CI j~tiztute Rfeport No. 314 D rTICt-LECTE

Mu~agenic Potential of 12b4-NPina -oloxymetly1) Pyridinium] Ethanei Dichloride

t-iemihydrate in t he Ames SalrnonelMuMamma'inMCrosome Mutagenicity Test

Suzatme E. Sebastian, B3A, SPC-, USAand

Don T. Korte, rPhD, M-,NSC

-* - GENETIC TrOXICOLOGY BRANCHDIVISION OF TOXICOLOGY

BESTI AVAILABLE COPYOctober 19.M

Troxilology Scrics: 193

LETTERMAN ARMY INSTITUTE OF RESELARCH -PUrSI 010 OF SAN FRANCISCO. CALIFOdNIA 94129,,,

B 23

St oj: Poentiha of 1, 2 -[l[i4-(N-Pinacoloynethyl)Pyridiniunt Iflihane Dichiuride!! 'it~ihy't!ate in the Ames SalroneMl/Mammalian Microwome Mutagenicitv Test (ToxicologySeries 193)--Sebastian and Korte

This document has been npproved for public release Ind sale; its disitribution is

Devsrov this report when it is no longer needed. Do not return to the originator.

Citation of Irade names in this report does not :onstitute an official endorsementor approval of the use of such items.

T1iis material has been reviewed by Letterman Army Institute ofResearch and there is no objection to its presentation and/orptibtication. The 6pinions or assertions contained herein are theprivate views of the author(s) and are not to be construed as officialor as reflecting the views of the Department of the Army or theDepartment of Defense. (AR 360-5)

Edwin S. Beatrice (date)COL, MCCommanding

UNCLASSIFIED

SECURITY CLASSIFICATION OF THIS"O PAE1C_________________ Form Approved

REPORT DOCUMENTATION PAGE OMBNO. 0704-01w

Is. REPORT SECURITY CLASSIFICATION lb RESTRICTIVE MARKINGS

2. SECURITY CLASSIFICATION AUTHORITY 3 DISTRIBUTION/AVAILABILITY OF REPORT.... IApproved for public release; distribution

Zb. DECLASSIFICATIONI DOWNGRADING SCHIEDULE is unlimited.

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

Institute Report No. 314

G. NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a NAME OF MONITORING ORGANIZATIONGenetic Toxicology Branch I) Walter Reed Army Institute of ResearchDivision of Toxicology ISGRD-ULE-T

6c. ADDRESS (Cty, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)Letterman Army Institute of Research Washington, D.C. 20307-5100Presidio of San Francisco, CA 94129-6800

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

Research & Development CommandSk. ADDRESS (City, State, .nd ZIP Code) 10. SOURCE OF FUNDING NUMBERSFort Detrick PROGRAM PROJECT ITASK WORK UNITFrederick, MD 21701-5012 ELEMENT NO NO NO. CCESSION NO.

67234 A875 BC DA CH036611. TITLE (include Secufty Classification) 'Mutagenic potential of 1,2-bis[4- (N-pinacoloxymethyl)pyridinium]ethane dichloride hemihydrate in the Ames Salmonella/mammalian microsome mutagenicity test

12. PERSONAL AUTHOR(S) Suzanne E. Sebastian and Don W. Korte, Jr.

13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 15. PAGE COUNTInstitute FROM 4/21/86 TO S1Z41861,I 19

16. SUPPLEMENTARY NOTAnONToxicology Series 193

17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP Mutagenicity, Genetic toxicology, Ames Test, 1,2-BIS[4-(N-

PINACOLOXYMEWYL) PYRIDINIUM]"ANE DI(CILORIDE BfIHYDRATE,oxime

19. ABSTRACT (Continue on reverse if necessaly and identify by block number)The mutagenic potential of 1,2-BIS4-N-PINACOLXYETiYL)PYRIINIUETANE DILORIDE

HVIIIHYDRATE was assessed by using the Ames Salmonella/Mammalian Microsome MutagenicityTest. Tester strains TA97, TA98, TAoo, TATn"T- SS , TA1537, and TAIS38 were exposedto doses ranging from 1.0 mg/plate to 0.00032 mg/plate. The test compound was not muta-genic under conditions of this test.

20. DSTRIBUTION/AVAILABILITY OF ABSTRACT 121 ABSTRACT SECURITY CLASSIFICATIONMUNCLASSIFIED/UNLIMITED C3 SAME AS RPT 0 DTIC USERS iunclassified

22a. NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (Include Area Code) 22c. OFFICE SYMBOLEdwin S. Beatrice, COL MC 415-561-3600 SGRD-ULZ

DD Form 1473, JUN 36 Previous edtions are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

UNCLASSIFIED

ABSTRACT

The mutagenic potential of 1,2-BIS(4-(N-PINACOLOXYMETHYL) PYRIDINIUM] ETHANE DICHLORIDE HEMIHYDRATEwas assessed by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Test. Tester strains TA97, TA98, TA100, TA102,TA1535, TA1537, and TA1538 were exposed to doses ranging from1.0 mg/plate to 0.00032 mg/plate. The test compound was notmutagenic under conditions of this test.

Key Words: Mutagenicity, Genetic Toxicology, Ames Test, 1,2-BIS [4- (N-PINACOLOXYMETHYL) PYRIDINIUM]ETHANE DICHLORIDEHEMIHYDRATE, oxime.

Accesior) F-or

NITIS CRA&?

4TI T,,E

J u s..f -. -. .. .

By.

D~s i, ';,".. . 'I

61-Ii-J

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 CommandWalter Reed Army Institute of Research

Washington, D.C. 20307-5100

PROJECT/WORK UNIT/APC: 3MI62734A875/308/TLEO

GLP STUDY NUMBER: 86002

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

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

REPORT AND DATA MANAGEMENT:

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

TEST SUBSTANCE: 1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE

INCLUSIVE STUDY DATES: 21 April - 24 May 1986

OBJECTIVE:

The objective of this study was to determine themutagenic potential of 1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE (LAIR CodeTP62) by using the Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

iii

ACKNOWLEDGMENTS

MAJ John W. Harbell, PhD, MSC; SGT Lillie D. Witcher,BS; and Ms. Joanne Wong provided research assistance.

iv

SIGNATURES OF PRINCIPAL SCIENTISTS INVOLVED IN THESTUDY

We, the undersigned, declare that GLP Study 86002 wasperformed under our supervision, according to the proceduresdescribed herein, and that this report is an accurate recordof the results obtained.

DON W. KORTE, Jr, Y1D / DATEMAJ, MSCStudy Director

SU ,EAE. SEBASTIAN, BA / DATE

Principal Investigator

CONRAD R. WHEELER, PhD / DATEDACAnalytical Chemist

vI

DEPARTMENT OF THE ARMY

LETTERMAN ARMY INSTITUTE OF RESEARCH

' 4 M PRESIDIO OF SAN FRANCISCO, CALIFORNIA 94129-6800

EIPLV TO

ATTENTION Or

SGRD-ULZ-QA 1 November 1988

MEMORANDUM FOR RECORD

SUBJECT: GLP Compliance for GLP Study 86002

I. This is to certify that in relation to LAIR GLP Study86002, the following inspections were made:

15 April 1986 - Protocol Review21 May 1986 - Plate Incorporation (TP62)17 March 1987 - Plate Incorporation (TP64)20 March 1987 - Plate Counting (TP64)

2. The institute report entitled "Mutagenic Potential of 1,2-Bis[4-(N-Pinacoloxymethyl) Pyridinium] Ethane Dichloride Hemihydratein the Ames Salmonella/Mammalian Microsome Mutagenicity Test,"Toxicology Series 193, was audited on 23 April 1987.

CAROLYN' M. LEWISChief, Quality Assurance

vi

TABLE OF CONTENTS

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

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 ................................ 1

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

Test Compound ......................................... 2Test Solvent ......................................... 2Chemical Preparation .................................. 2Test Strains ......................................... 2Test Format .......................................... 31L'a Interprecation ................................... 5Deviations from the Protocol/SOP ..................... 5Storage of the Raw Data and Final Report ............. 5

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

DISCUSSION ............................................... 10

CONCLUSION ............................................... 10

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

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

Appendix A: Chemical Data .......................... 13Appendix B: Individual Plate Scores ................ 14

OFFICIAL DISTRIBUTION LIST ................................ 19

vii

Mutagenic Potential of 1,2-BIS(4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIRYDRATE in the AmesSalmonella/Mammalian Microsome Mutagenicity Test--Sebastian and Korte

INTRODUCTION

1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANEDICHLORIDE HEMIHYDRATE was synthesized for a United StatesArmy Medical Research and Development Command program chargedwith developing more effective oximes for treatment of nerveagent poisoning. The Ames Test is one of a series of testsin which these compounds will be evaluated to determine theirrelative potential for further development.

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 aninexpensive yet highly predictive and reliable test fordetectingj mutagenic activity and thus carcinogenic potential(1)

This evaluation of 1,2-B1514-(N-PINACOLXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE utilizes a revisionof the Ames Salmonella/Mammalian Microsome Mutagenicity Test(2). Two new tester strains, a frame-shift strain (TA97) anda strain carrying an ochre mutation on a multicopy plasmid(TAl02), are added to the standard tester set.

Objective of the Study

The objective of this study was to determine themutagenic potential of 1,2-BIS[4-(N-PINACOL0XYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE (liAR Code TP62) byusing th- revised Ames Salmonella/Mammalian MicrosomeMutagenicity Test.

Sebastian and Korte--2

MATERIALS AND METHODS

Test Compound

Chemical Name: 1,2-EPIS[4- (N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYPATh

LAIR Code Numrber: TP62

Physical State: White crystalline solid

Source: SRI International, Menlo Park, CA

Storage: IL,2-BIS[4- (N-PINACCLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE was received from SRIinternational, 333 Ravenswood Ave., Menlo Park, CA 94025 andassigned the LAIR Code number TP62. The test compound wasstored at room temperature (210C) until used.

Chemical Properties/Analysis: Data provided by SRII:.tornational characterizing the chemical composition and,rity of the test. material are presrnted in Appendix A calonaw L confirmotory analynis of the test materi.-, r,.r fom.d hy

t I. 1'ivision of T:-:icozlgy, LAIR (Presidio ol Sn ro

The positive control z-hemicals were dissolved in grade I<:!methyl sulfoxide (lot 113F-0450) obtained from SigmaC!.e-nical Co. (St. Louis, MO). The test chemical wasdissolved in glass distilled water. Reagent grade water ufdin this assay ",s first passed through a Technic Model 301Reverse Osmosis U:it (Seattle, WA), then through a Co:ninoM-I Mega Pure System glass distillation unit (Corning GlassWorks, Corning, NY) (3)

Chemical Preparation

On the day of dosinc, 300 mg zf the test compound wasreasured into a sterile via]. 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, TAI00, TA102, TA1535,TA1537, and TA1538 obtained directly from Dr. Bruce Ames,University ot California, Berkeley, were used. These strainswere maintained in our laboratory in liquid nitrogen.

Sebastian and Korte--3

Quality control tests were run concurrently with the testsubstance to establish the validity of their special featuresand to determine the spontaneous reversion rate.Descriptions of the strains, their genetic markers, and themethods for strain validation are given in the LAIR SOP, OP-STX-1 (4).

Test Format

1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANEDICHLORIDE HEMIHYDRATE was evaluated for mutagenic potentialaccording to the revised Ames method (2). A detaileddescription of the methodology is given in LAIR SOP, OP-STX-l(4).

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 1,2-BIS(4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICILORIDE HEMIHYDRATE ranging from 1.6 x 10

- 3

iwq/plate to 5 mg/plate, and approximately 108 cells of TAl00per plate. Top agar containing trace amounts of histidineand biotin was placed on the plates. Strain verification wasconfirmed on the bacteria, along with a determination of thespontaneous reversion -ate. After incubation, the growth onthe plates was observed. Since the highest dose showed adecreased number of macrocolonies (below the spontaneousrate) and an observable reduction in the density of thebackground lawn, the highest dose selected for themutagenicity test was 1.0 mg/plate.

Mutagenicity Test:

The test substance was evaluated over a 1000-fold rangeof concentrations, decreasing from the minimum toxic level(the maximum or limit dose) by a dilution factor of 5, bothwith and without 0.5 ml of the S-9 microsome fraction. TheS-9 (batch R-315) was purchased from MicrobiologicalAssociates Inc. (Bethesda, MD) . The optimal titer of this S-9, as determined by Microbiological Associates Inc., was0.75 mg protein/plate. After all the ingredients were added,the top agar was mixed, then overlaid on MGA plates. Theseplates contained 2% glucose and Vogel Bonner "E" concentrate(5). Plates were incubated upside down in the dark at 37'Cfor 72 hours (Maron 1985, personal communication). Plateswere prepared in triplicate, and the average revertant countswere recorded. The average number of revertants at each doselevel was compared to the average number of spontaneous

Sebastian and Korte--4

revertants (negative control). The spontaneous reversionrate (with and without S-9) was monitored by averaging thecounts from two determinations run simultaneously with thetest compound. The spontaneous reversion rate was determinedby inoculating one set of plates before and one set after thetest compound plates so that any change in spontaneousreversion rate during the dosing procedure would be detected.This spontaneous reversion rate was also compared withhistorical values for this laboratory and those cited in

Mar n and Ames (2). Sterility and strain verificdtioncont.rols were run concurrently. All reagents, te';tcompounds, and media were checked for sterility by platingsamples of each on MGWA media and incubating them at 37'C withthe test plates. The Salmonella strains were verified by astandard battery of tests. The integrity of the differentSalmonella strains used in the assay was verified by thefollowing standard tests:

-Lack of growth (inhibition) in the presence of crystalviolet which indicates that the prerequisite alterationof the lipopolysaccharide layer (LP) of the cell wall ispresent.

-Growth in the presence of ampicillin-impregnated diskswhich indicates the presence of an ampicillin-resistantR Factor in all strains except TA1535, TA1537, andTA1538.

-Lack of growth (inhibition) following exposure toultraviolet light which indicates the absence of the DNAexcision-repair mechanism (for all strains exceptTA102).

Five known mutagens were tested as positive controls toconfirm the responsiveness of the strains to the mutationFrccess. Each strain must be tested with at least onepositive control but may be tested with several. Thcscompounds, benzo[a]pyrene (lot 18C-0378), 2-aminofluorene(lot 021547), 2-aminoanthracene (lot 020797), mitomycin-C(lot 015F-0655), and N-methyl--N'-nitro-N-nitrosoguanidine(lot 127C-0342), were obtained from Sigma Chemical Co. (St.Louis, MO). The test compound and mutagens were handledduring this study in accordance with the standards publishedin NIH Guidelines for the Laboratory Use of ChemicalCarcinogens (DHHS Publication No. (NIH) 81-2385, May 1981).

Sebastian and Korte--5

Data Interpretation

According to Brusick (6), 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 (2,4). A strong correlated dose response in strainTA100 without a doubling of the individual colony count mayalso be considered positive.

Maron and Ames (2) 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. This gave the colonies an additional 24 hours to grow,thus enabling all revertant colonies, especially those ofTAI02, to be detected with the colony counter.

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 1,2-BIS[4-(N-PINACOLOX'?METHYL)PYRIDINIUMIETHANE DICHLORIDE HEMIHYDRATE wasdetermined (Table 1). For this experiment all sterility,strain verification and negative controls were normal (Table1). Exposure of the tester strain (TA100) to the highestdose showed a decrease in the number of macrocolonies and anobservable reduction in the density of the background lawnindicating chemical toxicity. Therefore, the highest doseselected for the mutagenicity test was 1.0 mg/plate. Normalresults were obtained for all sterility and strainverification tests during the Ames Test performed on 21-24 May1986 (Table 2). 1,2-DIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE did not induce any appreciableincrease in the revertant colony counts relative to those ofthe negative control cultures (Table 3).

Sebastian and Korte--6

TABLE 1: TOXICITY LEVEL DETERMINATION FOR TP62

GLP STUDY NUMBER 86002

TOXICITY DTERMATON REVERTANT PLATE COUNT (TAIQD)0

CONCENTRATION MEAN +IS BACKGROUND LAWW.*

START RUN NEGATIVE CONTROL 77 7.5 NL5.0 mg/plate 1 1.0 NG1.0 mg/plate 49 9.6 ST0.2 mg/plate 62 5.9 NL0.04 mg/plate 74 10.5 NL0.008 mg/plate 61 22.5 NL0.0016 mg/plate 74 12.5 NLEND RUN NEGATIVE CONTROL 92 10.1 NL

STRAT VERIFICATION OR TOXICITY DETERMIN T,-N

TA00*

HISTIDINE REQUIREMENT NGAMP ICILLIN RESISTANCE GUV NGCRYSTAL VIOLET SENSITIVITY NGSTERILITY CONTROL NG

STERILITY CONTROL FOR TOXICITY DETERMINATION

MATERIAL TESTED OBSERVATIONAMINIMAL GLUCOSE AGAR PLATES NGTOP AGAR NGDILUENT WATER NGNUTRIENT BROTH NGTEST COMPOUND (HIGHEST DOSE) NG

*NL=Normal Lawn, G=Growth, NG=No Growth, ST=Slight Toxicity

Sebastian and Korte--7

TABLE 2: STRAIN VERIFICATION AND STERILITY TESTINGFOR THE MUTAGENICITY DETERMINATION OF TP62

GLP STUDY NUMBER 86002

STRAIN VERIFICATION

OBSERVATIONS*

HISTIDINE AMPICILLIN UV CRYSTAL STERILITYSTRAIN REOUTREMENT RLFTTA1C REPAIR WIOLFT ONTIR

TA97 NG G NG NG NGTA98 NG G NG NG NGTAI00 NG G NG NG NGTA102 NG G G NG NGTA1535 NG NG NG NG NGTA1537 NG NG NG NG NGTA1538 NG NG NG NG NG

STERILITY CONTROL FOR MUTAGENICITY DETERMINATION

MATERIAL TESTED OBSERVATI N*

MINIMAL GLUCOSE AGAR PLATES NGTOP AGAR NGDILUENT WATER NGNUTRIENT BROTH NGTEST COMPOUND (HIGHEST DOSE) NGS-9 NG

*G= Growth, NG = No Growth

S

Sebastian and Korte--8

10NU 'n r4C 1 ) DmC -

** w -40- -414C

cN C - -4 C-4 4 - 4~ M 11 N r- -4 Q-H +1 +1+1 +1 +1+1 +1 +1 -+1 +1+1+1+1H C

(a

14 - -4 4 -4-4 r4 1- N1 .- 4 N-4 .- I C-I

E-0 0

S0 m C1 CC~)( LO I -

U A& -+1 + +1+1 +1+1 +i +1 +1+1+1 +1 +1++1 1 +( +1

HLO C J~(N. (N r h NW040 0 -' 0 C ON0Mr-C

-4 M04 -4 -- 40 r- -4mq L 1

(1) 0d

C> r OWMC> - N0 *N1 )cc MW - -4M ) 17 4 0- lMMNC 0UlO.4(N Kr -4U

-~ +1+ 1+-1+H11+ H-H+ 1+ 1+ 1+ 1 M4

- toL 0D-I 'I r- W )(10 0 , I M ) C'( 1 4-i 1

k ''r- i -4(j -g4-04r4 CNFO CJ 04 J(N C4-4 C

o m ITa% N 10) (1)

>v r-(' C) CN U-O*0 *) - a

0 n N CC C14 CNW .(

N ~ ~ -_;- - 4 r- - 0 10)+i +1 +1H+1 +1 +1 +1+1 -H + -H H+1-H +i-H +1I

~E4 4

U') %D

Q-)

43~~~0F Z 1E0)EM E 0 13)e C, 0-4E 110-) tl 01Ol 0)1:;)0) 5J4-

5 =L=L EE co c)FE L = =LE E m -i O(r. IT C > CD-T C)S- C

>4 c C-) LOC )C% DC C o( C ,C C 4C ( DC

0(D(Ne'.C; e C- . C t N ( NC;CC. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ U >1- 0J.c~~cl1 E'-E4-E'- -

Se~bastian and Korte--9

Lr +1 +1 +1+1 -H--H +1 +1++II +:lI3+

E- 0

4-4.

00

U 4--

'H %0 mA, ) CD -qC3()LO0

44.4 r-0 L

Lr +1+ + + ++1H+1 +1 +141+1 +1+1 +11 +1 Ms.-1)

0

z E~-P4 O(

-W 'D M D C C'JC 440

0 N 70 C 4CN -44i1

>10

0r 0 0)liU U (0)

:3 CDCN.- aj o

:,,bi:t tian rid KoI Le--10

A tabulaz prebentation of the raw data is included inAppendix B.

DISCUSSION

Certain test criteria must be satisfied before an Ames:est can be considered a valid assessment of a compound'smutagenic potential. First, the special features of the Ames.rains must be verified. These features includedeonstration of ampicillin resistance, alterations in the LFlayer, and deficiency in DNA excision-repair (except 7A102) .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. Tfthese 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, 1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE was evaluated in theAres Test. Criteria for a positive response include both acorrelated dose response over three dose concentrations, anda revertant colony count at least two times (TA97, TA98,TAl00, TAl02) (1,6) or three times (TA1535, TA1537, TA1538)(2,4) the spontaneous revertant colony count. 1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUM]ETHANE DICHLORIDE HEMIHYDRATE didnot induce the requisite dose-response relationship or theincrease in revertant colony counts necessary for a positiveresponse. Thus, the results of this test indicate that 1,2-IS[4-(N-P INACOLOXYMETHYL)PYRIDINIUM]ETHiANE DICHLORIDE

HEMThYDRATE is not mutagenic when evaluated in the Ames test.

CONCLUSION

1,2-BIS[4-(N-PINACOLOXYMETHYL)PYRIDINIUMIETHANEDICHLORIDE HEMIHYDRATE was evaluated for mutagenic potentia2ir the Ames Test, in both the presence and the absence ofmetabolic activition, and did not induce a positive mutagenicresponse under conditions of this study.

Sebastian and Korte--li

REFERENCES

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

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

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

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

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

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

Sebastian and Korte--12

APPENDICES

APPENDIX A: Chemical Data....................................13

APPENDIX 9: Individual Plate Scores......................... 14

Sebastian and Korte--13

APPENDIX A: Chemical Data

Chemical Name: 1,2-Bis[4-(N-pinacoloxymethyl)pyridinium]

ethane dichloride hemihydrate

SRI Reference Number: 6868-16

LAIR Code Number: TP62

* Chemical Structure:

(CH3 )3CH(CH 3)OCH 2- N CH2 -- 2C 1/2H 2 0

2

Molecular Formula: C26H42N202C12 1/2 H20

Molecular Weight: 494.5

Physical State: White crystalline solid

Analytical Data:NMR (300 MHz, D20): 50.63 (s, 9 H, C(Cl13)3), 0.96 (d,

J = 6.6 Hz, 3 H, CH(CI3)-O), 3.27 (d, J = 6.3 Hz, 1 H,Ci(CH3)-O, 3.34 (s, 2 H, N--CH2), 5.74 (m, J = 5.7 Hz, 2 H,O-CH2-N), 7.85 (d, J 7 1.2 Hz, 2 H, aromatic protons meta topyridinium nitrogen), 8.75 (d, J = 7.2 Hz, 2 H, aromaticprotons ortho to pyridinium nitrogen).* The NMR spectrumobtained upon receipt of the compound corresponded closely tothe spectrum provided by the source (obtained in DMSO). Anydiscrepancies were due to the difference in solvents as wellas the higher field strength and greater resolution of theNMR used to analyze the compound in our lab. No peaks otherthan those attributable to the compound were observed in theNMR spectrum.

Stability:NMR data demonstrate that the compound is stable in

water (D20) for at least 8 days.t

Source: Clifford D. BedfordSRI InternationalPhysical Sciences DivisionMenlo Park, CA

Wheeler CR. Toxicity Testing and Antidotes for ChemicalWarfare Agents. Laboratory Notebook #85-12-024, pp 3-4.Letterman Army Institute of Research, Presidio ofSan Francisco, CA.

Sebastian and Korte--14

C' C4

a4 E

tL 0 m >O

ElE

EJ E-' E-4

00

044

04 w 2z -

:3 H 00a 4 ENO0 1

H 0F"

HN~ z oD r- m r-0E H

z >4 0w x 004 -0

Q: 0

IOL4z-

V~

0-4 1-4 CN ML 0 - N

C4 w.: O4(JC l 0 .- i" 0) 0E-4 - P x E E-4E- 4 0

0 11 M0 0 111l (a

C) n401 0 Q A N04 g

Seb~astian and Korte-- 15

, w. C ' 4 I DC N C -3 -.C14 .N ( 14 CN.N

>44

0

Ui -4 .- LinoA.D - D-O%N C'1N cqC%N ( (N NN M

cc 0

4E-

.1 ON4 -4 C3- - ~

N li C1 ') CN(NC1 c'i(NC1

zE-4

>4 1-4 r-4 -4r-

x C)

z

C4

H3CDC

OF.; C

V)90

0~

Sebastian and Korte--16

IN -%r D' CoLflr

CLICIt-

>40)

0X

-- 4 ~ ~ ~ ~ ~ ' l INM-TN"4C4 y

.A W

* 00

)-4A

0 O (N OOc0 O.D ON -

<U CT\ C0

>: ~ NG ' 14 ) ojC-4I- (NO Iv

o r-CN(" 0~(('

~4-J

zW -U) 0

I C.)

(1:J 0- U)

c'

Sebastian and Korte--l'7

-q~iU (J (N -4 -4 _q -4 -1 -4 -4 -4 -4 -4 -4

a4

14

0Cc'j4a% C 0CDc OC14 C> "co O D LID ('4LA) 00 ri() " C14 .- CN CN N 14NC14 4~

-E-

0- LO (D <D c O C U") U') ma (Y)uC) r- o 10 Lr .- 0%

-4 -4 -i 4 -q .- 4 - 4-4 - 4-4 -4-4 -4 -4-4-4 1

'-4

4 --4 Cj -- 444 -4- -4 -

4-)

o >4

>4

H

0C.) FE

0:4 4 Ez S NOH r.4P Ei k m

E 0 0- C

(N~0 C( N( N (N (%D0 to t.0

E-4-E-HE

Sebastian and Korte---''?

E-41

-4--

E-l

C4 W M: I4J-

r-4 LnOC4 Lt 4 r-I -4 4 -4-4 tf)C -I-4 4

F-I

1.

z

C%4 N (N C% N 4 l (N (1 ( N N

f-I

Sebastian and Korte--19

OI1.ICIAI. DISTRIBUTION LISTCommanderUS Army Medical Research Commander

& Development Command US Army Medical Bioengincering

ATTN: SGRD-RMS/Mrs. Madigan Research & Development LaboratoryFort Detrick. MD 21701-5012 ATTN: Library

Fort Derrick. Bldg 568Dcfense Technical Information Center Frederick. MD 21701-5010ATTN: DTIC/DDAB (2 copies)Cameron Station CommanderAlexandria, VA 22304-6145 US Army Research Institute A

of Environmental MedicineOffice of Under Secretary of Defense ATN: SGRD-UI-RSA

Research and Engineering Kansas Street

ATTN: R&AT (F&LS), Room 3D12Q Natick. MA 01760-5007The PentagounWashingtonl. DC 2(1)0 -3080 Coman iai nder

LIS Army Rescaich Institute ofDASG AAFJMI. Surgical ResearchArmy/Air Force Joint Medical Library Fort Sam Houston, TX 78234-6200Offices of the Surgeons General5109 Lecsburg Pike. Room 670 CommanderFalls Church, VA 22041-3258 US Army Research Institute of

Chemical DefenseHQ DA (DASG-ZXA) ATTN: SGRD-UV-AJWASH DC 20310-2300 Aberdeen Proving Ground, MD 21010-5425

Commandant CommanderAcademy of Health Sciences US Army Aeromedical ResearchUS Army LaboratoryATTN: IISIIA-CDM Fort Rucker. AL. 36362-5000Fort Sam louston, TX 78234-6100

AIR FORCE Office of ScientificUniformed Services University of Research (NI-)

Health Sciences Building 410. Room A217Office of Grants Management Boiling Air Force Base, DC 20332-64484301 Jones Bridge RoadBethesda, MD 20814-4799 USAF School of Aerospace Medicine

Document SectionUS Army Research Office USAFSAMITSKDATTN: Chemical and Biological Brooks Air Force Base, TX 78235-5301

Sciences Division'() Box 12211 ]head. Biological Stiences DivisionRe-warch Triangle Park. NC 277)9 2211 ()FFIIC' OF NAVAl RFSFAR(II

800 North Quincy StreetDirector Arlington. VA 22217-5100ATTN: S(;RI) UWY-I.Walter Reed Army Institute of ResearchWashington. DC. 20307-5100

CommanderUS Army Medical Research Institute

of Infectious DiseasesATTN: SGRD-ULZ-AFort Detrick. MD 21701-5011

CommanderUS Army Medical Bioengineering Researchand )evclopment L.aboratoryArTN S;IRI)-UI)G N1:ort I)ctrick, Bldg 568Frederick. MI) 21701-5010

4/NN