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Institute Report No. 267

Acute Oral Toxicity ofGuanidine Nitrate In Rats

DT1C 'Lawrence Mullen, BS, SP5"-

Earl W. Morgan, DVM, MAJ, VC ELECTE,Carolyn M. Lewis, MS JUN 2 8 1988

Conrad R. Wheeler, PhD JN2 19and S

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

MAMMALIAN TOXICOLOGY BRANCHDIVISION OF TOXICOLOGY

0

May 1988 Toxicology Series: 81

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129.

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Acute oral toxicity of guanidine nitrate in rats (Toxicology Series 81)--Mullen et al.

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Z' 02-20A 8_1)AB IDA 3391311. TITLE (include Security Classification)

\cut.,, Or.al Toxicity of Guanidine Nitrate in 1'ats

12. PERSONAL AUTHOR(S)MIullen L, Mforgan 13W, Le,!i, (CM, Wheeler CR, and Korte M)W Jr

13a. TYPE OF REPORT 13b. TIME COVERED 14 DATE OF RE vQH T (Year. Month, Day) 15. PAGE COUNT

Institute FROM _____TO N___ ay 1988 64116. SUPPLEMENTARY NOTATION

17 COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP cute Oral Toxicity, Guanidine Nitrate, Nitroguanidine,

Munit ion, Rat

19. ABSTRACT (Continue on reverse if necessary and identify by block number)The acte oral1 toxic itv Of igIuI.nidine nitrate was detenained inl Hiale and female albino

S, rague-Dawlex- rats administered a single dose by oral gavage. ['he median lethal doses (NLD)* ere 989.6+08.7 mig/kg in male and '729.8+34.3 mig/kg in female rats. The primary category of

clinical signs wa.s behavioral (e.g., inactive, irritable, disoriented, hyperactive, ataxic)w: ich was observed in 63 of 99 animnals dosed with guanidine nitrate. Other categories offrequenitlIy observed clinical signs were gastrointestinal (e.g., mlaterial in mouth, perianal:,tainin, inceased salivation, diarrhea) which was observed in 37 of 99 animals, and respi-

* ratury (egreddish nasal discharge, increased rate and/or decreased depth of respiration)* Iwh1ich was observed in 26 of 99 animals. These findings suggest that guanidine nitrate is

asl~ghtly- toxic comnpound with a primary effect on thie central niervous/neuromuscular system.

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ABSTRACT

The acute oral toxicity of guanidine nitrate wasdetermined in male and female albino Sprague-Dawley ratsadministered a single dose by oral gavage. The median lethaldoses (MLD) were 989.6 ± 68.7 mg/kg in male and 729.8 ± 34.3mg/kg in female rats. The primary cateqory of clinical signswas behavioral (e.g., inactive, irritable, disoriented,hyperactive, ataxic) which was observed in 63 of 99 animalsdosed with guanidine nitrate. Other categories of frequentlyobserved clinical signs were gastrointestinal (e.g., materialin mouth, perianal staining, increased salivation, diarrhea)which was observed in 37 of 99 animals, and respiratory(e.g., reddish nasal discharge, increased rate and/ordecreased depth of respiraticn) which was observed in 26 of99 animais. These finaings suggest that guanidine nitrate isa slightly toxic compound with a primary effect on thecentral nervous/neuromuscular system.

*Key Words: Acute Oral Toxicity, Guanidine Nitrate,Nitroguanidine, Munition, Rat

oassion For JNTIS GRA&tI M"

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PREFACE

TYPE REPORT: Acute Oral Toxicity GLP Study Report

TESTING FACILITY:

US Army Medical Research and Development CommandLetterman Army Institute of ResearchDivision of Research SupportPresidio of San Francisco, CA 94129-6800

SPONSOR:

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

WORK UNIT/APC: WU 180, Environmental Healtn Effects of ArmyMaierials/APC: TLB0

GLP STUDY NUMBER: 84001

STUDY DIRECTOR: MAJ Don W. Korte, Jr, PhD, MSCChief, Division of Toxicology

PRINCIPAL INVESTIGATOR: Lawrence Mullen, BS, SP5

CO-PRINCIPAL INVESTIGATOR: Carolyn M. Lewis, MS

PATHOLOGIST: LTC Lance 0. Lollini, DVM, VC, Diplomate,American College of Veterinary Pathologists

REPORT AND DATA MANAGEMENT: A copy of the final report, studyprotocols, raw data, SOPs and analiquot of the test compound willbe retained in the LAIR Archives.

TEST SUBSTANCE: Guanidine Nitrate

INCLUSIVE STUDY DATES: 29 February - 11 April 1984

OBJECTIVE: To determine the acute oral toxicity of guanidinenitrate in Sprague-Dawley rats.

S:

ACKNOWLEDGMENTS

SP5 Thomas P. Y-ellner, BA, and SP4 Steven K. Sano, BS,proviW.I-d rez-earc - :--sis---ance; Richard D. Spieler, SusanHernandez, and Mich-ael Sands provided animal care andfacility management-; and Callie B. Crosby, MXA, and BrendaGoce Drovided secre~rarial assistance.

0i

SIGNATURES -OF PRINCIPAL SCIENTISTS AND MANAGERS INVOLVED -IN THE STUDY-

We, the undersigned, declare that GLP Study 84001 was performedunder our supervision, according to the procedures described herein,and that this repoli is an accurate record of the results obtained.

-L j -1-.

DON W. KORTY JR., hD / DAYE EARL W. MORAN, DV DATEMAJ, MS CPT, VCStudy Director Co-Author

-'b 2 5 ", j, . ...ENCE MULLEN, B / DATV Ij LANCE 0. L6LLINI, DMV / DATE

SPUSA LTC, VCPrincipal Investigator Pathologist

CAROLYN 1. LEWIS, MS I DATE CONRAD R. WHEELER, PhD /DATEDAC DACCo-Principal Investigator Analytical Chemist

"'4

6.

I

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 9412946800

SGRD-ULZ-QA 22 April 19%;

MEMORANDUM FOR RECORD

SUBJECT: Report of GLP Compliance for GLP Study 84001

1. I hereby certify that in relation to LAIR C-L? Study

84001, the following inspections were made:

14 Mar:n 1984 - Dosing and Observations

2. The report and raw data for this study were audited on

21 April 1987.

WALTER G. BELLSFC, USAQuality Assurance Auditor

Vi

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TABLE of CONTENTS

Ab stract .................................................... i

Pre face ................................................... iiiAcknowledgments ............................................ ivSignatures of Principal Scientists .......................... vReport of Quality Assurance Unit ........................... viTable of Contents ......................................... vii

BODY OF REPORT

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

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

MATERIALS ................................................... 1

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

METHODS ..................................................... 2

Acclimation and Group Assignment ....................... 2Compound Preparation ................................... 3Dose Levels ............................................ 3Test Procedure ......................................... 3Observations ........................................... 4Necropsy ............................................... 4Statistical Analysis ................................... 4Duration of Study ...................................... 4utviations irom FrQtocci ............................... 4Storage of Raw Data and Repert ......................... 5

RESULTS ..................................................... 5

Mortality .............................................. 5Lethal Dose Calculatizns 5............................5

0 Clinical Observations .................................. 6Pathology Report ...................................... 12

DISCUSSION ................................................. 12

CONCLUSIONS ................................................ 12

REFERENCES ................................................. 13

vii

6

TABLE OF CONTENTS (cont.)

APPENDICES ................................................. 11

Appendix A. , emcal Data ............................Appendix B. -nimal Data ..Appendix C. Historical Listing of Study Events ....... 22Appendix D. Cumulative Mortality Data ................ 24

.* Appendix E. Individual Animal Histories ..............Appendix F. Tndividual Body Weights ..................Appendix G. P±thology Report.......................... )6

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Acute Oral Toxicity of Guanidine Nitrate in Rats-Mullen et al

INTRODUCTION

Cuanidine nitrate is an intermediate prcd.,uut in the-~synthesis of nitrogu anidine. Nlitroguanici rl.es a primary

ccrnonent of US Army triple-;base propellants cind is now beinoProduced in a Governmenit-owned contractorr-ct 1erated ammunitionp1 ant . The US Army Biomedical Research ,.nd 1), velopment!Lacoratory (USABRDL) , as part of its missioni *,oc evaluate theenvironmental and health hazards of military-uniquepropelants generated by US Army mun.,it'ions,-manufacturinigfacities, reviewed the nitroguanidine data base andidentl'fed significant gaps in the toxicity data (1). The

0 Divisicn of Toxicology, LAIR, was tasked by TUSABRDL todevelop a genetic and mammalian toxicity profile fornitroguanidine, related intermediates/by-products of itsmanufacture, and its environmental degradation products.

Oblective of the Study

% The objective of this study was to determine the acute oraltoxicity of guanidine nitrate in Sprague-Dawley rats.:'-

MATERIALS

Chemical name: Guanidine Nitrate

Chemical Abstracts Service Registry Number: 506-93-4

Structural formula:

H2N\ +C NH, N0 3

Moleculaz formula: CH6N3-N03H Additional chemical information appears in Appendix A.

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Compound Preparation

Depending upon the dose level, various amounts ofguanidine nitrate were weighed in a Mettler AK 160 ElectronicBalance and were added to 50 ml of the vehicle for males and25 ml of the vehicle for females to yield the desired dosingconcentration. Complete analyses of the dosingsolutions/suspensions for homogeneity and ve: ification ofconcentrations appear in Appendix A.

Doqep Levels

The Approximate Lethal Dose (ALD) was 750 to 1000 mg/kgfor both male and female rats. Based on these data, testdosages were selected (Table 1). The 610-mg/kg dose levelwas added to the female dose groups to define the lowerlimits of the dose-response curve. A data point below 50%mortality is necessary to obtain a proper statisticalderivation of the MLD value.

TABLE 1

Guanidine Nitrate Dosages (mg/kg)

Male- Females

683* 610826 7181000 8471210 10001470 1180

1390

* By chemical analysis of this solution it was4 determined that these animals actually received

311 mg/kg.

Test Procedure

4 This study was conducted in accordance with EPAguidelines (2) and LAIR SOP-OP-STX-36 (3). All doses werecalculated by using a program developed for the Hewlett-Packard 9815A programmable calculator. The volumesadministered ranged from 1.9 to 2.6 ml for males and 1.2 to1.8 ml for females depending on the animal's weight and dose

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group. Each rat -n tie vehicle control group received 2.0ml. All dcsinu iraterial was a(dr.inistered with Perfektum

-

(Popper & Sons, Inc., New Hyde Park, NY) stainless steeLanimal feeding tubes. Before each dosing, the suspension wasvortexed to ensure adequate distribution. The volume of eachdose was drawn from the middle of each test chemical vialwith 3.0-mi plastic, disposable, sterile syringes. Thedosing procedures were conducted without animal sedation oranesthesia.

Cbservatinns

Debservation-' for mortality and signs of acute toxicityweie performed caiv according to the following procedure:(a) animals were cbserved undisturbed in their cages, (b)

* animals were removed from their cages and given a physicalexamination, and (c) animals were observed after beingreturned to their cages. On the day of dosing, the animalswere checked intermittently throughout the day. At least one

0 observation was recorded during the first 4 hours after

A second "walk through" observation was performed daily withonly significant observations recorded. Body weights wererecorded weekly during the course of the study.

Necropsy

Animals t.hnt died during the observation period weresubmitted for a complete gross necropsy. Those whichsurvived the 14-day study period were submitted for necropsyimmediately after ter- tcn by barbiturate overdose.

~~~~Statist f : , - ...

Statistic.i analyses were performed on the studyresults. Select-ed lethal dose values were derived by probitanalysis accordina tc Tie maximum likelihood method, as

* described by Wney (4). The program, PROBIT, developed forthe Data Gener7 Computer, Model MV8000, was used todetermine the crobit curve and lethal dose values.

., Duration of _ t'iy

0 Appendix C' .s a complete listing of historical events.

Deviations from Protocol

Due t, the length of time required to complete dosing ofthe animals, the. multiple observations scheduled for thefirst 4 hours after dosing could not be accomplished.

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Mullen et al-5

However, at least one observation was recorded for eachanimal during the first 4 hours after dosing. Pilot studyresults did not provide an adequate indication of thetoxicity observed in the female animals. Consequently, anadditional group was added to the female study so that a doselevel with less than 50% mortality could be achieved. Onanalysis, the 683 mq/kg dosing soluticn was a 311-mg/kqsolution. Since this was the low dose group and nomortalities had occurred, this group had no impact on thecalculated MLD values. Steam outages occurred from 0700 to1800 hours on 3 March 1984 and from 2200 hours on 22 March1984 to 1400 hours on 23 March 1984. These days wereselected by the building engineers for routine maintenance.During these outages, the relative humidity went up to 64% inroom RS1418 and 74% in RS1408, and the temperature fell to18.9'C in RS1409. These changes did not appear to have anyeffect on the outcome of the study.

Storace of Raw Data and Report

A copy of the final report, study protocols, raw data,retired SOPs, and an aliquot of the test compound will beretained in the LAIR Archives.

RESULTS

Mortait

Forty-six (74%) of the deaths occurred between 1 and 8hours after dosing. An additional 13 (21%) deaths occurredbetween 8 and 24 hours after dosing. The remaining threeanimals were found dead on the morning of the second day

(between 36 and 47 hours after dosing). Table 2 lists thecompound-related deaths by group and the percent mortality.Appendix D is a tabular presentation of cumulative mortality.

Lethal Dose Calculations

- Lethal dose values were calculated by probit analysis,and the equation for the probit regression line was:Y = -19.6 + 8.2 log X for males and Y = -32.5 + 13.1 log Xfor females, where X is the dose and Y the correspondingprobit value. Misdosed animals were excluded fromstatistical analysis and eliminated from the study. Figures

* 1 and 2 graphically present the actual data points and theregression line. Lethal doses calculated from the equationfor the probit regression line are presented in Table 3.

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M,.len et al-6

TABIE 2

Guanidine Nitrate-Related Deaths by Group

GROUP Dose Level Deaths/ Percent(mg/kg) Number in Group Mortality

MALES

1 683- 0/10 02 826 3/10 303 1000 3/8> 384 1210 7/8> 885 1470 9/10 906 Vehicle 0/5 0

'EMtALES

6A 610 1/7> 141A 718 5/9> 562A 847 6/9> 673A 1000 10/10 1004A 2180 10/10 1005A -'390 8/8> 1007 Vehicle 0/5 0

Due to an error in th : preparation of this solution theseanimals actua1 ' c, ved 311 mg/kg.

> Reduced numbe.-,; in groups were due to one or more misdosedanimals which were x:;:luded from statistical analysis andremoved from the study.

Clinical ObserviI-an

Guanidine nitrate produced clinical signs at each doselevel. The most frequently observed signs were behavioral(63 of 99 animals dosed with test compound), gastrointestinal(GI) tract symptc'rrv,: (37 of 99), and respiratory (26 of 99)siqns. Most cliw..i signs were recordeai within the first 48hours, alt-hoiiqh one behavioral sign, irritability, wasobserved in a lox animals intermittently throughout thestudy. However, irritability tended to decrease as doselevels increasey1, and it was also present in 7 of the 10vehicle control innimals.

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TABLE 3

' Calculated Lethal Doses (LD) of G ianidire Nitrate

A Effect Calculated Dose* 95t Conifidence LimitsLevel (mg/kg) (mg/kg)

MALES

LD10 690.8 ± 104.5 (324.1, 838.7)LD50 989.6 + 68.7 (793.0, 1137.8)LD90 1417.8± 166.0 (1210.8, 2473.6)

FEMALES

LD10 582.4 ± 50.9 (414.5, 657.1)LDS0 729.8 ± 34.3 (640.7, 799.4)LD90 914.5 ± 60.3 (829.3, 1161.4)

Calculated dose ± standard error

Behavioral signs were present in all dose groups. Adose-response relationship was observed for this group ofsigns. The dose-response relationship is more apparent ifone deletes those animals that died before clinicalobservation could be made. For the highest two female dosegroups, all animals with recorded symptoms displayed one ormore behavioral signs. Behavioral signs included ataxia,inactivity, changes in preening behavior, disorientation,irritableness, aggressiveness, hyperactivity, jumping,prostrate condition, tremors, and twitching.

GI symptoms included increased salivation, reddishmaterial present in mouth, diarrhea, and perianal staining.These symptoms were more prevalent at the lower dose levels.The apparent lower frequencies of all these signs in thehigher dose groups are attributed to the fact that theseanimals rapidly progressed to the more severe signs and/ordied before any of the signs could be observed. Table 4contains a summary of the clinic.,i ni.qris observed. Append ix. contains the individiii[ tniin.il iIi-Lo ; . We ight gains ofsurvivors were not significantly ,itecLed by dosing. Table 5presents the mean body weights by groups. Appendix Fcontains weight tables for individual rats.

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TABLE 4

Incidence of Clinical Signs in Rats AdministeredGuanidine Nitrate

MALES

Clinical Group 1 2 3 4 5 6(Dose) Control 683 826 1000 1210 1470

(n 5 80 10 0_ _

Behavioral* 3 8 10 4 5 6

Gastrointestinal> 0 2 5 2 3 5Respiratoryt 2 1 4 0 1 4Ocular< 0 0 2 0 0 2Rough coat 0 0 1 0 0 0Hunched posture 0 0 0 0 2Normal throughout 1 2 0 0 0 3

FEMALES

Clinical Group I 6A 1A 2A 3A 4A 5ASians (Dose) Ccntrol 610 718 847 1000 1180 1390

(n=) 5 7 i 9 9 _J0 1_ 8

Behavioral* 5 5 9 6 9 8 3

Gastrointestinal> 1 5 6 2 2 5 1RespiratoryT 4 2 4 5 0 1Ocular< 0 1 0 1 0 0Rough coat 0 1 5 3 1 2 1Normal throughout 0 1 0 0 0 0 0

* Includes ataxia, disorientation, hyperactivity, jumping,irritableness, inactivity, changes in preening, prostratecondition, aggressiveness, somnolence, tremors, andtwitching.> Includes increased salivation, material in mouth, diarrhea,and perianal staininq.t Includes reddish rasai discharge and stains on head,increase in respiratory rate, and decrease in respiratorydepth.< Includes reddi>,iJ material around eyes (chromodacryorrhea)and conjunctivitl,;.

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Mullen et al-li

Table 5

Mean Body Weights in Grams ± S.E.*

Dose Receipt Day 0 Day 6 t Day 14

MALES

683 152.4 223.9 287.0 290.2mg/kg ±3.7(li0) ±5.4(10) ±6.8(10) ±8.0(10)

826 159.5 222.6 .53.7 274.3

mg/kg ±3.6(10) ±5.4(10) ±8.1V7) ±7.1(7)

1000 158.5 224.9 283.2 289.8mg/kg ±4.5(10) ±5.6(10) ±7.6(5) ±6.1(5)

1210 149.3 224.0 251.0(1) 269.0(1)mg/kg ±4.2(10) ±5.6(10)

1470 155.0 226.1 217.0(1) 222.0(1)g 4.1(10) ±4.8(10)

Vehicle 156.2 235.8 306.8 314.4±3.8(5) ±4.6(5) ±5.2(5) ±8.4(5)

FEMALES

610 144.1 146.5 176.5 191.6mg/kg ±5.3(8) ±4.3(8) ±5.8(6) ±4.8(6)

718 136.6 154.8 217.3 209.0mq/kg ±1.4(10) ±3.1(10) ±10.3(4) ±8.9(4)

847 136.7 152.9 192.7 182.7mg/kg ±2.9(10) ±3.0(10) ±7.3(3) ±15.3(3)

1000 141.1 156.3mg/kg ±3.4(10) ±2.9(10)

1180 138.9 154.7mg/kg ±2.9(10) ±3.5(10)

1390 140.6 155.6mg/kg ±2.6(10) ±2.8(10)

Vehicle 151.6 178.8 215.4 207.4±2.4(5) ±4.1(5) ±4.9(5) ±4.1(5)

* Number in parenthesis = number of animals.t For the females (except controls) this is Day 7.

2atholoy Re-ort

The presence of multiple red foci in the thymuses of thefemales was the o:ly gross lesion attributable to the testcompound. The preence of foci exhibited a dose-responseeationship. Thf patho togi st's report is presented in

Ae ce ,di x G.

DISCUSS ION

The calculated MLD for auanidine nitrate in Sprague-Dawley rats was 94--.6 ± 68.7 mg/kg for males and 729.8 ± 34.3.rnr'kg for females. These MLD values are within the slightlytcxic range (5). The major category of clinical signsobserved was behavioral, which included inactive, irritable,disoriented, and hyperactive. These findings are consistentwith an effect on the central nervous/neuromuscular systemand the reported action of guanidine as a striated muscle

0 stimulant (6).

Similar resultIs were also reported for guanidinehydrochloride (7). However, guanidine hydrochloride produceda considerably more profound effect on the CNS-NM system (80of 86) and GI tract (53 of 86) than did guanidine nitrate.The clinical signs produced by guanidine hydrochloride werenot only observed more frequently but were also more severethan those produced by guanidine nitrate. The oral MLDs inmale and female rats for the hydrochloride salt were 556.5mg(base)/kg and 474.6 mg(base)/kg, respectively. Forcomparison, the oral ML['- in male and female rats forguanidine nitrat .... !-e 478.6 mg (base)/kg and 352.9mg (base) /kgre: -tively. These values are relatively closeto those obtained for the hydrochloride salt. Therefore, onecan attribute thtu toxicity of these two salts to theguanidine base.

CONCLUSIONS

Guanidine nDrate is a "slightly toxic" compound thatproduces behavior<ti, gastrointestinal, and respiratory signs.Calculated MLD v ': s were 989.6 + 68.7 mg/kg in male and729.8 ± 34.3 mn, > in feale Sprague-Dawley rats.

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REFERENCES

1. Kenyon KF. A data baDe ass-s.m:.L CL .:vironmenta!fate aspects of nitroguanidine. Fred.rck, MD: US ArmyMedical Bioengineering Research and Deve lopmentLaboratory, 1982. DTIC No. ADA125591.

2. Environmental Protection Agency. Office of Pesticides4 and Toxic Substances, Offices of Toxic Substances (TS-

792). Acute exposure, oral toxicity. In: Healtheffects test.guidelines. Washington, DC: EnvironmentalProtection Agency, August 1982; EPA 560/6-82-001.

3. Acute oral toxicity study (ALD and LD50) . LAIR StandardOperating Procedure OP-STX-36, Letterman Army Instituteof Research, Presidio of San Francisc:, CA. 22 December1982.

4. Finney DJ. Probit analysis. 3rd ed. Cambridge:Cambridge University Press, 1971: 20-80.

°.- 5. Hodge HC, Sterner JH. Tabulation of toxicity classes.Am Ind Hyg Assoc Q 1943; 10: 93-96.

6. Windholz M, ed. Merck Index. 10th ed. Rahway, NJ:Merck and Co, 1983:657.

7. Morgan EW, Sano SK, Korte DW. Acute oral toxicity(LD50) of guanidine hydrochloride in rats. Presidio ofSan Francisco, CA: Letterman Army Institute ofResearch, 1985; Institute Report No. 204.

0j

Mul en et, al-14

Appendix A. Chemical Data.................................... 15

Appendix B. Animal Pat<....................................... 21

Appendix C. -i i-Lca-I Listing of Study Events.............. 22

Appendix D. Cumulative Mortality Data........................ 24

Appendix E. Individual Animal Histories...................... 25

* Appendix F. Individual Body Weights......................... 43

Appendix G. Pat,,,, ociy Report................................. 56

Mo

Mullen et al-15

Appendix A: CHEMICAL DATA

Chemical Name: Guanidine Nitrate

Lot Number: 123820

Chemical Abstracts Registry Number: 506-93-4

LAIR Code: TW030

Chemical Structure:H 2N\ +

C - NH-, NO 3

H2 N

Molecular Formula: CH 6N3 .NO3

Molecular Weight: 122.1

Physical State: White crystalline powder

Melting Point: 214 0 CI

Analytical Data:

Infrared spectrophotometry was performed and the spectrumobtained 2 was identical to the Sadtler spectrum 3 for GuanidineNitrate. Major absorption peaks were observed at 3400(broad), 3200, 1665, 1575, 1400, 1385, and 825 cr - . Thegrade of material obtained for this study is referred to asthe Ultralog Grade by the manufacturer. The label on thebulk container states that the purity is at least 99.99%.

Source: Chemical Dynamics CorporationHadley Road, PO Box 395South Plainfield, NJ

1Windholz M, ed., The Merck Index. 9th ed., Rahway, NJ:Merck and Co., Inc., 1976: Monograph Number 4414.2Wheeler CR. Nitrocellulose-Nitroguanidine Projects.Laboratory Notebook #84-05-010.2, p. 62. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.3Sadtler Research Laboratory, Inc., Sadtler standard spectra,Philadelphia: The Sadtler Research Laboratory, Inc., 1962:Infrared Spectrogram #14498.

S1

.'.: ' a 1* ,2* 1 -

Appendix A (cont.): CHEMICAL DATA

7,L:si-vsis of iosnr ' t i, S',spensions and Deteiminatio! ofStability

Posirvg siutll..5!suspensions of guanidine nitrate were- rtexed to ensur-, urns on of particulate material and

sarmpes wero renoved from the top, middle, and bottom..a .... ± were tranoe:orred to screwcapped tubes and store(n

:w "-o, prior - analysisro :w I- crorthe

Fcr -: iv1 .... ion of the sample was necessary. Thc:st s,.iuo ..,.... mr ished by heating the 1 ml sam to

51C in a water _- _ dissolve suspended material, and thenquickly cooling : r',; temperature. Before the guanidinenitrate could crys'al-- out of solution, 0.5 ml was'ransferreu to a 'il'metric flask and diluted to vol,.?mewi h water.

A second dilution of 1:i00 was performed for a totaldilutior of 1:10, .Aliquots (2 ml) of the final dilutionwere assayed usi: - '.fication of the Voges-Proskauerassay for guanidc, .1 iuantitation was accomplished bymeasuring the abs :rrancc, of a colored guanidine derivative ata waveIerith of 5 35 nm.

For the fi;- an-lysis, seven samples were chosen thatrepresentea te ---ire range of concentrations used fordosing.- The re:' lts 'ndicated that homogenous suspensionscar. be prepared ,, ncyml. (Table 1) . As a result ofthis determi .;dbs, quent analyses were performedwith roc'i , ( .,., the top, middle, and bottomsarnl,;( cbt aioe ,r osinq solutions/suspensions wereheated to dissolv- suspn, ded material and pooled) .3 Theseresults are pre>'.trid in Table 2. Of the ten suspensionsanalyzed, nine wc-e determined to be within 6% of the target.The concentratir- f.he.- 68-mg/ml solution, however, showed adeviation of 54. itbe.a the target value. This reflects, inall probability. ditutyun error in making the solution.

.Micklus 1-1J, St. ". !' colo.... determination ofmono- and dJ*-ut :,uanidines. Anal Biochem 1973;53 ::-45-553..Wheeler CR. N>.r 1 u ,I ose-Nitroquanidine Projects.

Laboratory Noter, - #P'-)5J-0 10.2, p 49-51, 56. LettermanArm, n:; :'. h, Presidio of San Francisco, CA.%i 5 i , -'

Mullen et al-17


Analytical Method: Stock Solutions -ind CdJibration Plot

Stock Solution - 50 g/mi in water:

The stock solution was prepared by weighing out 50.0 mgof guanidine nitrate and transferring this amount to a1000-ml volumetric flask. The compound was diluted tovolume with water and mixed well.

Standard Curve (calibration plot):

To generate the standard curve, two ml of guanidinenitrate solution were prepared at a variety ofconcentrations as follows:

Final ml of stock added to mls of waterConcentration

2 jg/ml 0.08 ml stock 1.92 ml water5 0.20 1.80

10 0.40 1.6015 0.60 1.4020 0.80 1.2025 1.00 1.0030 1.20 0.80

Calculations:

Linear regression was used to calculate the standardcurve. In all cases a correlation coeffic nt (r) ofgreater than 0.999 was obtained.

assay value = a/ml found x dilution factor(mg/ml) 1000

where jg/ml found is determined by linear regression

dilution factor 1 10,000

0

S.

Len e u al- I


TAF3Lt 1: An .i' I .; o sng '1';uspension:; for iomog, ,2ty

Date Date Scurce ct Target Conc. Actual Conc. 2 Targ2rMixed Analyzed Sample* mg/ml mg/ml

6 Mar 18 May T 66 > 67.4 102.1M 65.5 99.2B 65.7 99.5

Mean 66.2 100.3

14 Mar 18 May T 83 > 80.5 97.0M 75.7 < 91.2B 77.2 93.0

Mean 77.8 93.7

8 Mar 18 May T 100 96.0 96.0M 99.8 99.8B 97.6 97.6

Mean 97.8 97.8

8 Mar 18 May T 133 122.2 91.9M 131.2 98.6

122.8 92.3Mean 125.4 94.3

8 Mar 18 May T 166 162.5 97.9M 165.3 99.6B 158.7 95.6

Mean 162.2 97.7

6 Mar 18R May 200 184.5 92.3M 188.0 94.0

197.0 98.5Mean 189.8 94.9

*The letters T, M, -'r B refer to the top, middle, and bottom of thedosing soution/su.:ison.

>These samples were -,'ution:3.

<This sample was o- -:1niy assayed on 18 May and a low value of 67.9mg/mi was d-etermined Peanalysis on 29 May 84 gave a value of 75.7mg/ml. As a check fir consictency, the samples prepared on 6 Mar and 27

Mar were also reanj, .-.ed cr ,79 May. The values obtained for thesesamples were within o respectivo values obtained on 18 May.

4I

Mullen et al-19


TABLE 2: Verification of Guanidine Nitrate :oncentrations*

Date Date Target Conc. Actual Conct % TargetMixed Analyzed mg/ml mg/ml

14 Mar 6 Jun 68.0 31.0 ± 0.5 45.683.0 79.7 ± 1.1 96.1100.0 93.8 ± 1.1 93.8121.0 113.3 ± 1.3 93.6147.0 137.9 ± C.8 93.8

21 Mar 30 May 71.8 67.2 ± 1.3 93.6* 84.7 78.6 ± 0.2 92.8

100.0 92.3 ± 0.2 92.3118.0 110.3 ± 0.6 93.5139.0 129.8 ± 0.2 93.4

27 Mar 61.0 not analyzed

*Wheeler CR. Nitrocellulose-Nitroguanidine Projects.Laboratory Notebook #84-05-010.2, p. 57-59. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.

tMean and standard deviation of three analyses.

'p,

0 N

-In]len et: a!-20


Stability:

The stability of guanidine nitrate in aqueoussolution is demonstrated by the absorbance valuesobtained for a standard solution containing 20 Ag/mi ofguanidine nitrate. This solution was prepared on 25 Mayand kept at rccm temperature over the period ofanalysis. From 25 May to 6 June, four assays of thissolution were performed yielding statistically identical

absorbance values.1 Since the Voges-Proskauer assay isspecific for unsubstituted and mono-substitutedguanidines, the d,,!ta demonstrate that aqueous solutionsof guanidine nitrate are stable for a period of at least12 days (Table 3).

TABLE 3: Stability Assay of a 20 gg/ml Standard Solution ofGuanidine Nitrate

Date of Araivsis Absorbance Values*

25 May 84 1.74 ± 0.0229 Maw 84 1.76 ± 0.0530 May 34 1.76 ± 3.02

6 Jun 84 1.76 ± 0.02

* Values are yr w - ror three replicates.

1 Wheeler CR. Nitrocellulose-Nitroguanidine Projects.

Laboratory Notebook #84-05-010.2, p 55-57,59. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.

4

%,:j.0

Mullen et al-21

Appendix B: ANIMAL DATA

Species: Rattus norvegicus

Strain: Sprague-Dawley

Source: Bantin-KingmanFremont, CA

Sex: Male and Female

Date of Birth: Males: 23 January 1984Females: 31 January 1984

8 February 1984 (for animalsnumbered 84D00-863, -865, -867,-868, -871, -886, -896, -900)

Method of Randomization: Weight bias, stratified animalallocation

Condition of animals at start of study: Normal

Body weight range at dosing: Males: 190 to 255 gFemales: 140 to 174 g

Identification Procedures: Ear tag. Tag numbers--Males:84D00596-84D00667, Females:84D00693-84D00794 and 84D00863-84D00900 with exclusions.

Pretest conditioning: Quarantine/acclimation. Males 29 Feb-7 Mar 84. Females 14-21 Mar 84, and21-28 Mar 84 for the additional group.

Justification: The laboratory rat has been proven to be asensitive and reliable system fordeterminations of lethal dose.

0

01

Mullen et al-22

Appendix C: HISTORICAL LISTING OF STUDY EVENTS

FEMALES

Date Event

29 Feb 84 Received initial shipment of animals.Five females were used for vehiclecontrols; their dates are the same asthose of the events for males (nextpage).

20 Mar 84 Fifty-two female animals were receivedfrom GLP Study 84016. Animals wererandomized and allocated to groups lA-2A.Food was removed by 2200 hours.

21 Mar 84 Animals in Groups IA-5A were weighed,dosed, and observed. All animals thandied were submitted for necropsy.

22 Mar-10 Apr 8-1 All animals were observed daily formortality and clinical signs.

23 Mar 84 Eight female animals, Group 6A, receivedfrom GLP Study 84015, constituted anadditional dose group.

27 Mar 84 Food was removed from Group 6A by 2200hou -s.

28 Mar 84 Animals in Groups IA-5A were weighed.Group 6A animals were weighed, dosed, andobserved. All animals that died werenecropsied.

4 Apr 84 All surviving animals in groups IA-5A* were observed, weighed, sacrificed, and

necropsies performed. Group 6A animalswere weighed.

11 Apr 84 All surviving animals in Group 6A wereobserved, weighed, sacrificed, andnecropsies performed.

,0

1,0R 11 WR

Mullen et al-23

Appendix C (cont.): HISTORICAL LISTING OF STUDY EVENTS

MALES

Date Event

29 Feb 84 Severity-two male Sprague-Dawley rats werereceived at LAIR. Rats were housedindividually and their left ear w:istagged. Animals were weighed and twoanimals were submitted for qualitycontrol necropsy.

5 Mar 84 Animals were randomized, divided intodose groups, and weighed.

14 Mar 84 Animals were weighed, dosed, andobserved. All animals that died weresubmitted for necropsy.

14-28 Mar 84 All animals were observed daily formortality and clinical signs.

20 Mar 84 All animals were weighed.

28 Mar 84 All surviving animals were observed,weighed, sacrificed, and necropsiesperformed.

0

I R0I titJ1V .. _

Y eri et: al-2'

Appendix 0: CUMULATIVE MORTALITY DATA (deaths/group)

T ime Aft t *r Do'; iml(

Ank~ Uic i-,, IZ 4~ sHor A

FEMALES

713 q 39 1 1 3 5 5 5 5 5 5347 9 1 1 4 4 4 6 6 6 6 6 6 6

1000 10 0 0 7 7 7 1010o10 10 10 10 101180 10 0 2 9 10 10 10 10 10 10 10 10 101390 8 0 4 583 8 8 8 888 8Vehicle 5 Ci 00a0 0 00 0 00 0

M ALES

683 10 0 0 0O 0 00 0 00 0826 10 C' 0 1 2 2 2 3 3 3 3 3 3

1000 8 j90 122 3 33 3 33 31210 8 0 2'- 2 3 7 7 7 7 7 7 71470 10 0167 -16 9 C99 9 9 9Vehicle 5 0 0 0 0 000 00 0

GNV.R

Mullen et al-25

Appendix E: INDIVIDUAL ANIMAL HISTORIES

MALES: Vehicle Control

Animal Clinical Signs Dates Observed SeverityNumber (1984)

84D00605 Irritable March 15 Slight

84D00606 None Observed N/A N/A I

84D00622 Nasal Discharge, Red March 22 Slight

84D00655 Irritable March 15 ModerateMarch 16-28 Slight

Nasal Discharge, Red March 22 Slight

84D00665 Inactive March 15 ModerateIrritable March 15 Moderate

4 'I

,Mullen et :l-26

Appendix E (cont.) • INDIVIDUAL ANIMAL HISTORIES

3 F ,LI : ,;,11Irninidine NitraLe

Animal Clinical 2Iqns Dates Observed SeverityNumber (1984)

84DO0597 Material Peria:nal, Brown March 14 SlightIrritaki> March 15,17, Slight-

22,23

84D00600 Material Mollth, Clear March 14 SlightInactive March 15 Slight

84D00602 None Ob -erved N/A N/A

-1 84D00617 Irritable March 15,16 SI1 nht

% 84D00635 Irr it - March 15-19 SlightMarch 20,21 Mode rMarch 22-25 ioht

84D00636 Hveractive March 15 SlightIrritable March 19,20 Slight

84D00642 Hyperactive March 15 Slightirritabi.e March 15 Slight

March 16 Moderate

84D00645 Lrrita t< March 16,17 SlightInactive March 16 SlIght

Nasal Discharge, Red March 22 Slight

84D00651 None )i .2erved N/A N/AS

84D00662 inact- :e March 15 MarkedMarch 16,17 SlightMarch 20,21 Slight

Irrit~a: March 15 SlightIncr c-. Te.>rLature March 15 Marked

- --

Mullen et al-27

Appendix E (cont.): INDIVIDUAL ANIMAL HISTORIES

MALES: 826 mg/kg Guanidine Nitrate

Animal Clinical Signs Dates rbserved SeverityNumber (1984)

84D00596 Material Mouth, Red March 14 SlightMaterial Perianal, Yellow March 14 ModerateAtaxia March 14 SlightSomnolence March 14 SlightDeath March 14 6 hrs

84D00611 Nasal Discharge, Red March 15 SlightDecreased Preening March 15 ModerateStain Perianal, Yellow March 15 MarkedIrritable March 15,16 Moderate

March 19 Slight

84D00615 Inactive March 16 Moderate

84D00618 Tremors March 14 SlightMarch 15 Marked

Ataxia March 14 SlightDecreased Preening March 15 MarkedChromodacryorrhea March 15 Marked

* Conjunctivitis March 15 MarkedIncreased Salivation March 15 MarkedStain Perianal, Yellow March 15 MarkedMoribund March 15 N/ADeath March 16 48 hrs

84D00632 Hyperactive March 15 MarkedIrritab-lo March 20,21 Sliqht

84D00633 Rough CoaL March 14 SlightIrritable March 15 ModerateInactive March 16 Moderate

84D00634 Prostrate March 14 N/ADecreased Resp. Rate March 14 SlightIncreased Resp. Depth March 14 SlightDeath March 14 5 hrs

13

Mi.len et al-28

Appendix E (cont.) INDIVIDUAL ANIMAL HISTORIES

MALES: 826 ma/kq Caianidine Nitrate (cont)


84DO0639 Material Pezianal, Yellow March 14 Sight,itch March 15 Marked

Tremors March 15 MarkedHyperactive March 15 ModerateMaterial Head, Red March 15 MarkedStain Perianal, Yellow March 15 MarkedDecreased Preening March 15 M. rked

March 16 Moderate

84D00640 Irritable March 15 ModerateHyperactive March 15 Slight

84D00643 Inactive March 14 3lightDecreased Preening March 15 MarkedTremors March 15 VlarkedChromodacryorrhea March 15 MarkedMaterial Head, Red March 15 MarkedStain Perianal, Yellow March 15 ModerateIrritable March 16 Moderate

..

.4

4

Mullen et ai-29


MALES: 1000mq/kg Guanidine NitraLe

Animal Clinical Signs Ditues Obsurved SeverityNumber (1984)

84D00598 Material Mouth, Red March 14 Slight

84D00604 Material Mouth, Red March 14 SlightAtaxia March 14 SlightInactive March 16 Slight

84D00624 Irritable March 15,16 Moderate

84D00625 Hyperactive March 15 ModerateIrritable March 16,17, Slight

18,21Inactive March 20 Slight

84D00627 Ataxia March 14 ModerateJumping March 14 MarkedDeath March 14 5.5 hrs

84D00631 None Observed N/A N/A

84D00637 Misdose

84D00638 Ataxia March 14 SlightDeath March 15 24 hrs

84D00658 Death March 14 5 hrs

84D00659 Misdose

,u en er a1-}


M L.: 1210 mg/kg Guanidine Nitrate

-Animal Clin'cai Siqns Dates Observed Sve r i tNumber (1984)

84,700607 Hunched Posture March 15 ModeratDecreased Preening March 15 MocerateMterial bMouth, Red March 15 Marke-MaterPIC Perianal, Yellow March 15 Sligh,

yp Hyerac. 1ve March 15 Moderane

84f80612 fiunche, I u r,, March 14 .S1 ,IhtAt axia March !4 S i iMaterial Mouth, Clear March 14 Slight

• Death March 15 24 hrs

84D00614 Prostrate March 14 N/AIncreased Resp. Rate March 14 SlightDecreased Resp. Depth March 14 SlightDeath March 14 7 hrs



84D00644 Death March 14 3 1irs

84D0065C daxs C'

8;D00654 Ataxia March 14 SlightInact iv,, March 14 SlightDeath March 15 24 hrs

84D.06C1 Material Pprianal, Brown March 14 SlightMaterial Piaht 11ind Leg March 14 SlightAtaxi z~March 14 Slight

Death March 15 24 hrs

14"2 0664 Mi " ",i

1 Z4

Mullen et al-31


MALE: 1470 mq/kg Guanidine Nitrate

Animal Clinical Signs Dates Observed SeverityNumber (19d4)

84D00601 Inactive March 14 ModerateMaterial Mouth, Clear March 14 SlightIncreased Resp. Depth March 14 SlightDecreased Resp. Rate March 14 SlightDeath March 14 5.5 hrs

84D00609 Material Mouth, Clear March 14 ModerateInactive March 14 SlightDeath March 14 3.5 hrs

84D00610 Material Mouth, Red March 14 SlightInactive March 14 SlightIncreased Resp. Depth March 14 SlightDecreased Resp. Rate March 14 SlightDeath March 14 3.5 hrs

84D00613 Material Mouth, Clear March 14 SlightInactive March 14 MarkedMateria Eye, Red March 14 Slight

, Twitchi..g March 14 SlightAtaxia March 14 ModerateDeath March 14 7 hrs

84D00616 Material Mouth, Clear March 14 SlightInactive March 14,20,21 SlightHunched Posture March 14 MarkedMaterial Eye, Red March 14 SlightTwitching March 15 Marked

. Decreased Preening March 15 Moderate* March 16 Slight

Nasal Discharge, Red March 15 ModerateStain Mouth, Red March 15 SlightFeces Perianal March 15 ModerateIrritable March 16 Moderate

March 17,19 Slight* Material Perianal, Brown March 16 Slight

84D00626 Increased Resp. Depth March 14 SlightDeath March 14 2 hrs

- ia<

0_. ,:,ul len e-t ai-32


MALE: 1470) mgikg Guanidine Nitrate (cont.)

.\,imal Clinical Signs Dates Observed SeverityNumber (1934)

84D00630 Death March 14 3.5 hrs



84D00663 Ataxia March 14 SlightDeath March 15 24 hrs

-- - - - - -- - - - -

Mu [len (t il-33


FEMALES: Vehicle Controls



84D00695 Material Nose, Red March L5 MarkedMarch 16 Slight

Increased Resp Rate March 15 MarkedStain Perianal,Yellow March 15 Marked

March 16-18 ModerateWheezing March 15 ModerateDecreased Preening March 16 ModerateIrritable March 21 Slight

84D00706 Irritable March 15 MarkedMarch 17 ModerateMarch 18,19 Slight

84D00714 Hyperactive March 15 Moderate


N"

0B

Appendix E (cont.) : INDIVIDUAL ANIMAL HISTORIES

YEMALLi 610 mg/kg Guanidine Nitrate

Amimal Ciinicai Signs Dates Observed SeverityNu be r (1984)

84D008 7 1ntiv March 28 Moderat,7.ateria Mcuth, Clear March 28 ModeI-At,:

:sori.0ed March 28 Slight-increa't, i Pesp. Depth March 28 M-C.d,-Deciea--;&.* : esp. Rate March 28 ModerateTwitchisn. March 28 MarkedMoribund March 28 N/ADeath March 28 2.5 hrs

84DOC865 None Observed N/A N/A

S S4D00867 Material M:,uth, Clear March 28 Si1-7ht

84D0C868 Inactive March 28 ModerateIrritablE March 28 ModerateDecreasecd Resp. Rate March 28 Sligh:increased Resp. Depth March 28 SliohtMaoer-.-tK Nose, Red March 28 Slight

84D00871 Misdose

84D00886 Inactive March 28 ModerateApril 1-3 Moderate

rrit;' March 28,29 ModerateMarch 31, SlightApril 3 Slight

Disorin'ted March 28 SlightMarch 29 MarkedMarch 30 Moderate

Stain th, Fed March 28 N/A* Diarr" - March 29 Marked

-Sa in -,ianaI, Yellow March 29,31 MarkedMarch 30 ModerateApril 5 Slight

]Stain PR'. , ~ March 29 MarkedApril 5 Slight

* Stair; .F 'd, Clear March 30 MarkedRough ',>At March 30,31 Moderate

April 1 ModerateApril 2,3 Slight

Inctreasci Pesp. Rate April 1 Slight

S • nu'

Mullen et al-35


FEMALES: 610 mg/kq Guanidirie NitraLe (cont.)


84D00896 Inactive March 28 ModerateIrritable March 28 SlightStain Mouth, Red March 28 N/AExcited March 29 Moderate

84D00900 Disoriented March 28 SliqhtIncreased Resp. Rate March 28 SlightDecreased Resp. Depth Marrh 28 SlightStain Mouth, Red March 28 SlightAggressive March 28 N/A

* Irritable March 28,29 SlightInactive March 28 Slight

%r -U


FEMAIES: 718 mg/kg Guanidine Nitrate


84P00750 Ac1crc s sve March 21 SliqhtHyperaP ve March 21 liqhtSnactlv< March 21 Mla uked

March 22 ModeratoTwitchin4 March 21 SlightMaterial Mouth, Red March 22 ModerateRough Coat March 22 ModerateIncreased Resp. Rate March 22 SlightDecrease- Reso. Rate March 22 Slight

84D00751 Aggressive March 21 Slight:Irritable March 21,22 SlightDisoriest.ed March 22 Slight

84D00753 Inactive March 21 SliahtStain Ncuth, Clear March 21 SllihtDeath March 21 '4 hrs

84D00756 Disorje:'ted March 21 SlightRough Coat March 21 SlightIncreased Sal vation March 21 SlightTwitcr', March 21,22 SlightStnin. -, CLear March 21,22 MarkedStain head, Clear March 21,22 SlightMaterial Perianal, Yellow March 21,22 ModerateMateriL>- zbdomen, Yellow March 21,22 ModerateIncreased Resp. Rate March 22 SlightDeath March 23 48 hrs

84D00760 Rough March 21,22 ModerateStain M1 h, Red March 21 ModerateInact v March 21 Moderate

March 22 MarkedStain . , Clear March 22 Moderate

4 Death March 23 48 hrs

Mullen et al-37


FEMALES: 718 mg/kg Guanidine Nitrate (cont.)

Animal Clinical Signs Dates Observed Sever'twNumber (1984)

84D00762 Hyperactive March 21 SlightIrritable March 21,22 SlightInactive March 22 Slight

84D00764 Hyperactive March 21 SlightTwitching March 21 ModerateRough Coat March 21 ModerateMaterial Mouth, Clear March 21 SlightDeath March 22 24 hrs

84D00765 Hyperactive March 21 SlightInactive March 21 MarkedTwitching March 21 SlightDeath March 22 24 hrs

84D00772 Misdose

84D00793 Irritable March 21 ModerateMarch 22 Slight

Inactive March 21 Marked

Stain Mouth, Clear March 21 ModerateMaterial Eye, Clear March 21 MarkedRough Coat March 22 Slight

I , _. ,!- ,:

MW*~*~ - UV

' 11e n e t ai7-3_98


FEMALLS 847 mg/kg Guanidine Nitrate

Animal Clinical Signs Dates (1984) Severity


84D00743 Death March 21 4 h-s

84D00755 Irritable March 21 SiDeath March 21 4 hrs

84D00761 Rough Coat March 21 SlightIncreased Resp. Rate March 21 SlightHyperactive March 21 SlightInactive March 21 ModerateMaterial Mouth, Red March 21 SliahtDeath March 22 24 hrs

84D00767 Misdose

84D00770 "Iyperactive March 21 SiightIrritable March 21,22 SldnhtInactive March 22 SlightRough Coat March 22 SlightDecreased Preening March 22 SlightIncreascd Resp. Rate March 22 Slight

84D00781 Disoripnt-ed March 21 SlightInact' ,> - March 21 SlightIrritable March 22 Slight

April 1 SlightStain Nose, Red March 26 Slight

84D00782 Disoriented March 21 SlightInactive March 21 SlightMaterial Mouth, Red March 21 SlightIrritaV,' March 22 SlightRough 1im March 22 Slight

84D00788 inactiv March 21 SlightTremoi, March 21 SlightIncrea:-,! Resp. Rate March 21 SlightIrritable March 21 ModerateDeath March 22 24 hrs

84D00789 Death March 21 1 hr

0Ii

am0NR

.Mullen et al-39


FEMALES: 1000 mg/kg Guanidine Nitrate

Animal Clinical Siqns Dates Observed SeverityNumber (1984)

84D00747 Disoriented March 21 SlightHyperactive March 21 ModerateDeath March 21 4.5 hrs

84D00749 Hyperactive March 21 SlightIncreased Resp. Depth March 21 ModerateDeath March 21 4.5 hrs

84D00758 Moribund March 21 N/ADeath March 21 4.5 hrs

84D00766 Increased Salivation March 21 MarkedIncreased Resp. Rate March 21 SlightDisoriented March 21 SlightDeath March 21 4.5 hrs

84D00775 Inactive March 21 SlightDisoriented March 21 ModerateIncreased Resp. Rate March 21 ModerateDeath March 21 4.5 hrs

84D00777 Disoriented March 21 SlightHyperactive March 21 SlightIrritable March 21 MarkedInactive March 21 ModerateAggressive March 21 SlightDeath March 22 24 hrs

84D00780 Irritable March 21 SlightInactive March 21 SlightIncreased Resp. Rate March 21 SlightDeath March 21 4.5 hrs

84D00783 Inactive March 21 ModerateDeath March 21 4.5 hrs


V"EMALE <: ',J.J rg 7 Guarlidiize Nitrate (cont.)

Anmai C±i.rl±ccl Signs Dates Observed Severity

Nuib e r (1984)

34D00786 Disori±-7ted March 21 SlightTremors March 21 SlightI nact i" March 21 SIiaht-Mater J-I Mouth, Clear Marcn 21 SliahtDeath March 22 24 hrs

84D00787 Rough Co March 21 SlbahtTremors March 21 SlightTncreas<J ?esp. Rate March 21 SihtInactiv, March 21 S ii ,t

at er 1 1 Eye, Red March 21 SlihtDeath March 22 24 hrs

U __________ ____________ ___________________________________

%'

.*1*:

"

Mullen et al-41


FEMALES: 1180 mg/kg Guanidine Nitrate



84D00746 Irritable March 21 SlightDisoriented March 21 SlightStain Mouth, Clear March 21 ModerateDeath March 21 4.5 hrs

84D00748 Disoriented March 21 SlightHyperactive March 21 SlightDeath March 21 4.5 hrs

84D00759 Rough Coat March 21 SlightIncreased Salivation March 21 ModerateDisoriented March 21 Slight

Death March 21 5 hrs

84D00768 %iwitching March 21 SlightIncreased Salivation March 21 ModerateInactive March 21 ModerateDeath March 21 4.5 hrs

84D00774 Disoriented March 21 SlightIncreased Preening March 21 SlightTremors March 21 ModerateInactive March 21 MarkedMaterial Mouth, Clear March 21 SlightDeath March 21 6 hrs

84D00778 Hyperactive March 21 SlightRough Coat March 21 SlightDeath March 21 5 hrs

84D00784 Disoriented March 21 SlightDeath March 21 4.5 hrs

84D00785 Inactive March 21 SlightTremors March 21 ModerateIncreased Salivation March 21 ModerateDeath March 21 4.5 hrs


.k .


'EM.ALE2: 130 .m,-'kc: Cuanidine Nitrate

-?riina I Clinical Daces Observed Sever tNumber (1984)

F4P00745 Death March 21 2.5 hr-

84D00754 Misdose


84D00763 Rough Coat March 21 ModerateIncreased Salivation March 21 MarkedDisorie--od March 21 ModerateDeath Iarch 2i 5.5 h-,


84D00773 Death March 21 2.E ilrs

84D00776 Hyperac:ive March 21 Si.,htIncreae, Resp. Rate March 21 SlightDeath March 2] 4.5 hrs

84D0077D ... Cdose

84D00790 Inact March 21 r kedTr-o March 21 Mark-dT rihn' " March 21 N/ADeath March 21 4.5 hrn


0~

-L j0-o'*

.J.>

Mullen et al-43

Appendix F: INDIVIDUAL BODY WEIGHTS

MALES: Vehicle Controls

Animal At Tmination WeightNumber Receipt Dosinq Day 6 Day 14 Change

(g) (g) (g) (q) (g)

84D00605 158 233 304 311 78

606 152 229 293 294 65

622 146 230 307 309 79

655 156 233 305 313 80

665 169 254 325 345 91

Mean 156.2 235.8 306.8 314.4 78.6

StandardDeviation 8.5 10.3 11.5 18.7 9.2

StandardError 3.8 4.6 5.2 8.4 4.1

-P

Appendix F (cont.): INDIVIDUAL BODY WEIGHTS

- M.ALES: 683 mg/ki

Animal At Termination Weigir-N. u,',,er Receipt Dosing Day 6 Day 14 u:a ng

V (g) (g) (g) (g)

. 8owD0 05 97 1t 210 260 270

600 175 255 321 323 68

602 168 242 304 311 69

617 144 206 264 257

* 635 144 113 294 293 75

636 160 217 272 265 48

642 137 205 261 262 9

645 147 219 287 289 ,

651 15? 242 305 316 74

662 146 225 302 316 91

Mean 152.4 223.9 287.0 290.2 66.3


StandardError p. '.4 6.8 8.0 4.0

m-

0"

Mullen et ai-45


MALES : 326 mg/kg

Animal At Tci:mination WeightNumber Receipt Dosing Day 6 Day 14 Change

(g) (g) (q) (g) (g)

84D00596 L67 253 Dead

611 137 198 236 251 53

615 159 219 26c 273 54

618 152 198 Dead 1

632 157 215 258 268 53

633 160 232 300 310 78

634 173 240 Dead

" 639 151 228 256 282 54

640 175 227 280 275 48

% 643 164 216 247 261 45

Mean 159.5 222.6 263.7 274.3 55.0


StandardError 3.6 5.4 8.1 7.1 4.1

p'

Appendix F (cont.) INDIVIDUAL BODY WEIGHTS

MLES: 1000 mg/kg

.kr1im a A1 Termination WeightNumb er Receip- Dosing Day 6 Day 14 Cnanw.,

(g)(g) (gA)

604 17 243 31037

624 134 220 270 291

625 163 228 287 294 66

* 627 1J10Dead

631 164 225 268 269 .14

637 150 21*7 Misdose

638 222 Dead

658 1> 255 Dead

6'59 14"I21 Misdose

M~r 135224.9 283.2 289.8 59. 6

StandardDeviation 1217.6 16.9 13.7 10.9

* StandardErrr .~5.6 7.6 .14.9

Eror6.

Mullen et al-47


MALES: 1210 mg/kg

Animal At Termination WeiqhtNumbe r Receipt Dos i nq Da y 6 Poy 14 ClkI1(eW

(g) (g)(g(c)()

84D00607 1-41 208 251 269 61

612 173 263 Dead

614 138 215 Dead

619 135 200 Dead

620 162 224 Dead

644 146 223 Dead

650 145 237 Misdose

654 149 234 Dead

661 138 213 Dead

664 166 223 Misdose

Mean 149.3 224.0 251.0 269.0 61

StandardDeviation 13.2 17.7

StandardError 4.2 5.6

-e


M-ALES: 1470 mq/kq

AnAt Termination WeightNner Receipr Dosing Day 6 Day 14 Change

8420E01 16 4 /-28 PCdd

609 ]r- 242 Dead

610 17/3 251 Dead

613 171 239 Dead

616 140 204 217 222 1

626,, t 15i 2 18 Dead

630 150 ,29 Dead

649 146 2C5 Dead

656 13-1 222 Dead

663 151 223 Dead

Man1,12C,217.0 222.0 18


StandardError*;4.

14ullen et al-49


FEMALES: Vehicle Controls

Animal At Tfermination WeightNumber Receipt Dosing Day 6 Day 14 Change

(g) (g) (g) (g) (g)

84D00693 155 169 207 202 33

695 155 188 214 217 29

706 150 186 228 208 22

714 143 169 203 195 26

737 155 182 225 215 33

Mean 151.6 178.8 215.4 207.4 28.6


StandardError 2.4 4.1 4.9 4.1 2.1

0

0

wI

0

Appendix F (cont.) INDIVIDUAL BODY WEIGHTS

FEMALES: 61f) ma/kg

inimnal At Termination WihNumber Receipt Dosing Day 6 Day 14 Change

(a ()(g) (g) (a)

84D00863 148 147 Dead

865 13"! 140 169 18b 45)

867 147 15 0 195 204 54

868 116 124 156 173 49

*871 150 145 Misdose

886 165 167 170 196 29

896 156 152 183 190 38

900 134 144 186 207 59

Mean "16.56 176.5 191.8 45.7

StandardDeviation 15912.1 14.1 11.8 10.9

StandardError 534.3 5.8 4.8 4.5

li0' l 14C

Mullen et al-51


FEMALES: 718 mg/kg

Animal At Termination WeightNumber Receipt Dosing Day 6 Day 14 Change

(g) (g) (g) (g) (g)

84D00750 138 144 189 184 40

751 145 171 232 219 48

753 138 154 Dead

756 141 157 Dead

760 134 141 Dead

-1762 133 171 233 224 53

764 139 154 Dead

765 135 153 Dead

772 131 148 Misdose

793 132 155 215 209 54

Mean 136.6 154.8 217.3 209.0 48.8


StandardError 1.4 3.1 10.3 8.9 3.2

.

0

SI


17EM~~Pl2: 612rj,kg

:%-i ma~ 1 Termination ehLNu-br~e ~ ci Dosing Day 6 Day 14 Chance

(0) (g)(g) (g) g

84D00742 12 160 Dead

74 16 -

755 140 149 Dead

761 12 5 144 Dead

07671 137, 1149 M isdr)s e

770 127 137 178 172 35

781 127 145 200 188 43

7182 13- 15E)6 200 188 4

788 1Y 166 D ead

72'9 14p4 i"6 Dead

Mean 1> - 152.9 192.7 182.7 36.7

StandardDevi 4tin ~ 9.5 12.7 9.2 5.7

StandcardError ;.3.0 7.3 5.3 3.3

Mullen et al-53,p


FEMALES: 1000 mg/kg


(g) (g) (g) (g) (g)

84D00747 144 157 Dead

749 138 160 Dead

758 146 156 Dead

766 143 158 Dead

775 156 163 Dead

777 123 145 Dead

780 127 142 Dead

783 156 174 Dead

786 141 157 Dead

137 151 Dead

Mean 141.1 156.3



V

%


°EMAIES: ~i10 mg/kg

.AI'a At Terminati-n eiql.tNumber Recei:t: DosinC Day 6 Day 14 r aL a

( (g) (g) (g)

0 1 4 1 1112 Dead

746 III 159 Dead

4 48 135 153 Dead

759 13 148 Dead

* 768 14? ,I7 Dead

774 123 139 Dead

778 141 152 Dead

784 i 75 Dead

785 136 154 Ded

!) %4 1 (8 Dead

Mean 13 o 154.7

StandardDeviation 9.", 10.9

StandardF.rr cr 3.53

0

Mu] lern et aJ-55

Appendix F (cont.) : INDIVIDUAL BODY WEIGHTS

IFEMALES: 1390 mg/kq


(g) (g) (g) (g) (g)

84D00745 1,44 164 Dead

754 144 158 Misdose

757 136 154 Dead

763 146 161 Dead

771 131 146 Dead

773 158 171 Dead

776 141 155 Dead

779 140 159 Misdose

790 132 146 Dead

791 134 142 Dead

Mean 140.6 155.6



0t

06

Appendix G: PATHOLOGY REPORT

Patholgy Report

GLP Study 84-001

Oral Lethal Dose (LDs 0 ) Test in Rats

of fiinidine Nitrate (CH5N3.HNO 3 ) ((AS No. 506-93-4)

History:

iFift-itht- io iid 59 female Sprague-Dawley rats, 6 weeks of age.were placied The tcliowtng test -roups:

Croup No. Dose m/kg No. Rats Sex

I Vehicle Control 5 male2 b83 10 male

R26 10 male4 1000 t0 male

5 1 "2) 8 mile

6 1470 10 male

I Vehicle Control 5 female1A 71 9 female2A 847 9 female3A 1000 10 female

* 4A 1180 10 female5A 1390 9 female

6A 610 7 fema le

The test cu-pound wa!s dissolved in sterile water and dosed by oralgava;'e. Forty-nine rats died and were necropsied on the dosing day.An additl eal 14 rats died within 24 hoirs, and 2 died within 48 hours.The remaining rats iurvlved until coctpletion of the study and werekilled by .trappritorne,i ir.ject!ri of sodium pentobarbital.

GrosS er.,p sv 7c, rrrs

I ndIl , r: I dt t I groupi data are separated in Tables I and IT.

lItrogenrr C ,iits d". r,, vsophageal pretoration or tracheal tears and

depositjj A Ol lie test ,bsitance In the thcrax occurred In 2 mkiles andI fem le. [hirtren feale had multiple red foci in the thymus. Noneof the miles hid this IeFton.

Sumiry:

Sixty-five rats (24 rules, 41 freailes) died during testing. Three

rats, 2 r: V.s and I lerlry, wire inappropriately dosed resulting iniatr-geni, , h-r t.ier Ie ire, 62 rats can be considered to have diedas a resiuI: -f rll' tesr :-iopund. The only lesions attributable to the

to po :nd ' :,:tipi|e rei foci in the thymus. This was confined to

Mullen et al-57

Appendix G (cont.): PATHOLOGY REPORT

pathology Report - GLP Study 84-001

the females and appeared to be dose dependent. The remaining lesionswere considered incidental findings which were not compound related.No lesions were seen in the vehicle control rate.

LANCE 0. LOLLINI. DOIDipiomate. ACVPLTV. VC

Chief, Pathology Services Group

Appendix G (cont.) PATHOLOGY REPORT

TabiL I

0,ral LID in Rats (CAS No. 506-93-4)

GLC Study 4o. 34001

Si) 3J:, XXX G;re, Cr,,, IA Group 2A Group 3A

b 7 777 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

' ,, 5 6 5 5 6 9 4 4 5 8 6 8 7 88 4 5 6 7 7 8 9 8 8

, '.5 00 1 2 3 2 3 5 9 1 8 0 1 2 7 9 8 6 5 7 0 3 o I

Surv!vai to

C 3plet io. + + + + 0 0 0 3 0 ++ + 0 0 0 0 0 0 + + + 3 0 0 0 0 0 C 0 D

Thinau,'!. PIc red

* iooi 00'.D0 0 0 0 Q 0 0 0 0 0 00+00000 0 0 0 0 0 0 +C 0

Red tocus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 000 Or v

Sc3 all Intestine

Lu-Ltnai Contents

Dark red C " f 1X O++ 00 0 0 0 0 0 0 0

00000 0 0 00 ) 0

rown (0),0 C, u 0 000 0

000 0 0 + 0 0 0 0 0 0

5ur\Ch - red

foci Z , 1. '. 0 0 0 f) 0 o ).. 0 0 0 0 0 0 0 0 0 ( 0 0 0 0 0 0 0 0 0 0 0

Lumtna l con-

tent3 - cirk

red +0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

00 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Pertcraton

Thorax

Clear fluid

present 0 0 f 0 0 0 0 0 0 0 0 0 00 0 00000000 0 0 0 0 0 0 0 0 0 0

Bladder

'all thickened 0 ) 0 0 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Calculi 0 ") 0 ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

KidneySllatll rdl 11.1ated• crlv i Il d 0 000000000 0000 0 0 0 0 0 0 0 0 0 0 0 0 0

* Ureter - blila-

.Literal d! l..d .j j 0 000000000 0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0

0

01 Ij

Mullen et aI-59


Table 1A

Orl 5D0 In RatS (CAS No. 506-93-4)

GLP Study No. 84001

Fe ma les

ID 084D00XXX Group 4A Group 5A Group 6A

7 77 77 77 7 77 7 77 77 7 777 8 88 88 89

Survival toCompletion 00 00 0 0 000 0 00 00 0 00 00 0 . . . .

ThymsusMultiple redfoci 0 0 0 + 0 + t 0 0 + 0 +- 0 + + 0 +- 0 + 0 C () 0 0 0 0 0

LungRed focus 0 00 00 00 0 00 00 0 00 00 00 0 000 00 0

Small1 Intestir:Luminal Contents

W ark red 0 0 0 000 0 000 0 0 00 00 000 00 0 00 00

Bloodbrown 0 00 00 0 00 00 00 00 00 0 00 00 00 00 0

Stomach - redfoci multiple 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

luminal con-tents - darkred 0 00 0 00 00 00 00 0 000 0 00 00 00 00 0

EsophagusFerforation 0 00 00)0 00 0 00 00 0 0 +00 00 00 a00

ThoraxIpresent 0 00 00 0 00 00 00 0 000 + 00 00 0 000 0

B ladderwall thickened 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0

Calculi 0 00 0 00 00 00 00 00 00 0 00 00 00 0+ 0

Bilateral dilated :pelvis0 00 00 00 0 00 00 00 00 0 00 00 00 0 +0

Ureter

Biaea iae 0 000000000 1

Appendi>- G (cont.): PATHOLOGY REPORT

Table 1B

Oral LDo in Rats (CAS No. 506-93-4)

GLP Study No. 84001

Males

, D ZOOCK:X Group Group 2 Group 3

6 6 6 6 6 5 6 6 6 6 6 6 6 6 6 5 6 6 6 6 6 6 6 6 6

0 u 26 5 900 1 334456 9 3 1 1 33344

IIS" 6,, 2 5 5 7 0 2 7 5 6 2 5 1 2 6 4 8 1 5 2 3 9 0 3

Survt',ai to

u omnpletion + +-, + + + + 4- + + + + 0 -+4

% 31oo.J - brown 0 )0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 0 0 0 0 0 0

Esop'ageal

perforation 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Trac-al ter 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Thcrax fluid 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Rena'

pe'!s dilated

un l-:eral 0 0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

" C-,rticaj. cy.ts s 2 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

FN.. . t !r I

r:.r, 0 op0cit' ,1000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4d;

[.!.

:0

Mullen et al-61


Table 1-B (Cant)

.9Oral LD 50 in Rats (CAS No. 506-93-4)

GU' Study No. 84001

Males

ID #84000XXX Group 4 Group 5 Group 6

6 6 66 665 6 666 6 66 66 6 66 6 6 6 6666 66 63 2 553 9 02 23 12 4 1 156 0 0 0 1 12345 6 1

7,178 98 84 4 51 4 049 2 4 17 1 90 3 609 63 6

Survival toCompletion 0 0 00 0+.+... 0 00 00 0 0+ 0 0 000 00 00 +

Blood -brown 0 00 00 00 0 00 0 00 0 0 000 0 00 00 00 00 0

EsophagealPerforation +00 00 0 00 00 0 00 0 0 000 0 00 00 00 00 0

Tracheal tear 000 +0 0 0 000 0 00 0 00 00 0 00 00 00 0 00

Thorax fluid +00 +0 00 0 00 0 0 000 0 00 0 0 000 00 00 0

* RenalpelIvs dilatedunilateral 0 00 00 00 0 00 000 00 0 00 0 00 00 00 00 0

Cortical cysts 0 00 0 000 0 00 000 00 0 00 0 00 00 00 00 0

Eye unilateralperipheralcorneal opacity 0 0 000 00 00a0 0 00 00 0 +0 0 ooo0o00 0oo0

0p

SRIIM X


Table II

Oral LDso -n Rats (CAS No. 506-93-4) - GLP Study No. 84001

Group Data - Males

1V lu 7 1 4 6

No. Ari ils,'Croup 5 10 10 10 8 10

Vehicle

Do'.e mg/kg Coltrol 683 826 1000 1210 1470

Died 0 3 5 7 9

Les ions-thynmsmultiple redfoci 0 0 0 0 0 0

Lung

red focus 0 0 0 0 0 0

Small intestinered contents 0 0 0 0 0 C

Blood - brown 0 0 1 0 0 0

Stomach

red foci 0 0 0 0 0 0

R 'vtents 0 0 0 0 0 0

Esophagea 1preforat ton 0 0 1 0 0

.A Traceal tear 0 0 0 1 0 0

Thoracic fluid 0 0 0 2 0 0

Bladder

wall thickened 0 0 0 0 0 0

CalcuIt 0 0 0 0 0 0

Kidney - dilat, .p'lvls biltor!( (1 0 0 0 0 0

LI lar .. 0 1 0 0 0 0

0: 1 0 0 0 0

~Urete.rdil-itd Al lr., 0 0 01 0 0 0

Ey.

',irn, * 1J, t, ( 0 ) 0 1 0

0

rrw wI inr

NI

Mullen eL a]-63

% Appendix G (cont.): PATHOLOGY REPORT

Table 11 (Cont)

Oral LD50 i m: ; A No. 5 ju-,-4) - .L- SLldy N-. 6400

Group Data - Females

Group 1 1A 2A 3A 4A 5A 6A

No. Animls/Group 5 9 9 10 10 9 7

VehicleDose mg/kg Control 718 847 1000 1180 1390 610

- Died 0 5 6 10 10 9 1

Les ions-t hymusmut iple red

foci 0 1 1 2 4 5 0

k-Lung

% red focus 0 1 0 0 0 0 1

Small intestine

redcontents 0 2 0 0 0 0 0

Blood - brown 0 0 0 1 0 0 0

Stomach

red foci 0 1 0 0 0 0 0

Red contents 0 1 0 0 0 0 0

Esophageal

preforation 0 0 0 0 0 1 0

Tracheal tear 0 0 0 0 0 0 0

Thoracic fluid 0 0 0 0 0 1 0

Bladder

wall thickened 0 0 0 0 0 0 1

Calculi 0 0 0 0 0 0 1

Kidney - dilated

pelvis bilateral 0 0 0 0 0 0 1

Unilateral 0 0 0 0 0 0 0

Cysts 0 0 0 0 0 0 0

Ureter

* dilated bilateral (1 0 0 0 0 0 1

corneal opacity 0 0 0 0 0 0 0

0

S

Distribution List

Co man d er CommandantUS Army Biomedical Research and Academy of Health Sciences

DevCopmnent Laborat.-'r- (27) United States ArmyATTN: SGRD-UBZ-C ATTN: Chief, Environnic.;'ajFort Detrick. Frederick, MD 21701-5010 Quality Branch

Preventive Medicine DivisionDctense Technical hi ormation Ccntcr (HSIIA-IPM)

(DFIC) (2) Fort Sam Houston, TX 78234.AVTN: DTIC-DLACameron Station Commander US Army MaterielAlexandria, VA 22304-6145 Command

ATTN: AMSCGUS Army Medical Rcscarch and 5001 Eisenhower Avenue

cv .... ,...... ..... . 12) Alexandria, VA 22333'ATFN: SGRD-RMI-S

Fort Detrick, Frederick, MD 21701-5012 CommanderUS Army Environmental -ygiem.

Commandant AgencyAcademy of Health Sciences, US Army ATTN: Librarian, HSDH-AD LATTN: AHS-CDM Aberdeen Proving Ground, >1ID 21010Fort Sam Houston. TX 78234

DeanChicf School of MedicineUSAEHA Regional Divikion, West Uniformed Services University of theFitzsimmons AMC Health SciencesAurora, CO 80045 4301 Jones Bridge Road

Bethesda, MD 20014Chict

USAEIIA Regional Division, North CommanderFort George G. Meade, MD 20755 US Army Materiel Command

ATTN: AMCEN-AChief 5001 Eisenhower AvenueUSAVIIA Rcglo;i,a ! .. ion, South Alexandria, VA 22333Bld1g. ISOFort McPhcrson. GA -';30 ItQDA

ATTN: DASG-PSP-ECommander Falls Church, VA 22041-3258US.\ Health Service,*.A [TN: IISPA-P HQDAt:ort Sam Houston, TX 7:'5,14 ATTN: DAEN-RDM

20 Massachusetts, NWWashington, D.C. 20314

dt1c · 2011. 10. 11. · 1 1 file copy lc) institute report no. 267 acute oral toxicity of...

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