i.;;LMR - DTIC · Hiatt et a]--2 Vehicle for Test Substanc _ Sterile isotonic saline (Travenol Laboratories, Deerfield, IL) was used as the vehicle for guanidine nitr.5e. The expiration

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uNCLASSIFIEDSECURITY CLASSIFICATION OF THIS PAGE

REPORT DOCUMENTATION PAGE NO.Z0%0 18Ia. REPOR SC4-1Fa. REPORT SECURITY CLASSIFICATION lb RESTRICTIVE MARKINGS

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Institute Report No. 282

6a. NAME OF PERFORMING ORGANIZATI N 6b. OFFICE SYMBOL 7a NAME OF MONITORING ORGANIZATIONManmalian Toxicology Branch (if applicable) US Army Biomedical Research and DevelopmentDivision of Toxicology SGRD-ULE-TO Laboratory6c. ADDRESS (City, State, end ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)Letterman Army Institute of Research Ft. DetrickPresidio of San Francisco, CA 94129-6800 Frederick, MD 21701-5010

Ba. NAME OF FUNDING /SPONSORING 8b. OFFICE SYMBOL 9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION US Army Medical (If applicable)

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Ft. Detrick PROGRAM PROJECT ITASK WORK UNITFrederick, MD 2170-5012 ELEMENT NO. NO. NO. CCESSION NO.

62720A 835 AB DA 30391311. TITLE (include Security Classfication)

Dermal Sensitization Potential of Guanidine Nitrate in Guinea Pigs

12. PERSONAL AUTHOR(S)

Gerald F.S. Hiatt, Earl W. Morgan, and Don W. Korte, Jr.13.. TYPE OF REPORT 13b. TIME COVERED 114. DATE OF REPORT (Year, Month, Day) 1S. PAGE COUNT

Institute FROM3 Jul 84To 27 Aug84 July 1988 2516. SUPPLEMENTARY NOTATION

1.'. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUjP Dermal sensitization; Mammalian toxicology,- Guanidine nitrate

,SABuehler test; Guinea pigs,- Nitroguanidine; Munitions;, k )T_

19 ABSTRACT (Continue on reverse if necessary and Identify by block number)

)Atanidine nitrate, an intermediate product in the synthesis of the triple-base propel-Iait component, nitroguanidine, was evaluated for its potential to produce dermal sensiti-zation in male guinea pigs. The Buehler test, which utilizes repeated closed patch induc-tions with the test compound, was used for this evaluation. No evidence of guanidinenitrate-induced sensitization was obtained in the study., •

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ABSTRACT

Guanidine nitrate, an intermediate product in thesynthesis of the triple-base propellant component,nitroguanidine, was evaluated for its potential to producedermal sensitization in male guinea pigs. The Buehler test,which utilizes repeated closed patch inductions with the testcompound, was used for this evaluation. No evidence ofouanidine nitrAt-induced sensitization was obtained in thestudy.

Key Words: Dermal Sensitization, Mammalian Toxicology,Guanidine Nitrate, Buehler Test, Guinea Pigs,Nitroguanidine, Munitions

AcceSSion For

NTI1S GRA&IDTIC TABUnannounced E o

justificatio CP

Distribution/

Availability COes

IAva an or

;Dist Specal

PREFACE

TYPE REPORT: Dermal Sensitization GLP Study Report

TESTING FACILITY:

US Army Medical Research and Development CommandLetterman 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: Gurida Reddy, PhD

PROJECT/WORK UNIT/APC: 3E162720A835/180!TLB0

GLP STUDY NO.: 84019

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

PRINCIPAL INVESTIGATOR: Gerald F.S. Hiatt, PhD

CO-PRINCIPAL INVESTIGATOR: Earl W. Morgan, DVM, MAJ, VCDiplomate, American College ofVe erinary PreventativeMedicine, American Board ofToxicology.

REPORT AND DATA MANAGEMENT: A copy of the final report,study protocols, raw data,retired SOPs, and an aliquotof the test compound will beretained in the LAIRA-chives.

TEST SUBSTANCE: Guanidine Nitrate

INCLUSIVE STUDY DATES: 3 July - 27 August 1984

OBJECTIVE: The objective of the study was to evaluate thedermal sensitization potential of guanidinenitrate in guinea pigs.

iii

ACKNOWLEDGMENTS

SGT Steven K. Sano, Max Goldnan, PhD, and Joy Bausermanassisted with the research. Richard D. Spieler, RichardKatona, and Charlotte Speckman provided animal care andmanaged the facilities. Callie B. Crosby, Lynda Araiza, andJoAnn Nishimoto provided office mdnagement during tlheperformance of the study and prepayration of the report.

.4.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS INVOLVED IN THESTUDY S

We, the undersigned, declare that GLP Study 84019 wasperformed under our supervision, according to the proceduresdescribed herein, and that this report is an accurate recordof the results obtained.

DON W. KORTE JR., PD / DATEMJ, MS -Study Director

GERALD F.S. HIATT, PhD, / DATE VDACPrincipal Investigator

CPT, VCCo-Principal Investigator

CONIAD R. WHEELER, PhD / DATE'(DACAnalytical Chemist

Ve

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DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO. CALIFORNIA 94129-6800

REPY T'

ATTENTION OF

SGRD-ULZ-QA (70-in) 13 July 198R

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 84019

1. This is to certify that in relation to GLP Study84019, the following inspections were made:

6 March 1984 - Protocol ReviewI August 1984 - Dosing1 August 1984 - Patch/Removal

9 August 1984 - Scoring

2. The institute report entitled "Dermal SensitizationPotential of Guanidine Nitrate in Guinea Pigs, " ToxicologySeries 100, was audited on 23 March 1987.

CAROLYN M. LEWISChief, Quality Assurance

Vi

V vV9r V *~%~'!

TABLE OF CONTENTS

Abst act ... ... ... .... ... ... ... ... .... ... ... ... ..

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

Acknowledgments............................................... ivSignatures of Principal Scientists............................ vReport of Quality Assurance Unit............................. viTable of Contents............................................ vii

BODY OF REPORT

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

Objective of Study........................................1I

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

Test Substance............................................ 1Vehicle for Test Substance............................... 2Positive Control.......................................... 2Vehicle for Positive Control............................. 2Animal Data............................................... 2Husbandry................................................. 3

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

Group Assignment/Acclimation............................3Dosage Levels............................................. 3Compound Preparation..................................... 3Test Procedures........................................... 4Deviations from Study Protocol........................... 5R-aw Data and Final Report Stnrage .... .................. 6

RESULTS....................................................... 6

Guanidine Nitrate......................................... 6Positive Control.......................................... 9Negative Control.......................................... 9

DISCUSSION................................................... 9

Dermal Irritation and Sensitization...................... 9Guanidine Nitrate........................................ 11

CON4CLUSION.................................................. 11

I E F-' l E 2................................................. 12

4.,

TABLE OF CONTENTS (cont.)

APPENDICES ................................................. 13

Appendix A. Chemical Data ............................ 14Appendix B. Animal Data .. . . .. .. 8Appendix C. H;lsto, ical Listinq of Events ..............Appendix D. Individual "rrr i Sco-,es.................. ..

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

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Dermal Sensitization Potential of Guanidine Nitrate inGuinea Pigs--Hiatt et al

INTRODUCTION

Guanidine nitrate is an intermediate product in thesynthesis of nitroguanidine. Nitroguanidine is a primarycomponent of US Army triple-base propellants and is now beingproduced in a Government-owned contractor-operated ammunitionplant. The US Army Biomedical Research and DevelopmentLaboratory (USABRDL), as part of its mission to evaluate theenvironmental and health hazards of military-uniquepropellants generated by US Army mrnitions-manufacturingfacilities, reviewed the nitroguanidine data base andidentified significant gaps in the toxicity data (1). TheDivision of Toxicology, LAIR, was tasked by USABRDL todevelop a genetic and mammalian toxicity profile fornitroguanidine, related intermnediates/by-products of itsmanutacture, and its environmental degradation products.

Objective of Study

The objective of this study was to evaluate the dermalsensitization potential of guanidine nitrate in guinea pigs.

MATERIALS

Test Substance

Chemical name: Guanidine Nitrate

Chemical Abstracts Service Registry No.: 506-93-4

Chemical structure:

H2N \ +

C = NH 2 NO 3

H2 N

Molecular formula: CH5N3HNO3

Other test substance information is presented inAppendix A.

P

I %,' ' ?.,.', > N, " : .. ......... , - ... .... . .. . . .. ..

Hiatt et a]--2

Vehicle for Test Substanc _

Sterile isotonic saline (Travenol Laboratories,Deerfield, IL) was used as the vehicle for guanidine nitr.5e.The expiration date for this lot (7C950X0) was Octobe 1963.

Positive Control

Chemical name: Dinitrochlotobenzene (DNCB)

Chemical Abstracts Service Registry No. : 97-00-7S

Chemical structure:

CIN02

0NO2

Molecular formula: C6H3N2O4Cl

Other positive control substance information ispresented in Appendix A.

Vehicle for Po itivControl

The vehicle for DNCB was a propylene glycol (3%) andisotonic saline (97%) mixture. Propylene glycol (lot number36485) was obtained from Certified Laboratories, Inc.,(Philadelphia, PA). Saline was the same as for the guanilinenitrate vehicle.

Animal Data

Forty-six male guinea pigs, Hartley strain (Charles-River Breeding Laboratories, Wilmington, MA) we-,. studied.They were identified individually '.ith ear tags numbered84E047 to 84E092, inclusive. Two animals were selected torquality control necropsy evaluation on receipt. Four of theanimals were selected for a pilot study to determine anonirritating dose level. Animal weights on receipt (3 Jul84) ranged from 178 to 225 g. Additional animal data appearin Appendix B.

%S

Hiatt et al--3

L-3ban

Guinea pigs assigned to this study were cagedindividually in stainless steel, wire mesh cages in racksequipped with automatically flushing dump tanks. The diet,fed ad libitum, consisted of Certified Purina Guinea Pig ChowDiet 5026 (Ralston Purina Company, Checkerboard Square, St.Louis, MO); water was provided by continuous drip from acentral line. Temperature within the animal room wasinitially maintained in the range 18.9 to 22.2*C. Inresponse to evidence of respiratory infection in a few of thetest animals, room temperature was increased on 25 Jul 84 andmaintained in the range of 20.0 to 23.8'C. Relative humiditywas maintained in the range of 42 to 69%, with occasionalspikes as high as 88% during steam line adjustments and roDmwashing. The photoperiod ;as 12 hours of light per day.

METHODS

This study was conducted in accordance with LAIR SOP-OP-STX-82 "Buehler Dermal Sensitization Test" (2) and EPAguidelines (3).

Group Assignment/Acclimation

The guinea pigs were quarantined for 15 days beforeadministration of the first induction dose. During thequarantine period, they were checked daily for signs ofillness and weighed once a week. Ten animals were assianedto each of four groups by a stratified randomizationtechnique based on their body weights.

Dosage Luvels

Three animal groups comprise the basis for this report.Dermal sensitization potential was evaluated in a test groupreceiving three weekly induction doses of 10% guanidinenitrate and, after a two-week delay, a challenge dose at thesame concentration. Dinitrochlorobenzene, a known potentsensitizing agent (4), was applied to another group, at a0.1% concentration, as a positive control. A negativecontrol group received 10% guanidine nitrate only on the daycf challenge dosing.

Compound Preparation

Guanidine nitrate was moderately soluble in isotonicsaline resulting in a milky solution with some finesuspension. The dinitrochlorobenzene dosing solution was

A-

Hiatt et al--4

prepared by first adding 30 mg DNCB to 1.0 ml of propyleneglycol and heating until it dissolved (approximately 400C).To this, 29 ml of 0.9% sodium chloride solution were added,to give a final concentration of 0.1% (w/v). This solutionwas heated to 650C and vortexed before application to keepthe DNCB in solution. DNCB solutions were prepared fresh foreach application day.

Test Procedures

The closed patch dermal sensitization test proceduresutilized in this study were developed by Buehler and Griffitx(5-7) to mimic the repeated-insult patch test for humans.Test compounds were applied for six hours under a closedpatch once a week for three weeks during the induction phase.The same application site was used for each induction dose.To distinguish between reactions from repeated insult andsensitization, duplicate patches of the challenge dose wereapplied, one on the old site and one on a new site. Todistinguish between reactions from primary irritation andsensitization, a negative control group was added whichreceived only the challenge dose.

During the induction phase, the test and positivecontrol groups were dosed with 0.5 ml of the appropriatesolution/suspension applied topically under a 2.5-cm 2 gauzepatch. This procedure was performed for three consecutiveweeks (18, 25 Jul, and 1 Aug for the DNCB-positive controlgroup; 25 Jul, 1 Aug, and 8 Aug for the 10% guanidine nitratetest group). Twenty-four hours before each dosing an 8-cm 2

area on th)e left flank of the animal was clipped withelectric clippers (Oster® Model A5, size 40 blade, SunbeamCorp., Milwaukee, WI) and then shaved with an electric razor(Norelco® Speed Razor Model HP1134/S, North American PhillipsCorp., Stamford, CT). The patch was taped with Bleriderm®hypoallergenic surgical tape (3M Corp., St. Paul, MN) to thesame site each time, and the animal was wrapped several timeswith Vetrap® (3M Corp., St. Paul, MN) . The patch was left inplace for six hours. When the wrap and patch were removed,the area under the patch was marked off with a felt--tipsurgical marking pen for ease of scoring.

Animals were challenged two weeks (15 Aug for the DNCB-positive control and negative control groups; 22 Aug for the10% guanidine nitrate group) following the third inductiondose. Test group and positive control group animals receivedtwo 0.5-ml doses each, one applied to the old site on theleft flank and the other to a new site on the right flank.Negative control animals received only a single 0.5-ml dose,

PON.

Hiatt et al--5

applied to the left flank. Procedures for clipping, shaving,and wrapping and the exposure period remained the same.

In Buehler's procedure, skin reactions are scored 24 and48 hours after the challenge dose only. In the presentstudy, skin reactions were scored 24 and 48 hours after eachinduction dose as well. Skin reactions were assigned scoresaccording to Buehler's grading system: 0 (no reaction), 1(slight erythema), 2 (moderate erythema), and 3 (markederythema). Results are expressed in terms of both incidence(the number of animals showing responses of 1 or greater ateither 24 or 48 hours) and severity (the sum of the testscores divided by the number of animals tested). Resultsfrom the left flank are compared with right flank and withthe negative control group.

Some modifications of Buehler's procedures were made.Instead of placing animals in restraint during the 6-hourexposure period, the animals were wrapped several times withan elasticized tape to hold the patch in place.Consequently, the animals were able to move Ebout freely intheir cage during the exposure period. Buehler aid Griffith(7) also recommended depilating the day before the challengedose. For consistency with induction procedures, this stepwas replaced by clipping the animals.

A historical listing of study events appears in

Appendix C.

Deviations from Study Protocol

This study was conducted in accordance with the protocoland applicable amendments with the following exceptions:

A 0.5 level (very slight erythema) was added to thescoring system to allow for borderline responses.

The DNCB solution was maintained at approximately 65*Cbefore dosing the guinea pigs. This was necessary to keepthe DNCB in solution but did not result in thermal insult tothe animals' skin as the aliquot for dosing cooled quicklyduring pipetting and application to the patch. Appreciablesensitization was produced by DNCB with this method.

A pilot study using 100%, 10%, 1% and 0.1%concentrations of guanidine nitrate, to evaluate the acutedermal irritation potential of guanidine nitrate in guineapigs, was performed in four animals before the formal study.Irritation produced by the 100% suspension was equivocal inthis pilot study; the initial formal induction dosing was

Hiatt et al--6

therefore performed at the 100% level. However, significantirritation occurred in the test animals in response to thisfirst induction application, and this group of animals wasremoved from the study. In accordance with the SOP, avehicle control group had received saline only at this firstinduction dosing. These animals from the vehicle controlwere substituted for the animals removed from the study,forming a new test group. This new test group was dosed wiltha 10% suspension which was nonirritating.

Two animals died during the study period, one (84F065)from the DNCB-positive control group and the other (84FG84)from the negative control group. A third animal (84F075)from the discontinued 100% guanidine nitrate group also died.Postmortem findings revealed preexisting adrenal and hepaticlesions for animal 84F065, viral pneumonia for animal 84F075,and pulmonary edema and myocardial hemorrhage for animal84F084.

It is believed that these deviations from the protocoldid not adversely affect the study, as reflected by theresults in the negative and positive control groups.

Raw Data and Final Report Storage

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

Guanidine Nitrate

Tables 1 and 2 summarize the incidence of reactions 24and 48 hours after each dose. No reaction was observed inresponse to guanidine nitrate, either at 24 or 48 hours.This lack of response is reflected in Tables 3 and 4, whichreport the severity of skin reactions at 24 and 48 hours.Response severity for each group is calculated by summing thescores of responding animals and dividing by the total numberof animals within that group. For guanidine nitrate noresponses were obtained, and therefore severity scores werezero at all times.

NZMV .QM

Hiatt et al--7

TABLE 1

Incidences of Skin Reactions after 24 Hours

Induction ChallengeTest Group First Second Third Left Right

GuanidineNitrate 0/10 0/10 0/10 0/0 0/10

NegativeControl* 1/9 ---

DNCB> 1/10 7/9 9/9 9/9 9/9

The Negative Control Group received only a challenge dose

of the test compound. Group size decreased due to non-compound-related death.

> Group size decreased due to non-compound-related death.

TABLE 2 0

Incidences of Skin Reactions after 48 Hours

Induction ChallenaeTest Group First Second Third Left Right

GuanidineNitrate 0/10 0/10 0/10 0/10 0/10

Negative

C o nt ro l * ........ . 0/ 9

DNCB> 0/10 5/9 7/9 6/9 7/9

The Negative Control Group received only a challenge dose

of the test compound. Group size decreased due to non-compound-related death.

> Group size decreased due to non-compound-related death.

h N

Hiatt et al--8

TABLE 3

Severity of Skin Reactions after 24 Hours

Induction Chalenge

Test Group First Second Third Left Right

GuanidineNitrate 0.0 0.0 0.0 0.0 0.0

NegativeControl* --- 0.06

DNCB 0.1 0.78 1.22 1.11 1.33

* The Negative Control Group received only a challenge doseof the test compound.

TABLE 4

Severity of Skin Reactions after 48 Hours

Induction ChallengeTest Group First Second Third Left Right

GuanidineNitrate 0.0 0.0 0.0 0.0 0.0

NegativeControl* ......... 0.0 ---

DNCB 0.0 0.56 0.78 0.78 1.11

The Negative Control Group received only a challenge dose

of the test compound.

Hiatt et al--9

Positive Control

Dinitrochlorobenzene produced a marked response at alltime points after the first induction dose. Between 70% and100% of the DNCB-treated animals exhibited a response 24hours following the second or third induction and challengedoses. Between 50% and 80% of these animals still exhibiteda response 48 hours following the same doses; the reactionswere therefore persistent. Severity scores for theseresponses to DNCB ranged from 0.7 to 1.33 at the 24-hourscoring period (Table 3). The highest score, 1.33, wasobserved on the right (non-induction) flank in response tothe challenge dose. By 48 hours the reactions had subsidedsomewhat; consequently, the severity range decreased tobetween 0.56 and 1.11 (Table 4).

Negative Control

Only one response was observed in the negative control(challenge dose of guanidine nitrate) group, a 0.5(borderline) score on one animal at 24 hours.

Individual 24-hour and 48-hour scores for all animalsappear, by group, in Appendix D.

DISCUSSION

Dermal Irritation and Sensitization

Most skin reactions occurring from contact withchemicals can be classified as either irritation orsensitization. Both reactions present as inflammation of theskin; the difference between irritation and sensitization isthe mechanism responsible for this inflammation. Primaryirritation is direct inflammation in response to injury tothe skin produced by the eliciting chemical. Irritation is alocally mediated response ranging from mild reversibleinflammation to severe ulceration progressing to necrosis.Sensitization is manifested as indirect inflammation mediatedby components of the immune system in response to activationby the eliciting chemical (8). Dermal sensitization isusually a delayed hypersensitivity or cellular immunologicreaction. Although both types of reactions can appeargrossly similar in experimental animals and may even beproduced by the same agent, it is possible to distinguishbetween them. Irritation is an immediate response and can beproduced upon first contact with the chemical, whereassensitization requires at least one innocuous "conditioning"exposure before a reaction can be elicited.

Hiatt et al--lO

Irritative responses usually rcquire a relatively highconcentration or dose of the offending chemical, whereassensitization reactions may occur in response to minutequantities. Essentially all individuals in a population willexpress an irritative response to a reactive chemical,provided the dose is high enough, whereas only a fraction Ythe population normally becomes sensitized to the samechemical. A fully developed response can be produced by lirstcontact with an irritant, but initIal contact with asensitizer produces no reaction (a conditioning exposure isnecessary). Unless there is accumulation of damage,subsequent exposures to an irritant produce inflammation ofessentially similar intensity/severity, whereas the reactionto a sensitizer often increases over 2 to 4 exposures afterthe initial contact. An irritant produces inflammation ofrapid onset with short duration, whereas a sensitizationreaction is somewhat delayed and prolonged. The inflammatoryresponse to an irritant may spread beyond the area of contact,whereas sensitization reactions are usually circumscribed.

The features of irritation and sensitization wereapplied by Buehler and Griffith (5-7) to establish guidelinesfor differentiation between the two. In evaluating a dermalsensitization study they recommend comparing the results froma challenge dose in the experimental group with those for thenegative control group:

Irritative Responses:

- occur in a large proportion of test animals.- develop in response to the first or second exposure.- usually fade within 24 to 48 hours, unless damage i s

severe.- may be stronger at challenge to a previously unexposed

area of skin (contralateral flank).

Sensitization Reactions:

- occur in only a few animals, unless the compound is apotent sensitizer.

- are absent after the initial (conditioning) exposure,but appear in response to subsequent exposures.

- develop slowly, the intensity/severity of inflammationoften is greater at 72 to 96 than at 24 to 48 hours.

- increase in intensity/severity from one exposure tothe next (at sites previously exposed or unexpoeo) .

Dermal irritancy potential is evaluated by the method ofDraize et al (9) in which the chemical is applied once, athigh concentration, and the resulting acute inflammatoryreaction is graded. Evaluation of sensitizing potential is

- r '" ' ' , 0,

Hiatt et al--ll

accomplished by repeated application, at lower non-irritatingconcentrations, over a few weeks. There is then a latentperiod, usually two weeks, to allow the immune system toelaborate and increase its specific response to the chemical.A challenge dose is then given, and the resultinginflammatory response is graded. Analysis of the incidence,severity, and timing of the response to the challenge doseestimates the sensitizing potential of the study compound.

Guanidine Nitrate

Guanidine nitrate was evaluated for its ability toelicit a delayed-hypersensitivity or cellular immunologicreaction via contact with the skin. Guanidine nitrateproduced no response indicative of the potential to elicitdermal sensitization when evaluated according to the method(5-7) of Buehler and Griffith. This finding closelyparallels the result of an earlier study on the hydrochloridesalt of guanidine (10). In that study, guanidinehydrochloride exhibited no sensitizing potential in theBuehler Dermal Sensitization Test. B

Sensitization produced by guanidine nitrate would havebeen detected by this study. A hypersensitivity-typeresponse was reliably elicited by DNCB in the present groupof animals. This response to DNCB was characteristic of thatobserved previously within the Institute (10). Although DNCBis capable of producing primary irritation, thecharacteristics of the responses observed in this study areindicative of a reaction due to sensitization. Theconcentration of DNCB used for induction and challenge is toolow to produce primary irritation. Also the response to DNCBwas observed primarily after two or more exposures, and theseverity generally increased with the number of previousexposures.

CONCLUSION

Guanidine nitrate, based on a zero percent sensitizationrate in this study, exhibited no potential for inducingdermal sensitization.

Ug

Hiatt et al--12

REFERENCES

1. Kenyon KF. A data base assessment of environmental f.aspects of nitroguanidine. Frederick, MD: US Army Medic aLBioengineering Research and Development Laboratory, 1982;DTTC No. ADA125591.

2. Buehler dermal sensitizaltion test. LAIR StandardOperating Procedure OP-STX-82. Presidio of San Francisco,CA: Letterman Army Institute of Research, 18 May 1984.

3. Environmental Prottction Agen'y. Office of Pesticrii.-and Toxic Substances, Office of Toxic Substances ('1-792)Dermal sensitization. In: Health effects test guidelines.Washington, DC: Environmental Protection Agency, August II-YZEPA 560/6-82-001.

4. Landsteiner K, Jacobs J. Studies on sensitization ofanimals with simple chemical compounds. J Exp Med 1935;61: 643-656.

5. Buehler EV. Delayed contact hypersensitivity in theguinea pig. Arch Dermatol 1965; 91:171-175.

6. Griffith JF. Predictive and diagnostic teting forcontact sensitization. Toxicol Appl Pharmacol 1969; Suppl3:90-102.

7. Buehler EV, Griffith JF. Experimental skin sensitizationin the guinea pig and man. In. Maibach HI, ed. Animalmodels in dermatology. Edinburgh: Churchill Livingstone,1975:56-66.

8. Mathias CGT. Chemical and experimental aspects ofcutaneous irritation. In: Marzulli FN, Maibach HF, eds.Dermatotoxicology. Washington, DC: Hemisphere PublishingCorporation, 1983: 167-168.

9. Draize JH, Woodard G, Calvery HO. Methods for the studyof irritation and toxicity of substances applied topically tothe skin and mucous membranes. J Pharmacol Exp Ther 1944;82.3/7-390.

10. Hiatt GFS, Morgan EW, Korte DW. Dermal sensit-izationpotential of guanidine hydrochloride in guinea pigs.Tuxicology Series 84. Presidio of San Francisco, CA:Letterman Army Institute of Research. Institute Report No..i0, January 1986.

Hiatt et al--13

Appendix A. Chemical Data................................... 14

Appendix B. Animal Data..................................... 18

Appendix C. Historical Listing of Events.................... 19

Appendix D. Individual Dermal Scores........................ 22

Hiatt et al--14

Appendix A: CHEMICAL DATA

Chemical Name: Guanidine Nitrate

Lot Number: 123820

Chemical Abstracts Service Regist-ry No.: 506-93-4

LAIR Code: TP030

Chemical Structure:H 2N\ +

C NH2 NO 3

H 2N

Molecular Formula: CH 6N3 -NO3

Molecular Weight: 122.1

Physical State: White crystalline powder

Melting Point: 214OC 1

Analytical Data:

Infrared spectrophotometry was performed and the spectrumobtained 2 was identical to the Sadtler spectram 3 for GuanidineNitrate. Major absorption peaks were observed at 3400(broad), 3200, 1665, 1575, 1400, 1385, and 825 cm-1 . Thc'grade 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.

2 Wheeler CR. Nitrocellulose-Nitroguanidine Projects.

Laboratory Notebook #84-05-010.2, p. 62. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.

3 Sadtler Research Laboratory, Inc., Sadtler standard spectra,Philadelphia: The Saduler Research Laboratory, Inc., 1962:Infrared Spectrogram #14498.

Hiatt et al--15

Appendix A (cont.): CHEMICAL DATA

Stability of Dosing Formulations:

The stability of guanidine nitrate in aqueoussolution is demonstrated by the absorbance valuesobtained for a standard solution containing 20 pg/ml ofguanidine nitrate. This solution was prepared on 25 Mayand kept at room temperature over the period ofanalysis. From 25 May to 6 June, four assays of thissolution were performed yielding statistically identicalabsorbanco values. 4 Since the Voges-Proskauer assay isspecific for unsubstituted and mono-substitutedguanidines, the data demonstrate that aqueous soiu, :ns 0of guanidi- - nitrate are stable for a period of at least12 days (Table 1).

TABLE 1: Stability Assay of a 20 gg/ml Standard Solution ofGuanidine Nitrate

Date of Analysis Absorbance Values*

25 May 84 1.74 ± 0.0229 May 84 1.76 ± 0.0530 May 84 1.76 ± 0.026 Jun 84 1.76 ± (.02

• Values are mean ± S.D. for '-hree replicates.

4 Wheeler CR. Nitrocellulose-Nitroguanidine Projects.Laboratory Notebook #84-05-01,).2, pp 55-57,59. LettermanArmy Institute of Research, Fresidio of San Francisco, CA.

U0

. .. . _ r, .,... . ,... .. - ., .. ., .,: .,. .,2 " .'< V ¢2 g¢: .W 2 .¢ <[ji%

Hiatt et al--16

Appendix A (cont.) : CHEMICAL DATA

POSITIVE CONTROL

Chemical Na:me: 1--11hic-:-, 4-Jinitrcbenzene

Alternate Chemical Nam&,:2,-ilt ohroeLCU

Ch-emical Abstracts Serv cc, P-q, stry N,,mber:9VC

Chemical Striicture:

0INO2

0NO2

-Molecolar orua:C 6 "H3 N2 C04C1

Physical State: Yellow crystals

Melting Point : 2-540 C1

Purity:Thte compound was designated as 951. pure by source,.

Analytical Data:Chemical1 analysis was performed as follows:

In frared spect r ,- were obtaid ned with a Perk in-Flmor 9h3 2spectrometer. ,~e o'C~ n maqne'-,ic resonanc,-e (NrMR) pwere onod~ f a Varian X1,300 inst uinent- wi tnt-t ramethyls -' in- i: r iit-r-nai standard ind chemici

5fl ~ ~ ~ J j)(1''~di~pa sp' million (d) ] lxwresolut in n *. analystis wa crformef:d wi, Fa 1 aXMS-2SOFFA (30 LB, 1 capi lr:C ALuMr-) el

The f ol Lrwinq ,i err obtaine-d;3 10,4, 2 P 7 7, 13, -i F IE 17 50C, 175, 1 I , I51542, 1349, 1246, 1i. 6, 1046, 917, 902, 4732 cm-1 . The IF o rr wa-s very close e-~~'''reference spectru.m. Dft er-nc-: we-e d tfiner speclral ro u 4rbc eion. the 3--instrument. NXPR (CTC1 3 ) d *7.78 (1 11, ,1 j .11I:5.38 (1 H, q, Jurtho - 8.'7 Jmeta =3.6 fi7), 0.i-74(1 H, d, Jmeta =2.4 H,,) Thie spectrum of 1>308C wasidentical to thp Al. dricL re ~orence spectrum. (uC-MSAnalysis: A plot- o4f he1, tot ii ion current ver-SU' ScCn-

Hiatt et al--17

number showed one major peak for DNCB with only tracesof other compounds (not identified) . Molecular ionmasses (rn/z) of 202 and 204 confirmed the identity ofthe major peak as DNCB.7

Lot Number: 11F-0543

Source: Sigma Chemical Co.

St. Louis, MO

lWinciholz M, ed. The Merck Index. 10th ed. Rahway, NJ:Merck and Co., Inc., 1983:300.

2Wheeler CR. Toxicity Studies of Water Disinfectant.Laboratory Notebook #85-12-021, pp. 9-10. Letterman ArmyInstitute of Research, Presidio of Sari Francisco, CA.

3Ibid. pp. '-1-12.

4 1ld. pp. 13-16.

5'Sadtler Research Labot:atory, Tinc., Sadt 1er standard spectra.Philadelphia: The Sadtler Research Laboratory, Inc., 1962:Infrared spectrogram #964.

6Pouchert CJ. The Aldrich Librar :. -f NMR Spectra. V.ol. 1,2nd ed. Milwaukee: Aldrich Chemical Co., 1981:1173,spectrum D.

7WheeLer CR. Toxicity Studies of Water Disinfectant.Laboratory Notebook #85-12-021, pp. 13-15. Letterman ArmyInstitute of Research, Presidio of San Francisco, CA.

XS

Hiatt et al--18

Appendix B: ANIMAL DATA

Species: Cavia porcellus

Strain: Hartley

Source: Charles River B,eedini !iborator-esWilmington, tLA

Sex: Male

Date of Birth: 15 June 1984

Method of randomization: Weight nias, stratified animalallocat ion

Animals in each group: 10 male 1 :imals

Condition of animals at start of -tudy: Normal

Identification procedures: Ear tag, tag numbers 84E047 to84E09? inclusive.

Pretest conditioning: Quarantine/acclimation 3-18 July 1984

Justification: The laboratory guinea pig has proven to be as--sitive and reliable model for detection ofdelayed hypersensitivity from dermal contact.

'Ui

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Hiatt et al--19

Appendix C: HISTORICAL LISTING OF EVENTS

DateEvn

3 Jul 84 Forty-six animals arrived at LAIR. Animalswere examined, placed in cages, and fed.Animals were ear tagged and weighed. Twoanimals were submitted for necropsy as qualitycontrols.

3 Jul - Animals were checked daily.20 Aug 84

10,17,24, Animals were weighed.31 Jul,14,21 Aug 84

10 Jul 84 Four pilot animals were shaved. Pilot dosing

was solution prepared.

11 Jul 84 Pilot animals were patch tested.

12 Jul 84 Pilot animals were scored for 24-hour skinreaction.

13 Jul 84 Pilot animals were scored for 48-hour skin •reaction.

16 Jul 84 Pilot results were evaluated, testconcentration was determined, animals wererandomized into groups.

18 Jul 84 Test animals, except negative control group,were given first induction dose.

19 Jul 84 Test animals, except negative control group,were scored fcr 24-hour skin reaction.

20 Jal 84 Test animals, except negative control group,were scored for 48-hour reaction. v

20 Jul 84 100% guanidine nitrate suspension wasdetermined to be too irritating for inductiondosing. Test animals were replaced with thosefrom saline control group. One test animalwas found dead (stress, pneumonia).

I

iiiatt et a7--20

Appendix C (cont.): HISTORICAL LISTING OF EVENTS

Datevnt

24 Jul 84 All animals, except iegative control group,were clipped and shaved.

25 Jul 84 Positive control animals were giver secondinduction dose. Tes': (10% guanidine nitrate)animals were given first induction dcse. OneDNCB-treated animal ,.as found dead (adren, nihepatic lesions, str .ss) . Room temperatureSett inc was increase !.

26 Jul 84 Test and positive cotrol groups were scored t0224-hour skin reactio:i.

27 Jul 84 Test and positive cotrol qroups were scored fre48-hour skin reaction.

31 Jul 84 All animals, except negative control group,were clipped and shaved.

1 Aug 84 Positive control animals were given thirdinduction dose. Tesc (10% guanidine nitrate)animals were given second induction dose.

2 Auq 84 Test and positive control groups were scored for24-hour skin reaction.

3 Aua 84 Test and positive control groups were scored for48-hour skin reaction.

7 Aug 84 Test grcp was clipp, d and shaved. Fr ir pilotaniIaI1: wvt< sa' ifi-ed.

8 Aug 84 Test group (10% guan-dine nitrate) was giventhird induction dose.

9 Aug 84 Test group was scored for 24-hour skin reaction.

10 Aug 84 Test group was score i for 48-hour ski reaction.

14 A.g 84 Positive and nega.ivi, control grouns wereclipped and shaved.

15 Aug 84 Positive arld negative control groups were giv,nchallenge dose. One negative control animalwas found dead (stress).

Hiatt et al--21

Appendix C (cont.): HISTORICAL LISTING OF EVENTS

Date Event

16 Aug 84 Positive and negative control groups were scoredfor 24-hour skin reaction.

17 Aug 84 Positive and negative control groups were scoredfor 48-hour skin ieaction.

21 Aug 84 Test group was clipped and shaved.

22 Aug 84 Test (10% guanidir:e nitrate) group was givenchallenge dose.

23 Aug 84 Test group was scored for 24-hour skin reaction.

24 Aug 84 Test group was scred for 48-hour skin reaction.

27 Aug 84 Thirty-seven study animals were sacrificed.

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