urine10-11 23-24 Jan 87 Brown material on forepaws

11 24 Jan 87 Diarrhea13-15 26-28 Jan 87 Brown material on forepaws

14 27 Jan 87 Diarrhea16 29 Jan 87 Diarrhea

16-18 29-31 Jan 87 Orange-rust colored urine20-21 2-3 Feb 87 Small amount of feces

23 5 Feb 87 Orange-rust colored urine28 10 Feb 87 Orange-rust colored urine

S

S-

Coppes et al -- 58




8GF312 7-9 26-28 Jan 87 Thick/foamy urine9 28 Jan 87 Orange-rust colored urine

13 1 Feb 87 No urine under cage14 2 Feb 87 Orange-rust colored urine

14-15 2-3 Feb 87 Thick/foamy urine16 4 Feb 87 Red material under cage17 5 Feb 87 Small amount of urine

Small amount of feces19 7 Feb 87 Yellow-stained nose

Yellow-stained forepawsNo feces under cageDeprived of water

19-20 "-8 Feb 87 Hypertonia20 8 Feb 87 Small amount of feces22 10 Feb 87 Small amount of feces

22-23 10-11 Feb 87 Orange-rust colored urine

86F315 7 26 Jan 87 Small amount of feces8 27 Jan 87 Thick/foamy urine

Red material in urine8-9 27-28 Jan 87 Orange-rust colored urine

9 28 Jan 87 Cool to touch10 29 Jan 87 Inactive

Hunched postureSmall amount of fecesHypertonia

11 30 Jan 87 Death

86F317 5 26 Jan 87 Yellow-stained nose7-19 28 Jan-9 Feb 87 Thick/foamy urine7-29 28 Jan-19 Feb 87 Nasal discharge

8 29 Jan 87 Orange-rust colored urine9 30 Jan 87 Small amount of feces

10-17 31 Jan-7 Feb 87 Orange-rust colored urine11-12 1-2 Feb 87 Small amount of feces18-20 8-10 Feb 87 Small amount of feces27-28 17--18 Feb 87 Small amount of feces

Coppes et al -- 59



Maternal StudyID Dayis) Date(s) Signs

86F319 7-12 27 Jan-i Feb 87 Thick/foamy urine9-10 29-30 Jan 87 No feces under cage

11 31 Jan 87 Hypertonia12-13 1-2 Feb 8"7 Small amount of feces12-14 1-3 Feb 87 Red stained nose and mouth13-14 2-3 Feb 87 Hypertonia

15 4 Feb 87 Death

S

0|

t

Coppes et al -- 60

Appendix HMATERNAL GROSS NECROPSY FINDINGS AT CESAREAN SECTION

Control Animals

Maternal ID Finding

86F212 Dark red fallopian tubesFragile uterus

86F218 Small, pale liver

86F231 Cyst on rignt fallopian tubeDark red right fallopian tuibe

86F235 Cysts on fallopian tubesDark red fallopian tubes

86F297 Cysts on left fallopian tube

86F303 Dark red mass on pancreas

81 '304 Cyst on right fallopian tubeTwo dark red masses on pancreasSlightly dilated renal pelvis of rightkidney

86F309 Cyst on left fallopian tube

86F316 Cyst on right fallopian tube

86F321 Cyst on left fallopiJan tube

Hard brown wart-like mass on liver

0

Coppes et al -- 61

Appendix H(Cont.)MATERNAL GROSS NECROPSY FINDINGS AT CESAREAN SECTION


Maternal ID Finding

86F202 Cysts on right fallopian tubeDark red fallopian tubesTwo white spots on left kidney

86F225 Cyst on left fallopian tubeCavity adjacent to vessel in left kidneymedulla

Blotchy mottled liver

86F226 Dark red left fallopian tube

86F230 Dark spot on right ovary 0

86F232 Cysts on fallopian tubesDark spot on left ovary

86F239 Dark red fallopian tubes

86F292 Cysts on fallopian tubes


86F310 White spot in cortex of left kidney

86F311 Red mass in pancreasWhite/gray mass on lower lobe of left lung

86F320 Cysts on fallopian tubesDark red mass on pancreas

I

Coppes et al .-- 62



Maternal ID Finding

86F215 Cysts on fallopian tubesRed inflamed fallopian tubes

86F217 Cysts on fallopian tubesDark red fallopian tubesDark spot on left ovary


86F224 Cysts on fallopian tubesDark red fallopian tubes




86F284 Cysts on right fallopian tubeDark spot on left ovary

86F285 Slightly dilated renal pelves of kidneys


86F314 Cyst on right fallopian tubeThree dark spots on pancreasBlotchy right kidney


Coppes et al -- 63



Maternal ID Finding

86F221 Cysts in adipose tissue surrounding ovaries

86F228 Dark red fallopian tubesBlotchy liver

86F240 Cysts on right fallopian tube

86F288 Cyst on left fallopian tube

86F290 Cyst on left fallopian tubePale kidneys


86F302 Cysts on fallopian tubesCavity adjacent to vessel in left kidney

medullaSlightly dilated renal pelvis in right

kidney

86F312 Dark red spot on pancreasBlotchy right kidney

86F317 60% of tissue in upper left lobe of lung Anecrotic, adhered to chest wall; 60% oftissue in middle lobe of right lungnecrotic, cyst on lobe

A

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I

Coppes et al.- 64

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Appendix 0

INCIDENCE Or FETALMALFORMATIONS AND VARIATIONS

Control Animals

Maternal Number Malformed VariantsID Examined No. % No. %

86F212 8 0 0 3 3886F214 8 0 0 5 6386F218 10 0 0 1 1086F231 12 0 0 5 4286F235 10 0 0 3 3086F236 8 0 0 5 6386F237 8 0 0 3 3886F297 9 0 0 7 7886F303 8 0 0 2 2586F304 10 0 0 1 1086F309 10 0 0 9 9086F316 11 0 0 0 086F321 9 0 0 2 22

'3

I

COPPes et a!. -53

Appendix O(Cont.)

INCIDENCE OF FETALMALFORMATIONS AND VARIATIONS

100 mq/kg/day Nitroguanidine Animals


8GE'202 7 0 0 0 086F203 9 0 0 5 5686F210 9 0 0 0 086F225 9 0 0 9 10086F226 6 0 0 2 3386F230 6 0 0 1 1786F232 11 0 0 11 10086F239 10 0 0 4 4086F292 11 0 0 3 2786F295 8 0 0 1 1386F300 10 0 0 2 2086F308 10 1 10 7 7086F310 10 0 0 7 7086F311 10 0 0 7 71086F320 9 0 0 6 67

Coppes et al -- 154

Appendix O(Cont.)

INCIDZNCE Or FZTLMALFORMATIONS AND VARIATIONS



86F215 7 0 0 0 086F217 11 0 0 6 5586F220 9 0 0 4 4486F223 10 0 0 10 10086F224 12 0 0 11 9286F227 9 0 0 1 1186F234 9 0 0 3 3386F238 8 0 0 2 2586F284 8 0 0 3 3886F285 7 0 0 2 2986F298 10 0 0 2 2086F301 9 0 0 5 5686F313 8 0 0 5 6386F314 3 0 0 3 10086F322 11 0 0 5 45

0

C.-ppes ec l-

Appendix O(Cont.)

INCIDENCE OF FETALMALrORATIONS AND VARIATIOPS



86F221 9 0 0 5 5686F228 9 0 0 9 10086F240 ).0 0 0 3 3086F283 11 0 0 4 3686F288 9 0 0 7 7866F294 6 0 0 6 10086F296 10 0 0 4 4086F302 4 0 0 1 2586F312 11 0 0 5 4586F317 8 1 13 8 100

Ccppres et al.--15O

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