AD-A203 802

Institute Report No. 320

Mutagenic Potential ofPhysostigmine Salicylate in the Ames

Salmonella/Mammalian Microsome Mutagenicity Test

Suzanne E. Sebastian, BA, SPC, USAand

John W. Harbell, PhD, MAJ, MSC

DTICGENETIC TOXICOLOGY BRANCH E L ECT E

DIVISION OF TOXICOLOGY JN51~S JAN 2 5 8

H

December 1988 Toxicology Series 203

LETTERMAN ARMY INSTITUTE OF RESEARCHPRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129

D!~jSTINSAEMNTA89 0- 0 7Apprved for publbe reloas;

Dlstibution UnDimited

UNCLASSIIElSECURITY CLASSIFICATION OF THIS PAG

REPORT DOCUMENTATION PAGE Form Approved

la. REPOR' SFCURITY CLASSIFICATION b RESTRICTIVE MARKINGS OSN.00- 8

1UNCLA-D-SIFIED2a. SECURITY CLASSIFICATION AUTHORITY 3. DISTRIBUTION /AVAILABILITY OF REPORT

2b. DECLASSIFICAT ION IDOWNGRADING SCHEDULE APPROVED FOR PUBLIC RELEASE;_____________________________ DISTRIBUTION IS UNLIMITED.

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

Insti1.ute Report No.: 3206a. NAME Ci1: PERFORMING ORGANIZATION 6b. OFFICE SYMBO0L 7a. NAME OF MONITORING ORGANIZATIONGenetic Toxicology (if applicable) US Army Medical ResearchDivisicr. of Toxicology jSGRD-ULE-T Institute of Chemical Defense6c. ADDRESS (City, State, and ZIP Code) 7b. ADDRESS (City. State, and ZIP Code)Letterman Army Institute of Research Aberdeen Proving GroundPresidin of San Francisco, CA 94129-680C Maryland 21010-5425

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

Research & Development Co dand8c. ADDRESS (City, State, and ZIP Code) 10 SOURCE OF FUNDING NUMBERSFort Detrick PROGRAM PROJECT TASK WORK UNITFrederick, Maryland 21701-5012 ELEMENT NO NO NO. ACCESSION NO.

________________________________62734 A875 IBC IDAOH0366

11. TITLE (include Security Classification)(U.1) Mutagenic Potential of Physostigmie S, i1cylate in the Ames .Salmonella/M;s-mralian Microsome Mutagenicitk Test12 PERSONAL AUTHOR(S)

SE Sebastian and Harbell13a. TYPE Of REPORT 13b. TIME COVERED 114. DATE OF REPORT (Yea, Month, Day)' IS. PAGE COUNTIn s t it-U t e FROm30JAN87 Td3APR8P Decembr 198216 SUPPLEMENTARY NOTATION

Tozic ilogy Series No. 203 %17. COSATI CODES 18. SUBJECT T%&)MS (Continue on reverse if necessary and identify by block number)

FiELDI GROUP ISUB-GROUP Physostigmine Salicylate, Mutagenicity, GeneticIToxicology, Ames Test ,>4*

I ~I .

19,ABSTRMT (Conti nue on reverse if necessary and identify by block number)The alutagenic potential of PHYSOSTIGMINE SALICYLATE was assessed by using

icesSalmonella/Mammalian Microsome Mutagenicity Test. Tester strainsSTA98, TAlOO, TA1O2, TA1535, TA1537, and TA1538 were exposed to doses

;.rc frorni 0.2 mg/plate to 0.00064 mg/plate. The test compound was not2itagenLc 1uoder conditions of this test.

I20 DISTRIBUTION /AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY CLASSIFICATION'COUNCLASSIFIED/JNLIMITED 0 SAME AS RPT C3 DTIC USERS UNCLASSIFIED

22a NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (include Area Code) 22c OFFICE SYMBOL

IF-WUN S. BEATRICE 'COL, MC_41)5130 ';,DQ.DO Form 1473, JUN 86 Previous editions are obsol eto. SECURITY CLASSIFICATION OF THIS PAGE

UNC'LASSIVIED

ABSTRACT

The mutagenic potential of PHYSOSTIGMINE SALICYLATE wasassessed by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Test. Tester strains TA97, TA98, TAI00, TAI02,TA1535, TA1537, and TA1538 were exposed to doses ranging from0.2 mg/plate to 0.00064 mg/plate. The test compound was notmutagenic under conditions of this test.

Key Words: Mutagenicity, Genetic Toxicology, Ames Test,PHYSOSTIGMINE SALICYLATE

Aocession For

NT IS GPA&I moor

DTIC TABUnaxnunced ElJus ruation

By_Distribution/

Availebility Code93

Dist j Speciali6 Lri_1

PREFACE

TYPE REPORT: Ames Test 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 Medical Research Institute of Chemical DefenseAberdeen Proving Ground, MD 21010-5425Project Officer: LTC J. von Bredow, PhD, MSC

PROJECT/WORK UNiT/APC: 3M162734A875/308/TLEO

GLP STUDY NUMBER: 87001

STUDY DIRECTOR: MAJ John W. Harbell, PhD, MSC

PRINCIPAL INVESTIGATOR: Suzanne E. Sebastian, BA, SPC, USA

REPORT AND DATA MANAGEMENT:

A copy of the final report, study protocol, retiredSOPs, stability and purity data on the test compound,and an aliquot of the test compound will be retained inthe LAIR Archives.

TEST SUBSTANCE: PHYSOSTIGMINE SALICYLATE

INCLUSIVE STUDY DATES: 30 January 1987 - 3 April 1987

OBJECTIVE:

The objective of this study was to determine themutagenic potential of PHYSOSTIGMINE SALICYLATE (LAIRCode TW73) by using the Ames Salmonella/MammalianMicrosome Mutagenicity Test.

iii

u I I II

ACKNOWLEDGMENTS

SGT Lillie D. Witclier, BS5, USA, and SGT Gayle Orner, BS,USA provided restarch assistance. MAJ Don W. Korte, Jr.,PhD, MSC provided pro,;ram rvlidance and facilitated the-induct of the stutdy -nd thle pub.Lication of tche final report.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS INVOLVED IN THESTUDY

We, the undersigned, declare that GLP SLudy 87001 wasperformed under our supervision, according to the proceduresdescribed herein, and that this report is an accurate recordof the results obtained.

jQHN W. HARBELL, PhD / DateMAJ, MSStudy Director

Principal Investigat L

CONRAD R. WHEELER, PhD / DATEDACAnalytical Chemist

v

N.'" DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITUTE OF RESEARCH

PRESIDIO OF SAN FRANCISCO, CALIFORNIA 941129

REPLY TO

ATTENT(ON OF

SGRD-ULZ-QA q December 1988

MEMORANDUM FOR RECOM;)D

sUBJEcr GLP Compl iance for GL" Study 871'91

1. This is to certify that in relation to LAIR GLP Study 87001,the following inspections were maie:

26 January 1987 - Protocol Review13 .'ebruary 1987 - Plate Counting (Pilot)31 March 1987 - Dosing (Final Assay)

2. The institute report entitled l"Mutagenic Potential ofPhysostigmine Salicylate in the Ames qalmone~la/MammalianMicrosome Mutant.nicity Test, " Toxicology Series 203, was audite.'on 24 November 1987.

CAROLYN M. LEWIS, MSDiplomate, American Board of Toxico-<'ChieF, Qu.il.ity Assurance

vi

TABLE OF CONTENTS

Abstract....................................................1i

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

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

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

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

Table of Contents......................................... vii

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

Objective of the Study................................. 2

MATERIALS AND METHODS....................................... 2

Test Compound.......................................... 2Test Solvent........................................... 2Chemical Preparation................................... 3Test Strains........................................... 3Test Format............................................ 3Data Interpretation...................5Deviations from the Pooo/O............Storage of the Raw Data and Final Report.............. 5

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

DISCUSSION.................................................. 7

CONCLUSION.................................................. 7

REFERENCES................................................. 11

APPENDICES................................................. 12

Appendix A: Chemical Data............................ 13Appendix B: Individual Plate Scores................. 15

OFFICIAL DISTRIBUTION LIST................................. 20

vii

Mutagenic Potential of PHYSOSTIGMINE SALICYLATE in theAmes Salmonella/Mammalian Microsome Mutagenicity Test-Sebastian and Harbell

INTRODUCTION

Soman, the primary nerve agent utilized by threat forces,is refractory to the standard antidotal therapy, atropine andpralidoxime (2-PAM), fielded by the US Army. Consequently,the highest priority has been placed on fielding a moreeffective treatment regimen. A regimen incorporatingpyridostigmine as a prophylactic agent, combined with standardatropine/2-PAM therapy, has proven extremely effective inreducing mortality of Rhesus monkeys exposed to multilethalconcentrations of soman (1). However, these animals require aprolonged period of recovery during which they are completelyincapacitated. This has been attributed to the quaternarynature of pyridostigmine, which does not cross the blood-brainbarrier and thus only protects the peripheral nervous system.Consequently, a tertiary carbamate, PHYSOSTTGMTNE, has beenproposed for the prophylactic regimen since it would protectthe central nervous system in addition to the peripheralnervous system. Experimental studies support this hypothesisas animals pretreated with physostigmine before exposure tosoman recover at a faster rate than animals pretreated withpyridostigmine (2,3). An enhanced rate of recovery ofsoldiers from a multilethal exposure to soman would produce adecided advantage in maintaining a fully functional militaryunit during a future conflict.

Although PHYSOSTIGMINE has been available for more than acentury (4), little directed research on its mutagenicpotential has been conducted. Consequently, the Division ofToxicology, Letterman Army Institute of Research, was tasked bythe US Army Medical Research Institute of Chemical Defense toprovide a mutagenicity profile of PHYSOSTIGMINE SAIICYLATE.This report describes the results of a mutagenicity study ofP1Y.';:;T1GMlNE SALICYLATE in th' Ames test.

The Ames Salmonella/Mammalian Microsome MutagenicityTest is a short-term screening test that utilizes histidineauxotrophic mutant strains of Salmonella typhimurium todetect compounds that are potentially mutagenic in mammals.A mammalian microsomal enzyme system is incorporated in thetest to increase sensitivity by simulating in vivo metabolicactivation of the test compound. The Ames Test is an

I -,t i an A ;d 11 C1 - e I I

inexpensive yet highly predictive and reliable test ford-t ecting MUtaer.ic ictivity and thuF; rrcinoqenic pote(ntial

This evaluan :cn of PY9TGIESAL1CYLATE utilizes a~aisonof the A-es SairT ,r11 /Mammaiian Microsorne

'..tagenicity Test (6) .Two new tester strains, a framce-shift5s..rain (TA97) and a _--,rain -arrying an cchre mutation con anl.ilticopy plasrnid (TAIO2) , ore added t~o -he standard toster

C "Jective of the Study:

rhe objective of this study was to determine therm,-tageflic potential of PHYS(GSTIGMITNE SALICYLATE (LAIR Code

'q7)by using theit revised A.-ioc- .co--lmonplla/Mammalian''urow-ome Mut aqeni cit y Tost.

M~kTERIALS AND METHODS

Sst- CcpLound

'Chemical Namea: zPiiYgC,"STTG%1TNE SALICYLATE

LAIR Code Number: TW*73

Physical State: Wh ite :-rysti'a1i:~ aid

Source: Division of Experimental TherapeuticsWRAIR, Washington, Dl.C.Requesi-ed by LTC vc~n B.- -.ow, USAMRICD

Storage: PPYSOSTIGMINE _A,,TCYLA'"E was received andci~iqod heLAI Cde umerTW. The test compound was

w.1ored in a desiccator at ~2oZuntil used.

Chemical Properties/Ana'Lsis: Data provided by WRAIRc'haracterizing the ch(--. cal omposition and purity of theL,2st material are pr'ed~ cin Appendix A along withcxifirmatory anialysis ot the If'ot material performed by theDivision of Toxicoloqy, LA. (Pros'dio of San Francisco, CA).

-,s- SolventL

The positive co~ntrol chemicals were dissolved in grade Idi-methyl sulfoxide (lot 113F-0450) obtained from SigmaC'iernical Co. (St. Louis, MU) . The test compound wasd ssolved in glass-distilled water. The glass-distilledwiter used in this assay was first passed through a Technic

Sebastian and Harbell--3

Model 301 Reverse Osmosis Unit (Seattle, WA), then through aCorning MP-l Mega-Pure System glass distillation unit(Corning Glass Works, Corning, NY) (7).

Chemical Preparation

On the day of dosing, 300 mg of the test compound wasmeasured into a sterile vial and dissolved in glass-distilledwater to achieve a 5% (w/v) solution. Aliquots of thissolution were used to dose the test plates.

Test Strains

Salmonella strains TA97, TA98, TAl00, TA102, TA1535,TA1537, and TA1538 obtained directly from Dr. Bruce Ames,University of California, Berkeley, were used. These strainswere maintained in our laboratory in liquid nitrogen.Quality control tests were run concurrently with the testsubstance to establish the validity of their special featuresand to determine the spontaneous reversion rates.Descriptions of the strains, their genetic markers, and themethods for strain validation are given in the LAIR SOP, OP-STX-I (8).

Test Format

PHYSOSTIGMINE SALICYLATE was evaluated for mutagenicpotential according to the revised Ames method (6). A detaileddescription of the methodology is given in LAIR SOP, OP-STX-l(8).

Toxicity Tests:

Toxicity tests were conducted to determine a sublethalconcentration of the test substance. This toxicity level wasfound by using minimal glucose agar (MGA) plates,concentrations of PHYSOSTIGMINE SALICYLATE ranging from 1.6 x10- 3 mg/plate to 5 mg/plate, and approximately 108 cells ofTAI00 per plate. Top agar containing trace amounts ofhistidine and biotin was placed on the plates. Strainverification was confirmed on the bacteria, along with adetermination of the spontaneous reversion rate. Afterincubation, the growth on the plates was observed. Since thetwo highest doses showed a decreased number of macrocolonies(below the spontaneous rate) and an observable reduction inthe density of the background lawn, the highest dose selectedfor the mutagenicity test was 0.2 mg/plate.

S~bastian and Harbell--4

Mutagenicity Test:

The test substance was evaluated over a 1000-fold rangeo concentrations, decreasing from the minimum toxic level<;he maximum or 1Y it dose) by a dilution factor of 5, both:h and withouc 0.5 ml of the S-9 microsome fraction. The

:;-9 (lot R-315) was purchased from MicrobiologicalA;2ociates, Inc. (Bethesda, MD). A standard S9 mix (4%) waslii;ked (6) . After ll the inqrodients were added, the top aqarw:is mixed, then overlaid on MGA plates. These platesc-ntained 2% glucose and Vogel Bonner "E" concentrate (9).Plates were incubated upside down in the dark at 370C for 48i:irs. Plates were prepared in triplicate, and the averagerevertant counts were recorded. The average number ofrevertants at each dose level was compared to the averagenumber of spontaneous revertants (negative control). Thespontaneous reversion rate (with and without 3-9) wasm:nIutored by averaging the counts from two determinations runsinultaneously with the test compound. The spontaneous:_ 7ersion rate was determined by inoculating one set of

-ites before and one set after the test compound plates soK t any change in spontaneo :s reversion rate during the4 sing procedure would be detected. This spontaneousrtversion rate was also compared with historical values forthis laboratory and those cited in Maron and Ames (6).S'.erility and strain verification controls were runconcurrently. All reagents, test compounds, and media werechecked for sterility by plating samples of each on MGA mediaand incubating them at 370C with the t l. lates. Thei-itegrity of the different Salmonella sLriins used in thea say was verified by the following standard tests:

-Lack of growth (inhibition) in the presence of crystal violetwhich indicates that the prerequisite alteration of thelipopolysaccharide layer (LP) of the cell wall is present.

-Growth in the presence of ampicillin-impregnated diskswhich indicates the presence of an ampicillin-resistant RFactor in all strains except TA1535, TA1537, and TA1538.

-Lack of growth (inhibitlon) following exposure toultraviolet light which indicates the absence of the DNAexcision-repair mechanism (all strains except TAl02).

Six known mutagens were tested as positive controls tocnfirm the responsiveness of the strains to the mutationp-ocess. Each strain must be tested with at least onep sitive control but may be tested with several. These

r',c-undn, benzo[ajpyrene (lot 18C-0378), 2-aminofluorene (lot!,47), 2-amino~inthracene (lot 020797), mit.omycin-C (lot* V-065), N-methyl-N'-nitro-N-nitrosoguariidine (lot 127C-

Sebastian and Harbell--5

0342), and 4-nitroquinoline-n-oxide (lot 84F-0572), wereobtained from Sigma Chemical Co. (St. Louis, MO). The testcompound and mutagens were handled during this study inaccordance with the standards published in NIH Guidelines forthe Laboratory Use of Chemical Carcinogens (DHHS PublicationNo. (NIH) 81-2385, May 1981).

Data Interpretation

According to Brusick (10), a compound is consideredmutagenic if a positive dose response (correlated doseresponse) over three dose concentrations is achieved with atleast the highest dose yielding a revertant colony countgreater than or equal to twice the spontaneous colony countfor the tester strains TA98 and TAI00, or three times thespontaneous colony count for strains TA1535, TA1537, andTA1538. A strong correlated dose response in strain TA100without a doubling of the individual colony count may also beconsidered positive.

Maron and Ames (6) consider a compound mutagenic intester strains TA97 and TA102 if a correlated dose responseover three concentrations is achieved with the highest doseyielding a revertant colony count greater than or equal totwice the spontaneous colony count.

Deviations from the Protocol/SOP

A 72-hour rather than a 48-hour incubation period wasused for strain TA102 only. This gave the colonies anadditional 24 hours to grow thus enabling all revertantcolonies to be detected with the colony counter (Maron 1985,personal communication). Colony counts for all other strainswere recorded after 48 hours.

Storage of the Raw Data and Final Report

A copy of the final report, study protocols, raw data,SOPs, and an aliquot of the test compound will be retained inthe LAIR Archives.

RESULTS

On 16 May 1986, the toxicity of PHYSOSTIGMINE SALICYLATEwas determined (Table 1). For this experiment all sterility,strain verification and negative controls were normal (Table1). Exposure of the tester strain (TAl00) to the two highest.doses showed a decrease in the number of macrocolonies, and an

.-bastian and Harbell--6

TABLE 1: TOXICITY LEVEL DETERMINATION FORPHYSOSTIGMINE SALICYLATE

GLP STUDY NUMBER 87001

TOXICITY DETERMINATION REVERTANT PLATE COUNT (TA1001

"ONCENTRATION iAN j BACKGROUND LAWN*

START NEGATIVE CONTROL 76 4.7 NL5.0 mg/plate 0 - ST.0 mg/plate 21 7.6 ST

).2 mg/plate 84 10.6 NL!.04 mg/plate 80 10.4 NL

).008 mg/plate 86 7.8 NL).0016 mg/plate 71 7.0 NL.,D NEGATIVE CONTROL 91 7.2 NL

STRAIN VERIFICATION FOR TOXICITY DETERMINATION

?iISTIDINE REQUIREMENT NG!MPICILLIN RESISTANCE G

i\ NG

"RYSTAL VIOLET SENSITIVITY NG'ERTLITY CONTROL NG

STERILITY CONTROL FOR TOXICITY DETERMINATION

"ATERIAL TESTED O V *•INIMAL GLUCOSE AGAR P; ATES NG70P AGAR NGTILUENT WATER NGN:UTRIENT BROTH NG-EST COMPOUND (HIGHEST DOSE) NG

SNL=Normal Lawn, G=Growth, NG=No Growth, ST=Slight Toxicity

Sebastian and Harbell--7

observable reduction in the density of the background lawn,indicating chemical toxicity. Therefore, the highest doseselected for the mutagenicity test was 0.2 mg/plate. Normalresults were obtained for all sterility and strain verificationtests during the Ames Test performed on 13-15 August 1986(Table 2). PHYSOSTIGMINE SALICYLATE did not induce anappreciable increase in the revertant colony counts relative tothose of the negative control cultures (Table 3). A tabularpzesentation of the raw data is included in Appendix B.

DISCUSSION

Certain test criteria must be satisfied before an AmesTest can be considered a valid assessment of a compound'smutagenic potential. First, the special features of the Amesstrains must be verified. These features includedemonstration of ampicillin resistance, alterations in the LPlayer, and deficiency in DNA excision-repair (except TAI02).Second, the Salmonella strains must be susceptible tomutation by known mutagens. Third, the optimal concentrationof the test compound must be determined by treating TA100with a broad range of doses and observing the potential toxiceffects on formation of macrocolonies and microcolonies. Ifthese tests are performed and expected data are obtained,then the results of an Ames Test can be considered valid.

After validation of bacterial strains and selection ofoptimal sublethal doses, PHYSOSTIGMINE SALICYLATE wasevaluated in the Ames Test. Criteria for a positive responseinclude both a correlated dose response over three doseconcentrations, and a revertant colony count at least twotimes (TA97, TA98, TAl00, TA102) (5,10) or three times(TA1535, TA1537, TA1538) (6,8) the spontaneous revertantcolony count. PHYSOSTIGMINE SALICYLATE did not induce therequisite dose-response relationship or the increase inrevertant colony counts necessary for a positive response.Thus, the results of this test indicate that PHYSOSTIGMINESALICYLATE is not mutagenic when evaluated in the Ames Test.

CONCLUSION

PHYSOSTIGMINE SALICYLATE was evaluated for mutagenicpotential in the Ames Test, in both the presence and theabsence of metabolic activation, and did not induce apositive mutagenic response under conditions of this study.

Sebastian and Harbell--8

"ABLE 2: STRAIN VERIFICATION AND STERILITY TESTINGFOR THE MUTAGENICITY DETERMINATION OF

PHYSOSTIGMINE SALICYLATE

GLP STUDY NUMBER 87001

STRAIN VERIFICATION

OBSERVATIONS*

HIS'T )INE AMPIC I JL N UV CRYSTAL STERILITY:_'fj ElhLjREITNEREPAIE v I OT -CQNTIOL

'I'A97 NG G NG NG NGTA98 NG G NG NG NGYAi O 0 NG G NG NG NGA 102 NG G G NG NG7'A1535 NG NG NG NG NGTA1537 NG NG NG NG NGTA1538 NG NG NG NG NG

STERILITY CONTROL FOR MUTAGENICITY DETERMINATION

MATERIAL TESTED OBSERVATION*

INI MAl, GLUCOSF AGAR PLATES NG'l'0P AGAR NGP;UENT WATEI NGNUTRIENT BROTH NGTEST COMPOUND (HIGHEST DOSE) NGS-9 NG

G = Growth, NG = No Growth

Sebastian and Harbell--9

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REFERENCES

1. Kluwe WM, Chinn JC, Feder P, Olson C, Joiner R.Efficacy of pyridostigmine pretreatment against acutesoman intoxication in a primate model (Paper No. IX-l).In: Proceedings of the sixth medical chemical defensebioscience review. Columbia, MD (4-6 Aug) 1987:227-234.

2. Leadbeter L, Inns RH, Rylands JM. Treatment ofpoisoning by soman. Fundam Appl Toxicol 1985; 5:S225-S231.

3. Harris LW, McDonough JH, Sticher DL, Lennox WJ. Pro-tection against both lethal and behavioral effects ofsoman. Drug Chem Toxicol 1984; 7:605-624.

4. Karczmar AG. History of the research with anticholin-escerase agents. In: International Encyclopedia ofPharmacology and Therapeutics. Oxford and New York:Pergamon Press, 1970 (Section 13) 1:1-8.

5. Ames BN, McCann J, Yamasaki E. Methods for detection ofcarcinogens and mutagens with Salmonella/MammalianMicrosome Mutagenicity Test. Mutat Res 1975;31:347-364.

6. Maron DM, Ames BN. Revised methods for the SalmonellaMutagenicity Test. Mutat Res 1983;113:173-215.

7. Operation of the Technic Model 301 Reverse Osmosis Pre-treatment Water System and the Corning Model MP-1 GlassStill. LAIR Standard Operating Procedure OP-STX-94,Presidio of San Francisco, California: Letterman ArmyInstitute of Research, 29 July 1985.

8. Ames Salmonella/Mammalian Microsome Mutagenesis Test.LAIR Standard Operating Procedure OP-STX-l, Presidio ofSan Francisco, California: Letterman Army Institute ofResearch, 29 August 1986.

9. Vogel HJ, Bonner DM. Acetylornithinase of E. coli:Partial purification and some properties. J Biol Chem1956;218:97-106.

10. Brusick D. Genetic toxicology. In: Hayes AW, ed.Principles and methods of toxicology. New York: RavenPress, 1982:223-272.

Se-bastian and Harbell---12

AP'PENDICES

A?PENDIX A: Chemical Data.................................... 13

AvPENDIX B: Individual "late Scores......................... 15

Sebastian and Harbell--13

APPENDIX A: Chemical Data

Chemical Name: Physostigmine salicylate

Other Names: Eserine salicylate; Physostigmine, 2-hydroxybenzoate; 1, 2, 3, 3a, 8, 8a-Hexahydro-1, 3a, 8-trimethylpyrrolo[2,3-b]indol-5-ol methylcarbamate(ester), (3aS-cis)-, mono (2-hydroxybenzoate) (salt)

Lot Number: BL25591

Chemical Abstracts Service Registry Number: 57-64-7

LAIR Code: TW73

WRAIR Code: WR 6570AM

Chemical Structure:

CH 3

CH 3 II7 OyNH2oH+

I I HOCH 3 CH 3

Molecular Formula: C15H21N302 ° C/H603

Molecular Weight: 413.47

Analytical Data:

The test compound was analyzed by the sponsors and theidentity confirmed by UV and IR spectroscopy, high pressureliquid chromatography, mass spectrometry and elementalanalysis.' Based on HPLC analysis of this test compound incomparison with the USP physostigmine salicylate referencestandard, lot BL25591 contains 66.7% (100.1% of theory)physostigmine base and 33.7% (100.8% of theory) salicylicacid or 100.4% physostigmine salicylate. 1

HPLC analysis of physostigmine salicylate in this labwas performed using a Hewlett-Packard 1090 HPLC systemequipped with a diode array detector. The compound waschromatographed under the following conditions: silica

SEbastian and Harbell--14

APPENDIX A (cont.) : Chemical Data

column (4.6 x 100 mm, Brownlce Labs, Inc.); mobile phase, 15%acetonitrile/buffer (0.01M Na2HPO4 with 0.0025M tetramethyl-i:irmonium chloride); flow rate, 1.5 ml/min; wavelengthmcnitored, 210 rim. The compound eluted as two peaks withr~tention times of 0.9 min (salicylic acid), and 3.9 min( L1ysostigmine) 2

] (KBr): 3320(broad), 2964, 2325, 1744, 1629, 1594,l ', 1460, 1383, 1326, 1245, 1203, 1184, 1151, 1140, 1087,iCO6, 993, 944, 860, 807, 754, 704, 667, 382 cm-1. 3 The IRzpectrum was identical to that provided by the sponsors i .

Sct. rce: Bill EllisDivision of Experimental TherapeuticsWalter Reed Army Institute of ResearchWashington, DCRequested by LTC Jurgen von Bredow, PhD, MSC

V :,,nmr i V, Bon f,,z A, dn(I , im P. Assay of phynost iqmineSi cy tIto, WR- (W AM, L25591. Menlo Park, CA: .NI'

j.-ei-national, 1 November 1986; Report no. 553.

'2\hec.er CR. Toxicity testing of antidotos of chemicalwarfare agents. Laboratory notebook #85-12-024.1, pp 2-11.Letterman Army Istitute of Research, Presidio of SanFrancisco, CA.

3Wheeltr CR. Toxicity testing of antidotes of chemicalwarfare agents. Laboratory notebook #85-12-024.3, pp 10-11.Letterman Army Institute of csearch, Presidio of SanFrancisco, CA.

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